(Genetics, Genomics and Breeding of Crop Plants) Yi-Hong Wang - Tusar Kanti Behera - Chittaranjan Kole - Genetics, Genomics and Breeding of Cucurbits PDF

Handbook of
Molecular and Cellular

Methods in
Biology and Medicine
Third Edition
Handbook of
Molecular and Cellular
Methods in
Biology and Medicine
Third Edition
Edited by
Leland J. Cseke Ara Kirakosyan
Peter B. Kaufman Margaret V. Westfall
Boca Raton London New York
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Contents
Preface��ix
Editors................................................................................................................................................ xi
Contributors.................................................................................................................................... xiii
Part I DNA-Based Technology
Chapter 1 Isolation and Purification of DNA................................................................................ 3

Leland J. Cseke, Chuanfu An, and Chung-Jui Tsai
Chapter 2 DNA Cloning Strategies.............................................................................................. 29

Leland J. Cseke, Geetika Trivedi, Avinash Sreedasyam, Peter B. Kaufman,
and Ara Kirakosyan
Chapter 3 Polymerase Chain Reaction Methodologies................................................................ 37

Ara Kirakosyan, Peter B. Kaufman, Leland J. Cseke, and E. Mitchell Seymour
Chapter 4 Preparation of Nucleic Acid Probes............................................................................ 57

Leland J. Cseke and Luis Rogelio Cruz-Vera
Chapter 5 Southern Blot Hybridization....................................................................................... 77

Chung-Jui Tsai and Leland J. Cseke
Chapter 6 Genomic DNA Libraries.............................................................................................97

Leland J. Cseke, Ara Kirakosyan, and Peter B. Kaufman
Chapter 7 DNA Sequencing and Analysis................................................................................. 141

Leland J. Cseke and Lance Larka
Part II RNA-Based Technology
Chapter 8 Isolation and Purification of RNA............................................................................ 167

Chung-Jui Tsai, Leland J. Cseke, and Scott A. Harding
Chapter 9 Real-Time PCR and qRT-PCR Methodologies......................................................... 187

Ajay Kumar Pandey, Leland J. Cseke, Maria R. Davis, Peter B. Kaufman,
E. Mitchell Seymour, and Ara Kirakosyan
v
vi Contents
Chapter 10 Preparation of RNA Probes...................................................................................... 197

Peter B. Kaufman, Leland J. Cseke, Ara Kirakosyan, Avinash Sreedasyam,
and Geetika Trivedi
Chapter 11 Northern Blot Hybridization..................................................................................... 211

Ara Kirakosyan, Peter B. Kaufman, Chung-Jui Tsai, and Leland J. Cseke
Chapter 12 cDNA Libraries......................................................................................................... 217

Chapter 13 Differential Display................................................................................................... 261

Yuh-Shuh Wang, Scott A. Harding, and Chung-Jui Tsai
Chapter 14 Localization of RNA Expression.............................................................................. 273

Scott A. Harding, Chung-Jui Tsai, and Leland J. Cseke
Part III Protein-Based Technology
Chapter 15 Isolation and Purification of Proteins........................................................................ 291

Leland J. Cseke, Peter B. Kaufman, Ara Kirakosyan,
and Margaret V. Westfall
Chapter 16 Western Blot Hybridization....................................................................................... 313

Peter B. Kaufman, Ara Kirakosyan, Leland J. Cseke,
and E. Mitchell Seymour
Chapter 17 Localization of Protein Expression........................................................................... 331

Ara Kirakosyan, Peter B. Kaufman, Margaret V. Westfall, Michael Mashore,
Casey R. Lu, and E. Mitchell Seymour
Chapter 18 DNA Footprinting and Gel Retardation Assay......................................................... 337

Chapter 19 Protein–Protein and Protein–Ligand Interactions.................................................... 351

Marilyn D. Yoder
Part IV Metabolites
Chapter 20 Isolation and Purification of Metabolites.................................................................. 367

Ara Kirakosyan, Kathleen R. Noon, Maureen McKenzie, Leland J. Cseke,
and Peter B. Kaufman
Contents vii
Chapter 21 Analysis of Metabolites............................................................................................. 381

Kathleen R. Noon, Ara Kirakosyan, Maureen McKenzie,
and Peter B. Kaufman
Part V Genomics, Proteomics, and Metabolomics
Chapter 22 Bioinformatics and Data Analysis............................................................................. 409

Susan Holmes and Omar De la Cruz Cabrera
Chapter 23 Microarray Platforms................................................................................................ 437

Keerthi P. Venkataramanan, Leland J. Cseke, Gopi K. Podila,
Ara Kirakosyan, and Peter B. Kaufman
Chapter 24 Microarray Data Collection...................................................................................... 447

Ara Kirakosyan, and Peter B. Kaufman
Chapter 25 Microarray Data Analysis......................................................................................... 455

Peter B. Kaufman, and Ara Kirakosyan
Chapter 26 Proteomics: Data Collection and Analysis................................................................ 459

Evelyn H. Kim, David E. Misek, and Margaret V. Westfall
Chapter 27 Metabolomics: Data Collection and Analysis........................................................... 471

Taketo Okada and Akira Katoh
Chapter 28 Common Protocols on Membrane Lipid Analysis and Lipidomics.......................... 485

Arun Kumar Das
Chapter 29 Integrated Functional Genomics...............................................................................509

Leland J. Cseke, Feng Chen, Peter B. Kaufman, and Ara Kirakosyan
Part VI Manipulation of Biological Systems
Chapter 30 Inhibition of Gene Expression................................................................................... 521

Joy M. Agee, Lynn Boyd, and Leland J. Cseke
Chapter 31 Inhibition of Protein Synthesis.................................................................................. 549

Peter B. Kaufman, Ara Kirakosyan, and Leland J. Cseke
viii Contents
Chapter 32 Gene Transfer and Expression in Animal Cells........................................................ 557

Sarah E. Kampert, Eric Devaney, and Margaret V. Westfall
Chapter 33 Methods of Stem Cell Lineage Analysis In Situ and in Cell Culture....................... 579
Andrei B. Borisov
Chapter 34 Tissue Engineering of Fully Differentiated Tissue: Lessons from Work

on Cardiac Myocytes................................................................................................. 593
Tamara K. Stevenson and Margaret V. Westfall
Chapter 35 Plant Tissue and Cell Culture.................................................................................... 607

Nidhi P. Chanana, Leland J. Cseke, Ara Kirakosyan, and Peter B. Kaufman
Chapter 36 Gene Transfer and Expression in Plants.................................................................... 629

Ramesh C. Thakur, Leland J. Cseke, Ara Kirakosyan, and Peter B. Kaufman
Part VII Macromolecular Analyses
Chapter 37 Microscopy: Light and Confocal............................................................................... 657

Casey R. Lu, Peter B. Kaufman, and Leland J. Cseke
Chapter 38 Microscopy: Scanning Electron, Environmental Scanning Electron,

and Transmission Electron........................................................................................ 667
Casey R. Lu, Peter B. Kaufman, Ara Kirakosyan, and Leland J. Cseke
Chapter 39 Laser Capture Microdissection and Whole Genome Amplification......................... 687

Ramesh Buyyarapu, Venkateswara Rao Sripathi, Sarah Beth Cseke,
and Ramesh V. Kantety
Preface
Since the publication of the first edition of the Handbook of Molecular and Cellular Methods in
Biology and Medicine in 1995, there have been several milestones in the field of biology. Genome
level sequencing of higher eukaryotes has progressed at an unprecedented speed. Starting with
baker’s yeast (Saccharomyces cerevisiae) in 1996, the organisms whose genetic code has been com-
pletely sequenced now include human (April 14, 2003), Arabidopsis (2000), rice (Oryza sativa)
(2002), balsam poplar (Populus trichocarpa) (2006) with new completions for plants, animals, and
microorganisms being added on at an increasingly rapid pace. The invention of DNA microarray
technology and advances in bioinformatics have generated vast amounts of genomic data, signifi-
cantly boosting the throughput of biological research. Similarly, the development of RNAi technol-
ogy is allowing researchers to selectively knock out specific genes in a wide variety of organisms,
allowing high-throughput analysis of the functions of many thousands of genes. While the molecu-
lar and cellular methods presented in the first edition remain as valuable tools, it is clear that today’s
researchers need an updated tool kit that incorporates conventional as well as modern approaches
to tackle biological and medicinal research in the post-genomics era.
We have significantly revised this CRC Press handbook for the third edition in order to address these
recent changes. All protocols have been evaluated, revised, and sometimes replaced with more efficient,
reliable, or simpler ones. The book is also completely reorganized, making use of section headings to
keep the chapters focusing at different biological levels connected to one another. The basic logic behind
this sectional organization is the central dogma of biology (DNA → RNA → protein → metabolites).
After these more traditional approaches, we take up the topics of modern “omics” approaches, including
genomics, proteomics, and metabolomics. This is followed by topics dealing with the manipulation of
biological systems (including RNAi) and macromolecular analyses (focusing on the use of microscopy).
As in the first edition of this book, we have included, within each chapter, various notes and cau-
tionary considerations for potentially hazardous reagents. However, we also strongly recommend that
the reader take the time to observe the precautions detailed for each chemical on individual material
safety data sheets (MSDSs) prior to performing each procedure. Observing correct safety procedures
and developing good safety habits are perhaps the most important steps the reader can take toward
avoiding long-term health concerns for the user as well as the people working around him or her.
We thank all those from CRC Press/Taylor & Francis Group involved in this endeavor for their
patience and help with the preparation of the third edition of this handbook.
Leland J. Cseke
Ara Kirakosyan
Peter B. Kaufman
Margaret V. Westfall
For MATLAB® and Simulink® product information, please contact:
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Web: www.mathworks.com
ix
Editors
Leland J. Cseke, PhD, received his doctorate in plant cellular and molecular biology through the
Department of Molecular, Cellular, and Developmental Biology at the University of Michigan, Ann
Arbor, Michigan. His dissertation research included the molecular biology, evolution, and biotechno-
logical applications of terpenoid scent compound production in Clarkia and Oenothera species in the
laboratory of Dr. Eran Pichersky. Currently, Dr. Cseke is an assistant professor in the Department of
Biological Sciences at the University of Alabama, Huntsville, where he works in conjunction with the
Department of Energy and Argonne National Laboratory to determine the molecular mechanisms of
keystone species in forest ecosystem responses to environmental changes. In addition, Dr. Cseke is work-
ing with the United States Department of Agriculture to develop cost-effective molecular methods for
plant species identification and has helped to develop the national guidelines for plant molecular identifi-
cation. Other projects include the metabolic engineering of Jatropha curcas plants for the improvement
of biodiesel with emphasis on transcriptional regulators that help to control oil biosynthesis.
Dr. Cseke spent several years as an assistant research professor at Michigan Technological
University working to discover the function of aspen (Populus tremuloides) MADS-box genes in
wood and floral development. He has also been a postdoctoral fellow in the Department of Plant
Sciences at the University of Arizona in the laboratory of Dr. Richard Jorgensen. There, he worked
to elucidate the factors involved in functional sense and antisense suppression of genes involved in
anthocyanin biosynthesis. Dr. Cseke’s interests include the biosynthesis of plant chemical products,
environmental genomics, and metabolic engineering of plant metabolism. This led to his coauthor-
ing another book, Natural Products from Plants, Second Edition (CRC Press, 2006). In addition,
he has done some work in the study of methods for improving the separation of the cancer-fighting
diterpene, taxol, in Taxus species in the laboratory of Dr. Peter B. Kaufman, and his knowledge
of such subjects has been directed toward teaching classes that emphasize biotechnology and the
chemical principles of biology.
Ara Kirakosyan, PhD, DSc, is an associate professor of biology at Yerevan State University,
Armenia, and is currently a research scientist at the University of Michigan. He received his PhD
in molecular biology in 1993 and his DSc in biochemistry and biotechnology in 2007, both from
Yerevan State University, Armenia. Dr. Kirakosyan’s primary research areas include the impact of
phytopharmaceuticals on prevention of heart failure, challenges and pitfalls in antioxidant research,
and mechanisms of synergistic action of bioactive medicinal compounds at target sites. His research
on phytochemicals and antioxidants in foodstuffs has direct implications for the health industry,
specifically in the area of cardiovascular disease. He is also interested in plant biotechnology
research to produce enhanced levels of medicinally important, value-added secondary metabolites.
He carried out postdoctoral research at Gifu Pharmaceutical University, Gifu, Japan, under the
supervision of Professor Kenichiro Inoue. The primary research topic dealt with molecular biology
of dianthrones and triterpene glucoside biosynthesis. He also took part in visiting research investi-
gator positions in Germany. First, he was visiting scientist at Heinrich Heine University, Düsseldorf
(host scientist Professor Dr. W.A. Alfermann). The research there concerned a lignan anticancer
project, i.e., the production of cytotoxic lignans from Linum (flax). The second position involved
a carbohydrate-engineering project as a DAAD fellow in the Institute of Plant Genetics and
Crop Plant Research (IPK) Gatersleben, under the supervision of Professor Dr. Uwe Sonnewald.
Dr. Kirakosyan’s collaboration with U.S. scientists started with the USDA-founded project on plant
cell biotechnology for the production of dianthrones and phloroglucinol derivatives in Hypericum
perforatum. This research was carried out with Dr. Donna Gibson at USDA Agricultural Research
xi
xii Editors
Service, Ithaca, New York. In 2002, he was a Fulbright visiting research fellow at the University
of Michigan, Department of Molecular, Cellular, and Developmental Biology in the laboratory of
Professor Peter B. Kaufman. He is the coauthor of two other books, namely, Recent Advances in
Plant Biotechnology (Springer, 2009) and Natural Products from Plants, Second Edition (CRC
Press/Taylor & Francis, 2006). Dr. Kirakosyan is principal author of over 50 peer-reviewed research
papers in professional journals and several chapters in books dealing with plant biotechnology, bio-
chemistry, pharmacology, and molecular biology.
Peter B. Kaufman, PhD, received his BSc in plant science from Cornell University, Ithaca, New York,
and his doctorate in plant biology from the University of California, Davis, California. He is cur-
rently professor emeritus of biology at the University of Michigan, Ann Arbor, and research sci-
entist with the University of Michigan Integrative Medicine (UMIM) program. He is the author of
nine books and over 230 research publications. His most recent books include Recent Advances in
Plant Biotechnology (A. Kirakosyan and P.B. Kaufman, editors) (Springer Publishers, New York,
2009); Natural Products from Plants, Second Edition (CRC Press, 2006), coauthored with Leland J.
Cseke, Ara Kirakosyan, Sara Warber, James Duke, and Harry Brielmann; and Creating a Sustainable
Future: Living in Harmony with the Earth (Researchco Book Centre, New Delhi, India, 2002), coau-
thored with James Hoyt, Christopher Coon, Barbara Madsen, Sara Warber, J.N. Govil, and Casey R.
Lu. Dr. Kaufman is a fellow of the American Association for the Advancement of Science (AAAS).
He is also past president of the Michigan Botanical Club and past chairman of the Michigan Natural
Areas Council. He served as secretary-treasurer of the American Society of Gravitational and Space
Biology (ASGSB) and was the recipient of ASGSB’s Orr Reynolds Distinguished Service Award.
He is currently doing research on natural products of medicinal value in plants with support from
UMIM, the National Institutes of Health, Xylomed Research, Denali BioTechnologies, LLC, and
the Cherry Marketing Institute (CMI) of Michigan. He has performed research with colleagues at
Lund University, Lund, Sweden; University of Calgary, Alberta, Canada; Nagoya and Kyushu uni-
versities, Nagoya and Fukuoka, Japan; International Rice Research Institute, Los Baños, Philippines;
Michigan State University, East Lansing; University of Colorado, Boulder; North Carolina State
University, Raleigh; Purdue University, West Lafayette, Indiana; USDA Plant Hormone Lab,
Beltsville, Maryland; Hawaiian Sugarcane Planters’ Association, Aiea Heights, Honolulu; the Volcani
Agricultural Research Center, Bet Degan, Israel; Denali BioTechnologies, Homer, AK; and Yerevan
State University, Yerevan, Armenia. He recently (2007) established the Hazel S. Kaufman Integrative
Medicine Retreat Center at Manchester, Michigan (hskimrc.org), and established at this same site, sus-
tainable energy projects, including a geothermal heating system, a solar photovoltaic system (14 kW),
and a wind turbine (5 kW).
Margaret V. Westfall, PhD, is an associate professor of surgery and molecular and integrative physi-
ology and an assistant professor of biomedical engineering at the University of Michigan, Ann Arbor,
Michigan. She received her BA in biology from Colorado College, Colorado Springs, Colorado; her
MSc in microbiology from the University of Montana, Missoula, Montana; and her doctorate in phys-
iology from Loyola University, Chicago, Illinois. She held postdoctoral positions at the University of
Illinois at Chicago and at the University of Michigan, Ann Arbor, Michigan. Dr. Westfall has taught
skeletal muscle physiology, and molecular and integrative cardiac physiology for engineers. Her
research is focused on heart failure and includes studies on protein kinase C modulation of contractile
function; the role of the thin filament molecular switch protein, troponin I, in modulating contractile
function; and analysis of proteomic expression in end stage heart failure. Dr. Westfall’s research
has been funded by the National Institutes of Health and the American Heart Association. She cur-
rently serves on the editorial board for the American Journal of Physiology—Heart and Circulatory
Physiology and the Journal of Molecular and Cellular Cardiology. Dr. Westfall has published over
50 peer-reviewed research publications and is the author of several book chapters. She is a fellow of
the Council on Basic Cardiovascular Sciences for the American Heart Association.
Contributors
Joy M. Agee, MSc, is a scientist in training in the PhD program for biotechnology, science, and engi-
neering at the University of Alabama in Huntsville (UAH). She is currently conducting her doctoral
research in the laboratory of Dr. Richard M. Myers at HudsonAlpha Institute for Biotechnology. Her
research is aimed at understanding the molecular biology of triple negative breast cancer (TNBC), a
form of breast cancer that lacks receptors for estrogen, progesterone, and human epidermal growth
factor 2. Agee received her MSc in molecular biology from UAH. Her research focuses on iden-
tifying the expression of RNAi genes in the ectomycorrhizal fungus, Laccaria bicolor. Agee is a
National Science Foundation Graduate research fellow.
Chuanfu An, PhD, is a postdoctoral research scientist at the University of Florida. He received his PhD
in plant genetics and genomics from Mississippi State University, Mississippi State, Mississippi in 2008,
and was a postdoctoral researcher in the School of Forestry and Natural Resources at the University of
Georgia for two years. His research interests include plant secondary metabolism and defense.
Andrei B. Borisov, PhD, is an assistant professor at Wayne State University in Detroit. He received his
doctorate from the Institute of Cell Biology of the Russian Academy of Sciences, St. Petersburg, and
later worked at the University of Michigan, Ann Arbor, before accepting his current position at Wayne
State. Dr. Borisov’s research interests are focused on the interrelations of proliferation and differentia-
tion of precursor cells during development, regeneration, and malignant growth. Another field pertain-
ing to his studies is cell and molecular biology of aging and the mechanisms underlying the impairment
of the compensatory and regenerative capability of skeletal and cardiac muscle during senescence.
Lynn Boyd, PhD, is an associate professor at the University of Alabama in Huntsville. Dr. Boyd
received his BA in Latin from Wake Forest University in 1983, and later received his PhD from the
Department of Human Genetics at the University of Utah in 1992. Research in the Boyd laboratory
focuses on the role of the ubiquitin pathway in Caenorhabditis elegans development.
Ramesh Buyyarapu, PhD, is a research scientist working in the Molecular Technology

Development team in Trait Genetics and Technology at global headquarters of Dow AgroSciences
LLC based in Indianapolis, Indiana. He received his PhD and MSc in plant and soil sciences,
majoring in molecular genetics, genomics, and bioinformatics from Alabama A&M University,
Normal, Alabama. During his PhD, his research focused on the development of tools for compre-
hensive analysis of cotton genomes. He started his career as a postdoctoral research scientist at
Alabama A&M University. His current job responsibilities include marker enrichment for asso-
ciation with fiber traits and nematode resistance in cotton.
Nidhi P. Chanana, PhD, fellow, The Energy and Resources Institute (TERI), New Delhi, has more
than 10 years of experience in plant cell and tissue culture. She received her doctorate from the
University of Delhi under the guidance of Professor S.S. Bhojwani. She also worked with Dr. J.B.M.
Custers at Plant Research International, Wageningen, the Netherlands (1999–2000). Her present
interests include development of haploids as well as genetic transformation system in J. curcas
and sesame. She spent three months in the laboratory of Dr. Gopi K. Podila at the University of
Alabama, Huntsville, United States, where she worked on optimizing the protocol for genetic trans-
formation of J. curcas. As an adjunct faculty member at TERI University, she teaches and super-
vises PhD students in the field of plant biotechnology.
xiii
xiv Contributors
Feng Chen, PhD, is an associate professor of plant functional genomics in the Department of Plant
Sciences, University of Tennessee, Knoxville, Tennessee. He received his BSc in molecular biology
from Nankai University, Tianjin, China; his MSc in genetics from the Institute of Genetics, Chinese
Academy of Sciences, Beijing, China; and his PhD in plant biology from the University of California,
Davis, California. Dr. Chen’s research program focuses on use of an integrated genomics approach that
combines bioinformatic analysis, metabolomics, transcriptomics, and large-scale in vitro biochemical
assays to characterize the biosynthesis, biological function, and evolution of plant secondary metabolism.
Omar De la Cruz Cabrera, PhD, is a postdoctoral scholar in the Department of Statistics at

Stanford University. He received his doctorate in statistics at the University of Chicago in 2008.
Before starting graduate study in statistics, he worked in pure mathematics, receiving his doctorate
in set theory from the University of Florida in 2000. His interest is in the development and appli-
cation of new statistical methods to problems in data-intensive fields of biology. In particular, he
is interested in methods involving mathematical tools less frequently applied to statistics, such as
geometry.
Luis Rogelio Cruz-Vera, PhD, received his BSc in chemistry at Autonomous University of
Puebla, Mexico, in 1995; his MSc in genetics and molecular biology at the Center for Research
and Advanced Studies (CINVESTAV-IPN), Mexico, in 1996; and his PhD in genetics and molec-
ular biology at the Center for Research and Advanced Studies (CINVESTAV-IPN), Mexico, in
2000. His previous appointments include postdoctoral researcher, Department of Genetics and
Molecular Biology, Center for Research and Advanced Studies, Mexico City, Mexico (2000–
2003) and postdoctoral researcher, Department of Biological Sciences, Stanford University
(2003–2007). He currently serves as assistant professor, Department of Biological Sciences,
University of Alabama in Huntsville (2007–present). His research interests include regulation of
gene expression, especially on those mechanisms involving the regulation of ribosome function.
Sarah Beth Cseke, MSc, received her BSc in biology and MSc in plant molecular biology with
an emphasis on tissue culture from the University of Alabama in Huntsville (UAH). Since 2006,
she serves as the lead research assistant in the Center for Molecular Biology at Alabama A&M
University (AAMU). She has seven years of combined experience as a research assistant. During
this period, she has worked with diverse genes in transgenic poplar trees (P. tremuloides), cot-
ton species (Gossypium spp.), and reniform nematodes (Rotylenchulus reniformis). She has also
undertaken plant transformation for over-expression and RNAi in Arabidopsis, tobacco, Populus,
and Glycine species. She is familiar with the basic molecular biology research tools, including
PCR, RNA extractions, cDNA library preparation, and plant transformation. She has also taught
graduate, undergraduate, and high school students in formal and individualized sessions.
Arun Kumar Das, PhD, is currently affiliated with the Michigan Metabolomics and Obesity Center
(MMOC), Internal Medicine Department, University of Michigan, where he is acting head of the
Lipidomics Section. Dr. Das worked for many years at MHRI (Mental Health Research Institute, now
known as The Molecular and Behavioral Neuroscience Institute), University of Michigan, in the area
of membrane lipid research. He received his PhD in India at the University of Calcutta, where his
graduate training focused on organic chemistry specialization in lipid chemistry and analysis.
Dr. Das acquired an extensive knowledge and background on various aspects of chemistry, biochemis-
try/enzymology and molecular biology of membrane lipid and its metabolic enzymes. Over the years,
he made a number of discoveries in the area of chemical synthesis of phospholipids, enzymology of
lipids, effects of hypolipidemic drugs on lipid metabolism, cloning cDNA of lipid metabolic enzyme
and so on. Dr. Das has published 30 peer-reviewed research publications and is a coauthor of several
book chapters on lipid research. He became a member of the American Oil Chemist Society in 1992.
Contributors xv
His current interest at the MMOC is on studies of structural and functional aberration of membrane
lipids, lipid metabolites, and the enzymes involved in diabetes, obesity, and related physiological
dysfunctions.
Maria R. Davis, PhD, was an associate professor of molecular biology in the Department of
Biological Sciences at the University of Alabama in Huntsville. Unfortunately, we lost Maria in
the tragic shooting that occurred on the campus of UA-Huntsville on February 12, 2010. Dr. Davis
received her bachelor of engineering degree from the University of Michigan, Ann Arbor, in chemi-
cal engineering with a bio-option minor in 1981. She worked at E.I. DuPont DeNemours in the
Chemical, Dyes and Pigments Department in East Chicago, Indiana, as a process engineer until
1983. She completed her master of engineering degree in chemical engineering with an interdisci-
plinary minor at North Carolina State University, Raleigh, in 1985. She then pursued her PhD in
biochemistry to graduate in 1992 with a minor in plant pathology. Dr. Davis did her postdoctoral
research at Monsanto Chemical Corporation in St. Louis, Missouri, where her interest in plant
sciences and bioengineering of crop plants blossomed. She worked for six years at RESGEN (an
Invitrogen Company) as a senior scientist overseeing both production and research staff, developing
large insert libraries, testing RNA/DNA isolation kits, assembling vector constructs, and engineer-
ing Escherichia coli strains. Her most recent research interests were devoted to identification of
fungal pathogenicity factors to better understand the progression of fungal diseases in animals and
plants. She chose to use a variety of proteomic, genomic, metabolic methods together with molecu-
lar biology tools to identify new factors, then address how these factors may work to enhance the
attack of the fungus on the host organism. Additional research interests were in the area of cellular
transport in order to understand mechanisms used to localize proteins extracellularly. She received
research grants from the National Science Foundation and Binational Agricultural Research and
Development (BARD) Program. Dr. Davis had more than 15 publications in peer-reviewed journals,
including reports in conference proceedings. She taught an introductory course in biology and a
course in cell and developmental biology at the University of Alabama in Huntsville.
Eric Devaney, BA, MD, is an associate professor of surgery at the University of Michigan, where
he practices as a pediatric cardiac surgeon. Dr. Devaney graduated summa cum laude from the
University of Virginia and received his MD from the University of California, Los Angeles. His
general surgery training was undertaken at the University of California, San Francisco. He received
specialty training in cardiothoracic surgery and pediatric cardiac surgery at the University of
Michigan. Dr. Devaney is board certified in general and thoracic surgery. His clinical interests are
in the surgical repair of complex neonatal cardiac defects and in the use of ventricular assist devices
in the treatment of pediatric heart failure. His primary basic science research interests focus on the
sarcomeric basis of contractile dysfunction in heart failure and cardiomyopathy, and the genetic
modification of cardiac myocytes using adenoviral gene delivery.
Scott A. Harding, PhD, is a senior research scientist with the Warnell School of Forestry and Natural
Resources at the University of Georgia. Dr. Harding received his PhD in agronomy from Kansas State
University, Manhattan, in 1990. His research interests include plant development and metabolism.
Susan Holmes, PhD, was a tenured researcher specializing in biological applications at INRIA in
Montpellier, France until 1993. She came to Stanford University for a year to teach computational
statistics, was at MIT and Harvard for two years and at Cornell for two years, and then returned
to Stanford where she is currently a professor of statistics. All of her work focuses on multivariate
statistics applied to biology. She is currently working on the phylogeny and statistical analyses of
HIV drug resistance, the interaction between the immune system and cancer, codon usage bias and
the phylogeny of Dehalococcoides, and image segmentation for cell localizations in lymph nodes.
xvi Contributors
Sarah E. Kampert, BA, graduated from Boston University in 2008 with a degree in biochemistry
and molecular biology. She is currently a graduate student in the Program in Cellular and Molecular
Biology at the University of Michigan. Kampert is pursuing research to evaluate cellular structure
and function in response to adenoviral-mediated gene transfer of sarcomeric proteins.
Ramesh V. Kantety, PhD, is a professor of plant biology and genomics in the Department of Natural
Resources and Environmental Sciences at Alabama A&M University. He received his MSc in plant
breeding from Auburn University, his PhD in plant genetics and breeding from Purdue University,
and received his postdoctoral training on plant–pathogen interactions from the Boyce Thompson
Institute for Plant Biology at Cornell University. Later, he worked as a research associate in the
Department of Plant Breeding at Cornell University, where he was instrumental in establishing
plant genomics and bioinformatics programs in several key crops important to U.S. and world food
security. His research program’s focus is to improve the productivity of food, fiber, and energy crops
to meet current and emerging global needs utilizing integrated genetic and genomic approaches.
His graduate and undergraduate level teaching responsibilities include plant genetics, genomics,
and bioinformatics.
Akira Katoh, PhD, is a senior researcher at the Core Laboratory, Nara Prefectural Small and
Medium-sized Enterprises Support Corporation in Nara, Japan. He received his PhD from the
School of Agricultural Science, Nagoya University, Aichi, and later worked at the National Institute
for Basic Biology in Aichi, at the University of Tsukuba in Ibaraki, as well as at the Nara Institute
of Science and Technology in Nara before accepting his current position at the Core Laboratory.
Dr. Katoh’s research interests focus on metabolomics, pharmacological analysis based on transcrip-
tomics, herbal medicine, and plant physiology based on molecular biology.
Evelyn H. Kim, PhD, is a postdoctoral fellow in the Department of Surgery at the University
of Michigan, Ann Arbor, Michigan. She received her BSc and MSc in chemistry from Dankook
University, South Korea (1996, 1999), and her PhD in chemistry from the University of Michigan
(2009). Dr. Kim’s research work is in the areas of proteomic analysis of human disease, including
ovarian cancer and pancreatic cancer. She has published six peer-reviewed manuscripts and pre-
sented over a dozen talks at scientific conferences.
Lance Larka, PhD, is chief operating officer of iXpressGenes, Inc. at the HudsonAlpha
Institute for Biotechnology. He received his BSc in genetics in 1995 from the University of
California, Davis. Larka has worked in the field of molecular biology for public research institu-
tions, educational institutions, private for-profit companies, and international for-profit compa-
nies, focusing on laboratory automation, liquid handling processes, oligonucleotide synthesis,
process optimization, DNA (Sanger) sequencing, and laboratory design and construction. His
company focuses on protein production and purification, DNA sequencing for service, and pro-
ducing synthetic biology bio-parts. He is a judge for the new innovative product category at
LabAutomation.
Casey R. Lu, PhD, is a professor of biology in the Department of Biological Sciences, Humboldt
State University (HSU), Arcata, California. He received his BSc with honors in biology in 1980,
his MSc in biology in 1987, and his PhD in education and biology in 1993, all from the University
of Michigan, Ann Arbor. Dr. Lu has worked to enhance the electron microscopy facilities at HSU
since 1999. He has also received numerous grants from the University of California Office of the
President for establishing and directing the Redwood Science Project at HSU. At HSU, he teaches
introductory cellular and molecular biology, plant physiology, secondary science methods, plant
tissue culture, and electron microscopy. He has presented papers/posters at annual meetings of the
American Society of Plant Biologists, California Science Teachers Association, and the National
Contributors xvii
Science Teachers Association. His research interests include ultrastructural changes in plants chal-
lenged with heavy metals and physiology of the gravitropic response in plants.
Michael Mashore is the lead technician at the Veteran’s Administration San Diego Core for Micro
Imaging. He began studying microscopy with a focused interest in electron microscopy while com-
pleting his BSc in biology at Humboldt State University, Arcata, California.
Maureen McKenzie, PhD, is the chief executive officer of Denali BioTechnologies, Inc. (DBI),
the first and only company in Alaska dedicated to pharmaceutical and nutraceutical discovery and
development from boreal territories. Dr. McKenzie founded DBI to focus on drug discovery from
unique plants, microbes, and marine organisms that thrive in harsh, psychrophilic (cold-loving)
habitats. In 2005, Dr. McKenzie received USDA funding, in conjunction with the University of
Alaska–Fairbanks, to demonstrate the feasibility of a nutraceutical industry in frontier regions
of Alaska. Her work on this initiative culminated in invited sessions before the Natural Health
Products Research Society of Canada, International Convention on Biodiversity, and the United
Nations in 2007. Dr. McKenzie has received many awards for innovation in research and business
and is the author or coauthor of numerous patents on drug discovery technology and nutraceuti-
cal products, peer-reviewed articles, and book chapters. She has served as adjunct faculty in the
Department of Pharmaceutics at the College of Pharmacy of the University of Florida, Gainesville,
since 2004. Prior to that, she was an affiliate assistant professor in the Department of Physiology
and Pharmacology at the College of Veterinary Medicine of Iowa State University, Ames, and was
an assistant professor of chemical biology and pharmacognosy and member of the Laboratory for
Cancer Research in the College of Pharmacy at Rutgers, The State University of New Jersey, New
Brunswick. She received her BSc in nutrition/food technology from Iowa State University in 1978,
her MSc in food science from Rutgers in 1982, and her PhD in biochemistry through a joint pro-
gram of Rutgers, the University Medicine and Dentistry of New Jersey and Robert Wood Johnson
Medical School, and Princeton University in 1987.
David E. Misek, PhD, is a research assistant professor in the Department of Surgery at the
University of Michigan, Ann Arbor, Michigan. Dr. Misek received his BSc in microbiology from
Colorado State University (1979) and his PhD in pathology from SUNY Downstate Medical Center
(1986). He is a member of the University of Michigan Cancer Center, the University of Michigan
Gastrointestinal Peptide Research Center, and an associate member of the Early Detection Research
Network (EDRN) of the NIH/NCI. Dr. Misek’s research focuses on proteomic and genomic analy-
sis of human disease, including breast cancer and pancreatic cancer. His research has been sup-
ported by the National Cancer Institute within NIH and the Department of Defense. Dr. Misek has
published over 95 papers in peer-reviewed journals and books. He also serves as an ad hoc reviewer
for many journals.
Kathleen R. Noon, PhD, is director of the Innovation Center Mass Spectrometry Facility at the
Medical College of Wisconsin, Milwaukee. She received her BSc in medical technology from
Alverno College, Milwaukee, Wisconsin; her MSc in chemistry from the University of Wisconsin–
Madison; and her PhD in chemistry from Michigan State University, East Lansing. She has held
postdoctoral positions at the University of Utah, Salt Lake City, and the University of Michigan,
Ann Arbor. Prior to her current position, she was the manager of the Biomedical Mass Spectrometry
Facility in the Pharmacology Department at the University of Michigan. Dr. Noon’s research focuses
on separation science and biomedical applications of mass spectrometry, including small molecule
quantitative analysis and proteomics. She has trained over 50 graduate students in the theory and
operation of mass spectrometers. Dr. Noon has published more than 14 papers in peer-reviewed
journals and 1 book chapter. She is a member of the American Society of Mass Spectrometry and
the American Chemical Society.
xviii Contributors
Taketo Okada, PhD, is an assistant professor of molecular biology and biochemistry of medicinal
plants and oriental medicine in Tokushima Bunri University at Kagawa, Japan, since 2005. He
received his BSc in 1999 from Hoshi University, Tokyo, Japan; and his MSc in 2002 and his PhD
in 2005 from Chiba University, Chiba, Japan, in pharmacy. He is also a pharmacist licensed by the
Ministry of Health, Labour and Welfare of Japan in 1999. His research interests focus on the molec-
ular characterization of secondary metabolite biosynthesis in medicinal plants and on metabolome
analysis of medicinal plants and herbal medicines. He is also working at an herbal garden for culti-
vation research of medicinal plants.
Ajay Kumar Pandey, PhD, is a postdoctoral research associate at Foreign Diseases Weed
Science Research Unit (ARS-USDA), Fort Detrick, Maryland. Dr. Pandey received his BSc from
H.N.B. Garhwal University, India, with a major in botany and chemistry in 1996. He received his MSc
in biotechnology from Himachal Pradesh University, India, in 1998, and then pursued his PhD
in biotechnology and graduated in 2003 from Thapar Institute of Engineering and Technology,
Patiala, India. Dr. Pandey started postdoctoral research work at Academia Sinica, Taiwan, and
continued in this capacity at the University of Alabama in Huntsville with research interest in
molecular aspects of plant–microbe interactions. Currently, he is working on Asian soybean rust
(ASR) and utilizing virus-induced gene silencing (VIGS) assays to investigate the role of candi-
date defense genes in soybean.
Gopi K. Podila, PhD, was professor and chair of the Department of Biological Sciences at the
University of Alabama in Huntsville, Alabama. However, we lost Gopi during the tragic shooting
that occurred on the campus of UA-Huntsville on February 12, 2010. Until May 2002, Dr. Podila had
served as professor of biochemistry and molecular biology in the Department of Biological Sciences
at Michigan Tech University, Houghton, Michigan. He received his BSc in biological sciences from
Nagarjuna University in India, his MSc in plant pathology from Louisiana State University (1983),
and his PhD in molecular biology from Indiana State University (1987). Dr. Podila’s research dealt
with plant–fungus interactions, molecular biology of plant development, plant biotechnology, and
functional genomics. His research was supported by USDA, USFS, NSF, DOE, CPBR, and industry.
He published over 80 papers in peer-reviewed journals and books, and a book, Current Advances in
Mycorrhizae Research. Dr. Podila served on the editorial boards of Symbiosis, New Phytologist, and
Physiology and Molecular Biology of Plants, was an ad hoc reviewer of USDA, NSF, USDA-BARD,
and Italian Ministry of Education and Research grant proposals. He had visiting professor appoint-
ments at INRIA-Nancy, France; the University of Torino and the University of Urbino, Italy; and the
University of Helsinki, Finland. Dr. Podila organized and chaired several international symposia on
plant–fungus interactions and served as councilor-at-large for the International Symbiosis Society.
E. Mitchell Seymour, MSc, PhD, is a research associate. Dr. Seymour received his BSc in biol-
ogy from the University of Notre Dame and his PhD in biochemical and molecular nutrition from
Michigan State University. He has conducted extensive research in both eukaryotic and prokaryotic
cell and molecular biology. His research has included projects in molecular virology, environmental
microbiology, and cancer biology. His work has utilized several animal models and cell culture
lines. His research has been supported by the NIH, the California Table Grape Commission, the
Cherry Marketing Institute, the U.S. Highbush Blueberry Council, and the U.S. Apple Association.
His current research explores the impact of phytochemical-enriched diets on heart failure pathogen-
esis, metabolic syndrome, and atherosclerosis. He is currently the manager of the Cardioprotection
Research Laboratory at the University of Michigan Medical School, Section of Cardiac Surgery.
Avinash Sreedasyam, MSc, is a PhD student in the Department of Biological Sciences, University
of Alabama in Huntsville. He received his BSc in microbiology from Kakatiya University, India,
Contributors xix
and his MSc in biotechnology from the University of Wollongong, New South Wales, Australia. His
current research focuses on understanding defense response regulation during plant–fungal symbi-
otic interactions using high throughput transcriptomic approaches.
Venkateswara Rao Sripathi is a graduate research assistant in the Department of Natural

Resources and Environmental Sciences at Alabama A&M University. He received his MSc and BSc
from Acharya N.G. Ranga Agricultural University, Rajendranagar, India. Sripathi has established
a combinatorial approach for identifying novel microRNAs in the orphan genomes and metabolic
pathways. He worked as a research scholar at Applied Genomics Lab, ICRISAT, India, focusing
on marker-assisted backcrossing in Sorghum. He has been instrumental in standardizing the pro-
tocols for LCM-based sequencing at the Center for Molecular Biology, Alabama A&M University.
Currently, he is working on establishing protocols for chromosome-based sequencing in poly-
ploid crops.
Tamara K. Stevenson, BA, graduated from the University of Michigan in 2008. She is a research
associate in the Department of Surgery at the University of Michigan. She is currently pursuing
research on the functional response to pressure overload–induced hypertrophy and remodeling of
the adult heart.
Ramesh C. Thakur, PhD, received his PhD in forestry from the University of Horticulture and
Forestry, Solan, India, in 1989. Since then, he has worked as a member of the forestry faculty in
the Department of Tree Improvement and Genetic Resources at the University of Horticulture and
Forestry, Solan, India; as a postdoctoral fellow at the University of Freiburg, Germany; Forestry
and Forest Products Research Institute, Tsukuba, Japan; and currently holds the position of assistant
research scientist at Michigan Technological University, United States. Dr. Thakur has published
extensively in various journals, books, and conference proceedings. He has taught courses on tree
improvement and plant biotechnology. His research interests include tissue culture, genetic manipu-
lation, genetics and plant breeding, environmental pollution, and plant nutrition.
Geetika Trivedi, MSc, is a PhD student in the Department of Biological Sciences, University of
Alabama in Huntsville, Alabama. She received her BSc in biology in 2003 from C.S.J.M. University,
India, and her MSc in biotechnology in 2005 from Allahabad Agricultural Institute–Deemed
University, India. Her current research involves understanding the molecular basis of signaling and
metabolic re-programming during plant–fungal symbiosis.
Chung-Jui Tsai, PhD, is Winfred N. Haynes Professor and Georgia Research Alliance Eminent
Scholar of the Warnell School of Forestry and Natural Resources and the Department of Genetics
at the University of Georgia, Athens, Georgia. Dr. Tsai received her BSc and MSc from National
Taiwan University in Taiwan (1989 and 1991, respectively), and her PhD in forest science from
Michigan Technological University (1995). Her research is concerned with phenylpropanoid metab-
olism, wood formation, one-carbon metabolism, and growth-and-defense trade-offs in Populus.
Dr. Tsai’s research is supported through DOE, NSF, USDA, CPBR, and industry. He has published
over 50 peer-reviewed papers in journals and books.
Keerthi P. Venkataramanan, BTech, is a fourth year graduate student pursuing his PhD in the
Biotechnology Science and Engineering Program at the University of Huntsville in Alabama. His
research focuses on the anaerobic fermentation of biodiesel-derived crude glycerol using Clostridium
pasteurianum. His research interests include functional genomics and metabolomics. He received
his BTech in biotechnology from S.R.M. University, India, in 2007. He is expected to receive his
MSc in chemical engineering in the summer of 2011.
xx Contributors
Yuh-Shuh Wang, PhD, is currently a senior scientist in the Institute of Technology, University of
Tartu, Estonia. Dr. Wang received her PhD degree in forest molecular genetics and biotechnology
from Michigan Technological University, Houghton, in 2002; and her M.Sc. degree in botany at the
National Taiwan University, Taipei, Taiwan. Her research has focused on molecular mechanisms
of plant development. Using cDNA-AFLP and differential display, she isolated and characterized
suites of cDNAs that are differentially regulated during tuberization of sweet potato and vascular
development of quaking aspen as her graduate work.
Marilyn D. Yoder, PhD, is an associate professor of cell biology and biophysics in the School of
Biological Sciences, University of Missouri–Kansas City. She received her BSc in nutrition from the
University of Kentucky and her PhD in biochemistry from the University of California, Riverside.
Dr. Yoder’s research involves structural, functional, and evolutionary relationships of proteins using
x-ray crystallography and other biophysical techniques. Specific proteins of interest include plant
and bacterial pectate-degrading enzymes, eukaryotic phosphatidylinositol transfer proteins, human
semenogelin proteins, and bacterial cytolethal distending proteins.
Part I
DNA-Based Technology
GENETICS, GENOMICS
AND BREEDING OF
CUCURBITS

Genetics, Genomics and Breeding of Crop Plants
Series Editor
Chittaranjan Kole
Department of Genetics and Biochemistry
Clemson University
Clemson, SC
USA
Books in this Series:

Published or in Press:
• Jinguo Hu, Gerald Seiler & Chittaranjan Kole:
Sunflower
• Kristin D. Bilyeu, Milind B. Ratnaparkhe &
Chittaranjan Kole: Soybean
• Robert Henry & Chittaranjan Kole: Sugarcane
• Kevin Folta & Chittaranjan Kole: Berries
• Jan Sadowsky & Chittaranjan Kole: Vegetable
Brassicas
• James M. Bradeen & Chittaranjan Kole: Potato
• C.P. Joshi, Stephen DiFazio & Chittaranjan Kole:
Poplar
• Anne-Françoise Adam-Blondon, José M. Martínez-
Zapater & Chittaranjan Kole: Grapes
• Christophe Plomion, Jean Bousquet & Chittaranjan
Kole: Conifers
• Dave Edwards, Jacqueline Batley, Isobel Parkin &
Chittaranjan Kole: Oilseed Brassicas
• Marcelino Pérez de la Vega, Ana María Torres,
José Ignacio Cubero & Chittaranjan Kole: Cool
Season Grain Legumes
• Yi-Hong Wang, Tusar Kanti Behera & Chittaranjan
Kole: Cucurbit
• Michael Pillay, George Ude & Chittaranjan Kole:
Banana

GENETICS, GENOMICS
AND BREEDING OF
CUCURBITS
Editors
Yi-Hong Wang
Department of Biology
University of Louisiana at Lafayette
Lafayette
USA
Tusar Kanti Behera
Division of Vegetable Science
Indian Agricultural Research Institute
New Delhi
India
Chittaranjan Kole
Department of Genetics and Biochemistry
Clemson University
Clemson, SC
USA
Science Publishers
Jersey, British Isles
Enfield, New Hampshire

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Preface to the Series
Genetics, genomics and breeding has emerged as three overlapping and

complimentary disciplines for comprehensive and fine-scale analysis of
plant genomes and their precise and rapid improvement. While genetics
and plant breeding have contributed enormously towards several new
concepts and strategies for elucidation of plant genes and genomes as well
as development of a huge number of crop varieties with desirable traits,
genomics has depicted the chemical nature of genes, gene products and
genomes and also provided additional resources for crop improvement.
In today’s world, teaching, research, funding, regulation and utilization
of plant genetics, genomics and breeding essentially require thorough
understanding of their components including classical, biochemical,
cytological and molecular genetics; and traditional, molecular, transgenic
and genomics-assisted breeding. There are several book volumes and
reviews available that cover individually or in combination of a few of these
components for the major plants or plant groups; and also on the concepts
and strategies for these individual components with examples drawn
mainly from the major plants. Therefore, we planned to fill an existing gap
with individual book volumes dedicated to the leading crop and model
plants with comprehensive deliberations on all the classical, advanced and
modern concepts of depiction and improvement of genomes. The success
stories and limitations in the different plant species, crop or model, must
vary; however, we have tried to include a more or less general outline of
the contents of the chapters of the volumes to maintain uniformity as far
as possible.
Often genetics, genomics and plant breeding and particularly their
complimentary and supplementary disciplines are studied and practiced
by people who do not have, and reasonably so, the basic understanding of
biology of the plants for which they are contributing. A general description
of the plants and their botany would surely instill more interest among
them on the plant species they are working for and therefore we presented
lucid details on the economic and/or academic importance of the plant(s);
historical information on geographical origin and distribution; botanical
origin and evolution; available germplasms and gene pools, and genetic
and cytogenetic stocks as genetic, genomic and breeding resources; and

vi Genetics, Genomics and Breeding of Cucurbits
basic information on taxonomy, habit, habitat, morphology, karyotype,

ploidy level and genome size, etc.
Classical genetics and traditional breeding have contributed enormously
even by employing the phenotype-to-genotype approach. We included
detailed descriptions on these classical efforts such as genetic mapping
using morphological, cytological and isozyme markers; and achievements
of conventional breeding for desirable and against undesirable traits.
Employment of the in vitro culture techniques such as micro- and megaspore
culture, and somatic mutation and hybridization, has also been enumerated.
In addition, an assessment of the achievements and limitations of the basic
genetics and conventional breeding efforts has been presented.
It is a hard truth that in many instances we depend too much on a few
advanced technologies, we are trained in, for creating and using novel or
alien genes but forget the infinite wealth of desirable genes in the indigenous
cultivars and wild allied species besides the available germplasms in national
and international institutes or centers. Exploring as broad as possible
natural genetic diversity not only provides information on availability of
target donor genes but also on genetically divergent genotypes, botanical
varieties, subspecies, species and even genera to be used as potential parents
in crosses to realize optimum genetic polymorphism required for mapping
and breeding. Genetic divergence has been evaluated using the available
tools at a particular point of time. We included discussions on phenotype-
based strategies employing morphological markers, genotype-based
strategies employing molecular markers; the statistical procedures utilized;
their utilities for evaluation of genetic divergence among genotypes, local
landraces, species and genera; and also on the effects of breeding pedigrees
and geographical locations on the degree of genetic diversity.
Association mapping using molecular markers is a recent strategy to
utilize the natural genetic variability to detect marker-trait association and
to validate the genomic locations of genes, particularly those controlling the
quantitative traits. Association mapping has been employed effectively in
genetic studies in human and other animal models and those have inspired
the plant scientists to take advantage of this tool. We included examples of
its use and implication in some of the volumes that devote to the plants for
which this technique has been successfully employed for assessment of the
degree of linkage disequilibrium related to a particular gene or genome,
and for germplasm enhancement.
Genetic linkage mapping using molecular markers have been discussed
in many books, reviews and book series. However, in this series, genetic
mapping has been discussed at length with more elaborations and examples
on diverse markers including the anonymous type 2 markers such as
RFLPs, RAPDs, AFLPs, etc. and the gene-specific type 1 markers such as
EST-SSRs, SNPs, etc.; various mapping populations including F2, backcross,

Preface to the Series vii
recombinant inbred, doubled haploid, near-isogenic and pseudotestcross;

computer software including MapMaker, JoinMap, etc. used; and different
types of genetic maps including preliminary, high-resolution, high-density,
saturated, reference, consensus and integrated developed so far.
Mapping of simply inherited traits and quantitative traits controlled
by oligogenes and polygenes, respectively has been deliberated in the
earlier literature crop-wise or crop group-wise. However, more detailed
information on mapping or tagging oligogenes by linkage mapping or
bulked segregant analysis, mapping polygenes by QTL analysis, and
different computer software employed such as MapMaker, JoinMap, QTL
Cartographer, Map Manager, etc. for these purposes have been discussed
at more depth in the present volumes.
The strategies and achievements of marker-assisted or molecular
breeding have been discussed in a few books and reviews earlier. However,
those mostly deliberated on the general aspects with examples drawn mainly
from major plants. In this series, we included comprehensive descriptions
on the use of molecular markers for germplasm characterization, detection
and maintenance of distinctiveness, uniformity and stability of genotypes,
introgression and pyramiding of genes. We have also included elucidations
on the strategies and achievements of transgenic breeding for developing
genotypes particularly with resistance to herbicide, biotic and abiotic
stresses; for biofuel production, biopharming, phytoremediation; and also
for producing resources for functional genomics.
A number of desirable genes and QTLs have been cloned in plants since
1992 and 2000, respectively using different strategies, mainly positional
cloning and transposon tagging. We included enumeration of these and
other strategies for isolation of genes and QTLs, testing of their expression
and their effective utilization in the relevant volumes.
Physical maps and integrated physical-genetic maps are now available
in most of the leading crop and model plants owing mainly to the BAC,
YAC, EST and cDNA libraries. Similar libraries and other required genomic
resources have also been developed for the remaining crops. We have
devoted a section on the library development and sequencing of these
resources; detection, validation and utilization of gene-based molecular
markers; and impact of new generation sequencing technologies on
structural genomics.
As mentioned earlier, whole genome sequencing has been completed
in one model plant (Arabidopsis) and seven economic plants (rice, poplar,
peach, papaya, grapes, soybean and sorghum) and is progressing in an
array of model and economic plants. Advent of massively parallel DNA
sequencing using 454-pyrosequencing, Solexa Genome Analyzer, SOLiD
system, Heliscope and SMRT have facilitated whole genome sequencing in
many other plants more rapidly, cheaply and precisely. We have included

viii Genetics, Genomics and Breeding of Cucurbits
extensive coverage on the level (national or international) of collaboration

and the strategies and status of whole genome sequencing in plants for
which sequencing efforts have been completed or are progressing currently.
We have also included critical assessment of the impact of these genome
initiatives in the respective volumes.
Comparative genome mapping based on molecular markers and map
positions of genes and QTLs practiced during the last two decades of the
last century provided answers to many basic questions related to evolution,
origin and phylogenetic relationship of close plant taxa. Enrichment of
genomic resources has reinforced the study of genome homology and
synteny of genes among plants not only in the same family but also of
taxonomically distant families. Comparative genomics is not only delivering
answers to the questions of academic interest but also providing many
candidate genes for plant genetic improvement.
The ‘central dogma’ enunciated in 1958 provided a simple picture of gene
function—gene to mRNA to transcripts to proteins (enzymes) to metabolites.
The enormous amount of information generated on characterization of
transcripts, proteins and metabolites now have led to the emergence of
individual disciplines including functional genomics, transcriptomics,
proteomics and metabolomics. Although all of them ultimately strengthen
the analysis and improvement of a genome, they deserve individual
deliberations for each plant species. For example, microarrays, SAGE, MPSS
for transcriptome analysis; and 2D gel electrophoresis, MALDI, NMR,
MS for proteomics and metabolomics studies require elaboration. Besides
transcriptome, proteome or metabolome QTL mapping and application
of transcriptomics, proteomics and metabolomics in genomics-assisted
breeding are frontier fields now. We included discussions on them in the
relevant volumes.
The databases for storage, search and utilization on the genomes, genes,
gene products and their sequences are growing enormously in each second
and they require robust bioinformatics tools plant-wise and purpose-
wise. We included a section on databases on the gene and genomes, gene
expression, comparative genomes, molecular marker and genetic maps,
protein and metabolomes, and their integration.
Notwithstanding the progress made so far, each crop or model plant
species requires more pragmatic retrospect. For the model plants we need
to answer how much they have been utilized to answer the basic questions
of genetics and genomics as compared to other wild and domesticated
species. For the economic plants we need to answer as to whether they
have been genetically tailored perfectly for expanded geographical regions
and current requirements for green fuel, plant-based bioproducts and for
improvements of ecology and environment. These futuristic explanations
have been addressed finally in the volumes.

Preface to the Series ix
We are aware of exclusions of some plants for which we have

comprehensive compilations on genetics, genomics and breeding in
hard copy or digital format and also some other plants which will have
enough achievements to claim for individual book volume only in distant
future. However, we feel satisfied that we could present comprehensive
deliberations on genetics, genomics and breeding of 30 model and economic
plants, and their groups in a few cases, in this series. I personally feel also
happy that I could work with many internationally celebrated scientists
who edited the book volumes on the leading plants and plant groups and
included chapters authored by many scientists reputed globally for their
contributions on the concerned plant or plant group.
We paid serious attention to reviewing, revising and updating of the
manuscripts of all the chapters of this book series, but some technical and
formatting mistakes will remain for sure. As the series editor, I take complete
responsibility for all these mistakes and will look forward to the readers
for corrections of these mistakes and also for their suggestions for further
improvement of the volumes and the series so that future editions can serve
better the purposes of the students, scientists, industries, and the society of
this and future generations.
Science publishers, Inc. has been serving the requirements of science
and society for a long time with publications of books devoted to advanced
concepts, strategies, tools, methodologies and achievements of various
science disciplines. Myself as the editor and also on behalf of the volume
editors, chapter authors and the ultimate beneficiaries of the volumes
take this opportunity to acknowledge the publisher for presenting these
books that could be useful for teaching, research and extension of genetics,
genomics and breeding.
Chittaranjan Kole

Preface to the Volume
The Family Cucurbitaceae (cucurbits hereafter) has 118 genera and 825
species distributed primarily in the tropics and subtropics. It contains
some of the most nutritious, delicious and versatile food items in the
human diet. For example, watermelon contains 40% more lycopene than
tomatoes and lycopene is a powerful antioxidant that may lower the risk
of certain cancers and heart diseases. Melon is the main source of dietary
β-carotene together with carrots and broccoli. But only seven species of
four genera are economically important: Citrullus lanatus (watermelon),
Cucumis sativus (cucumber), Cucumis melo (melon), Cucurbita pepo (squash,
gourd, and pumpkin), Cucurbita maxima (squash), Cucurbita moschata
(squash and pumpkin), and Lagenaria vulgaris (L. siceraria, bottle gourd).
Less prominent are Luffa (Luffa acutangula and L. cylindrica), bitter melon
(Momordica charantia), and waxy gourd (Benincasa hispida).
The last two decades have proved to be the most exciting period in
cucurbit research although breeding effort and genetic/genomic studies
mostly focused on the economically more important cucurbits, i.e.,
cucumber, melon and watermelon. Cucumber becomes the first cucurbit
to be sequenced, after other field crops such as rice, sorghum, soybean
and maize. Its 26,682 predicted genes will facilitate genetic studies and
marker development in other closely related cucurbits such as melon and
watermelon. High-density genetic maps are now available for cucumber,
melon, watermelon and squash. More efficient and abundant marker
systems such as SNP and SSR are being developed. Genomic resources
such as sequenced ESTs, large-insert genomic libraries, high-throughput
sequencing have been or are being developed for cucurbits. Molecular
breeding using markers linked to agronomically important traits has become
an efficient tool in speeding up the new variety release.
This book provides an indepth review of the current state-of-the-art
of genetic and genomic research conducted in cucurbits. Each chapter is
authored by specialists in their field to report the latest trends and findings.
The chapters are well documented and illustrated. The hard work of all
contributors is greatly appreciated.
The book begins with an exhaustive description of cucurbits in terms of
classification, geographical distribution, production and their importance

xii Genetics, Genomics and Breeding of Cucurbits
in our diet (Chapters 1 and 2). These are followed by a discussion on how
traditional cucurbit breeding has produced a vast number of new varieties
that meet the needs of modern consumers (Chapter 3). Chapter 4 extends
the discussion to breeding of novelty cucurbits, i.e., squash for decoration
and pumpkins for Halloween, a popular tradition for children in the United
States. Chapter 5 describes the applications of genetic markers to diversity
analysis in cucurbits. Genetic mapping and map-based cloning of cucurbit
genes are described in Chapter 6. This is followed by a discussion in
mapping of monogenic traits and molecular breeding in Chapter 7. Chapter
8 expands the discussion to mapping of quantitative traits, which include
majority of agronomically important traits in cucurbits. The following three
chapters, Chapters 9, 10, and 11, describe the progress in research using
-omics in melon, watermelon and cucumber, respectively. Chapter 12 is
devoted into an important topic of cucurbits: sex expression as both genetic
and environmental factors can change sex expression of cucurbit flowers.
And finally, Chapter 13 provides perspectives on cucurbit research areas
that may become increasingly important.
This book is a testimony to the substantial progress made in the field of
cucurbit genetics, genomics and breeding, and the definite value of cucurbits
as a model system to study niche area such as sex expression. It is true that
the tools and concepts that are presented in the book will continue to evolve
rapidly and we hope this volume will provide a solid foundation for further
development in cucurbit genetics, genomics and breeding.
Lafayette, Louisiana, USA Yi-Hong Wang
New Delhi, India Tusar Kanti Behera
Clemson, South Carolina, USA Chittaranjan Kole

Contents
Preface to the Series v

Preface to the Volume xi
List of Contributors xv
Abbreviations xix
1. Major Cucurbit Crops 1
Yiqun Weng and Zhanyong Sun
2. Minor Cucurbits 17
T.K. Behera, A.K. Sureja, Sabina Islam, A.D. Munshi and A.S. Sidhu
3. Classical Genetics and Traditional Breeding 61
Stephen R. King, Angela R. Davis and Todd C. Wehner
4. Breeding Squash and Pumpkins 93
J. Brent Loy
5. Genetic Diversity Studies in Cucurbits Using 140
Molecular Tools
C. Esteras, F. Nuez and B. Picó
6. Molecular Genetic Mapping and Map-based Cloning 199
Yi-Hong Wang
7. Mapping and Molecular Breeding of Monogenic Traits 225
Yi-Hong Wang
8. Genome Mapping and QTL Analysis in Cucurbits 238
Hugo E. Cuevas, Jack E. Staub and Juan E. Zalapa
9. Genomic and Functional Genomic Resources of Melon 286
Zhangjun Fei and Yang Liu
10. Watermelon 309
Amnon Levi, W. Patrick Wechter, Judy. A. Thies, Kai-Shu Ling,
Umesh K. Reddy, Yong Xu, Shaogui Guo and Xingping Zhang
11. Cucumber Genomics 335
Zhonghua Zhang, Jun He and Sanwen Huang

xiv Genetics, Genomics and Breeding of Cucurbits
12. Sex Expression in Cucurbits 353

Rebecca Grumet and Jessica Taft
13. Future Prospects 376
Hiroshi Ezura
Index 387
Color Plate Section 393

List of Contributors
T.K. Behera
Division of Vegetable Science Indian Agricultural Research Institute,
New Delhi, India.
Email: tusar@rediffmail.com
Hugo E. Cuevas
Plant Genome Mapping Laboratory, Center for Applied Genetic,
Technologies, 111 Riverbend Road, Athens, GA 30602, USA.
Email: hcuevas@uga.edu
Angela R. Davis
Wes Watkins Agricultural Research Laboratory, USDA-ARS, PO Box 159,
Hwy.3 West, Lane, OK 74555, USA.
Email: angela.davis@lane-ag.org
C. Esteras
Instituto de Conservación y Mejora de la Agrodiversidada Valenciana
(COMAV), Universidad Politécnica de Valencia, Camino de Vera 14,
Valencia 46022, Spain.
Email: criesgo@upvnet.upv.es
Tel. +34 96 387 94 15
Hiroshi Ezura
Gene Research Center, Graduate School of Life and Environmental
Sciences, University of Tsukuba, Ten-nodai 1-1-1, Tsukuba 305-8572,
Japan.
Email: ezura@gene.tsukuba.ac.jp
Zhangjun Fei
Boyce Thompson Institute for Plant Research, Cornell University, Ithaca,
NY 14853, USA.
Email: zf25@cornell.edu
Rebecca Grumet
Department of Horticulture and Graduate Program in Genetics,
Michigan State University, East Lansing, MI 48824, USA.
Email: grumet@msu.edu

xvi Genetics, Genomics and Breeding of Cucurbits
Shaogui Guo
National Engineering Research Center for Vegetables Banjing, Beijing
100097, PO Box 2443, PR China.
Email: guoshaogui@nercu.org
Jun He
Institute of Vegetables and Flowers, Chinese Academy of Agricultural
Sciences, Beijing, 100081, China.
Email: hejun@caas.net.cn
Sanwen Huang
Email: huangsanwen@caas.net.cn
Sabina Islam
Indian Agricultural Research Institute, New Delhi, India.
Email: tokumkum@yahoo.com
Stephen R. King
Vegetable and Fruit Improvement Center, Department of Horticultural
Sciences, Texas, A & M University, College Station, TX 77843-2119, USA.
Email: srking@tamu.edu
Amnon Levi
USDA-ARS, U.S. Vegetable Laboratory, 2700 Savannah Highway,
Charleston, SC 29414, USA.
Email: Amnon-Levi@usda.ars.gov
Kai-Shu Ling
Email: kai.ling@ars.usda.gov
Yang Liu
Boyce Thompson Institute for Plant Research Cornell University, Ithaca,
NY 14853, USA.
Current address: College of Medicine, Texas A&M Health Science Center,
Temple, TX 76504, USA.
Email: lyang@medicine.tamhsc.edu
J. Brent Loy
Department of Biological Sciences, University of New Hampshire,
Durham, NH 03824,USA.
Email: jbloy@unh.edu

List of Contributors xvii
A.D. Munshi
Division of Vegetable Science Indian Agricultural Research Institute,
New Delhi, India.
Email: anilabhm@yahoo.co.in
F. Nuez
Instituto de Conservación y Meora de la Agrodiversidada Valenciana
Email: fnuez@btc.upv.es
B. Picó
Instituto de Conservación y Mejora de la Agrodiversidada Valenciana
Email: mpicosi@btc.upv.es
Umesh K. Reddy
Department of Biology, West Virginia State University, Institute, WV
25112, USA.
Email: ureddy@wvstateu.edu
A.S. Sidhu
Indian Institute of Horticulture Research, Bengaluru, India.
Email: amrik_sidhu1@rediffmail.com
Jack E. Staub
USDA-ARS, Forage and Range Research Laboratory, Utah State
University, Logan, UT 84322-6300, USA.
Email: jack.staub@ars.usda.gov
Zhanyong Sun
East-West Seed International Ltd., No.92-1 Minzu Avenue, Nanning,
Guangxi, 530022, P. R. of China.
Email: zhangyong.sun@eastwestseed.com
A.K. Sureja
Email: aksureja@rediffmail.com
Jessica Taft
Department of Horticulture and Graduate Program in Genetics,
Michigan State University, East Lansing, MI 48824, USA.
Email: taftjess@msu.edu

xviii Genetics, Genomics and Breeding of Cucurbits
Judy. A. Thies
Email: judy.thies@ars.usda.gov
Yi-Hong Wang
Department of Renewable Resources, University of Louisiana at
Lafayette, Lafayette, LA 70504, USA.
Email: yxw9887@louisiana.edu
W. Patrick Wechter
Charleston, SC, 29414, USA.
Email: pat.wechter@ars.usda.gov
Todd C. Wehner
Department of Horticultural Science, Box 7609, North Carolina State,
University, Raleigh, NC 27695-7609, USA.
Email: todd_wehner@ncsu.edu
Yiqun Weng
USDA-ARS Vegetable Crops Research Unit, Horticulture Department,
University of Wisconsin, Madison, WI 53706, USA.
Email: weng4@wisc.edu
Yong Xu
National Engineering Research Center for Vegetables Banjing, Beijing
100097, PO Box 2443, P. R. China.
Email: xuyong@nercv.org
Juan E. Zalapa
USDA-ARS, Madison WI; Dept. Horticulture, 1575 Linden Drive,
Madison, WI 53706, USA.
Email: jezlapa@wisc.edu
Xingping Zhang
Syngenta Seeds, 21435 Road 98, Woodland, CA 95695, USA.
Email: xingping.zhang@syngenta.com
Zhonghua Zhang
Email: zhangzhonghua@caas.net.cn

Abbreviations
A Andromonoecious
ACC 1-amino-1-cyclopropane carboxylate
ACO ACC-oxidase
ACS 1-Amino- cyclopropane-1-carboxylate synthase
AFLP Amplified fragment length polymorphisms
AG AGAMOUS
AP APETELA
ARO Agriculture Research Organization (Israel)
AVG Aminoethoxyvinyl glycine
AVRDC Asian Vegetable Research & Development Center
(presently The World Vegetable Center)
AWF Average weight fruit
BAC Bacterial artificial chromosome
BC Backcross
BLAST Basic Local Alignment Search Tool
BR Brassinosteroids
BSA Bulked segregant analysis
CAAS Chinese Academy of Agricultural Sciences
CAPS Cleaved amplified polymorphic sequence
CATIE Centro Agronomico Tropical de Investigacion y
Ensenanza
CC Coiled domain
cDNA Complementary DNA
CMV Cucumber mosaic virus
CNPH Embrapa Hortalicas
CENARGEN Embrapa Recursos Geneticos e Biotecnologia
cM Centi-Morgan
CP Coat protein (of virus)
cpDNA chloroplast DNA
CRC CRABSCLAW
CuGI Cucumber genome initiative (China)
CuLCrV Cucurbit leaf crumple virus
CVYV Cucumber vein yellowing virus
DTF Days to flower

xx Genetics, Genomics and Breeding of Cucurbits
DHL Doubled haploid line

DM Dry matter
ECCUDB European Central Cucurbits Database
EMS Ethylmethane sulfonate
ers ethylene response sensor
etr ethylene triple response
EST Expressed sequence tag
ET Ethylene
F Female
FAO Food and Agriculture Organization
FISH Fluorescence in situ hybridization
FW Fruit weight
FN Fruit number
FPC FingerPrinted Contigs
G Gynoecious
GA Gibberrelic acid
GMMV Green mottle mosaic virus
GO Gene ontology
GRIN Germplasm Resources Information Network (of USDA)
GSB Gummy stem blight
GxE Genotype x environment interactions
HFO-TAG High frequency oligonucleotides-targeting active genes
HICF High-information-content fingerprinting
HPLC High performance liquid chromatography
IARI Indian Agricultural Research Institute
IBL Inbred backcross line
ICuGI International Cucurbit Genomics Initiative
IGB Israel Gene Bank for Agricultural Crops
INDEL Insertion/deletion
INIFAP Instituto Nacional de Investigaciones Forestales,
Agricolas y Pecuarias
INRA National Institute of Agronomic Research (France)
IRTA Institute of Research and Technology in Agriculture
ISSR Inter-simple sequence repeats
ITS Internal transcribed spacer
IVF Institute of Vegetables and Flowers (CAAS)
KRIBB Korea Research Institute of Bioscience and
Biotechnology
LG Linkage group
LINE Long interspersed nuclear element
LOD Logarithm of odds
LOX Lipoxygenase
LRR Leucine-rich repeats

Abbreviations xxi
LTR Long terminal repeat

M Monoecious
MAS Marker-assisted selection
MLB Multiple lateral branching
MNSV Melon necrotic spot virus
mtDNA Mitochondrial DNA
MWMV Moroccan watermelon mosaic virus
NAM Nested association mapping
NBS Nucleotide binding site
NCBI National Center for Biotechnology Information
NIAS National Institute of Agrobiological Sciences
NIL Near-isogenic lines
NIVTS National Institute of Vegetable and Tea Science
NPGS National Plant Germplasm System
PAGE Polyacrylamide gel electrophoresis
PB Branch number
PI Plant Introduction
PIC Polymorphism information content
ORF Open reading frame
QTL Quantitative trait loci
PAGE Polyacrylamide gel electrophoresis
PCA Principle component analysis
PM Powdery mildew
PMF Percent of maturity fruit
PMR Powdery mildew resistance
PRSV Papaya ringspot virus
R Resistance
RAPD Random amplified polymorphic DNA
RFLP Restriction fragment length polymorphism
RGC R-gene candidate
RIL Recombinant inbred line
RKN Root-knot nematode
SAM S-Adenyosyl methionine
SBAP Sequence Based Amplified Polymorphism
SCAR Sequence characterized amplified region
SDS Sodium dodecyl sulfate
SFP Single-feature polymorphism
SNP Single nucleotide polymorphism
SqMV Squash mosaic virus
SqVYV Squash vein yellowing virus
SRAP Sequence-related amplified polymorphism
sRNA Small RNA
SSC Soluble solute content

xxii Genetics, Genomics and Breeding of Cucurbits
SSR Simple sequence repeats

STS Sequence-tagged site
TDF Transcriptome-derived fragment
TE Transposable element
TILLING Targeting induced local lesions in genomes
UTR Untranslated region
UzRIPI Uzbek Research Institute of Plant Industry
WGD Whole-genome duplication
WMV Watermelon mosaic virus
WVD Watermelon vine decline
ZYMV Zucchini yellow mosaic virus

1
Major Cucurbit Crops
Yiqun Weng1,* and Zhanyong Sun2
ABSTRACT
Cucurbit is a general term to denote all species within the Cucurbitaceae
family, which includes approximately 800 species in 118 genera.
Major cucurbit crops include cucumber, melon, watermelon, and
squash/pumpkin, which are all important vegetable crops that play
significant roles in human diet as well as rural economy. Nevertheless,
our understanding in phylogeny, genetics, biology, genomics and
many other fields of cucurbit crops, as compared with field crops and
model species, are lagging far behind. However, in recent years, with
technology innovations and instrumentation development, rapid
progress is being made in advancing our knowledge in major cucurbit
crops. In this chapter, we will give a brief review of research progress in
the past several years in four major cucurbits with focus on the economic
and biological importance of major cucurbits, the significant revision of
classical taxonomy of the genus Cucumis, new findings in domestication
and chromosome evolution of major cucurbits. We will also review the
current status of cucurbit germplasm conservation and utilization.
Keywords: Cucurbit, Cucumis, cucumber, melon, phylogenetics,
taxonomy, germplasm
1.1 Introduction
“Cucurbit” is a general term to denote all species within the Cucurbitaceae
family, which includes approximately 800 species in 118 genera. Cucurbits
are mostly annual, herbaceous, tendril-bearing and frost sensitive vines and
1
USDA-ARS Vegetable Crops Research Unit, Horticulture Department, University of Wisconsin,
Madison, WI 53706, USA; e-mail: weng4@wisc.edu
2
East-West Seed International Ltd., No.92-1 Minzu Avenue, Nanning, Guangxi, 530022, P. R.
of China.
*Corresponding author

2 Genetics, Genomics and Breeding of Cucurbits
are among the economically most important vegetable crops worldwide.

Cucurbits are growing primarily in the temperate and tropical regions.
Major cucurbit crops include cucumber (Cucumis sativus L.), melon (Cucumis
melo L.), watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai], and
squash or pumpkin (Cucurbita pepo L., Cucurbita maxima Duch. and Cucurbita
moschata Duch.).
An introduction to cucurbit crops has been provided in two full-length
books (Whitaker and Davis 1962; Robinson and Decker-Walters 1997). A
number of book chapters have periodical updates on research in cucumber
(Tatlioglu 1993; Staub et al. 2008), melon (McCreight et al. 1993; Pitrat 2008),
watermelon (Feher 1993; Wehner 2008), and squash/pumpkin (Ferriol
and Pico 2008; Paris 2008). This chapter will give a brief review of research
progress in the past several years in four major cucurbits with focus on
the economic and biological importance of major cucurbits, new findings
in cucurbit taxonomy, domestication and evolution, as well as germplasm
conservation and utilization.
1.2 Economic Importance of Cucurbit Crops

Among cucurbits, cucumber, melon, watermelon, and pumpkin are the
four most commonly cultivated crops. Minor cucurbits include West Indian
gherkin (Cucumis anguria L.) and African horned cucumber (Cucumis
metuliferus E. Mey) as well as ornamentals (hedgehog gourd, gooseberry
gourd), which are the topic of the second chapter of this book. Cultivars
and hybrids developed by breeders from cucumber, melon, watermelon
and pumpkin/squash are the basis for multi-billion dollar industries. Next
to tomatoes and onions, cucumbers and melons are the third most widely
cultivated vegetable crops in the world (Pitrat et al. 1999). According to the
United Nations’ Food and Agriculture Organization (FAO), the world total
areas of harvest for cucumber/gherkins and melon in 2008 were over 2.6M
ha and 1.3M ha, respectively, and China is the world’s leading producer
of these two crops (Table 1-1). Together, these two crops represented 7%
of the world’s total cultivated vegetable areas in 2001, ranking third after
tomato and watermelon (http://www.fao.org). Watermelon has the largest
cultivated areas among major cucurbit crops with over 3.7M ha harvest
acreage in 2008 worldwide. China, Turkey and Iran are the largest three
countries in watermelon production in 2008 (Table 1-1, see Chapter 10 of
this book for more details on watermelon).
Squash and pumpkins are unique in that they represent several species
for the same crop (Robinson and Decker-Walters 1997). Summer squash is
Cucurbita pepo, but winter squash may be C. pepo, C. moschata, C. mixta, or
C. maxima. The Jack O’Lantern type of pumpkin is C. pepo, but commercially
canned pumpkin pie mix is also made from C. moschata or C. maxima.

Major Cucurbit Crops 3
C. mixta and C. ficifolia are used for food in Mexico and in Central and South
American countries. Squash and pumpkin are usually grown for their fruit,
harvested immature for summer squash or mature for winter squash and
pumpkin. The world total area harvested for pumpkin was approximately
1.5M ha in 2008 (Table 1-1).
Table 1-1 Five leading countries in production of four major cucurbits in 2008*.
Crops Country Area harvested (ha)
Cucumber China 1,702,777
Cameroon 120,000
Iran 82,000
Russia 73,000
Egypt 67,810
World total 2,635,058
Watermelon China 2,162,456
Iran 135,000
Turkey 139,000
Russia 133,000
Brazil 93,600
Melon China 570,874
Turkey 103,000
Iran 80,000
Egypt 74,417
Spain 38,600
Pumpkin India 360,000
China 330,212
Cameroon 110,000
Cuba 73,038
Russia 53,000
*Data source: FAO Statistics 2010 (http://faostat.fao.org/).
Cucurbits are an important part of the human daily diet. Cucurbit

fruits are high in moisture and low in fat, which makes them popular
with consumers interested in healthy diets. Those with orange flesh like
muskmelon and winter squash are excellent sources of vitamin A. Orange-
fleshed pickling cucumber germplasm have also been developed (Simon
and Navazio 1997).
Cucurbit seeds can be classified as oil seeds because decorticated seeds
contain by weight 50% oil and 35% protein. The oil is unsaturated and
generally edible; however, the contents of conjugated trienoic fatty acids in
the oil of a few species preclude edibility but increase industrial values as
drying oils. Proteins of cucurbit seeds appear edible and supplementation
with certain amino acids increases the nutritional value of the protein (Jacks
et al. 1972; Mansour et al. 1993). For example, watermelon seeds, which are
used for food in various parts of the world, are low in moisture and high in

carbohydrates, fats, and protein. Varieties with very large seeds have been
developed especially for use as food in China, where more than 200,000
tons are produced annually on 140,000 ha land (Zhang 1996).
1.3 Biological Importance of Cucurbit Crops

All members of the Cucurbitaceae family have a lianous structure of
the plant body, the development of fleshy fruits, and a similar mode of
sex determination. These traits place cucurbits in a unique position for
understanding some important biological processes in plants.
The monoecious cucumber has long served as a model system for
sex determination studies driven by breeding programs for hybrid seed
production. Cucumber plants are mostly monoecious but can be dioecious or
hermaphroditic. Sex expression is controlled primarily by the F (femaleness)
and M (andromonoecy) loci in cucumber; and by the a (andromonoeocious)
and g (gynoecious) loci in melon, although environmental factors also
play important roles in this process. Genes involved in ethylene (ET)
biosynthesis/perception have been implicated in cucumber floral
development. CsACS1G encoding 1-aminocyclopropane-1-carboxylate
synthase (ACS) in the ET-biosynthesis pathway was mapped to the F
locus in cucumber (Kamachi et al. 1997; Trebitsh et al. 1997). Recently the
other three genes, M in cucumber, a and g in melon have all been cloned
(Boualem et al. 2008, 2009; Li et al. 2009; Martin et al. 2009), which will give
us a better understanding of the important processes of sex determination
in cucurbit crops and potentially contribute to better efficiency of hybrid
seed production. Readers can refer to Chapter 12 of this book for more
discussions of sex expression in cucurbits.
Major cucurbits have several-fold size differences in their mitochondrial
genomes (Ward et al. 1981). Watermelon possesses a relatively small
mitochondrial genome of 380 kb; squash has a larger one of 980 kb
(Alverson et al. 2010), while cucumber and melon mitochondrial genomes
are huge, at 1,500 kb and 2,400 kb, respectively. The chloroplast genomes
of melon, squash, and watermelon are maternally transmitted. While
the mitochondrial genomes of squash and watermelon are maternally
transmitted, those of melon and cucumber exhibit paternal transmission
(Havey 1997). Because the chloroplast, mitochondrial, and nuclear genomes
of Cucumis are differentially transmitted, this genus is an excellent system to
study the role of intergenomic transfer in the evolution of extremely large
mitochondrial genomes (Havey et al. 1998).
Phloem transport systems have been extensively researched because they
perform vital functions in plants, including distribution of photoassimilates,
nutrients, and signaling molecules to spatially separated organs. Cucurbits,
especially cucumber and pumpkin/squash are preferred models for phloem

physiology because of both the ease of sampling phloem sap and the facile
visualization of their large phloem sieve elements (Eschrich et al. 1971;
Clark et al. 1997; Zhang et al. 2010). Cucurbit phloem transport is unique
in that sucrose diffuses symplastically into intermediary cells (a form of
companion cell), where it is converted into raffinose-family oligosaccharide
sugars (Turgeon 1996).
Most cucurbits are consumed as either immature (for example
cucumber, summer squash) or mature (for example, melon, watermelon or
winter squash) fruits. A number of fruit development and ripening studies in
melon and cucumber have been conducted. Considering its morphological,
physiological, and biochemical diversity in flavor development and textural
changes during fruit ripening, melon was proposed to be a model plant
for the elucidation of key traits in fruit development (Ezura and Owino
2008).
Most species in the Cucurbitaceae family have basic chromosome
numbers of 7, 11, 12, 13, or 20, and relatively small genome sizes. The
chromosome numbers and genome size of the major cucurbit crops are listed
in Table 1-2. This also provides us a good opportunity to study chromosome
evolution in cucurbits.
Table 1-2 Chromosome number and genome size of major cucurbit crops.
Name Scientific name Chromosome # Genome size
(Mbp)*
Cucumber Cucumis sativus L. 2n = 2 x = 14 367
Melon Cucumis melo L. 2n = 2x = 24 450
Watermelon Citrullus lanatus Mats. & Nakai 2n = 2x = 22 430
Squash/pumpkin Cucurbita maxima Duch. 2n = 2x = 40 440
Cucurbita moschata Duch. 2n = 2x = 40 417
Cucurbita pepo L. 2n = 2x = 40 460–520
*Based on Arumuganathan and Earle (1991), and Tatum et al. (2006).
In the past two years, due to the use of next-generation sequencing

technologies, the whole genomes of many plants have been sequenced.
Among the four major cucurbits, the whole genomes of three, namely,
cucumber (Huang et al. 2009; Weng et al. unpubl. data), melon (Benjak et
al. 2010) and watermelon (Xu et al. 2009) have been sequenced or is near
completion. It is reasonable to say that cucumber (and other cucurbits)
genomics has come of age (see Chapter 11 for details). Thus, we can expect
more by exploring cucumber and other cucurbit genomics resources.
Particularly, cucumber may offer some advantages for genomic research
due to its economic importance, small genome size with relatively low
percentage of repetitive DNA, short life cycle and its unique position in
the phylogenetic tree of the Cucurbitaceae family.

1.4 Taxonomy, Origin and Domestication and Evolution of

Cucurbit Crops
The Cucurbitaceae family is well defined which includes 118 genera and
over 800 species (Jeffrey 1980). Cucurbitaceae can be divided into two
subfamilies: Zanonioideae and Cucurbitoideae. The food plants all fall
within the subfamily Cucurbitoideae. Further definition finds cucumber,
melon and watermelon belonging to the tribe Benincaseae and squash/
pumpkin was assigned to the tribe Cucurbiteae (Fig. 1-1). However,
molecular phylogenetic studies in recent years have significantly changed
our classical view of many species in this family, which is particularly true
for Cucumis that contains two very important cucurbit crops, cucumber and
melon. Our current view of Cucumis is illustrated in Fig. 1-1.
Figure 1-1 Major and minor cucurbit phylogenetic tree. Chromosome numbers and common
names follow each species name (when available). Molecular clock in million years ago,
if available, was shown on branching points. The tribe to which the species belongs was
shown to the right of vertical bars. Geographical occurrence of species: Green—America;
Black—mainland African; Red—Asia; Blue—Australia. The tree was redrawn after Schaefer
et al. (2009).
Color image of this figure appears in the color plate section at the end of the book.

1.4.1 Cucumber and Melon

The genus Cucumis traditionally contained 32 species that were further
divided into two subgenera, Melo and Cucumis (Kirkbride 1993). Subgenus
melo is centered in Africa with 30 species (2n = 24); subgenus Cucumis
includes the cultivated cucumber C. sativus (2n = 14) and its wild relative
C. hystrix (2n = 2x = 24), both of which have an Asian origin. However, in
recent molecular data-based phylogenetic trees, a number of other genera
like Cucumella, Mukia, Dicaelospermum, Myrmecosicyos and Oreosyce were
nested within Cucumis, thus, an expansion of Cucumis was proposed to
include these nested genera (Ghebretinsae et al. 2007a, b; Renner et al. 2007).
The revised genus Cucumis now has 52 species, which were grouped into
two subgenera: Humifructus (2 species, C. humifructus and C. hirsutus) and
Cucumis (the remaining 50 species) (Schaefer 2008). In the new phylogenetic
tree, C. hystrix remains the closest relative of cucumber, followed by a clad
containing D. ritchiei, Mukia javanica and Mukia maderaspatana (Ghebretinsae
et al. 2007a; Renner et al. 2007; Renner & Schaefer 2008; Schaefer et al. 2009).
Given the geographic distribution of these extant closest relatives, melon
(C. melo), which was traditionally assumed to have an African origin, was
proposed to have originated somewhere in Asia and then reached Africa
from there (Renner et al. 2007; Schaefer et al. 2009). More recently (Sebastian
et al. 2010), from molecular data for nearly 100 Cucumis accessions from
Africa, Australia, and Asia, it was shown that both melon and cucumber
are of Asian origin and have numerous previously overlooked species-level
relatives in Australia and around the Indian Ocean. Wild progenitors of C.
melo occur in India, but the Southeast Asian C. hystrix is the closest relative
of cucumber. Most surprisingly, the closest relative of melon is Cucumis
picrocarpus from Australia. Melon diverged from this Australian sister
species approximately 3 million years ago. Further revision of Cucumis is
likely. These new insights into the closest relatives of melon and cucumber
may have important implications for ongoing genomics and breeding
efforts in cucurbits.
1.4.2 Watermelon
The genus Citrullus is taxonomically complex and its composition is not
unanimously accepted by all taxonomists yet. Currently, Citrullus consists of
four diploid (2n = 2x = 22) species. C. lanatus var. lanatus is the domesticated
watermelon. Wild watermelon also known as citron is C. lanatus var. citroides
(L.H. Bailey) Mansf (but citron is known to be cultivated; Laghetti and

Hammer 2007). Three other wild species are Citrullus colocynthis (L.) Schrad.,
C. eccirrhosus Cogn and C. rehmii De Winter. The perennial C. colocynthis
grows in northern Africa, southwestern Asia and the Mediterranean,
whereas the perennial C. eccirrhosus and annual C. rehmii are endemic to the
Namib Desert (Levi et al. 2005; Dane and Liu 2006). Watermelon is thought
to have originated in southern Africa because it is found growing wild
throughout the area, and reaches maximum diversity there. Watermelon
may have been cultivated in Africa for over 4,000 years. C. colocynthis is
considered to be a wild ancestor of watermelon with small fruits and seeds,
and bitter flesh. Interspecific crosses of C. lanatus with C. colocynthis can
produce viable F1 hybrids. Although Citrullus species grow wild in southern
and Central Africa, C. colocynthis also grows wild in India. Thus, India and
China may be considered secondary centers of diversity for the genus. More
details regarding watermelon are discussed in Chapter 10.
1.4.3 Squash/pumpkin
The genus Cucurbita (squashes and pumpkins) is composed of 12–14 species
including five cultivated ones: C. pepo, C. maxima, C. moschata, C. ficifolia
and C. mixta. The latter two have less economic importance and a narrower
distribution (Robinson and Decker-Walters 1997).
While Cucurbita has an American origin in general, current genetic,
bio-geographic, and archaeological data suggest that the five cultivated
species were domesticated in different places, ranging from North America
to southern South America (Sanjur et al. 2002; Piperno and Stothert 2003).
Each species probably represents an independent domestication event from
different ancestor populations. Based on analysis of mitochondrial genes,
Sanjur et al. (2002) found that at least six independent domestication events
from distinct wild ancestors. C. mixta likely was domesticated from a wild
Mexican gourd, C. sororia. The wild ancestor of C. moschata is still unknown,
but will probably be found in lowland northern South America. C. andreana
may be the wild progenitor of C. maxima, but humid lowland regions of
Bolivia in addition to warmer temperate zones in South America from
where C. andreana was originally described should possibly be considered
as an area of origin for C. maxima.
Archaeological evidence of domestication of C. pepo in southern
Mexico dates back 10,000 years. Sanjur et al. (2002) suggested two separate
domestications in the C. pepo complex. The potential zone of domestication
for one of the domesticated subspecies, C. pepo subsp. ovifera, includes
eastern North America and should be extended to northeastern Mexico.
The wild ancestor of the other domesticated subspecies, C. pepo subsp. pepo,
is undiscovered but is closely related to C. pepo subsp. fraterna and possibly
will be found in southern Mexico.

1.4.4 Evolutionary Relationships Among Cucurbit Crops

Knowing the geographical origin of economically important plants is
important for genetic improvement and conservation. In a comprehensive,
multi-gene phylogenetic study including over 100 species in 114 genera,
Schaefer et al. (2009) revealed an Asian origin of Cucurbitaceae in the Late
Cretaceous, followed by the repeated spread of lineages into the African,
American and Australian continents via transoceanic long-distance
dispersal. North American cucurbits stem from at least seven range
expansions of Central and South American lineages; Madagascar was
colonized 13 times, always from Africa; Australia was reached 12 times,
apparently always from Southeast Asia. Overall, Cucurbitaceae underwent
at least 43 successful long-distance dispersal events over the past 60 million
years (Fig. 1-1).
The four major cucurbit crops have distinct chromosome numbers (Table
1-2). How the seven chromosomes of cucumber evolved from its ancestor
has long been an interesting and hot topic. Cross species transferability
of molecular markers among cucurbit crops has been well documented.
The availability of large numbers of molecular markers (Ren et al. 2009;
Cavagnaro et al. 2010) makes it possible to conduct comparative mapping
to reveal syntenic relationships among cucurbit genomes. By comparing
the melon and watermelon genetic maps to the cucumber genome, Huang
et al. (2009) were able to assign 348 (66.7%) of the 522 melon markers and
136 (58.6%) of the 232 watermelon markers onto cucumber chromosomes.
The comparison revealed that, except chromosomes 4 and 7, which are
largely collinear to melon chromosomes 7 and 1, respectively, each of the
remaining five cucumber chromosomes may result from a fusion of two
ancestral chromosomes (Huang et al. 2009) (also refer to Chapter 11 for more
detailed discussions). Fluorescence in situ hybridization of 45S rDNA and
CsCent1 repetitve DNA probes to cucumber pachytene chromosomes also
suggested that cucumber chromosomes 1 and 2 may have evolved from
fusions of an ancestral karyotype with 2n = 24 (Koo et al. 2010). Meanwhile,
during this process, many inter-chromosomal rearrangements including
centromere repositioning may likely have occurred (Han et al. 2009). The
seven meiotic chromosomes of C. sativus are larger than 12 of its wild
sister species or progenitor C. hystrix (Chen et al. 2004) and consist of six
metacentric and one submetacentric chromosome (Koo et al. 2005). It would
be enlightening to examine C. hystrix to look for its roles in the evolution of
modern cucumber. In addition, to gain a more complete picture, it will be
also helpful to investigate in more detail Dicaelospermumi and Mukia species
(Fig. 1-1) which were recently moved into Cucumis.

1.5 Cucurbit Germplasm Conservation and Utilization

1.5.1 Cucurbit Germplasm Conservation
Globalization, demands for greater dietary diversity, climate change will
have a big effect on the crop diversity in farmers’ fields. The development of
new varieties and cropping systems adapted to the socio-economic and new
environmental conditions will be crucial in order to take advantage of new
opportunities in some regions and limit yield losses in others. Attention has
been paid in some countries to increase genetic diversity within production
systems as a way to reduce risk, particularly in light of changes in climate,
pests and diseases. Plant breeders and farmers will need to be able to access
an even wider range of germplasm than today (FAO 2009).
Cucurbit genetic resources include cucurbit crops and their relatives.
Cucurbit gene banks are located on all continents, but there are relatively
fewer in Africa compared to the rest of the world. Around the globe,
cucurbit genetic resources are maintained in specified facilities known as
gene banks at the local and national level by governments, universities,
companies, farmers and others in the private and public sectors either
acting alone or networked with other institutions. In contrast to the gene
banks of the Consultative Group on International Agricultural Research
(CGIAR) Centers hold the major field crops, national gene banks are the
major repositories for cucurbit germplasm.
Few countries account for a larger percentage of the total world ex
situ cucurbit collection. Major holders of cucurbit crop genetic resources
are listed in Table 1-3. Many biotic and abiotic stresses are the most
limiting factors affecting cucurbit production around the world. There
will undoubtedly be an increase in demand for varieties that are adapted
to the new environmental conditions and pest and disease spectra. The
ability to access a wide range of genetic diversity is central to meeting this
demand, implying that in future there will be even greater interdependence
between countries and regions than is the case today. The concentration
of ex situ germplasm in a few countries and research centers highlights
the importance of mechanisms to ensure facilitated access. The ability of
a potential recipient to access a particular accession is often limited by
the size of a stored sample and its phytosanitary status, characterization,
adequate information systems, and international treaty. The most easily
accessible gene bank in the world is the USDA National Plant Germplasm
System (NPGS). Passport information is freely accessible through the web-
based Germplasm Resources Information Network (GRIN) (Table 1-3), and
germplasm is distributed free of charge and restriction. The establishment of
the European Central Cucurbits Database (ECCUDB) (http://www.comav.

Table 1-3 Major gene banks for cucurbit crops.

Genera Gene Bank* Accessions Reference
Cucumis AVRDC 628 Green et al. 2007
CAAS, China 2892 FAO 2009
ECCUDB 13899 Diaz et al. 2002
CNPH, Brazil 2400 FAO 2009
NIAS, Japan 4242 FAO 2009
NPGS, USA 7601 http://www.ars-grin. gov
Cucurbita AVRDC 642 Green et al. 2007
CATIE, Costa Rica 2612 FAO 2009
CENARGEN, Brazil 1897 FAO 2009
INIFAP, Mexico 1580 FAO 2009
Citrullus AVRDC 48 Green et al. 2007
IGB, ARO, Israël 840 FAO 2009
UzRIPI, Uzbekistan 805 FAO 2009
*AVRDC: The World Vegetable Center (Taiwan).
CAAS: Chinese Academy of Agricultural Sciences (Beijing, China).
CATIE: Centro Agronomico Tropical de Investigacion y Enseñanza (Costa Rica).
CNPH: Embrapa Hortalicas (Brazil).
CENARGEN: Embrapa Recursos Geneticos e Biotecnologia (Brazil).
ECCUDB: European Central Cucurbits Database.
IGB: Israel Gene Bank for Agricultural Crops (ARO, Volcani Center, Israel).
INIFAP: Instituto Nacional de Investigaciones Forestales, Agricolas y Pecuarias (Mexico).
NIAS: National Institute of Agrobiological Sciences (Japan).
NPGS: National Plant Germplasm System (USDA).
UzRIPI: Uzbek Research Institute of Plant Industry (Uzbekistan).
upv.es/eccudb.html) also facilitates germplasm use. The ECCUDB database

contains passport information of 22,815 accessions in 21 cucurbit geera and
more than 75 species from 33 institutions belonging to 19 European countries
(Díez et al. 2007). In contrast, the documentation and characterization of
many collections at national gene banks of developing countries are still
inadequate and much of the existing data is not accessible electronically.
To encourage increased use of the germplasm resources, documentation,
characterization and evaluation all need to be strengthened and harmonized
and the data need to be made more accessible. Access to and exchange of
plant genetic resources have become increasingly formal and more restricted
in the last 20 years. The International Treaty on Plant Genetic Resources
for Food and Agriculture has established a multilateral system to facilitate
access to germplasm of a number of crops. Unfortunately, cucurbit crops
are not included in this treaty.

1.5.2 Cucurbit Gene Pools and Utilization

The primary gene pool of cucumber consists of several botanical varieties
including C. sativus var. sativus (cultivated cucumber) and C. sativus var.
hardwickii (a wild relative of cultivated cucumber). C. sativus var. hardwickii
is cross-compatible with C. sativus var. sativus and possesses a multiple
fruiting and branching habit that is not common in cucumber (Horst and
Lower 1978). C. sativus var. hardwickii, therefore, represents the extreme in
variation in C. sativus germplasm (Dijkhuizen et al. 1996), and, thus, has
potential for increasing genetic diversity in commercial cucumber (Staub
et al. 1992).
Wild Cucumis species are cross incompatible with cucumber and melon,
which in many cases are themselves cross-incompatible (Kroon et al. 1979).
However, through embryo rescue, interspecific hybrids, amphidiploids
(Cucumis hystivus), aneuploidy derivatives as well as introgression lines
were successfully produced between cucumber and the wild, free-living C.
hystrix (Chen et al. 1997, 2000, 2004). C. hystrix has resistance to a number
of diseases such as gummy stem blight (Didymella bryoniae), downy
mildew (Pseudoperonospora cubensis) and southern root-knot nematode
(Meloidogyne incognita) (Chen et al. 2001, 2004; Zhou et al. 2008). Thus, C.
hystrix constitutes an important member of the secondary gene pool for
cultivated cucumber.
Despite many attempts, C. melo is not cross-compatible with any other
Cucumis species. However, C. melo may be the most variable of the genus
Cucumis and melon genetic resources are not threatened (Pitrat 2008).
Through recent work, Cucumis has been expanded to include 52 species
(Ghebretinsae et al. 2007a; Renner et al. 2007) and more could be added
(Sebastian et al. 2010). It is clear that significant work is needed to investigate
those newly added Cucumis species to see the possibility of using these
resources in cucumber or melon improvement.
Obtaining resistance to diseases and pests is a major objective in most
breeding programs of watermelon. However, because of limited resistance
within US plant introductions (PIs) of C. lanatus var. lanatus, limited progress
has been accomplished in this respect in watermelon. Although there is
great phenotypic diversity among watermelon cultivars developed in the
United States, they appear to have a narrow genetic background (Levi et
al. 2001).
The genus Cucurbita has 12–14 species including five domesticated
ones and their wild progenitors. These species are naturally distributed
in North and South Americas with the greatest diversity in Mexico. Both
domesticated and wild Cucurbita have a high potential value for breeding.
The five domesticated species are partially crossable allowing transferring
genes from one to another. Many wild species are also cross-compatible

with the domesticated ones thus being used for germplasm improvement
of cultivated species, e.g., powdery mildew and virus resistance, through
interspecific crossings. The zucchini yellow mosaic virus (ZYMV) resistance
gene(s) of C. moschata and the powdery mildew resistance gene of
C. okeechobeensis have been introgressed into C. pepo (Robinson and Decker-
Walters 1997; Whitaker and Robinson 1986). Multiple virus (ZYMV,
watermelon mosaic virus, and papaya ringspot virus) resistance genes,
derived from C. ecuadorensis, have been bred into C. maxima (Robinson and
Decker-Walters 1997; Herrington et al. 2001). The powdery mildew gene
of C. okeechobeensis spp. martinezii has been incorporated into C. moschata
(Robinson and Decker-Walters 1997; Cho et al. 2003).
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2
Minor Cucurbits
T.K. Behera,1,* A.K. Sureja,1 Sabina Islam,1 A.D. Munshi1
and A.S. Sidhu 2
ABSTRACT
Minor cururbitaceous vegetables are major sources of calories, minerals
and vitamins. Among these vegetables, pumpkin is a rich source of
carotene, bitter gourd is rich in vitamin C and iron and Luffa in minerals.
Pumpkin cultivars grow fruits with the strongest flavor, highest soluble
solids, and deepest flesh color that are preferred for canned and frozen
products. Besides being used as vegetables, bitter gourd possesses
antioxidant, antimicrobial, antiviral, antihepatotoxic, antiulcerogenic
properties, while also having the ability to lower blood sugar. The young
tender fruits of Luffa acutangula (ridge gourd) and Luffa cylindica (sponge
gourd) are edible and may be eaten sliced like cucumbers, or in soups
such as okra, or cooked like squash. Nutritional value like antioxidants,
carotenoids, tocopherol, minerals and ascorbic acid are also to be
considered while breeding these crops. In pumpkin, total carotenoid
content has a positive association with the fruit-flesh color intensity.
Dark orange fleshed varieties have high carotenoid content whereas
varieties with a bright yellow color flesh have high lutein content and
low carotene content. Use of wild species for transferring economically
important traits continues to be major objective for breeders especially
in cases where resistance genes for several pathogens and pests have
not been found within the cultivated species.
Keywords: Cucurbita moschata, Momordica charantia, Luffa spp, minor
cucurbits, germplasm enhancement, genetics and breeding.
1
2
Indian Institute of Horticulture Research, Bengaluru, India.
*Corresponding author: tusar@rediffmail.com, tusar@iari.res.in

2.1 Introduction
2.1.1 Distribution
Cucurbits are of tremendous economic importance and are cultivated
throughout the world from tropical to sub-temperate zones. China, Turkey,
India and Iran are important cucurbit growing countries. Pumpkin may
have been domesticated in Mexico and northern South America. Then it
migrated to the Caribbean islands and from there reached Florida, the
native Americans developed a distinct landrace called Seminole pumpkin.
More diversification on pumpkin has taken place in Asia and Africa. Bitter
gourd is a very important crop of India, the Phillipines, Malaysia, China,
Australia, Africa, the Middle East, Latin America and the Caribbean.
Momordica charantia was domesticated in eastern India or southern China.
Luffa cylindrica (smooth gourd) contains wild populations distributed from
southern Central Asia to north-eastern Australia and the South Pacific.
The domestic variety is cultivated in Asia, Africa and tropical America.
Luffa acutangula is mostly grown in South-eastern Asia and other tropical
countries (Table 2-1). In India, this diversity is concentrated in the Indo-
Gangetic plains, north-eastern regions, north-western Himalayas, the
Western and Eastern Ghats and sporadically in the tribal dominant belts
of Central India. More diversity occurs in Cucurbita spp. in the north-east
as also for ash gourd, bottle gourd while for Luffa, it is more concentrated
in the eastern peninsular tract. In case of Cucumis melo and round gourd,
it is more confined to north-western and Indo-Gangetic plains. In pointed
gourd, diversity is concentrated more in eastern part of the Indo-Gangetic
plains particularly in West Bengal and adjoining Bihar along eastern Uttar
Pradesh. Coccinia cordifolia (growing wild throughout India, raw fruits
used as vegetables), Cucumis sativus (distributed throughout India, in the
Himalayas as well), Lagenaria siceraria (African origin but domesticated
throughout India, tender fruits used as vegetables), Luffa cylindrica
(indigenous to India whereas acutangula found in western, Central and
southern India and is regarded as a wild form of cultivated species—tender
fruits used as vegetables), L. acutangula var. amara (occurs in peninsular
India and is a wild relative of cultivated spongegourd), L. echinata (western
Himalayas, Central India Gangetic plains) and L. graveolans (considered
as wild progenitor of L. hermaphrodita in Bihar and Sikkim) are potential
species. In addition, Momordica, M. balsamina occurs in semi-dry north-
western plains, northern parts of Eastern and Western Ghats, M. dioica
and M. cochinchinensis occur as wild/semi-wild in the Gangetic plains.
Trichosanthes has 21 species occurring in India and the major zones of species
concentration are (a) along the Malabar coast in the Western Ghats, (b) low
and medium elevation zones in the Eastern Ghats and the north-eastern

Table 2-1 Origin and geographical distribution of cultivated species of minor cucurbits of regional or local importance*.
Botanical name Common name(s) Origin Chromosome Geographical distribution Uses

no. (2n)
Benincasa Wax gourd, ash South-eastern 24 Tropical Asia Fruit, young leaf and bud
hispida gourd, white gourd, Asia as vegetable
petha
Citrullus Colocynth Africa 22 Tropical Africa, North- Seed is edible and used to
colocynthis western India make flour and cooking
oil
Coccinia indica syn. C. Little gourd, ivy Tropical Africa 22–48 Tropical Asia and Africa Fruit and young shoot as
grandis gourd, Kundru vegetable
Cucumis anguria West Indian gherkin Africa 24 Brazil, West Indies Young fruit as vegetable
and pickle
Cucurbita ficifolia Fig leaf, malabar Central Mexico 40 Central Mexico to Chile Mature fruit candied, seed
gourd is edible
Cucurbita foetidissima Buffalo gourd Mexico, S West 20 SW USA & Central Mexico Oil & protein rich seed
USA
Cyclanthera pedata Stuffing cucumber Tropical America 32 Andean countries Fruits as vegetable
Hogsonia macrocarpia Chinese Lard South Asia 18 South & SE Asia Fruits as vegetable’ seed
plant as cooking oil
Lagenaria siceraria Bottle gourd Lauki Africa 22 Trop. & Sub-trop. areas, Immature fruit & shoot as
Africa vegetable
Luffa acutangula Ridge gourd, Dhari India 26 India, East asia Young fruits as vegetable
tori
Minor Cucurbits 19
L. cylindrica Sponge gourd Chikni India 26 Throughout tropics, India Young fruits as vegetable,
tori mature for fiber
Table 2-1 contd....

Table 2-1 contd....
20
Botanical name Common name(s) Origin Chromosome Geographical distribution Uses
Genetics, Genomics and Breeding of Cucurbits

no. (2n)
Momordica charantia Bitter gourd Indo-Burma 22 Throughout tropics, India, S Young fruit, leaf as
Karela Region Tropical & SE Asia vegetable, seed as
Africa condiment
M. cochinchinensis Cochinchin gourd, India 28 South Asia Immature fruit as
sweet gourd vegetable, young leaf,
flower and seeds are
edible
M. dioica Kaksa, kakrol Southern tropical 28 Tropical Asia and Africa Fruit, shoot and leaf as
Asia vegetable
Praecitrullus fistulosuos Round melon, Indian India 24 North-West India Fruit as vegetable
squash, tinda
Sechium edule Chayote, chow-chow, Mexico, 24 Throughout tropics Young fruit and root
mirliton Guatemala especially Latin America (tuberous) as vegetable
Sicana odorifera Casabanana Brazil, Peru 40 Central America, northern As fruit
South America
Telfairia occidentalis Fluted pumpkin West Africa 22 Tropical Africa Leaf and shoot as
vegetable, seed is edible
T. pedata Oyster nut Africa 22 Tropical Africa Potential substitute for
almonds or Brazil nuts
Trichosanthes anguina syn. Snake gourd, India 22 South and South-East Asia Immature fruit, shoot and
T. cucumerina chichinda leaf as vegetable
T. dioica Roxb. Pointed gourd, parwal India 22 India Fruit as vegetable

*Sirohi et al. (2005).

Minor Cucurbits 21
hilly regions. Citrullus colocynthes exhibit much variation in north-western

plains. In Cucumis sativus, variation is concentrated in central and south-
western Rajasthan and Madhya Pradesh, southern India, along foothills of
the Himalayas and the north-eastern regions, for C. hardwickii, C. trigonus,
much variability occurs in the Himalayas. C. hystrix extends its range
from eastern plains to the north eastern hills in Assam, the Tura range of
Meghalaya and Mishmi Hills. In this chapter we discuss the minor cucurbits
like pumpkin (Cucuribta moschata), bitter gourd (Momordica charantia) and
Luffa spp. that are more commonly grown worldwide. They are often grown
in extensive monoculture typical of crop production in developed countries,
and also grown in traditional small gardens typified by low external inputs
in developing and underdeveloped countries.
2.2 Economic Importance

The cultivated pumpkins, squashes, and gourds, represent a very important
source of nutrition, not only in Latin American and Asian countries, but
also in many other regions worldwide. According to FAO (2010), 1,529,935
hectares of pumpkins, squash, and gourds are harvested annually with
a total production of 20,889,375 tons. Among the largest producers of
pumpkin are China, India, Russia, USA, Egypt, Ukraine, Italy, Iran, Mexico,
Turkey and Cuba. China remains the world’s leading producer of pumpkins,
squashes, and gourds with a production of 6,359,623 tonnes. About 64% of
the world’s production in 2008 was in Asia, which was led by China (30%)
and India (16.7%) (Table 2-2).
Table 2-2 World pumpkins, squash and gourds production in 2008.
Location Production (metric tons) Area Harvested (ha)

World 20889375 1529935
By Continent
Africa 1,714,474 242,955
Americas 2,536,052 203,191
Asia 13,390,541 936,590
Europe 2,937,503 129,949
Oceania 310,805 17,250
By Nation
China 6,359,623 330,212
India 3,500,000 360,000
Russian Federation 1,000,000 53,000
United States of America 786,980 34,720
Egypt 651,859 35,000
Ukraine 533,400 26,000
Italy 518,964 16,582
Iran 505,000 40,000
Source: FAO, FAOSTAT Agricultural Database. http://apps.fao.org. 2010.

2.2.1 Pumpkin
The common terms “pumpkin”, “squash”, “gourd”, “cushaw”, “ayote”,
“zapallo”, “calabaza”, etc. are often indiscriminately applied to different
cultivated species of the genus Cucurbita L.: C. pepo L., C. maxima Duchesne,
C. moschata Duchesne, C. argyrosperma C. Huber and C. ficifolia Bouche
(Ferriol and Pico 2008). “Pumpkin” is mostly used to refer to cultivars
with round fruits, which are used when mature for baking or for feeding
livestock. C. pepo is the most economically important species of Cucurbita
grown worldwide. C. maxima and C. moschata mainly includes cultivars
grown as “winter squashes” in developing countries under low-input
agricultural systems. In southern Latin America, C. maxima is largely grown
for immature fruit consumption (zapallito varieties) and some C. moschata
cultivars are also valued as “summer squashes”. C. argyrosperma and
C. ficifolia have less economic importance and a narrower distribution. In
both these species, the mature fruits are more valued, but some varieties
are eaten as a vegetable (Ferriol and Pico 2008).
Among cucurbitaceous vegetables, pumpkin (Cucurbita spp.) has been
appreciated for high yields, long storage life and high nutritive value.
C. moschata are eaten mature and can be baked, boiled, or microwaved. They
are low in saturated fat, cholesterol, and sodium, and are a good source
of dietary fiber, vitamins, and minerals (Nutrition Data 2006). Fruits with
yellow or orange flesh generally have high concentrations of carotenes,
some of which are the precursors of vitamin A (e.g., β-carotene) and play a
significant role in human nutrition, especially in tropical countries where
their consumption is high. C. moschata cultivars producing fruits with the
strongest flavor, highest soluble solids, and deepest flesh color are preferred
for canned and frozen winter squash. A variety of value added products such
as jam, jelly, marmalade, candy, puree, sauce, chutney, pickle and halwa are
prepared from pumpkin. Deep orange, carotene-rich fruits of C. moschata
and C. maxima are processed for use in the baby food industry. Pumpkin
flour can be used to supplement cereal flours in bakery products, soups,
instant noodles and natural coloring agent in pasta and flour mixes. Its
male flowers, young stems and leaves and young and ripe fruits are eaten
as a vegetable. The latter are also commonly used to prepare sweets and as
fodder. Its seeds are also consumed in many parts of Mexico and Central
America. The seeds are eaten whole, roasted or toasted and are ground
into different stews. They have high oil and protein contents. Plants of C.
moschata are also used as rootstocks for its resistance to diseases and abiotic
stresses, for the winter cultivation of watermelon, melon and cucumber
(Traka-Mavrona et al. 2000). Pumpkin seeds are a rich source of oil and
nutrients and can be consumed as food. The seed flour is used as a protein
supplement in bread and cookies. Pumpkin seeds have many health benefits

Minor Cucurbits 23
due to lower cholesterol and antidepressant qualities (Dhiman et al. 2009).

Naghii and Mofid (2009) reported the use of iron fortified ready-to-eat
pumpkin seed kernels improves the iron status. Pumpkin seeds are also a
good source of the elements K, P, Fe and β-carotene (Seo et al. 2005).
Abundant carotenoid, β-carotene, in several varieties of C. moschata
(Arima and Rodriguez-Amaya 1988, 1990), is an important precursor to
vitamin A, an essential vitamin for normal eye development and function.
Numerous epidemiological studies have implicated carotenoids in a
protective function against several cancers, cardiovascular disease and
cataracts, and function to enhance immune responses (Bendich 1993). Lutein,
a carotenoid prevalent in large amounts in C. moschata varieties (Arima and
Rodriguez-Amaya 1988; Khachik and Beecher 1988; Sommerburg et al.
1998), is one of the two principal pigments in the macular region of the
retina, and increased dietary intake increases pigment concentrations in
the macula and may reduce incidence of age-related macular degeneration
(Rodriguez-Carmona et al. 2006).
Pumpkin contains biologically active components that include
polysaccharides, para-aminobenzoic acid, fixed oils, sterol, proteins and
peptides (Buchbauer et al. 1998; Matsui et al. 1998; Appendino et al.
1999). The fruits are a good source of carotenoid and γ-aminobutyric acid
(Arima and Rodriguez-Amaya 1990; Gonzalez et al. 2001; Murkovic et
al. 2002; Zhang 2003). The hypoglycemic chemicals of pumpkin include
polysaccharides from the fruit pulp (Xiong 2000; Zhang and Yao 2002a, b),
oil from ungerminated seeds and protein from germinated seeds. These
chemicals are concentrated in fruits of pumpkin; therefore fruit of the
pumpkin has shown more pronounced hypoglycemic/antihyperglycemic
activity. Pumpkin extracts have broad spectrum antimicrobial activity and
hypo-cholesterolemic effect. In investigations on the antitumor activity of
pumpkin polysaccharide, increase of cell immune function was observed
(Guohua et al. 2000). Pumpkin has been used traditionally as medicine in
many countries such as China, Yugoslavia, Argentina, India, Mexico, Brazil
and America (Popovic 1971; Jia et al. 2003; Adolfo and Michael 2005). Some
of its common uses in most countries are for diabetes and treating internally
as well as externally for management of worms and parasites. Pumpkin has
antidiabetic, antihypertension, antitumor, immunomodulation, antibacteria,
antihypercholesterolemia, intestinal antiparasitia, antiinflammation and
antalgic properties (Caili et al. 2006). The fruit pulp and seeds of pumpkin
have shown hypoglycemic activity in normal animals and alloxan-induced
diabetic rats and rabbits. Both common and sugar-removed pumpkin
powder showed a significant reduction in blood glucose and an increase
in plasma insulin and protected the diabetic nephropathy (Ju and Chang
2001; Zhang and Bai 2004; Chen 2005). Yoshinari et al. (2009) reported
the anti-diabetic effects of pumpkin and its components, trigonelline and

nicotinic acid on rats. Reduction in blood glucose, serum total cholesterol

and triglyceride was observed in alloxan-induced diabetic rabbits applied
with pumpkin powder (Zhang 1998).
2.2.2 Bitter Gourd

Momordica charantia L., commonly known as bitter gourd, balsam pear,
bitter melon, bitter cucumber, and African cucumber, is a food crop with
many culinary uses especially in India, but is also grown as an ornamental,
and is used extensively in folk medicine. The fruits are cooked with other
vegetables, stuffed, stir-fried, or are added in small quantities to beans and
soup to provide a slightly bitter flavor. However, for most food preparations
fruits are blanched, parboiled, or soaked in salt water before cooking to
reduce the bitter taste. In addition to frying or cooking (e.g., for curries),
the fruits can be dehydrated, pickled, or canned. Fruits, flowers, and young
shoots are also used as flavoring agents in various Asian dishes. Young
Momordica shoots and leaves are also sometimes cooked and eaten as leafy
vegetables, and leaf and fruit extracts are also used in the preparation of
tea (Tindall 1983; Reyes et al. 1994).
Bitter gourd fruits are a good source of carbohydrates, proteins,
vitamins, and minerals (Desai and Musmade 1998) and have the highest
nutritive value among cucurbits (Miniraj et al. 1993). The vitamin “C”
content of Chinese bitter gourd varies significantly (440–780 mg·kg–1 edible
portion). The crude protein content (11.4–20.9 g·kg–1) of bitter gourd fruits is
higher than that of tomato and cucumber (Xiang et al. 2000). Considerable
variation in nutrients including proteins, carbohydrates, iron, zinc, calcium,
magnesium, phosphorous, and ascorbic acid has also been observed in
bitter gourd (Kale et al. 1991; Yuwai et al. 1991). The small-fruited gourds
usually have higher amounts of proteins, fats, carbohydrates, minerals
(Desai and Musmade 1998) and especially iron, calcium, and vitamin C
(Behera et al. 2006b).
Bitter gourd has been used for centuries in the ancient traditional
medicine of India, China, Africa, and Latin America. Bitter gourd extracts
possess antioxidant, antimicrobial, antiviral, antihepatotoxic, antiulcerogenic
properties, while also having the ability to lower blood sugar (Welihinda
et al. 1986; Raman and Lau 1996). These medical activities are attributed
to an array of biologically active plant chemicals, including triterpenes,
pisteins, and steroids (Grover and Yadav 2004). Ethno-medical reports of
M. charantia indicate that it is used in folkloric medicine for treatment of
various ulcers, diabetes, and infections (Gurbuz et al. 2000; Scartezzini
and Speroni 2000; Beloin et al. 2005). While the root decoctions have
abortifacient properties, leaf and stem decoctions are used in treatment
of dysentery, rheumatism, and gout (Subratty et al. 2005). In addition,

Minor Cucurbits 25
various injectable juice preparations of M. charantia drawn directly from

the fruit have traditionally been used for medicinal purposes worldwide.
Likewise, the extracted juice from leaf, fruit, and even the whole plant are
routinely used for treatment of sores/wounds, infections, parasites (e.g.,
worms), measles, hepatitis, and fevers (Behera et al. 2008c).
2.2.3 Luffa spp.

The two well known members of the genus Luffa are Luffa cylindrica (sponge
gourd) and Luffa acutangula (ridge gourd). They are very popular vegetables
in the tropical and subtropical regions. In India, they are eaten boiled or in
curry (mixed with potato or solo). The tender fruits along with khus seeds
make an excellent dish. In Japan, the young fruits are sliced and dried and
kept for future use. The young insipid leaves are consumed in Malaysia
(Porterfield Jr. 1955). In African countries, leaves are used as leafy vegetable
and seeds are used in several soup and sauce preparations (Adebooye
2009). The fiber obtained from mature fruits have great commercial use
and are generally used for cleaning glassware, kitchen utensils, floors
without scratching, washing ships and decks and manufacturing slippers
or baskets, used as shoe mats, inner cloth of bonnets. In combination
with other, toys, matting and hats are made. Combined with plaster and
varnished over it make materials soundproof and heat-proof wall boarding
(Porterfield Jr. 1955). In a more aesthetic sense, the fiber is painted and cut
into different shapes and used in home décor and fetch good return out of
small investments. The fiber is made into bath sponges. In Hungary, they
are used by masseurs in therapeutic baths. Loofa sponge is a lignocellulosic
material composed mainly of cellulose (60%), hemicelluloses (30%) and
lignin (10%) (Rowell et al. 2002; Mazali and Alves 2005). They are useful
material for stuffing pillows, mattresses, saddles, shoulder pads and
stiffening material. They can be effectively utilized to rub down painted
surface, because of their scoring property.
The fruit is rich in vitamin A, C and Fe (Yawalkar 1985) and has
considerable medicinal importance. Its abortifacient, antitumor, ribosome
inactivating and immunomodulatory activies have been reported (Yeung et
al. 1991; Ng et al. 1992; Wang and Ng 2002). Ridge gourd (Luffa acutangula)
leaves are used as poultice in haemorrhoids, leprosy and splenitis. The juice
of fresh leaves is reported to be useful in granular conjunctivitis in children.
Leaf decoction is used for treating anaemia and amenorrhoea in Java. Ripe
seeds are bitter and are reputed to possess emetic and purgative properties
(Porterfield Jr. 1951). Fresh fruit is considered to be cooling and beneficial
to the intestines, warming to the stomach and tonic to the genital organs.
Leaves are used in skin diseases and orchitis, the vine and root in decayed
teeth, ozena and parasitic affections. Wilson (1913) stated that the fiber is

valued as medicine in China. Juice from the above ground stem of sponge
gourd is used against respiratory problems in Japan. It is also used as an
ingredient for cosmetics (Lee and Yoo 2006). They are used for bathing,
removing toxins and regenerating the skin. They help varicose veins and
cellulite by stimulating circulation. The tender sponge gourd is considered
a diuretic and lactagog. It is also considered good against diabetes (Bal et
al. 2004a). Oral administrations (2 g/kg) of ethanolic extract of L. aegyptiaca
seeds decreased blood glucose level in streptozotocin diabetic rats (Fiky et
al. 1996). The ripe fruit after burning and pulverizing is used as carminative
and anthelmintic. The fruit juice is considered a purgative. Mature seeds
are bitter, emetic and cathartic. The seed oil is said to be useful for skin
affections and in Brazil, they are suggested as possible substitutes for olive
oil (Porterfield Jr. 1955).
Research in modern medicine system involves treating disease at the
gene level, aims at blocking expression of proteins, which are responsible
for disease. A wide variety of compounds (inhibitors) have been
isolated from plants and the most extensively studied are the ribosome
inactivating proteins (RIP). The RIP luffins have been isolated from Luffa
and characterized (Kishida et al. 1983). Besides, polypeptides of about 5
kDa molecular mass, such as thionins, have been reported to inhibit cell-
free protein synthesis, probably by a nonenzymatic mechanism (Garcia-
Olmedo et al. 1983). Ramakrishnan et al. (1989) purified a protein with
molecular weight of 30 kDa from L. aegyptiaca seeds, which inhibited cell free
translation at picomolar concentrations. Chemical linkage of the protein to
a monoclonal antibody reactive to transferring receptor resulted in a highly
cytotoxic conjugate. ChangYun and ZuChuan (1998) isolated and purified a
group of novel small ribosome inactivating proteins, LuffinS1, LuffinS2 and
Luffin S3 from L. cylindrica seeds. Parkash et al. (2002) reported presence of
luffacyclin, a ribosome inactivating peptide with antifungal activity against
Mycospharella arachidicola and Fusarium oxysporum from L. cylindrica seeds.
Luffaculin 1, a ribosome inactivating protein purified from L. acutangula is
reported to possess anti-HIV1 activities (Jing et al. 2008).
2.3 Botanical Descriptions

2.3.1 Pumpkin
The genus Cucurbita belongs to the tribe cucurbiteae and includes 12 or
13 species spread throughout the Americas (Jeffrey 1990a). The species
of the Cucurbita genus contain 20 pairs of chromosomes (2n = 40) and are
secondary polyploids with the basic number x = 10 (Singh 1979; Weeden and
Robinson 1990). Results from electrophoretic analyses also helped confirm

Minor Cucurbits 27
this genus to be polyploid (Kirkpatrick et al. 1985) or, more specifically of

allotetraploid (Weeden 1984) origin.
C. moschata are herbaceous, creeping, annual, monoecious plants, lightly
and densely pubescent, with short and long uniseriate trichomes and
caulescent vegetative apices that are fairly reflexed. Leaves have petioles of
30 cm or more length, are broadly ovate-cordate to suborbicular, measure
20–25 x 25–30 cm, are slightly lobate with three to five ovate or triangular
lobules, have an obtuse apex that is briefly apiculate, serrate-denticulate
margins and three to five ramified tendrils. Flowers are pentamerous,
solitary and axillary. Male flowers have 16–18 cm pedicels and a very short
calyx, are broadly campanulate to pateriform, expanded or foliaceous
towards the apex, 5–14 cm long, with five divisions for up to one-third
of their length. Female flowers have thick pedicels 3–8 cm long, and a
globose, ovoid, oblate, cylindrical, piriform, conical, turbinate ovary. They
have a very small calyx and sepals that are more often foliaceous than
in the males, measure up to 7.5 cm in length and are of thickened style.
Stigma is three lobed. C. moschata is distinguished by its big flowers, with
long slender androecium, foliaceous sepals, relatively soft pubescence
on the foliage, mottling leaves, and distinctly-colored seed margins. The
peduncles are compact and flared (Decker-Walters and Walters 2000). Fruits
are highly variable in shape, size, ribbing and rind texture. Rind color is
both thickened and durable and soft and smooth, and of a very variable
color—light green to uniform dark green or with cream spots, light to dark,
or completely white. The flesh is light or bright orange to greenish, ranges
from light to very sweet, is soft and generally not fibrous. It has numerous
seeds, which are ovate/elliptical, measuring 8–21 x 5–11 mm and have a
yellowish-white surface.
Among the domesticated species, C. moschata survive best in hot, humid,
low-elevation (usually under 1,500 m above sea level) climates of the mid-
latitudes. However, while it is true that this species is preferentially grown
within these limits, variants have been found above 2,200 m in Oaxaca,
Mexico. Diversity for various fruit characters exists in Mexico, Central
America, the western United States, India and Asia Minor. In Colombia
varieties with small fruits and dark seeds are abundant. In Japan, fruits are
frequently covered with warts. In the USA and Europe, only three groups
of cultivars, viz. Neck, Cheese and Bell, are commercialized (Whitaker
and Davis 1962; Robinson and Decker-Walters 1997). There is considerable
morphological diversity in C. moschata with regard to its seeds and fruit
color, shape, and thickness of rind (Bates et al. 1990; Robinson and Decker-
Walters 1997). Fruits are frequently furrowed and sometimes warty, have
a different rind color and high quality flesh, ranging from deep yellow to
orange.

Sisko et al. (2003) analyzed the genome sizes of 11 Cucurbita species

by flow cytometry. The 2C values ranged from 0.686 pg in C. foetidissima to
0.933 in C. ficifolia. The genome sizes of five cultivated species were 0.864 pg
(C. pepo), 0.887 pg (C. maxima), 0.933 pg (C. ficifolia), 0.708 pg (C. moschata)
and 0.748 pg (C. argyrosperma).
2.3.2 Bitter Gourd

The genus Momordica belongs to subtribe Thladianthinae, tribe Joliffieae,
subfamily: Cucurbitoideae of the family Cucurbitaceae (Jeffrey 1990b).
Schafer (2005) consider the genus Momordica as comprising 47 species, with
eight Asian species (which are all dioecious) and of the 39 African species,
20 are dioecious and 19 monoecious. According to de Wilde and Duyfjes
(2002), 10 species are reported in South East Asia, of which six each occur in
Malaysia and India, where M. balsamina L., M. charantia L., M. subangulata
Blume (ssp. renigera (G. Don) W. J. de Wilde) and M. cochinchinensis (Lour.)
Spreng. are common. Phylogenetic analyses of chloroplast gene, intron,
and spacer sequences reveal that Momordica is monophyletic and sister to
the dioecious Asian Thladiantha and Baijiania, (i.e., eight Asian Momordica
species are dioecious) (Schafer 2005).
The genus Momordica has six cultivated species, which can be grouped
under two heads: M. charantia L. and M. balsamina L. representing the
monoecious group and M. dioica Roxb., M. sahyadrica Joseph & Antony,
M. cochinchinensis (Lour.) Spreng., and M. subangulata Blume (ssp. renigera
(G. Don) W.J.J.deWilde) representing the dioecious group. The genus,
as circumscribed here, do not include Momordica cymbalaria Fenz. [Luffa
cymbalaria=M. tuberosa (Roxb.) Cogn.)], though few workers still treat it
under Momordica.
Chakravarty (1990) classified bitter gourd into two botanical varieties
based on fruit size, shape, color and surface texture: 1) M. charantia var.
charantia has large fusiform fruits, which do not taper at both ends,
and possess numerous triangular tubercles giving the appearance of a
“crocodile’s back”, and; 2) M. charantia var. muricata (Wild), which develops
small and round fruits with tubercles, more or less tapering at each end. Both
varieties are widely cultivated throughout tropical and subtropical regions.
Yang and Walters (1992) classified bitter gourd into three horticultural
groups or types: 1) a small-fruited type where fruits are 10–20 cm long,
0.1–0.3 kg in weight, usually dark green, and very bitter; 2) a long-fruited
type (most commonly grown commercially in China) where fruit are
30–60 cm long, 0.2–0.6 kg in weight, light green in color with medium size
protuberances, and are only slightly bitter, and; 3) a triangular-fruited type
where cone-shaped fruit are 9–12 cm long, 0.3–0.6 kg in weight, light to dark
green with prominent tubercles, and moderately to strongly bitter. Reyes

Minor Cucurbits 29
et al. (1994) reclassified Indian and Southeast Asian M. charantia botanical

varieties based on fruit diameter [(M. charantia var. minima Williams & Ng)
< 5 cm and (M. charantia var. maxima Williams & Ng) > 5 cm].
The cytogenetic studies confirmed the diploid chromosome number
(2n = 22) of M. charantia L (Table 2-3) . Another species of Momordica (M.
dioica; 2n = 28), a dioecious cucurbit, has a more asymmetrical karyotype
than the other two species, M. charantia and M. balsamina with 2n = 22,
but the meiosis in these species is regular (Bhaduri and Bose 1947; Roy
et al. 1966; Trivedi and Roy 1972). The monoecious species (M. charantia
and M. balsamina) are similar in their range and frequency of bivalents
and chiasmata, whereas, M. dioica (dioecious species) has less than half
as many chiasmata per chromosome (Singh 1990). Incompatibility exists
between 2n = 22 and 2n = 28 species in the genus when M. charantia and
M. balsamina crossed with M. dioica (Singh 1990). The cross, M. charantia x
M. dioica or its reciprocal, failed to set fruit when normal pollen was used
(i.e., ovary shriveled and was reabsorbed in 3 days) (Vahab and Peter 1993).
However, when such crosses were made using pollen of M. dioica stored at
10ºC and then crossed with M. charantia, the percentage of success increased
dramatically to greater than 90%.
Table 2-3 Related species of Momordica charantia L. and their chromosome numbers.
Species Chomosome Number (2n)

Momordica balsamina L. 22
M. charantia L. 22
M. cohinchinensis (Lour.) Spreng. 28
M. denudata C.B.Clarke 28
M. dioica Wall. 28
M. subangulata Bl. 28
M. subangulata ssp. renigera 56
M. sahyadrica 28
M. rostarata 22
M. foetida 44
Bitter gourd morphotypes include 5–7 lobed leaves, possess slender

and sub-glabrous or slightly hairy stems with simple tendrils, and produce
climbing vines that extend up to 5 m. Tendrils are unbranched, reaching
up to 20 cm in length. Plants develop flowers having yellow corollas that
are born solitary on peduncles with foliaceous bracts. Typically, green and
white fruits have longitudinal ridges, and a very irregular surface studded
with protuberances. Oblong fruits are 5 to 45 cm long, with muricate surface.
When mature, the three valves of the fruit dehisce, and recurved from the
apex to expose yellowish brown seeds that are attached to an orange-red,
pulpy endocarp. The seeds are between 13 to 24 mm long arils, that are
ovate-oblong, sculptured, and sub-tridentate at the base and apex.

The pistillate flower of bitter gourd consists of an inferior ovary and a

three-lobed, wet stigma that is attached to a columnar, hollow style (Pillai
et al. 1978). The ovary contains three carpels typical of many cucurbits, each
with 14–18 ovules, surrounded by an ovary wall. Although the number of
ovules in an ovary can be up to 60, the average is 40. Anatropous ovules
are attached to parietal placenta in two irregularly aligned rows in each
carpel. Unlike other cucurbits, however, no more than four ovules can be
seen in the ovary cross-section. Typically, pollen tubes penetrate papillae
tissue within one hour of pollination arriving at the ovary cavities about
six hours after pollination, and thus fertilization is accomplished within
18–24 hours post-pollination (Chang et al. 1999).
2.3.3 Luffa
Luffa is a member of the subfamily Cucurbitoideae, tribe Benincaseae,
subtribe Luffinae (Jeffrey 1962, 1980a). It is the only member of the subtribe
and has species in both the Old and New Worlds. In some respects, it
resembles members of the Cyclantherinae, an entirely New World subtribe
of the Siceyeae. It thus may be the connecting link of these two tribes. In the
most comprehensive taxonomic treatment of the genus presently available,
Cogniaux and Harms (1924) accepted eight species. One of these species
Luffa variegata Cogn., has since been transferred to Lemurosieyos (Keraudren
1965) and two others, L. forskalii Schwein. Ex Harms and L. umbellata (Klein)
M.J. Roem., have been reduced to synonymy or varietal status under L.
acutangula (L.) Roxb. (Jeffrey 1980a; Heiser and Schilling 1988). Five species,
four from the Old World and one in the New World, have generally been
accepted in recent years (Jeffrey 1980b; Heiser and Schilling 1988 ). Heiser
et al. (1988), however, proposed that three species are present in the New
World.
All Luffa species are vines and bear solitary pistillate flowers and
racemes of male flowers. Analyses of the flavonoids, breeding system,
leaves, flowers and fruit resulted in the grouping of L. acutangula and
L. aegyptiaca Mill. into a single clade, apart from the other five species (Heiser
and Schilling 1990). This result is not entirely supported by chloroplast DNA
makers (Chung et al. 2003).
2.4 Description of Germplasms and Gene Pools, and Potential

Wild Allied Species as a Resource of Donor Genes
2.4.1 Pumpkin
Correct establishment of the taxonomic identity of the germplasm collections
and their accurate characterization is vital for efficient maintenance and

Minor Cucurbits 31
utilization and reducing the frequency of unintended duplication. Initially

morphological and isozyme markers were utilized to characterize Cucurbita
germplasm collections. Using three enzyme staining systems, Puchalski
and Robinson (1978) reported general agreement with an earlier taxonomic
treatment of relationships among Cucurbita species (Bemis et al. 1970).
Extensive surveys of isozyme variation among Cucurbita species (Puchalski
and Robinson 1990) and, more specifically, in wild, weedy, domesticated
Mexican Cucurbita presented more complex views of genetic differentiation
and gene flow among taxa (Wilson 1989). Isozyme analyses by Decker-
Walters et al. (1990) did not support traditional infraspecific classifications
for C. maxima, C. argyosperma, or C. moschata.
In recent years, different classes of DNA markers have gained importance
to characterize Cucurbita germplasm. Juvik and Palmer (1984) conducted an
initial survey of 12 accessions representing four genera: Citrullus, Cucumis,
Cucurbita, and Lagenaria using restriction endonuclease site polymorphisms
in chloroplast DNA. Extensive analysis of chloroplast DNA of Cucurbita
(Wilson et al. 1992) provided valuable results in elucidating relationships
between domesticated taxa and their wild progenitors. Mitochondrial DNA
sequence analysis was conducted on a similar set of Cucurbita accessions
(Sanjur et al. 2002) built upon Wilson et al. (1992) findings and at least six
independent domestication events were suggested. Ferriol et al. (2003)
reported that sequence-based amplified polymorphisms (SBAPs) were
superior to random amplified polymorphic DNAs (RAPDs) in revealing
patterns of genetic diversity in C. maxima. They observed that the results
of RAPD analyses conformed neither to groupings by morphology nor
geographic origin. Ferriol et al. (2004a) also evaluated other Cucurbita species
with SRAPs and amplified fragment length polymorphisms (AFLPs) and
noted that SRAPs were generally more concordant with morphological
variation and agronomic traits. However, AFLPs grouped C. maxima
accessions by geographic origin, reflecting the bottleneck that occurred
with the introduction of this species to the Old World (Ferriol et al. 2004b).
Restrepo and Vallejo (2008) carried out molecular characterization of 121
introductions of C. moschata by AFLP markers and observed high genetic
diversity and genetic structure between most of the introductions.
The germplasm evaluation descriptors are often used to obtain
structure and comparability for germplasm characterization and evaluation.
Development of new descriptor systems for Cucurbita was reported by
Vinter et al. (2004). ECPGR has developed “Minimum descriptors for
Cucurbita spp., cucumber, melon and watermelon” in 2008.
Gene pools serve as a tool for conceptualizing the ability of plant
populations to cross with nonspecific populations and those of other species,
usually of the same genus (Harlan and deWet 1971). The primary gene
pool consists of interfertile populations, generally of a specific biological

species and may include other species that are fully cross-compatible. The
secondary gene pool is represented by all other populations that can be
crossed with the crop; the gene flow is possible but is connected with a
reduction of fertility within hybrid generations. Species from the tertiary
gene pool cannot be crossed with the crop species except through special
biotechnological approaches because the resulting hybrids often express
abnormalities and are usually lethal or completely sterile.
Generally, the five domesticated Cucurbita species are reproductively
isolated from one another. The primary gene pools of each species are
represented by their landraces and commercial cultivars as well as by their
infraspecific taxa (Table 2-4). Experimental crosses can be made among them
but with difficulty, and interspecific progenies are usually either sterile or
sparingly fertile (Merrick 1995). Spontaneous crosses between the cultivated
Cucurbita species are uncommon, but natural interspecific hybrids can be
occasionally detected in landraces mostly from Mexico (Decker-Walters et
al. 1990; Merrick 1990). Kristkova (1991) reported a spontaneous hybrid of
C. maxima × C. pepo under Central European field conditions.
Table 2-4 Gene pools of cultivated Cucurbita species.
Cucurbita spp. Gene pools

Primary Secondary Tertiary
C. argyrosperma C. argyrosperma C. moschata C. pepo
subsp. sororia C. maxima
subsp. argyrosperma C. foetidissima
(sensu lato)
C. ficifolia C. ficifolia C. pedatifolia C. lundelliana
C. foetidissima C. maxima
C. pepo
C. maxima C. maxima C. ecuadorensis C. lundelliana
subsp. maxima C. argyrosperma
subsp. andreana C. ficifolia
C. pepo
C. moschata C. moschata C. argyrosperma C. lundelliana
C. maxima
C. pepo
C. pepo C. pepo C. argyrosperma C. lundelliana
subsp. pepo (sensu lato) C. okeechobeensis C. ficifolia
subsp. fraterna C. moschata C. maxima
subsp. texana C. ecuadorensis
Adapted from Lira-Saade (1996).
Of all the domesticates, C. moschata is considered to be the extant species

with the most ancestral-like genome and displays wide cross-compatibility
(Merrick 1995). The domesticated species C. argyrosperma, C. pepo, and
C. maxima can be crossed with their wild or feral relatives (Merrick 1995;
Lira-Saade 1996). In the Americas, pairs of closely related domesticated

Minor Cucurbits 33
and wild species can occur sympatrically, and genetic interchange between
them takes place, providing a natural source of variation within populations
(Wilson 1990; Merrick 1991).
Hybridization experiments and field observations involving
C. argyrosperma and other wild and cultivated Cucurbita taxa have revealed
(Lira-Saade 1996) that, among the cultivated species, C. moschata has the
highest degree of compatibility with C. argyrosperma, placing it into its
secondary gene pool. The next level of cross-compatibility involves the wild
and cultivated taxa of C. pepo, some cultivars of C. maxima, and the wild
perennial species C. foetidissima, which collectively represent the tertiary
gene pool. The wild species that have shown some degree of compatibility
with C. argyrosperma possess genes of resistance to some viral diseases that
have a high incidence in the cultivated species (Lira-Saade 1996).
Greatest diversity in C. moschata occurs among the innumerable
landraces of the American tropics (Andres 2004a). Isozyme variation studies
in Cucurbita by Andres (1990) and Decker-Walters et al. (1990) revealed
moderate amounts of genetic differentiation within C. moschata. In the New
World, C. moschata is cultivated in a wide range of elevations, which suggests
that this species has evolved diverse adaptations to various environmental
conditions (Andres 2004b). The species is highly polymorphic (Andres
2004a) with considerable morphological diversity of its seeds and fruit
(color, shape, thickness, and durability of the fruit’s skin). The landraces
from northern Peru also exhibit high diversity. This includes the special
type called “Loche”, which typically has warty fruits and deep, orange
flesh valued highly by the locals as a flavoring for stews (Andres 2004a).
The existence of varieties with life cycles of differing phenology and the
breadth of its numerous cultivars developed in other parts of the world
(Gwanama et al. 2000) and of local varieties with excellent horticultural
characteristics strongly suggest that its collective genetic variation is very
extensive (Lira-Saade 1996).
Variation of C. moschata populations is also observed outside its center of
origin. For example, a landrace native to Nigeria, represents the only source
of resistance to certain viral diseases (Provvidenti 1993). The gene pool of
C. moschata is represented by the numerous commercial cultivars that have
been mainly developed in the United States and, to a lesser extent, in Brazil.
Some of these commercial cultivars have also different levels of resistance
and/or susceptibility to certain diseases, indicating a wide genetic variation
of this species (Lira-Saade 1996). Some of the Cuban landraces are a source of
genetic material for tolerance to marginal growth conditions (Andres 2004a).
Significant diversity is found in landraces from warmer regions of Asia or
Africa (the warty and wrinkled Japanese fruits; the Indian landraces with
large, soft-skinned fruits; the abundance of barbell-shaped fruits in Asia
Minor, the Nigerian landraces resistant to diseases, etc.) (Lira-Saade 1996;

Decker-Walters and Walters 2000). The possibilities of hybridization that

C. moschata has shown with other cultivated species (e.g., C. maxima) suggest
that that there are good prospects for the improvement of other Cucurbita
species (Lira-Saade 1996). C. moschata also crosses with wild species such
as subsp. C. sosoria and C. lundelliana, thus increasing its genetic pool.
RAPD markers were used to analyze the genetic diversity among
C. moschata landraces from Korea, southern Africa and other geographical
origins (Youn and Chung 1998; Baranek et al. 2000; Gwanama et al. 2000).
In all the cases, the accessions were grouped according to the agroclimatic
regions of origin and not according to the morphological traits. Esteras et
al. (2008) studied the diversity in Spanish landraces of C. moschata using
SSR markers. They reported high variability for fruit size, rind and flesh
color in the landraces and identified nine different morphotypes (flattened,
round, heart-shaped, oblong, cylindrical, pear-shaped, butternut, dumbbell,
and crookneck).
There are about 10 wild species of Cucurbita ranging from mesophytic
to xerophytic and from annual to perennial, which are naturally distributed
from the Central United States to Central Argentina, with the greatest
diversity in Mexico. Many wild species are also cross-compatible with the
domesticated species and increase their genetic diversity through natural
crossings (Lira Saade 1996; Montes-Hernandez and Eguiarte 2002).
C. ecuadorensis, endemic to Ecuador is easily crossed with C. maxima
and is being used as a source of disease resistance in breeding programs
and is well-adapted to drought. It is also related to C. moschata. C. moschata
is also partially cross-compatible with C. lundelliana, which is reportedly
resistant to diseases. Another wild species, C. okeechobeensis subsp. martinezii
L.H (Bailey), endemic to Mexico, is partially crossable with some of the
domesticated spp. and is a source of viral and fungal resistance. C. ficifolia
has been reported to be resistant to diseases, and tolerant to salinity, low
temperatures and to long storage periods. It is widely cultivated in small
gardens at high altitudes from Mexico to Central Chile, where it is used
to prepare candied squash. These wild species can be used as a source in
breeding for improvement of the domesticated Cucurbita species.
2.4.2 Bitter Gourd

The morphological documentation (e.g., passport data) and characterization
(assessment of genetic diversity) of bitter gourd germplasm (both cultivated
and wild types) was initially undertaken (1965 to 1972) in Germany, the
USA, Japan, China, Thailand, the Philippines, and India (i.e., National
Bureau of Plant Genetic Resources, Indian Agricultural Research Institute,
Kerala Agricultural University, Indian Institute of Horticultural Research).

Minor Cucurbits 35
More recently molecular markers [i.e., RAPD (Dey et al. 2006), ISSR (Singh
et al. 2007), and AFLP (Gaikwad et al. 2008)] have been used to assess the
genetic diversity of Indian bitter gourd genotypes including two promising
gynoecious lines (DBGy-201 and DBGy-202). A wide range in genetic
diversity was detected, indicating that a standard accession reference
array for future analyses might include “Pusa Do Mausami-green”, “Pusa
Do Mausami-white”, DBTG-2, Mohanpur Sel-215, and Jaynagar Sel-1.
Regardless of marker analyses type, however, Mohanpur Sel-125, DBTG-
101, and Jaynagar Sel-1 from West Bengal (an eastern state of India) are
genetically distinct (genetic similarity, GS) from other common landrace
accessions from North Indian states (GS = 0.57 to 0.72). Genetic differences
between M. charantia var. charantia (both exotic and indigenous) and M.
charantia var. muricata (indigenous) accessions are indicative of their use
as potential parents for the establishment of narrow (indigenous) and
wide-based (both exotic and indigenous) mapping populations. The exotic
(obtained from AVRDC-World Vegetable Centre) populations may be
informative for the characterization of qualitative and quantitative traits
in other cucurbit species (Serquen et al. 1997; Zalapa et al. 2007).
In bitter gourd, gynoecy is particularly interesting for hybrid
development (e.g., gynoecious x monoecious lines) and their commercial
production. The commercial deployment of gynoecy in hybrid technology
avoids the tedious step of pinching of male flowers during the production of
monoecious x monoecious hybrids. The utilization of such gynoecious lines
allows for the production of gynoecious or predominantly gynoecious lines
that provide early, uniform, high yield potential (Dey et al. 2010; Ram et al.
2002a). The hybrid, “Cuilli No.1” (China), for instance, was developed by
utilizing a gynoecious line as a maternal parent (Zhou et al. 1998). Likewise,
gynoecious lines originating in India were identified by Behera et al. (2006a;
lines DBGy-201 and DBGy-202) and Ram et al. (2002b; line Gy263B) for use
in hybrid development programs.
Commercial bitter gourd varieties and a few accessions/lines with
potentially important horticultural traits have been deposited and registered
in national germplasm collections. However, the National Bureau of Plant
Genetic Resources (NBPGR), New Delhi, India possess some unique
accession such as IC 256185, IC 248256, IC 213311, IC 248282, IC 256110 and
IC 248281 (Dhillon et al. 2005; resistant to fruit fly), NIC-12285 and VRBT-39
(resistant to downy mildew), IC 202195 (high yield and long fruited type),
TCR 404 (high yield and white fruited type), EC 399808 (high yield and
larger number of fruits), and INGR 03037 (gynoecious sex with high yield),
which can be used directly by plant breeders. Wild bitter gourd ecotypes and
botanical varieties (e.g., M. charantia var. muricata) are also important sources
of economically important traits (e.g., resistance against Dacus cucurbitae;

Dhillon et al. 2005). In the case of resistance to D. cucurbitae host response

varies dramatically among the cultivars (Yadav et al. 2003), where fruit fly
infestation has been shown to be lowest in “PBIG-123” (12.08%) and “Pusa
Do Mausami” (13.39%), and highest in “JMC-4” (41.49%).
2.4.3 Luffa
Cytogenetic investigations have been conducted among the Old World
species and chromosome counts in all the species were found to be the
same (2n = 26) with basic chromosome number of 13 (x = 13). The New
World species L. astorii also have 26 chromosome counts, but the presence
of numerous globular inclusions in the pollen mother cells of L. operculata
and L. quinquefida made it impossible to make exact counts in these species.
From the analysis of some of the hybrids, it is apparent that the chromosome
number of both the species is n = 13. Comparative morphology of the Old
World wild and cultivated species and chromosome pairing in interspecific
hybrid suggests that L. graveolens is the prime species, which has given rise
to the two cultivated monoecious species L. acutangula and L. cylindrica (Dutt
and Roy 1971). Pathak and Singh (1949) have given a detailed account on
the cytological behavior of interspecific hybrids between L. acutangula ×
L. cylindrica. These hybrids exhibited reduced fertility. Fertility is restored
when F1 hybrid is crossed with either of the parents. Cytological studies
reveal that each chromosome of haploid complements of the two species
is sufficiently homologous, but non-homologous segments are present in
normally pairing chromosomes of F1 hybrid between the two species. The
interspecific combinations between L. graveolens, L. aegyptiaca/cylindrica,
L. acutangula and Luffa echinata resulted in sterile hybrids (Dutt and Roy
1969, 1971). In a study on interspecific hybrids between L. acutangula and
L. graveolens and their amphidiploid (2n = 4x = 52), Dutt and Roy (1976)
reported frequent occurrence of univalents and bivalents at diakinesis and
metaphase-I. The univalents ranged from 0.0–18.0, the mean being 5.83 per
pollen mother cell (PMC). The rod bivalents varied from 0.0–14.0, whereas
the ring bivalents were 8.0–20.0 per cell. The mean number of chiasmata
per PMC was 37.43 as against 17.12 in the F1. Chromosome associations,
higher than bivalents were frequently scored, trivalents in 33.3% of the cells
and quadrivalents in 30.0% of the PMCs were observed. Low frequency
(<1%) of higher quadrivalents were also noticed. At anaphase-I, regular
disjunction of chromosomes along with few irregularities like unequal
separation and lagging chromosomes were found, which resulted in empty
pollen formation and subsequent pollen sterility of the amphidiploids.
Autotetraploidy was induced in L. acutangula (4x = 52), however, such

Minor Cucurbits 37
autotetraploids have no economic value (Roy and Dutt 1971). Artificial

hybrids between Old World species, i.e., L. aegyptiaca × L. astorii, L. aegyptiaca
× L. operculata, L. echinata × L. operculata and L. operculata × L. graveolens
were obtained and they were all sterile. L. quinquefida as the female parent
crosses readily with L. graveolens, but no seeds are obtained in the fruits.
Hybrids between various accessions within the New World species were
readily obtained. The hybrids between L. astorii and L. operculata were fully
fertile whereas those between L. quinquefida × L. operculata showed some
reduction in pollen stainabilty (46–71%), but seed set was equal to that of
the parental accessions. The hybrids between L. astorii and L. operculata was
obtained with great difficulties and only one viable seed was secured and
gave rise to a plant with abnormally developed flowers that produced no
anthers and no fruits were secured when it was pollinated by the parental
species. Hybrids between L. quinquefida and L. astorii were readily obtained
with L. quinquefida as the female parent. Four plants were grown to maturity.
Cytological analysis in one plant revealed 13 bivalents, pollen stainabilities
were 0, 7 and 18% and one plant had aborted anthers and produced no
pollen. No seed was obtained. The most fertile interspecific hybrids are those
between L. operculata and L. quinquefida, with L. quinquefida as female parent.
Four plants were grown. In one hybrid 13 bivalents were observed, and in
another one, anaphase-I was found to be regular with equal distributions
of chromosomes. Pollen stainabilities ranged from 20–52% with a mean of
37%. Fruits were readily secured when the L. operculata and L. astorii were
used as pollen parents, but the hybrids produced only one third to one
fourth the number of seeds in the parent.
Local cultivars and landraces of the two most domesticated species
L. acutangula and L. cylindrica are open-pollinated and hence populations
are variable. In India, National Bureau of Plant Genetic Resources (NBPGR)
systematically collects and maintains genetic resources of Luffa containing
genes for quality traits, adaptability to diverse agro-ecological zones,
resistance to diseases and pests as well as tolerance to stress environments.
The collections vary in plant type, sex forms, fruit skin color, fruit skin
glossiness, vine length, fruit length, fruits per plant, average fruit weight,
flesh texture, maturity days and other morphological and biochemical
traits. Apart from NBPGR, many Indian Council of Agricultural Research
(ICAR) Institutes and State Agricultural Universities working on Luffa
maintain genetic resources of Luffa. Internationally NACGRAB, Ibadan
(Nigeria), Asian Vegetable Research and Development Centre (AVRDC-
World Vegetable Centre), Taiwan, Southern Regional Plant Introduction
Station (SRPIS-USDA), Georgia (USA) are maintaining Luffa germplasm
collections.

2.5 Genetics and Breeding

2.5.1 Pumpkin
The major objectives for pumpkin and squash breeding programs are
earliness, environmental adaptation, improvement of fruit color and
morphology, high fruit gloss, a small blossom scar, high productivity,
enhanced nutritional value (β-carotene), pest and disease resistance and
abiotic stress (salinity, low temperature, drought) resistance or tolerance.
Besides, small, upright, open plant habit with shortened internode length for
reduced vining or for bush plant habit are also important. The pyramiding
of genes for resistance is a major goal of future breeding in these crops.
Fruit quality of pumpkin and squashes vary according to the species
and cultivar group and final use of the product (fresh market, canning, baby
food, seeds, etc.). Fresh market quality of pumpkin and squash usually
refer to fruit size, shape, color, eating acceptability (flesh color, consistency,
flavor, and sweetness) and flesh quality. Flesh quality is directly related to
the dry matter (DM) content of mesocarp, which is responsible for flesh
dryness. DM of flesh varies according to the balance of starch and sugar
content. Flesh sweetness increases upon fruit maturity, both on the plant
or during storage, however flesh dryness increases with maturity in the
field and decreases during storage. If the DM is too low, the cooked texture
of the fruit will be moist and fibrous. In contrast, a too high DM may lead
to dry flesh and low sweetness. Tropical cultivars of C. moschata, which
are frequently used in sauces, soups, stews, cakes and puddings do not
require high DM, and a smooth, pasty texture is popular (Daniel et al.
2005). C. moschata cultivars for canning industry should be the tastiest with
deepest-colored fruits. Processing varieties should have a soft and bright
orange rind and moderate-to-low levels of DM (9–11%). High sweetness
is preferred for baby food but is not necessary for production of pies. A
reasonable consistency with a high enough sugar level is necessary for the
frozen food industry (Loy 2004).
Pumpkin seed oil is economically important in many countries
of Central Europe. Selection for cultivars with a good seed chemical
composition, such as increased unsaturated fatty acid and tocopherol
content is required (Bavec et al. 2002). Tocopherols are antioxidants and
have the potential to reduce the risk of cancer and cardiovascular diseases.
Pumpkin seeds rich in phytosterol are desirable. Phytosterols reduce the
levels of blood cholesterol, decrease the risk of certain types of cancer and
enhances the immune system.
The genetics of the traits of Cucurbita have been reviewed by Paris
and Brown (2005). Fruit size is highly polygenic. The rind texture and
hardiness and the fruit cucurbitacin content are controlled by single genes

Minor Cucurbits 39
(Hi, Hard rind inhibitor and Bi, Bitter fruit in C. maxima). In C. maxima and
C. moschata, the Butternut and Buttercup varieties are popular because they
lack a hard shell and can be easily cut with a kitchen knife. Fruit shape is
polygenic, but is controlled by a major gene in C. moschata (Bn, Butternut).
Many genes control rind color (some affecting flesh color). However, other
allelic and non-allellic genes have also been reported in C. maxima and
C. moschata. Some of these genes are multiple-allelic and interact with each
other (Paris and Brown 2005). In C. maxima Bmax (Bicolour), derived from the
subsp. andreana, is responsible for precocious yellow fruit pigmentation,
and Rd (Red skin) for the red color of “Turk’s Caps”, dominant to other
colors. Knowledge of genetics of these traits facilitates their use in breeding
programs for improvement of Cucurbita.
Local Cucurbita germplasm has been subjected to horticultural and
agronomic evaluation worldwide with the objective of identifying valuable
accessions for breeding, viz. Bulgaria (Stefanova et al. 1994), Brazil (Choer
1999; Romao et al. 1999; Ramos et al. 2000), Cuba (Rios Labrada et al. 1998),
China (Chen 1993; Zhou et al. 1995) and India (Babu et al. 1996; Kale et al.
2002; Kumar et al. 2006). Transfer of economically important characteristics
such as host-plant resistance to diseases and pests or stress tolerance from
wild Cucurbita to their cultivated counterparts is challenging for breeders.
An interspecific cross is often used to introgress such characters. In the
genus Cucurbita, several attempts have been made to produce interspecific
hybrids among five cultivated species (C. pepo, C. maxima, C. moschata,
C. argyrosperma, C. ficifolia) and a number of non-cultivated ones. Using such
an approach, one could expect novel traits to be introgressed or genotypes
with recombined characters of involved species to be created.
Generally wild Cucurbita species have viny growth habit. The Bu
allele (Bush habit), which is responsible for bush vine in C. pepo, is also
responsible for the bushy habit in C. maxima, but the conversion from
compact to a more spreading growth occurs much faster in bush-vine
heterozygotes of C. maxima than those of C. pepo. A different gene confers
determinant plant growth in C. moschata (de, determinate) (Kwack 1995).
Bushy or semibushy habit has been introduced in many varieties of C.
pepo, C. maxima and C. moschata. The bushy habit has been introduced
into some cultivars of C. maxima by developing productive short- × long-
vined hybrids. The introduction of the bushy habit in C. moschata was
first transferred to temperate types by interspecific hybridization between
C. pepo and C. moschata followed by backcrosses to C. moschata. Some
compact hybrids have been derived from these bushy C. moschata cultivars
and long-vined types with tropical fruit characteristics (adapted to tropical
conditions and resistant to diseases) have been developed (Maynard et
al. 2002). These hybrids showed higher yields, but smaller fruits than the
long-vine landraces (Chesney et al. 2004). Bush plants are characterized

by early flowering, a high ratio of female to male flowers, early maturity

and a higher ratio of fruit to vegetative biomass than long vine varieties.
They are more amenable to high-density planting, sustainable weed control
and have rapid leaf canopy closure. These early types are much easier to
mature in cooler areas and can be marketed earlier. Higher ratio of female/
male flowers of the bushy plants is controlled by both environmental and
genetic factors. The intraspecific variation of this trait has been reported
in several species and transferred between them. High female lines of
C. moschata and C. maxima have been obtained from selected C. pepo varieties
(Kwack and Fujieda 1985; Gwanama et al. 2001; Loy 2004).
Bridge crosses exploit the natural potential of crossing ability among
species. Within the genus Cucurbita, C. moschata is crossable to many
wild and cultivated species, and it has been exploited as a bridge species
(Provvidenti et al. 1978). Bridging species is used to correct the sterility in
interspecific hybrids. Whitaker (1959) reported that C. lundelliana could
be crossed with each of the cultivated species of Cucurbita. Rhodes (1959)
developed an interbreeding population from crosses involving C. lundelliana
and the cultivated species C. pepo, C. argyrosperma, C. moschata, and
C. maxima. Thus, C. lundelliana served as a bridge to transfer genes between
species that would otherwise be difficult to cross (Whitaker and Robinson
1986).
Germplasm collections of C. maxima and C. moschata have received
attention for evaluation, both as sources of pathogen and pest resistance
and abiotic stress tolerance. Provvidenti (1982, 1986, 1990, 1993) reviewed
screening Cucurbita germplasm for reaction to viral pathogens. Munger
(1993) reviewed the genetic control of viral resistance in cucurbit breeding.
A devastating epidemic of zucchini yellow mosaic virus (ZYMV) in 1997
destroyed half of the pumpkin harvest in Austria, and development of
resistant varieties is the crucial factor to increase production (Pachner and
Lelley 2004). Evaluation for resistance against destructive viruses CMV
and watermelon mosaic virus-2 (WMV -2) discovered new sources of
resistance in C. maxima (Lebeda and Kristkova 1996; Kristkova and Lebeda
2000). Horvath (l993) conducted a screening of four Cucurbita species for
virus resistance.
Cucurbita ecuadorensis is resistant to a number of viruses, including
ZYMV, the watermelon strain of papaya ringspot virus (PRSV-W), and
watermelon mosaic virus (WMV) (Cutler and Whitaker 1969). Multiple
virus resistant C. maxima cultivars derived from C. ecuadorensis have been
developed at the Cornell University, USA. “Redlands Trailblazer” and
“Dulong QHI”, winter squash resistant to ZYMV, WMV, and PRSV, were
bred in Australia from the same interspecific cross (Herrington et al. 2001).
Resistance to different strains of aphid-transmitted ZYMV in C. ecuadorensis
is controlled by a recessive gene (zymecu), with modifiers, which has been

Minor Cucurbits 41
introgressed into C. maxima. Different genes of resistance have been reported

in C. moschata, such as the zymmos gene, derived from the tropical cultivar
“Soler”, and oligogenic resistance, which is controlled by three genes, Zmy-1,
Zmy-2 and Zmy-3, from the “Menina” landrace from Portugal (reviewed in
Paris and Brown 2005). High levels of resistance to ZYMV, PRSV-W, and
WMV were identified in a landrace of C. moschata “Nigerian Local” (Munger
and Provvidenti 1987). Resistance to ZYMV in this landrace is controlled by
a single incomplete dominant major gene (Zym-0) with modifiers. “Nigerian
Local” has also been used in breeding C. moschata as a source of resistance
to watermelon mosaic virus (WMV), controlled by the dominant Wmv,
papaya ringspot virus-W (PRSV-W), controlled by the recessive prv, and
Cucumber mosaic virus (CMV), controlled by the dominant Cmv gene (Brown
et al. 2003). C. ecuadorensis also provides resistance to WMV (Wmvecu), which
has been transferred to C. maxima, along with a low level of tolerance to
squash mosaic virus (SqMV) (Provvidenti 1997). Resistance to squash leaf
curl virus (SqLCY) (SIc gene) has been reported in C. moschata, and has been
transferred from C. ecuadoresis and C. lundelliana to C. maxima. Resistance
to these and other important viruses has been reported in some C. maxima
introductions (Kristkova and Lebeda 2000).
Considerable work has been done on screening for reaction to downy
mildew (Pseudoperonospora cubensis) and powdery mildews (Golovonomyces
cichoracearum and Podosphaera xanthii) and, to a lesser extent, on many
other fungal pathogens. Evaluations of Cucurbita germplasm for resistance
to powdery mildew have been well summarized by Jahn et al. (2002).
Field resistance has been reported in C. maxima landraces. Resistance to
G. cichoracearum has been reported in C. lundelliana (controlled by a dominant
gene, Pm) and in C. moschata (controlled by two genes, pm-1, from the
tropical cultivar “La Primera”, and pm-2, from “Seminole”) (reviewed in
Paris and Brown 2005). Resistance to P. xanthii, derived from C. okeechobensis
subsp. martinezzi, is governed by a single dominant gene with modifiers.
This resistance has been transferred to C. pepo (Cohen et al. 2003) using
C. moschata as a bridge species. This resistance has also been used to breed
resistant oriental varieties of C. moschata (Cho et al. 2003). Three-way
crosses have been used to transfer resistance genes for powdery mildew
and CMV from C. martinezii to C. pepo. Initially, C. martinezii was crossed
with C. moschata, then the resulting hybrid was crossed to C. pepo (Whitaker
and Robinson 1986).
Downy mildew is more prevalent in temperate and humid areas.
Lebeda and Widrlechner (2004) published the results of downy mildew
screening on wild and weedy accessions of Cucurbita. Resistance from
C. ecuadorensis and C. okeechobeensis was introgressesd into C. pepo, C. maxima
and C. moschata producing germplasm useful for breeding mildew-resistant
squashes (Robinson and John 1987). Lopes et al. (1999) presented results

from an inoculation of 150 Cucurbita accessions with fruit rot causing

fungus P. capsici.
Silver-leaf disorder caused by silverleaf whitefly, Bemisia argentifolii
in Cucurbita makes the plants less vigorous and silvered fruits produced
are commercially unacceptable. The resistance to this disorder found in
C. moschata (the Paraguayan landrace PI-162889 and Butternut types) is
controlled by a single recessive gene (sl, silverleaf resistance) (Gonzalez-
Roman and Wessel-Beaver 2002). Fruit fly (Dacus cucurbitae) resistance in
C. maxima is controlled by the Fr gene (Fruit fly). Resistance to pickle worm
in introductions of C. pepo, C. moschata and C. maxima, and tolerance to
squash vine borer in C. moschata have been reported (Whitaker and Bemis
1976; Paris and Brown 2005).
Haploidization is the process of producing haploid plants in a single
generation. Following haploidization, the chromosome number of haploid
plants may be doubled (dihaploidization) to obtain complete fertile
homozygous lines for use in breeding programs and genetic research.
Pollen irradiation (UV, gamma rays, and X-rays) is the most widely used
technique to induce in situ parthenogenetic haploid plants. Gamma
rays are commonly used in haploid programs because of their simple
application, good penetration, reproducibility, high mutation frequency,
and low disposal (lethal) problems. The irradiated pollen technique is an
effective method for the induction of haploid embryos in pumpkin (Kurtar
2009) who obtained haploid embryos and plants from pumpkin (Cucurbita
moschata Duchesne ex. Poir) pollinated with irradiated pollens at 50 Gy
and 100 Gy and observed the highest parthenogenetic response from 50
Gy gamma ray doses. Highest necrotic embryos were obtained in delayed
harvest times (5 and 6 weeks after pollination) and the best harvest time
was about 3 weeks (between the 18th and 24th day) after pollination. Kurtar
and Balkaya (2010) induced parthenogenetic haploid embryo via irradiated
pollen technique and produced haploid plantlets in winter squash (Cucurbita
maxima Duchesne ex Lam.).
Techniques such as embryo and callus culture, bridge crosses, ploidy
manipulation, protoplast fusion have been attempted for breaking the
presyngamic and post-syngamic crossing barriers, thus improving the
success of interspecific hybridization. To overcome the problems of sterility
and poor seed development in interspecific crosses, embryo culture is done.
Among Cucurbita species, conventional interspecific pollination followed
by in vitro culture of hybrid embryos can result in the production of fertile
hybrids (Sisko et al. 2003). Hybridization efficiency among Cucurbita species
and techniques to overcome crossing barriers and hybrid sterility have been
summarized by Robinson and Decker-Walters (1997) and Lira-Saade (1996).
Poorly developed seeds from interspecific hybrids or their progeny often
germinate better when their seed coats are removed (Whitaker and Robinson

Minor Cucurbits 43
1986). Ovule culture has been reported for use when making compatible
crosses in Cucurbita species (Hong and Hyo-Guen 1994). The Sakata Seed
Company of Japan offers the interspecific hybrids of C. moschata × C. maxima
(known as Kabocha) under different cultivar names (e.g., Alguri, Kikusui,
Tetsakabuto). Fruits are reported to combine favorable characters from
both species, and there is considerable heterosis for degree of female sex
expression and for yield (Whitaker and Robinson 1986).
Production of interspecific Cucurbita hybrids have been attempted using
embryo rescue, following techniques reviewed by Rakoczy-Trojanowska
and Malepszy (1989a) and Rakoczy-Trojanowska et al. (1992). Hybrid
genotypes with novel traits or entirely new characteristics may result. In
general, success of hybridization within this genus varies from combination
to combination. Successful rescue of Cucurbita hybrid embryos has
been reported for many interspecific combinations, including C. pepo ×
C. moschata (Wall 1954; de Oliveira et al. 2003; Sisko et al. 2003), C. maxima
× C. pepo (Rakoczy-Trojanowska and Malepszy 1986, 1989b; Sisko et al.
2003), C. moschata × C. maxima (Kwack and Fujieda 1987). C. ficifolia ×
C. maxima (El-Mahdy et al. 1991; Sisko et al. 2003), C. maxima × C. foetidissima
(Rakoczy-Trojanowska et al. 1992), and C. argyrosperma × C. moschata
(Sisko et al. 2003). Limited success in obtaining fully developed seeds that
germinated and produced viable plants has also been reported for a few
interspecific combinations, including C. andreana × C. ficifolia (Whitaker
1954), C. lundelliana × C. moschata (Whitaker 1959), and C. maxima ×
C. ecuadorensis (Paran et al. 1989).
The hybrid nature of plantlets obtained from interspecific crosses need
to be tested, since partenocarpic development or accidental pollination with
parental species cannot be fully excluded. Several methods are available to
confirm the hybrid nature of regenerants, such as differentiation in plant
morphological characters, chromosome counting, or the use of various
DNA-based molecular marker techniques. The genome sizes can be
compared and the hybrid nature of the rescued plants can be determined
early in the rescue process by using flow cytometry. Sisko et al. (2003)
showed that differences in genome size within the genus Cucurbita are big
enough to be used efficiently in determination of interspecific hybrids.
The main utility of genetic maps in plant improvement is their deployment
in marker-assisted selection (MAS) and breeding. The predictive value of
genetic markers used in MAS depends upon their inherent repeatability,
map position, and linkage with economically important quantitative or
qualitative traits (Staub et al. 1996). Until recently, the genetic mapping in
the genus Cucurbita was hampered by the lack of available sequence-specific
markers. Based on anonymous RAPD and AFLP markers, two maps using
interspecific crosses between C. pepo and C. moschata (Lee et al. 1995; Brown
and Myers 2002) were developed that did not allow comparative analysis

between species. Recently, Gong et al. (2008a) developed 500 microsatellite

markers from SSR-enriched genomic libraries, which showed a high
transferability among species within the genus Cucurbita. Well developed
SSR maps are of great value for comparative genetics to study genome
structure, studies on Cucurbit evolution, accelerate breeding programs
using marker-assisted selection, and for future gene isolation programs.
The first SSR-based genetic linkage map of Cucurbita moschata was created
by Gong et al. (2008b) by integrating the maps of two F2 populations with
one common parent developed from the crosses Waltham Butternut (WB)
× Nigerian Local (NL) and ZHOU (a hull-less type) × WB. The integrated
C. moschata map comprised 205 SSR markers and two morphological traits,
Gr (Green rind) and n (hull-less or naked seeds). The map was composed
of 27 linkage groups with a marker density of 7 cM. Comparing the
C. moschata map with the published Cucurbita pepo map, they found a high
level of macrosynteny. Seventy-two of the 76 common SSR markers between
C. moschata and C. pepo were located in homologous linkage groups. These
markers in general had conserved orders and similar genetic distances
and they represented orthologous loci. This high level of macrosynteny
between C. moschata and C. pepo will enable easy transfer of markers and
genes tightly linked to important traits and facilitate the construction of
a reference map for the whole Cucurbita genus. They obtained a reference
map based on these SSRs. No major chromosomal rearrangement between
the two species could be detected.
2.5.2 Bitter gourd

A wide range of quantitatively and qualitatively inherited phenotypic
variation is present in Asian bitter gourd (Behera 2004). Manipulation of
these traits could form the basis for breeding program goals. The most
important points of consideration in this regard are:
• Cultivars must meet international export standards—e.g., fruits must
be green, 20–25 cm long, and possess a short neck;
• Cultivars should possess characteristics that reduce malnutrition—e.g.,
high vitamin (carotenoids and ascorbic acid) and mineral (iron and
calcium) content;
• Improved abiotic stresses resistance (high temperature, water
deficiency, salt tolerance);
• Non-bitter cultivars, which will provide medicinal benefits—e.g.,
proteins (charantin), polypeptides (polypeptide-K), glycoalkoloids,
phenolics and other antioxidants;
• Improved germplasm with high yield potential should be
gynoecious;

Minor Cucurbits 45
• Improved germplasm (e.g., transgenic) should possess disease-pest

resistance (e.g, virus, powdery and downy mildew and red pumpkin
beetle, and;
• Improved germplasm should possess better fruit quality—e.g., later
seed maturity, minimized ridges, uniform green color in a range of
fruit size (small, medium, and large).
Light brown seed (lbs) coat color is recessive to dark brown (Srivastava
and Nath 1972). Large seed (ls) size is recessive to small seed size (Srivastava
and Nath 1972), white epicarp (w); which is recessive to green (Suribabu
et al. 1986; Vahab 1989), spiny fruit (traingular tubercles) is dominant over
smooth (Vahab 1989). Since immature bitter gourd fruits are sliced during
the preparation of various Asian meals, exceptional internal fruit quality
and uniform green peel color are desirable. Liu et al. (2005) reported high
heritability of fruit color (green vs. white) is controlled by two genes where
green is dominant to white (Miniraj et al. 1993; Liou et al. 2002; Hu et al.
2002). Fruit should be firm without excessive seed development, and free
of internal defects such as decay and splitting. Fruit color also governs
its marketability as the color preference differs from region to region. For
example, green-fruited types are in demand in southern China, while
white-fruited types are preferred in Central China. Similarly, dark green
to glossy green fruits are favored in northern India, whereas white fruits
are preferred in southern India.
Singh and Ram (2005) determined that complementary epistasis and
dominance x dominance interactions were important genetic determinates
of yield. Given these facts, Devadas and Ramadas (1994) recommended that
selection and hybridization (i.e., reciprocal recurrent selection) would be an
appropriate breeding strategy for improvement of fruit triterpinoid content.
The genetic analysis of a large fruited (M. charantia var. charantia/maxima) x
large fruited (M. charantia var. muricata/minima) population suggested that
the small fruit was partially dominant over large fruit (Kim et al. 1990), but
that fruit length was incompletely dominant and controlled by a minimum
of five genes (Zhang et al. 2006). Likewise, the dramatic role of epistatsis
in the development of fruits suggests that breeding for this trait will be
challenging (Sirohi and Choudhary 1983; Choudhury and Kale 1991).
Breeding for nutritional/medicinal quality in crop species typically
emphasizes accessions with relatively high vitamin C content (Behera et
al. 2006b). For example, bitter gourd lines DBTG-3, DBTG-8, DBTG-6, and
DBTG-9 contain >1,000 mg·kg–1 vitamin C in edible plant parts as compared
to ~500 mg·kg–1 in standard cultivated types (Behera et al. 2006b). These
high vitamin C lines are aggressively used in breeding programs whose
focus is on the development of cultivars with high nutritional values. High
quality is also found in Indian bitter gourd cultivars possessing high total

soluble solid content (> 3.10 0Brix; “MC-84”, “Preethi”, “RHRBG-5” and
“PBIG-1”), and vitamin C (> 950 mg·kg–1; “Konkan Tara” and “Hirkani”)
and fruit protein (>1.5%; ‘DVBTG-1’, “Preethi”, “Hirkani” and “Konkan
Tara”) content (Kore et al. 2003).
Polyploids can be produced by treating the seedlings at the cotyledon
stage with an emulsion of 0.2% colchicine. The seed treatment with 0.2, 0.4%
colchicine or 0.003% amiprophos-methyl was effective for chromosome
doubling, among which the treatment with 0.4% colchicine was most
effective. Amiprophos-methyl treatment also produced octoploid plants
with a high rate of seed germination. Multiple shoot treatments with
0.05% colchicine for 12 and 24 hours, and 0.1% colchicine for 24 hours also
produced octoploid plants. Leaf and guard cell size were bigger, and leaf
shape index (leaf length/leaf width) was lower in the octoploid as compared
to tetraploid plants. Leaves of the octoploid plants were uneven on the
surface with clear serrations.
Triploid plants of M. charantia were obtained by crossing the tetraploid
(colchicine-induced) and diploid plants. In seedlings of bittergourd,
colchicine at 0.2% for 18 hours to the shoot tip produced tetraploids (Kadir
and Zahoor 1965). However, polyploids were inferior to diploids in terms
of economic characters.
In M. charantia, progeny (M1) derived from radiation mutagenesis can
possess economically important unique characters, which are controlled by
single recessive genes. One such bitter gourd variety, MDU 1, developed
as a result of gamma raditation (seeds treatment) of the landrace MC
103 was found to possess improvement for yield (Rajasasekharan and
Shanmugavelu 1984). Likewise, the white bitter gourd mutant “Pusa
Do Mausami” (white-fruited type) was developed through spontaneous
mutation from the natural population “Pusa Do Mausmi” (green-fruited
type) at the Indian Agriculture Research Institute.
2.5.3 Luffa
Protein variation has been widely used for genetic studies in cucurbits
and seed protein is used mainly for species or variety differentiation as
they do not change in dry mature seed (Ladizinsky and Hymowitz 1979).
Tolentino et al. 1997 employed SDS-PAGE of seed proteins to determine
genetic diversity and taxonomic relationship of Luffa species. The similarity
indices of L. cylindrica and L. acutangula of accessions were 80.0 and 71.1%,
respectively. Domestication of the smooth gourd may have contributed to
its lesser diversity. A similarity index of 68.2% between the two species
suggests that the genome of smooth and ridge gourds do not differ
very much from each other. Contrary to this, LiLin et al. (2007) based
on morphological characters and RAPD analysis suggested that genetic

Minor Cucurbits 47
similarity between L. cylindrica and L. acutangula was 0.17, indicating the

two species are genetically diverse. However, they were in conformity with
Tolentino et al. (1997) regarding genetic diversity among the accessions of
L. cylindrica and L. acutangula. Allozymic, morphological and phonological
diversity in cultivated Luffa acutangula from diverse geographical regions
were reported by Marr et al. (2005). The allozyme analysis revealed that
L. acutangula and L. aegyptiaca are fixed for different alleles at nine loci,
indicating that they are completely reproductively isolated from each other.
Pandey et al. (2007) employed SDS-PAGE of seed proteins for biochemical
characterizations of 48 Indian genotypes of sponge gourd. JunHui and
ChangPing (2008) also employed morphological and RAPD markers to
study the genetic diversity in luffa. They observed 86.21% polymorphism
across 26 luffa accessions and the average Shannon’s information index
of RAPD loci was 0.325. The results of RAPD markers were not identical
those with morphological markers. Many wild relatives of Luffa are grown
in Bangladesh and so far 106 local landraces were reported (Rabbani 2007).
RAPD markers were used for assessment of genetic relationship among
landraces of Bangladeshi ridge gourd. Genetic variation statistics for all
loci estimated the average gene diversity value as 0.278 and the Shannon’s
Information Index as 0.415 (Hoque and Rabbani 2009).
Matsumoto et al. (1987) reported the procedure for successful isolation
and culture of protoplast from suspension culture of Luffa cylindrica. Seeds
of cultivar Futo was surface sterilized, germinated in MS agar medium and
calli were induced by placing cotyledon segments of 2–3 weeks old axenic
seedlings on MS agar supplemented with 2, 4-D and kinetin. For protoplast
isolation, cells were collected by centrifugation. The growth phase of culture
strongly influenced the yield and viability of protoplasts and the highest
yield of protoplast was obtained at 6 days with approximately 90% viability.
The plating efficiency of protoplasts was improved by using 0.5 mg/l
kinetin. The first division occurred within 5–7 days and after 10 days 35%
protoplasts divided at least once. Clusters of several cells were formed from
about 1/3rd of the divided protoplasts and later these developed into visible
colonies. Ito et al. (1991) produced hybrid cell lines between L. cylindrica
and Gynostemma pentaphyllum by protoplast fusion. The hybridity was
confirmed by their possession of both parental homodimeric isoenzymes
of glucose-6-phosphate isomerase and chromosome counts.
Evolution of nucleotide sequences in the plant kingdom has been
associated with considerable alterations of genome organization. The
cucurbit genome has been investigated extensively (Pasha and Sen 1995).
The three species of Luffa, L. acutangula, L. cylindrica and L. hermaphrodita
showed similar patterns of reassociation kinetics indicating considerable
similarity of their genome organization patterns. Luffa acutangula exhibited
29% highly repeated (Cot value of 10–3–10–1) and 23% moderately repeated

sequence (Cot value > 10–1–102) of total nucleotides in DNA and 48% unique
sequence (Cot value > 102), whereas L. cylindrica exhibited 30, 20 and 50%
highly repeated, moderately repeated and unique sequences, respectively.
The ratio of repeated to unique sequence was 1.08 for L. acutangula and 1.0
for L. cylindrica (Pasha and Sen 1995).
Hyashi et al. (1999) cloned cDNA encoding cycloartenol synthase
from L. cylindrica and deposited the sequence in the gene bank. Nucleotide
sequence of a cDNA putative oxidosqualence cyclase from L. cylindrica is
also present in the gene bank (Hyashi et al. 2000).
A novel circulating loop bioreactor with cell immobilized in L. cylindrica
sponge has been used for the bioconversion of raw cassava starch to ethanol
(Roble et al. 2002). Loofa sponge has been used as a medium for the culture
of human hepatocyte cell line (Chen et al. 2003). L. cylindrica showed a
very good performance as a solid substrate for the development of biofilm
aggregating microorganisms capable of metabolizing both organic and
inorganic compounds adsorbed in it, particularly those responsible for
nitrification (Tavares et al. 2008).
Cell immobilization technique using biological materials are eco-
friendly and have many advantages over suspended cell systems. Many
studies have shown that the adsorption method for immobilization of
microorganisms has many advantages over the entrapment method using
gel beads (Ogbonna et al. 1994; Fujii et al. 2001). L. cylindrica sponge is an
excellent carrier for immobilization of microorganisms, plants and animal
cells (Liu et al. 1999; Ogbonna et al. 2001; Roble et al. 2002; Chen et al. 2003).
In order to use a loofa sponge as an immobilization carrier in systems
containing/producing cellulose enzymes, a method of protecting loofa
from cellulose by acetylation has been developed (Akhiro et al. 2007). Loofa
sponge-immobilized fungal biosorbent have been used extensively for the
biosorption of heavy metals from olive oil mill wastewater (Ahmadi et al.
2006a) and other wastewater. Loofa sponge is a suitable natural matrix for
immobilization of P. chrysosporium. Immobilized P. chrysosporium has been
successfully used as biosorbing agent for removal of cadmium and lead
(Iqbal and Edyvean 2004, 2005; Ahmadi et al. 2006b).
Recently production of renewable energy from biomass materials
have been emphasized as a means of solving current environmental
problems such as global warming. In this regard, production of ethanol
from lignocellulosic material has great potential. The simultaneous
saccharification and fermentation process seemed to be one of the most
promising options (Ye and Jiayang 2002; Itoh et al. 2003).

Minor Cucurbits 49
2.6 Conclusion
Studies to elaborate taxonomic and phylogenetic relationships among
most wild and cultivated types are still required, and information about
biogeography and ecobiology of wild relatives is rather limited.
In spite of the progress, in many germplasm collections, basic
characterization data of these species are incomplete, limiting the utility
of these collections. Broadening the genetic diversity is essential for
development of commercial cultivars with novel traits in these species.
The germplasm collections have received considerable attention for
evaluation for pathogen, pest, and abiotic stress resistance. A complex of
viruses continues to pose a serious threat to the crop of these species in the
tropics and subtropics, necessitating the identification and pyramiding of
genes for resistance to these viruses in elite cultivars. Extensive germplasm
evaluations have to be conducted for tolerance to high temperatures, salinity,
and drought.
Transfer of economically important traits from wild species to their
cultivated counterparts continues to be challenging for breeders especially in
cases where resistance genes for several pathogens and pests have not been
found within the primary gene pools. Careful biosystematic and gene-pool
studies should continue to help in selection of genotypes for increasing the
probability of successful interspecific hybridization.
Genetic diversity analysis, genetic mapping and map construction is still
in its infancy in all of these species. The development of high-throughput
DNA markers in recent years has created a fertile ground for genetic
mapping and genomics in these crops. Current advancements in sequencing
technologies, functional genomics, and metabolomics will play important
roles in research and development in these crops in the coming decades.
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3
Classical Genetics and Traditional
Breeding
Stephen R. King,1,* Angela R. Davis2 and Todd C. Wehner3
ABSTRACT
Much advancement has been made in traditional breeding and classical
genetics of cucurbit crops, although most significant advancement have
been related to qualitative traits. Significant improvement in many
quantitative traits have been much harder to achieve, and typically
result from several incremental advances that occur over a long period
of time. Molecular techniques have the potential to overcome many
of the obstacles presented by traditional breeding techniques, but it is
imperative that the development and utilization of these new molecular
technologies work in conjunction with traditional breeders who have
the skill set necessary to evaluate the germplasm resulting from these
new technologies.
3.1 Introduction
Cucurbit crops simultaneously bestow upon the breeder several advantages
and disadvantages. As pointed out by Whitaker and Bohn (1950), cucurbit
crops are easily grown, indeterminant plant types, which typically offer
plenty of large flowers to work with over a fairly long period of time.
Probably the greatest disadvantage is that cucurbit crops tend to be large
plants that require abundant field space for proper examination of most
agronomically important traits. Adding to the disadvantages, cucurbit
1
Vegetable and Fruit Improvement Center, Department of Horticultural Sciences, Texas A &
M University, College Station, TX 77843-2119, USA; e-mail: srking@tamu.edu
2
Wes Watkins Agricultural Research Laboratory, USDA-ARS, PO Box 159, Hwy.3 West Lane,
OK 74555, USA; e-mail: angela.davis@lane-ag.org
3
Department of Horticultural Science, Box 7609, North Carolina State University, Raleigh, NC
27695-7609, USA; e-mail: todd_wehner@ncsu.edu

crops must be hand-pollinated to prevent cross-pollination, and since most

selections are for fruit qualities, pollinations must be made before selection
of desired phenotype. Consequently, our genetic understanding has lagged
behind other crops that can be more easily self-pollinated and properly
evaluated in a smaller area, such as tomato.
The establishment of the Cucurbit Genetics Cooperative (CGC) and the
annual publication of Cucurbit Genetics Cooperative Report has facilitated
the dissemination of information on cucurbit genetics. For example, the
number of identified genes in watermelon has grown from a total of 25
prior to the establishment of the CGC in 1978, to 60 genes and 111 isozyme
markers in 2007, the most recent year in which the genes were reported
for watermelon (Wehner 2007). This annual report is an excellent resource
for cucurbit breeders since each crop’s gene list (watermelon, melon,
cucumber, squash and pumpkin, other genera and species) is updated
every five years.
3.2 Classical Genetics

3.2.1 Inheritance of Traits
Classical genetics have increased our understanding of cucurbit crops and
aided breeders in the development of new and improved varieties. Breeders
in the past have been able to make improvement without understanding
the genetic control of these traits, but improvement under these conditions
are painstakingly slow. Understanding how different genes affect a variety
of traits allow breeders to devote the proper resources needed to improve
a particular trait. For example, if a breeder is selecting for a trait controlled
by a single gene, the population size will likely be much smaller than if the
trait is controlled by multiple genes with a large environmental influence.
The application of Mendelian genetics using classical techniques has
facilitated the discovery of a number of genes and their inheritance in
cucurbit crops.
3.2.1.1 Watermelon
Watermelon is a useful crop species for genetic research because of its small
genome size, and the many available gene mutants. The genome size of
watermelon is 424 million base pairs (Arumuganathan and Earle 1991).
Like some of the other cultivated cucurbits, watermelon has much genetic
variability in seed and fruit traits. Genetic investigations have been made
for some of those, including seed color, seed size, fruit shape, rind color,
rind pattern, and flesh color.

Classical Genetics and Traditional Breeding 63
Many watermelon fruit quality genes have been identified. Fruit shape
is controlled by an incompletely dominant gene, resulting in elongate (OO),
oval (Oo), or spherical (oo) fruit (Weetman 1937; Poole and Grimball 1945).
A recessive gene (f) controls furrowed fruit surface (Poole 1944). Explosive
rind (e) causes the fruit rind to burst or split when cut (Porter 1937); these
fruit are easily crushed and are sometimes used as pollenizer cultivars
not intended for harvest. Tough rind (E) improves shipping ability. The
single recessive gene su (Chambliss et al. 1968) eliminates bitterness in C.
lanatus, and is allelic to the dominant gene (Su) for bitter flavor in Citrullus
colocynthis.
Watermelon flesh color is controlled by several genes to produce scarlet
red, coral red, orange, salmon yellow, canary yellow, pale yellow, green,
or white. Genes conditioning flesh colors are B (Shimotsuma 1963), C
(Poole 1944), Wf (Shimotsuma 1963), y (Porter 1937), y-o (Henderson 1989;
Henderson et al. 1998), and py (Bang et al. 2010). There is some confusion
in the literature regarding flesh color inheritance, possibly due to the
potential presence of two different “white flesh” phenotypes (Bang et al.
2010). Wf is reported to control white flesh in watermelon and is reported
to be epistatic to B, where genotypes Wf--- are white, wfwfB- are yellow
and wfwfbb are red (Shimotsuma 1963). Henderson et al. (1998) reported
two genes separate red flesh and canary yellow flesh, C and i-C, where a
dominant C gives canary yellow flesh except in the presence of a dominant
i-C, which would give red flesh. Bang et al. (2007) demonstrated a single
gene distinguishing red and canary yellow flesh. They also showed a pale
yellow phenotype can result between crosses of canary yellow and red
(Bang et al. 2010). The recessive py gene seems to require the presence of
a dominant C gene. This py mutant may have been confused with white
flesh, which is caused by a dominant Wf. Interactions of Wf, B, C, and now
py need further study for clarification.
The coral red gene (Y) is dominant to salmon yellow (y), and orange
flesh (y-o) is a member of a multiple allelic system at that locus, where Y
(Coral red flesh) is dominant to both y-o (orange flesh) and y (salmon yellow),
and y-o (orange flesh) is dominant to y (salmon yellow). It is reported that a
dominant Scr produces scarlet red flesh instead of the recessive coral red
flesh (summarized in Wehner 2007). Scr is now believed to be part of the
multiple allelic system at the Y locus where Scr is another allele at the Y
locus (T Wehner, unpubl. data). More study is needed that includes classical
genetics combined with biochemical and molecular data to fully understand
the genes and inheritance of flesh color in this crop. This understanding is
critical since flesh color is indicative of carotenoid content, which impacts
the nutritional benefits of watermelon.
Several genes have been identified that affect the rind of watermelon.
The gene Sp produces spotted fruit (Poole 1944). Golden yellow (go) is a

single recessive gene that causes fruit to turn yellow at maturity (Barham
1956). Intermittent stripes (ins) produces narrow dark stripes at the peduncle
end of the fruit that become irregular in the middle and nearly absent at
the blossom end of the fruit (Gusmini and Wehner 2006). Yellow belly, or
ground spot, on “Black Diamond Yellow Belly” is controlled by a single
dominant gene (Yb). Weetman (1937) proposed that three alleles at a single
locus determined rind pattern. The allelic series was renamed to G, gs,
and g by Poole (1944). The g-s gene produces a striped rind, but the stripe
width (narrow, medium, and wide stripe patterns) has not been explained
as yet. Porter (1937) found that dark green was completely dominant to
light green (yellowish white, in his description). The watermelon gene p
produces pencilled rind pattern (Robinson et al. 1976) and the m gene for
mottled rind was first described by Weetman (1937).
3.2.1.2 Cucumber
Sex expression in cucumbers has played an important role in seed
production as well as development of new fruit types. This trait is affected
by several single-gene mutants. The F locus governs gynoecy, but is
modified by other genes and the environment, and interacts with a and m
(androecious and andromonoecious, respectively) (Rosa 1928; Tkachenko 1935;
Galun 1961; Shifriss 1961; Wall 1967; Kubicki 1969a). The F gene may also
be modified by an intensifier gene (In-F) which increases the femaleness
(Kubicki 1969a). Other genes that affect sex expression are gy for gynoecious,
m-2 for andromonoecious (Kubicki 1974) and Tr for trimonoecious expression
(Kubicki 1969b).
The discovery of parthenocarpy in cucumbers (Wellington and
Hawthorn 1928) has led to the development of seedless fruit when
combined with the gynoecious trait. There is dispute over the inheritance
of parthenocarpy. Pike and Peterson (1969) suggested an incompletely
dominant gene, Pc, affected by numerous modifiers. In contrast, de Ponti
and Garretsen (1976) explained the inheritance by three major isomeric
genes with additive action.
Bitterness in cucumbers can affect fruit quality, health potential, and
insect resistance. Eliminating bitterness in this crop can be accomplished
by selecting for the bitterfree allele (bi), which inhibits biosynthesis of
cucurbitacin (Andeweg and De Bruyn 1959). Cucurbitacins can be toxic
at high levels and they may act as an attractant for cucumber beetles, but
have been shown to repel spider mites and aphids.
Disease resistance is an important trait in cucumber as diseases can
reduce yield and quality. Currently there are 15 genes known to control
disease resistance in C. sativus. Three of these genes condition virus
resistance. Wasuwat and Walker (1961) found a single dominant gene

(CMV) for resistance to cucumber mosaic virus. However, others have

reported more complex inheritance (Shifriss et al. 1942). Two genes have
been found to condition resistance to papaya ringspot virus (Wang et al.
1984; Wai and Grumet 1995), and five different genes have been identified
for resistance to watermelon mosaic virus II (Cohen et al. 1971; Wai et al.
1997). Resistance to zucchini yellow mosaic virus has also been identified
(Provvidenti 1987; Kabelka et al. 1997).
Both resistance to scab and resistance to bacterial wilt are dominant
and controlled by Ccu (Bailey and Burgess 1934; Andeweg 1956; Abul-
Hayja et al. 1978) and Bw (Nuttall and Jasmin 1958; Robinson and Whitaker
1974), respectively. Other dominant genes providing resistance are: Cca for
resistance to target leaf spot (Abul-Hayja et al. 1978), Cm for resistance to
Corynespora blight (Shanmugasundarum et al. 1971), Foc for resistance
to Fusarium wilt (Netzer et al. 1977) and Ar for resistance to anthracnose
(Barnes and Epps 1952). In contrast, resistance to Anthracnose race 1 (Abul-
Hayja et al. 1978) and angular leaf spot (Dessert et al. 1982) are conditioned
by the recessive genes cla and psl, respectively.
Several reports have indicated that more than one gene controls
resistance to powdery mildew with interactions occurring among loci. The
resistance genes pm-1 and pm-2 were first reported by Hujieda and Akiya
(1962) in a cultivar which they developed and named “Natsufushinari”.
Kooistra (1968) using this same cultivar, later confirmed their findings and
identified one additional gene (pm-3) from USDA accessions PI200815 and
PI200818. Shimizu et al. (1963) also supported three recessive genes, which
are responsible for resistance of “Aojihai” over “Sagamihan”.
Currently, one gene, dm, has been identified, which confers resistance to
downy mildew (van Vliet and Meysing 1974). Inherited as a single recessive
gene, it also appeared to be linked with pm (van Vliet and Meysing 1977).
There are, however, indications that more than one gene may be involved
(Jenkins 1946).
3.2.1.3 Melon
Most melon cultivars are andromonoecious, but other expression patterns
are possible. Genes a (andromonoecious) and g (gynomonoecious) interact to
influence sex expression: a_g_ = monoecious; a_ gg = gynomonoecious; aa g_
= andromonoecious; and aa gg = hermaphrodite (Pitrat 2006). A third gene
was identified that creates a much more stable gynomonoecious phenotype
(gy), so that a+_ gg gygy = stable gynomonoecious.
Sterility is common in melon, as there are five different male-sterile
genes and two total plant sterility genes (Pitrat 2006). However, since there
are no seedling markers for these recessive male sterile genes, it is impossible
to tell which plants are sterile without growing the plants to flowering. If

the purpose is to use the male-sterile trait for hybrid seed production, by
the time you identify which plants are sterile, the fertile plants will have
contaminated your hybrid seed.
Fruit quality in melon is polygenic, but there are several genes that
have major effects. Some negative alleles for fruit quality include Bif for
Bitter fruit (Parthasarathy and Sambandam 1981), Me for Mealy flesh texture
(Ganesan 1988) and So for Sour taste (Kubicki 1962). Flesh color is dictated
by single genes but the intensity of color is quantitative. Fruit shape is also
influenced by genes for oval fruit shape (O), sutures (s) and spherical fruit shape
(sp), but sex expression may also have an influence as perfect flowers tend
to give round fruit while female flowers tend to give more elongated fruit
(Lumsden 1914; Bains and Kang 1963; Wall 1967).
There are many loci in melon for disease resistance. Fusarium wilt
resistance is provided by Fom-1 and Fom-3, which are alleles for independent
genes that provide resistance to races 0 and 2, and Fom-2 gives resistance to
races 0 and 1 (Risser 1973; Zink and Gubler 1985). Resistance to Alternaria
is provided by Ac (Thomas et al. 1990), and there are up to six genes that
provide a high to moderate level of resistance to gummy stem blight (Prasad
and Norton 1967; Zuniga et al. 1999; Frantz and Jahn 2004). There are up to
17 different genes that govern resistance to powdery mildew, depending
on the race/species involved (Jagger et al. 1938; Bohn and Whitaker 1964;
Harwood and Markarian 1968a, b; Kenigsbuch and Cohen 1989; Epinat
et al. 1993; Anagnostou et al. 2000; McCreight 2003). There are four genes
reported for downy mildew resistance (Cohen et al. 1985; Thomas et al.
1988; Epinat and Pitrat 1989; Kenigsbunch and Cohen 1992), and a fifth
gene is reported to act in combination with at least one other modifier gene
(Angelov and Krasteva 2000). There are also resistance genes for papaya
ringspot virus, zucchini yellow mosaic virus, and necrotic spot virus (See
Pitrat 2006 for gene names.).
Melon also has resistance genes for several insects. Gene Af provides
resistance to red pumpkin beetle (Vashistha and Choudhury 1974). Tolerance
to melon aphid is provided by Ag (Bohn et al. 1973), and Vat provides
resistance to viruses transmitted by aphids (Pitrat and Lecoq 1980). Melon
fruit fly resistance is provided by two genes, dc-1 and dc-2 (Sambandam
and Chelliah 1972). As with cucumber and squash, cucurbitacin content
also influences insect resistance/susceptibility, which in the case of melon
is governed by two genes, Bi and cb (Lee and Janick 1978; Nugent et al.
1984).
3.2.1.4 Cucurbita spp.

A single gene for gynoecious sex expression (G) has been identified in
C. foetidissima (Fulks et al. 1979; Dossey et al. 1981), but the gene has not

yet been transferred to cultivated squash. There are four male sterile genes
reported, two in each species, for C. pepo and C. maxima (Scott and Riner
1946; Eisa and Munger 1968; Superak 1987; Korzeniewska 1992), but as with
most of the other cucurbit crops there are no morphological markers, which
would allow them to be useful for seed production. There are two total plant
sterility genes, one in C. maxima and one in C. pepo (Carle 1997).
The reduced internode length that gives a bush habit in C. pepo and
C. maxima is governed by a single gene (Bu) (Shifriss 1947; Grebenšcikov
1958; Decker-Walters and Munger 1963; Wu et al. 2007). This trait is greatly
appreciated by gardeners with limited space. A unique gene found in the
Cucurbita spp. is the naked seeded trait where seed lack a lignified seed
coat and is typically used for the roasted pumpkin seed market (Schöniger
1952; Grebenšcikov 1954; Xianglin 1987; Zraidi et al. 2003, 2007).
There are far fewer identified disease resistance genes in squash than the
other major cultivated cucurbit crops. Resistance to powdery mildew (PM)
in C. okeechobeensis and C. lundelliana is controlled by a single dominant allele
and modifiers (Contin 1978; Paris and Cohen 2000; Cohen et al. 2003) and
two PM-resistant genes to race 1 and race 2 were identified in C. moschata
(Adeniji and Coyne 1983). A single gene has also been reported to provide
resistance to zucchini yellow mosaic virus. There are three complementary
dominant genes for resistance to Phytophthora capsici (Crown rot) (Padley et
al. 2009). Known virus resistance consists of one recessive gene for cucumber
mosaic virus (Brown et al. 2003), one recessive resistance gene to papaya
ringspot virus (Brown et al. 2003), two watermelon mosaic virus resistance
genes, one from C. moschata (Fulks et al. 1979; Brown et al. 2003), which may
be linked with or identical to Zym-1 and one from C. ecuadorensis (Shifriss
1989), and a total of six resistance genes and one modifier gene have been
reported for zucchini yellow mosaic virus in C. moschata, C. pepo, and/or C.
ecuadorensis (Mains 1950; Fulks et al. 1979; Paris et al. 1988; Robinson et al.
1988; Paris and Cohen 2000; Brown et al. 2003; Pachner and Lelley 2004).
There is one resistance gene reported for insects (Fr, for melon fruit fly
resistance) (Nath et al. 1976), although the gene cu (Sharma and Hall 1971),
which reduces foliar cucurbitacin content, will have a similar effect as for
the other cucurbit crops by reducing cucumber beetle preference while
making the plant more attractive to aphids and spider mites.
Fruit quality, shape and color are extremely diverse in the squash and
pumpkin species and a thorough review of the genes involved was compiled
by Paris and Kabelka (2009). A number of fruit color genes have been
identified. The B gene that was found in an ornamental gourd can be used
to give a yellow color and high vitamin A content. This same gene also has
pleiotropic effects on fruit and foliage and is affected by several modifier
genes (Ep-1, Ep-2 and Ses-B). The B gene is also complementary to L-2 to give
intense orange flesh instead of light yellow flesh color which also enhances

the carotenoid content. There are up to five different fruit bitterness genes,
but allelism tests have not been reported for all five genes.
3.2.1.5 Other Cucurbit spp.

There are a few reports on identified genes, linkages, and improvements
for other cucurbit crops. What is known is summarized in Taja and Wehner
(2008). In West Indian Gherkin (Cucumis anguria), four gene loci have been
described—a single dominant gene produces bitter fruit (Bt) (Koch and
Costa 1991); a dominant gene for resistance to cucumber green mottle mosaic
virus (Cgm) (den Nijs 1982); and two loci that control fruit spininess (S and P)
(Koch and Costa 1991). African horned cucumber (Cucumis metuliferus) has
two identified genes: watermelon mosaic virus-1 resistance is controlled by a
single dominant gene (Wmv) (Provvidenti and Robinson 1972), and a single
dominant gene for resistance to papaya ringspot virus (Prsv) (Provvidenti
and Gonsalves 1982). Luffa spp. have two reported genes: the gynoecious
gene (g) (Choudhury and Thakur 1965) interacts with andromonoecious
gene (a) to produce the phenotypes—monoecious or trimonoecious (AA GG),
andromonoecious (aa GG), gynoecious (AA gg), or hermaphroditic (aa gg)
plants. Melothria medraspatana has been reported to have a recessive gene
for small seed size (s), and the gene w that controls white seed coat color
if ww (Sing 1972). Bitter Melon (Momordica charantia) has four identified
genes—light brown seed (lbs) (Ram et al. 2006) is a single recessive to
dark brown; large seed (ls) is recessive to small seed size (Srivastava and
Nath 1972); white immature fruit skin (ww) is recessive to green epicarp
(Srivastava and Nath 1972); Ram et al. (2006) reported that gynoecism is
governed by a single recessive gene (gy-1).
Genes identified for bottle gourd (Lagenaria siceraria) include red
pumpkin beetle (Aulacophora faveicollis) resistance, a single dominant gene
(Af) (Vashishta and Choudhury 1972); bottle-shaped fruit (bb) is recessive
to disk-shaped fruit (BB); rr produces round fruit that is recessive to RR,
disk-shaped fruit. The gene db interacts with b to produce an F2 of 9 club:
3 round: 4 dumbbell-shaped fruit (Tyagi 1976). Dark green fruit color is
controlled by GG, which is dominant to light green fruit (gg) (Tyagi 1976).
Light brown seed coat (lb) is recessive to brown seed coat (Lb) (Tyagi 1976).
The single dominant gene (S) is responsible for segmented leaf shape
(Akhilesh and Ram 2006).
3.2.2 Classical Genetic Mapping Efforts

If one could select useful traits at the seedling stage, this would overcome
the disadvantages of needing large field plots and having to perform
large numbers of controlled pollinations. Complexity is encountered

when trying to select for quantitative traits that are heavily influenced
by the environment, such as yield. Because of environmental influences
on quantitative traits, large populations are needed to account for this
variability, which adds to time and space requirements, and cost for proper
evaluation. Breeders and geneticists have attempted to identify markers that
are associated with agronomically important traits, with the hope that the
marker can be: 1) identified at the seedling stage or shortly thereafter, and
2) have a small or no environmental influence. Traditional approaches to
identify traits early include morphological and isozyme markers, both of
which have been used to some degree in various cucurbit crops. It is likely
that both these markers have been used to maintain cultivar identities, but
there is little published information with regard to the extent that these
markers are used. Most major cucurbit seed companies have routinely used
isozyme markers to check hybrid purity (S. King, pers. comm.).
3.2.2.1 Watermelon
In watermelon 60 genes and 111 isozyme markers have been identified
(Wehner 2007), but there is only one linkage map. It includes two genes
(fruit bitterness and red flesh) and 22 isozymes that comprise seven linkage
groups covering 354 cM (Navot et al. 1990). While it is valuable to have
important fruit traits such as flesh bitterness and red flesh color linked to
markers, the utility has been limited since most breeding work is conducted
within germplasm that already lacks the bitter trait and is often conducted
within red fleshed cultivars.
Much work was performed to identify morphological markers
associated with genetic male-sterility in watermelon. This trait would
be extremely useful for the production of hybrid cultivars. It is essential,
during hybrid seed production, to identify which progeny contain the
male-sterile trait, since segregating populations are the only means to
maintain the genetic male-sterility trait. There are currently five genes for
genic male-sterility reported in this crop. One gene (gms) is associated with
glabrous foliage (Watts 1962, 1967; Ray and Sherman 1988), which makes
for an excellent morphological marker since it is simple to identify the trait
in young plants. A second male-sterility gene (ms-dw) is associated with
reduced internode length, another easy morphological marker (Huang et
al. 1998). However, these genes, as well as two other male-sterility genes,
are also associated with reduced female-fertility, limiting their suitability
for hybrid seed production. The fifth genetic male-sterility gene (ms-2) is
not associated with a reduction of female fertility (Dyutin and Sokolov
1990), but there are currently no morphological markers associated with
this gene, limiting its utility for hybrid seed production.

3.2.2.2 Cucumber
Since cucumber has just seven chromosome pairs and over 100 known
genes, it would seem that linkage maps would be fairly complete by now.
Unfortunately, we know of a few references reporting linkages of more than
two gene loci. Some of the difficulties linking genes in cucumber are due
to a portion of the nomenclature being unclear about possible duplication
of gene names originating from studies using different parental lines.
Additionally, some of the linkage relationships analyzed in previous studies
did not involve specific single genes but involved multigenic traits, or if a
single gene was involved, it was not specifically identified. Even with these
limitations, Wehner (2005) was able to summarize the literature for linkages
and describe the different linkage groups. This summary contained six
linkage groups and an assortment of linked genes that could not be placed
in any of the linkage groups. His work on cucumber linkages that include
traits is included below with modifications. The order in which the genes
were expressed in each group does not necessarily represent the order in
which they may be found on the chromosome and a question mark “?” will
follow each gene which has a questionable origin.
3.2.2.2.1 Linkage Group A

The largest linkage group in cucumber has 12 genes, composed of watermelon
mosaic virus-1 resistance (wmv-1-1), gynoecious (gy), glabrous (gl), delayed growth
(dl), divided leaf (dvl), determinate habit (de), Female (F), male sterile-2 (ms-2),
glabrate (glb), bitterfree (bi), delayed flowering (df), and Black spine-3 (B-3) or
Black spine-4 (B-4). In contributing to this grouping, Whelan (1974) noted that
ms-2 is linked with glb (rf = 0.215+.029) and de (rf = 0.335+.042) while being
independent of bi, gl, yc-1, yc-2, and cr. Gene de is linked with F (Odland
and Groff 1963b; Owens and Peterson 1982), which in turn is linked with
B-3 or B-4 (Cowen and Helsel 1983), gy (rf = 0.04) (Kubicki 1974), bi (rf =
0.375) and df (rf = 34.7) (Fanourakis 1984; Fanourakis and Simon 1987). Gene
de is also weakly linked with dl (Miller and George 1979), strongly linked
with dvl (Anon 1983), and independent of cp (Kauffman and Lower 1976).
Gene wmv-1-1 is linked with bitterfree (bi) but independent of Ccu, B, F or
pm? (Wang et al. 1987).
Two reports show that dvl is weakly linked with gl (rf = 0.40) and
independent of bi and Ccu (den Nijs and Boukema 1983), while Robinson
(1978d) originally indicated that gl was linked to yc and independent of
B, m, l, and yg as well as bi (den Nijs and Boukema 1983) and sp (den Nijs
and Boukema 1985), but more recently Robinson indicated that gl was
independent of yc (Robinson 1987a).

Completing linkage group A, Cowen and Helsel (1983) demonstrated

that the spine color genes (B-3 and B-4) were independent of the genes for
bitterness, and Whelan (1973) found that pl was independent of glb and bi,
while glb was independent of gl, bi, ls, yc, and cr, which further confirms
that gl (glabrous) and glb (glabrate) must indeed be separate loci.
3.2.2.2.2 Linkage Group B

Group B is composed of nine genes, negative geotropic peduncle response
(n), protruding ovary (pr), locule number (l), andromonoecious (m),opposite leaf
arrangement (opp), adromonoecious-2 (m-2), Bacterial wilt resistance (Bw), spine
size and frequency (s?) and a male-sterile gene (ms?) unless s? (Robinson
1978b) is the same as s from Hutchins (1940) and Poole (1944). If these were
the same, then linkage groups B and C will be joined for a total of 12 genes.
Of the first seven, two pairs have been defined with recombination values.
Youngner (1952) determined that m and l were linked with a recombination
frequency of 0.326 + 0.014 and Robinson determined that opp was linked
to both (Robinson 1987b). Iezzoni and Peterson (1979, 1980) found that m
and Bw were separated by only one map unit (rf = 0.011+0.003). Iezzoni
et al. (1982) also determined that m-2 was closely linked with both m and
Bw, and that Bw was independent of F from linkage group A (Iezzoni and
Peterson 1980).
Robinson (1978b, c), and Youngner (1952) found that linkages existed
between m, l, n, pr and spine number (s?) with the possibility of pleiotropy
being responsible for the m / pr relationship. They also demonstrated that B,
yg, and pm? were independent of the same genes (Youngner 1952; Robinson
1978b). Rounding out the linkage group is one of the male-sterility genes
(ms?). Robinson (1978c) found that it was linked with both m and l, but did
not identify which male-sterile gene it was.
3.2.2.2.3 Linkage Group C

Group C is the oldest and most mystifying linkage group. It is currently
composed of Red mature (R) for red or orange mature fruit color, Heavy netting
of fruit (H), Black or brown spines (B), cream mature fruit color (c), and spine size
and frequency (s) (Strong 1931; Tkachenko 1935; Hutchins 1940; Poole 1944).
However, there is speculation on the nature of this linkage group. Since
very few recombinants of the R, H, B and c, h, b linkage groups have been
reported, it is also felt that these characteristics may be the response of two
alleles of a single pleiotropic gene. There is also speculation that R and c are
different alleles located at the same locus (see earlier discussion).

Hutchins (1940) found that s was independent of B and H while linked

with R and c. If he was correct, then pleiotropy of H and B with R and c
is ruled out. His report also indicated that B and s were independent of
determinate habit (de) as was de of R, c and H.
A possibility exists that this linkage group may be a continuation of
group B through the s gene. Poole (1944) used the data of Hutchins (1940)
to determine that c and s are linked with a recombination frequency of
0.163 + 0.065. The question that remains is whether s (Hutchins 1940; Poole
1944) is the same as the gene for spine number in the findings of Robinson
(1978b). If Cowen and Helsel (1983) are correct in their finding that a linkage
exists between Female (F) and B then groups A and C may be on the same
chromosome. However, in this text they will remain separated based on
conclusions of Fanourakis (1984), which indicate that errors may be common
when attempting to distinguish linkages with F since classification of F is
difficult. This difficulty may also explain many conflicting reports.
3.2.2.2.4 Linkage Group D

Twelve genes, numerous spines (ns), small spines (ss), Tuberculate fruit (Tu),
Parthenocarpy (Pc), Dull fruit skin (D), uniform immature fruit color (u), tender
skin of fruit (te), compact (cp), downy mildew resistance (dm), Anthracnose
resistance (Ar), Corynespora cassiicola resistance (Cca) and powdery mildew
resistance expressed by the hypocotyl (pm? or pm-h) are in group D, but the
identity of the specific gene for powdery mildew resistance is elusive. Van
Vliet and Meysing (1947, 1977) demonstrated that the gene for resistance to
downy mildew (dm) was either linked or identical with a gene for resistance
to powdery mildew (pm?), but because the linkage between pm? and D
was broken while that of dm and D was not, pm? and dm must be separate
genes. The problem lies in the lack of identity of pm? because Kooistra (1971)
also found that a gene for powdery mildew resistance (pm?) was linked to
D. Further complicating the identity of pm, Fanourakis (1984) found that
pm-h was linked to te and dm, yet cp, which must be located at approximately
the same locus, was independent of te. He suggested that there were either
two linkage groups, ns, ss, Tu, Pc, D, U, te and cp, dm, Ar, located at distal
ends of the same chromosome with pm-h at the center, or the two groups are
located on different chromosomes with a translocation being responsible for
apparent cross linkages. However, evidence for the latter which suggested
that Female (F) was associated with the seven-gene segment is not probable
since there are few other supportive linkages between genes of this segment
and linkage group A. A more likely explanation is the occurrence of two
or more genes conditioning resistance to powdery mildew being found on
this chromosome.

Lane and Munger (1985) and Munger and Lane (1987) determined that
a gene for resistance to powdery mildew (pm?) was also linked with Cca
for susceptibility to target leaf spot but that linkage, though fairly tight,
was breakable.
The last four genes in this group are Tu, D, te and u (Strong 1931). Until
recently it was believed that each in the recessive form was pleiotropic and
consistent with European type cucumbers and each in the dominant form
was pleiotropic and consistent with American type cucumbers.
Fanourakis (1984) and Fanourakis and Simon (1987) reported that
crossing-over (R = 23.7) occurred between te and the other three genes,
which still appeared to be associated. However, using triple backcrosses
they demonstrated that there is a definite order for Tu, D and u within
their chromosome segment and that the Tu end is associated with the ns
and ss end.
3.2.2.2.5 Linkage Group E

Group E is currently composed of three genes long hypocotyl (lh), short petiole
(sp) and umbrella leaf (ul). The gene sp was strongly linked with lh and weakly
linked with ul (Zijlstra and den Nijs 1986). However Zijlstra and den Nijs
(1986) expressed concern for the accuracy of the sp and ul linkage data, since
it was difficult to distinguish ul under their growing conditions.
3.2.2.2.6 Linkage Group F

Group F is comprised of two genes, Fruit length (Fl) and Cladosporium
cucumerinum resistance (Ccu) which appear to be tightly associated. Wilson
(1968) concluded that pleiotropy existed between scab resistance and fruit
length because backcrossing scab resistance into commercial varieties
consistently resulted in reduced fruit length. However, Munger and
Wilkinson (1975) were able to break this linkage producing varieties with
scab resistance and longer fruit (Tablegreen 65 and 66, Marketmore 70 and
Poinsett 76). Now when these varieties are used to introduce scab resistance
long fruit length is consistently associated.
3.2.2.2.7 Unaffiliated Genes

Independent assortment data are as important in developing linkage
maps as direct linkage data and several researchers have made additional
contributions in this area. However, like linkage data, independent
assortment data care must be taken when utilizing them. For a complete
list of cucumber unaffiliated genes, see Wehner (2005).

3.2.2.3 Melon
There are approximately 186 identified genes and isozyme markers in
melon, making it the most saturated cucurbit crop in terms of identified
genes (Pitrat 2006). Linkages have been identified between several
agronomic and morphological traits. The linkages have been assigned to
eight linkage groups (Pitrat 1991).
3.2.2.3.1 Linkage Group 1

One of the genes for short internodes (si-1) was found to be linked to yellow
virescence (yv-2), which causes pale yellow cotyledons (Pitrat 1991).

Pitrat and Lecoq (1982) described linkages between virus aphid transmission
(Vat) and flaccida necrosis (Fn: wilting and necrosis in response to infection
with the F pathotype of Zucchini yellow mosaic virus), and Pitrat (1991)
added resistance to powdery mildew (Pm-w), and determined the order of
linkage to be Fn—Pm-w—Vat.

McCreight (1983) described linkages between the male sterile gene ms-1 and
red stem (r), which conditions red pigment under the epidermis of stems,
particularly at the nodes. Pitrat (1991) was able to add the glabrous foliage
gene (gl), and the chlorophyll deficient gene pale green (pa) to this linkage
group, and determined the order of genes to be: pa—gl—r—ms-1.

Pitrat and Lecoq (1984) described linkages between andromonoecious (a)
and Zucchini yellow mosaic virus resistance (Zym), and Pitrat (1991) added
halo cotyledons (h) and one gene for powdery mildew resistance, which was
identified as Pm-X. The order of the genes was not determined (Pitrat 1991).

One of the genes for resistance to Fusarium wilt races 0 and 2 (Fom-1) was
found to be linked to resistance to papaya ringspot virus (Prv) along with a
chlorophyll deficient marker (yv-x, which was later named yv-2, Pitrat et al.
1991). The exact order of the genes was not determined, but was reported
as either: Fom-1—Prv—yv-2, or Prv—Fom-1—yv-2 (Pitrat 1991).


Linkages were found between a second Fusarium wilt resistance gene
(Fom-2), reduced chlorophyll content in the yellow green gene (yg) and
a male-sterile gene (Ms-2). The order of the genes was determined to be:
Fom-2—yg—Ms-2 (Pitrat 1991).

Resistance to melon necrotic spot virus (nsv) was found to be linked to the
Pm-y gene for powdery mildew resistance (Pitrat 1991).

The chlorophyll deficient mutant flava (f) was found to be linked to the lmi
gene for long main stem internodes.
Pitrat (1991) identified five additional morphological and agronomic
trait genes that did not fit into any of the linkage groups and these have been
assigned to linkage groups 9 through 13 by Pitrat (1994) as follows: Group 9
= male sterile-4 (ms-4); Group 10 = dissected leaf (dl); Group 11 = virescent (v);
Group 12 = male sterile 3 (ms-3); Group 13 = male sterile-5 (ms-5). Acute leaf
apex (Ala) was linked with Lobed leaf (L) but was not assigned to a linkage
group (Ganesan and Sambandam 1985).
3.2.2.3.9 Isozyme Markers

Staub et al. (1998) were able to identify 30 isozyme markers in melon.
Eleven of these markers were assigned to two linkage groups (A and B).
The resulting map spanned 98 cM and had a mean linkage distance of 9 cM.
However, none of the isozyme markers were associated with agronomic
traits during the creation of the isozyme map.

Sanjur et al. (2002) listed up to 13 species in the genus Cucurbita and Robinson
and Decker-Walters (1997) suggested there are up to 15 species in this genus,
all of which are believed to have 20 pairs of chromosomes. This genus has a
total of 87 identified genes and 49 isozyme markers (Paris and Kabelka 2009).
The majority of the identified genes are from C. pepo (70), followed by C.
moschata (25) and C. maxima (19). The remaining genes are distributed across
four wild species and interspecific crosses. The isozyme markers were useful
for determining phylogenetic relationships, hybrid purity and cultivar
identity (Loy 1972; Ignart and Weeden 1984; Kirkpatrick et al. 1985; Decker

and Wilson 1987). There are also reports of genetic linkage between genes,
where dark stem (D) was found to be linked to mature orange fruit (mo-2)
in C. pepo (Paris 1997), mottled leaves (M) were linked to warty fruit (Wt) in
C. pepo (Paris et al. 2004), resistance to watermelon mosaic virus 2 (WMV-2)
with a plastid-specific aldolase (Aldo-p) in Cucurbita ecuadorensis, and bitter
fruit (Bi) was found to be linked to lobed leaves (Lo-2) in a C. ecuadorensis x C.
maxima interspecific cross (Herrington and Brown 1988; Paris et al. 2004).
While it is obvious how some of these linkages could be valuable in a
breeding program (e.g., select against lobed leaves to eliminate bitter fruit),
in practicality, three of these linkages have limited usage because there are
multiple genes for the trait as well as other modifier effects which affect
the phenotype which are not accounted for in the linkage.
3.2.3 Limitations of Classical Genetic Linkage Mapping and

Potential of Molecular Mapping
The primary limitation of current morphological and isozyme maps has
been the limited number of markers available, along with the relatively
few economically important traits associated with the markers. While the
maps have sometimes been useful to screen for hybrid purity during seed
production, only in rare cases are they used during the development of new
varieties. Morphological and isozyme markers are limited to expressed genes,
and because of this there is also the potential for environmental influences,
since gene expression patterns can be influenced by the environment. The
full potential of molecular maps can only be fully exploited when the entire
genome can be visualized on a map and important traits can be associated
with the map. Modern DNA based markers and their associated molecular
maps have the potential for overcoming the obstacles of morphological
and isozyme maps.
3.3 Traditional Breeding

3.3.1 Traditional Breeding Objectives and Achievements
Major goals for breeding programs are to develop high yielding cultivars
that have high quality fruit. Methods for achieving these goals differ
among breeders, as does his/her definition of quality fruit. Cucurbit
crops are naturally outcrossing, but often do not show heterosis in hybrid
combinations, and when there is heterosis, it usually is not as great as
it is for other outcrossing crops, such as onion or maize (Wehner 1999).
Cucurbit crops do not usually exhibit inbreeding depression, a factor
that may be related to the reduced heterosis (Rubino and Wehner 1986).
Studies in cucumber, melon, and squash indicate that in general, there

is little or no inbreeding depression but there is significant heterosis in

certain combinations (Robinson and Decker-Walters 1997; Whitaker and
Davis 1962). Robinson (1999) states that significant heterosis for earliness
and yield has been reported for cucurbits, including Benincasa, Lagenaria,
Luffa, Momordica, and Trichosanthes. He goes on to state that inbreeding
depression is not an important factor for producing seed of most hybrid
cucurbits cultivars.
3.3.1.1 Watermelon
Watermelon is unique among the cucurbit crops in that a significant portion
of current cultivars are seedless triploids, especially in the American market.
Triploid hybrids are produced by crossing tetraploid female with diploid
male inbred lines. Improving seedless watermelon involves selecting the
best diploid and tetraploid line, then testing them in hybrid combinations.
This method creates a new level of complexity for breeders since both
diploid and tetraploid lines must be managed. Initial breeding efforts on
tetraploids simply involved selecting the best diploids and using these to
create tetraploid parent lines. While this method has provided many current
triploid cultivars in the market, it has limitations since most diploids do not
make good tetraploid parents for triploid seed production. Fertility in the
tetraploid is an extremely important trait that has limited the use of many
tetraploid parents. Additionally, breeding within tetraploids is often more
complex than breeding diploids.
Watermelon cultivars are often monoecious, with older cultivars and
many wild accessions being andromonoecious. Watermelon is naturally
cross-pollinated like maize. However, there is little inbreeding depression
and heterosis in watermelon. It has been suggested that the lack of inbreeding
depression is due to the small population sizes used by farmers during the
domestication of the species, which forced out deleterious recessive alleles.
Therefore, even with monoecious sex expression and insect-pollinated
flowers, there would have been considerable inbreeding among the few
plants representing the population. Since there is little inbreeding depression
in watermelon, inbred lines are developed using self-pollination with little
loss of vigor from the parental population.
In studies of heterosis in watermelon, some estimates have shown a 10%
advantage of the hybrid over the high parent, but only for some parental
combinations (Wehner 1999). The small amount of heterosis observed in
watermelon makes it possible for growers to compete in the seeded market
using less expensive open-pollinated lines. However, hybrid varieties are
useful for combining multiple dominant traits from different parents.
Examples of such traits include red or canary yellow flesh, resistance to
Fusarium wilt and anthracnose, and resistance to powdery mildew. Hybrids

also protect proprietary breeding lines from unauthorized use. One of the
most important uses of hybrids is the production of seedless varieties.
Watermelon breeders today are less interested in studying heterosis or
measuring general (GCA) and specific (SCA) combining ability, because
hybrids have an advantage for protection of the proprietary breeding lines.
Furthermore, seedless cultivars are in high demand and can be produced only
as triploid hybrids. However, in the future it might be possible to develop
transgenic diploid seedless watermelons. In that case, the advantage of using
heterotic hybrids vs. inbred cultivars will again be questioned.
Environmental factors such as water availability may be important
in contrasting inbred cultivar vs. hybrid yields. A Florida study observed
that watermelon hybrids out-yielded inbred cultivars only in irrigated
fields, but quality was higher among the inbred cultivars in dry conditions
(Rhodes 1985).
Disease resistance has been, and continues to be a high priority of most
watermelon breeding programs. Resistance to Fusarium wilt has been
studied since the early 1900s (Orton 1911), and most modern cultivars have
resistance to most Fusarium races today (Henderson et al. 1970), although
the fungus continues to evolve in response to host plant resistance (Zhou
et al. 2010), necessitating continued breeding efforts. Resistance genes are
also used to provide protection from anthracnose (Layton 1937; Winstead
et al. 1959), papaya ringspot virus (Guner et al. 2008), and zucchini yellow
mosaic virus (Provvidenti 1991; Xu et al. 2004). Gummy stem blight
remains a high priority for watermelon research (King and Davis 2007),
but despite potential resistance in germplasm, protection does not hold
up in all locations.
Quality traits have been selected in watermelon for thousands of years.
These traits include size, shape, shelf-life, color, sugar content, and total
nutrient content. Watermelon is a dynamic plant with great potential for
alteration of quality traits. However, what is perceived as quality depends on
the country, demographics, and personal preference. Some people prefer the
mini melons, around 5 pounds, whereas others want giant watermelons over
100 pounds. There are unsweet, firm, white watermelons used for pickling
and preserves, and dark crimson watermelon with brix up to 14, even 15%.
More recently, breeders have been interested in phytonutrient content,
and breeding programs have focused on increasing total carotenoids,
lycopene, and improving citrulline contents (personal communication with
watermelon breeders).
3.3.1.2 Cucumber
Early studies on cucumber report considerable heterosis and/or inbreeding
depression within this crop, so long as the parents are not closely related

(Hayes and Jones 1916; Hutchins 1938; Ghaderi and Lower 1979a, b).
However, Rubino and Wehner (1986) indicated that inbreeding depression
was not important in cucumber and that midparent heterosis was noted
for most traits in many hybrids obtained from crossing S6 lines with a
gynoecious inbred line. In a similar but larger study, Cramer and Wehner
(1999) demonstrated in one out of four crosses, heterosis for fruit yield was
associated with a decreased correlation between percentage of fruit set
and fruit weight, an increased negative correlation between percentage of
fruit set and both the number of branches per plant and the percentage of
pistillate nodes, and an increased negative correlation between the number
of nodes per branch and total fruit weight. Inbreeding depression was
associated with a weakening of the strong negative correlations between
percentage of fruit set and the number of branches per plant, and between
the number of nodes per branch and total fruit weight. Those correlations
were associated with high-parent heterosis and inbreeding depression
only for the one cross, and would not necessarily apply to future crosses
in which heterosis may be observed for yield. More recently, Munshi et al.
(2006) suggest from their results that heterosis breeding is important for
effective utilization of non-additive gene actions; and Godoy et al. (2008)
showed both positive heterosis of hybrids over parent lines.
Cucumber breeders have combined several of the sex expression and
fruit quality genes to create improved varieties. It was discovered that
gynoecious sex expression created an earlier maturity, since there are no
male flowers. Many pickling cucumber hybrids were created that combined
gynoecious varieties with a monoecious variety to create a blended hybrid
composed of two distinct varieties. The monoecious variety provides pollen
to create fruit set in the gynoecious variety.
Gynoecious sex expression has also been combined with parthenocarpy
as a way to set fruit without pollen. It has been discovered that parthenocarpic
fruit has an improved shelf life and quality, and when combined with the
bitterfree allele the resultant fruit are of high quality. The combination of
gynoecious sex expression along with parthenocarpy is responsible for the
development of the extensive greenhouse cucumber industry, since this
eliminates the need for pollination and produces a superior quality fruit.
Gynoecious sex expression can also be used for hybrid seed production since
the female plants do not produce male flowers. Since it has been found that
sex expression can easily be changed with growth regulators, maintaining
the female line is relatively straight forward.
Cucumber breeders have been able to create varieties with multiple
disease resistances. Resistance to angular leaf spot, Anthracnose, downy
mildew, powdery mildew scab and target leaf spot are common in the
market. There are also varieties with multiple virus resistance available,
including cucumber mosaic virus, papaya ringspot virus, watermelon

mosaic virus and zucchini yellows mosaic virus. These disease resistant
varieties were created by screening thousands of segregating plants in
multiple generations using tedious inoculations.
3.3.1.3 Melon
Early reports differed in their analysis of whether melon showed heterosis
in yield and quality traits. The cumulative data suggests that well chosen
parental lines can produce hybrids with improved quality and yield (See
Robinson 1999 for a complete review of this subject in melons). More
recent findings demonstrated that in snake melon (C. melo L. var. flexuosus)
heterosis increased ascorbic acid and carotenoid content (Pandey et al.
2010), and heterosis could alter fruit shape in C. melo (Fernández-Silva et
al. 2009). An elegant study by Luan et al. (2010) demonstrated dramatic
performance differences between parents from diverse geographic origins
and among F1 hybrid progeny, a strong relationship between genetic
distance (determined using molecular techniques) and heterotic effects was
not consistently detected.
Breeders have created a diversity of types of melon, with well over a
dozen distinct forms currently on the market in various regions around
the world. The different types included a variety of shape, skin and flesh
color and texture. Breeders have also successfully combined a number of
quantitative traits such as sweetness and level of aromatic compounds,
despite the lack of efficient markers for these quantitative traits.
The discovery of gynoecious melons created a lot of excitement in the
seed industry, since the ability to produce gynoecious inbreds should be an
advantage for seed production in the same way it is for cucumber; however,
our experience has been that the gynoecious trait is influenced by genetic
background so that occasional perfect flowers may sometimes develop.
When a strong gynomonoecious genotype is identified, converting its sex
expression with hormones is much more difficult than with cucumber
(S King, unpubl. data).
Breeders have also created multiple disease resistant varieties of melon.
Resistance to Fusarium wilt and powdery mildew are common, but the
pathogens for these diseases continue to evolve making continued breeding
efforts necessary.

Many studies have demonstrated that C. pepo and C. maxima hybrids can have
superior yields (summarized in: Robinson 1999; see also Firpo et al. 1998;
Ahmed et al. 2003; López Anido et al. 2004). Stephenson et al. (2001) report
that sporophytic vigor (e.g., flower and fruit production) increased with the

level of heterozygosity and that the level of heterozygosity of the sporophyte

affects the in vitro and in vivo performance of the microgametophytes it
produces. In addition to yield, Gwanama et al. (2001) demonstrated that
heterosis in tropical pumpkin can increase soluble solids content.
As with the other cucurbit crops, there is a wide diversity of fruit types
within this group. The Cucurbita spp. are somewhat unique in that there are
different plant types and harvest stages for the fruit, some being harvested
as immature fruit while others are harvested as mature fruit.
Breeders have utilized the gene for bush habit found in Cucurbita
spp., and all modern summer squash varieties currently have the bush
habit. Recently, parthenocarpic summer squash varieties have come on the
market, with the potential for greenhouse production. While this market is
currently limited, the full potential is currently unknown.
There is a limited number of resistance genes currently available in
Cucurbita spp., so there are few resistant varieties available. Resistance to
powdery mildew is available in some varieties, and breeders have utilized
the precocious yellow gene, which prevents the expression of mottling
symptoms as a way to reduce damage caused by virus infection. There are
also a number of virus resistance genes which have been used in various
summer squash varieties, but their utility has been somewhat limited since
there are a number of different viruses that can affect squash, and each
resistance gene is specific for a particular virus. Virus resistant squash has
also been created using genetic engineering, which has the advantage that
multiple resistance can be stacked on a single construct so that resistance
segregates as a single gene, making breeding much less difficult.
3.3.2 Limitations of Traditional Breeding and Rationale for

Molecular Breeding
Traditional breeding has relied, either directly or indirectly, on
morphological markers to identify the trait of interest for selection in a
segregating population. Traditional methods include a direct measure of
the phenotype (e.g., flesh color), or an association of one phenotype with
another (e.g., lobed leaves with bitter fruit). Traditional breeding has been
extremely effective for making qualitative changes in cucurbit crops. Traits
such as lycopene containing watermelon (linked to red flesh), β-carotene
containing melon (linked to orange flesh) and parthenocarpy (linked to
seedless cucumbers) are examples of selection using phenotypic markers.
In addition to improvement in qualitative traits, there have been huge
changes in important quantitative traits through traditional breeding. These
include seed germination, seedling vigor, fruit yield, early maturity, fruit
size, sugar content, and freedom from defects.

Further improvement in quantitative traits using traditional strategies

will be time consuming. For example, selecting for higher carotenoid
concentration will be difficult using visual appearance as was done in the
past. It is fairly straight forward to select watermelon genotypes containing
lycopene from a segregating population, but it is difficult to distinguish
levels of lycopene based on color. Likewise, selecting orange fleshed fruits
in squash has led to squash fruits that contain carotenoids, but it has been
shown that quantitative differences in carotenoid content is controlled
by the complex interaction of alleles at many genes, some of which have
modifier effects (Paris 1994).
The most effective method for selecting a multiple allele trait is to utilize
multiple markers to identify a majority of the alleles. This is especially true
of the cucurbit crops, which require large amounts of space to evaluate.
However, morphological markers will have an intrinsic disadvantage if
the trait is influenced by the environment. The potential for morphological
markers is also limited by probability, since only coding regions of the
genome can be used as potential markers. The total genes, including
isozymes, available in cucurbit crops, are approximately 661, including
watermelon (171, Wehner 2007), cucumber (168, Wehner 2005), melon (186,
Pitrat 2006), and Cucurbita spp. (136, Paris and Kabelka 2009). Considering
the volume of traits and how few genes are identified in each species,
the potential for a morphological or isozyme tightly associated with any
particular trait of interest is quite low.
Breeding for disease resistance is often challenging for cucurbit crops.
Many disease resistance traits are quantitative, expression is often affected by
environment, a complex inoculation procedure may be required, and in some
diseases, reliable resistance has yet to be found. Gummy stem blight (GSB) is
a good example of a disease where developing new cultivars with resistance
has thus far proven difficult. GSB is a serious disease of watermelon, leading
to substantial yield losses (Keinath and Duthie 1998), and has been identified
by US watermelon producers as the number one problem needing further
research (King and Davis 2007). Host plant resistance should be an effective
method for control of GSB. However, despite numerous attempts, resistant
cultivars are not currently available for this disease in watermelon. Several
resistant sources have been identified (Sowell and Pointer 1962; Sowell 1975;
Gusmini et al. 2005), and “resistant” cultivars have been released (Norton
et al. 1986), but these cultivars do not withstand current disease pressure in
multiple locations (Hall and Sumner 1999; S. King and T. Wehner unpubl.
data). Resistance has been tracked using conventional screening methodology
which for GSB has proven unreliable, probably because of the significant
number of escapes using the current screening procedure. Progress in
breeding GSB resistant cultivars will only be achieved when a reliable method
to track resistance gene(s) is achieved.

Molecular markers have the potential for overcoming the limitations

associated with traditional selection strategies, since they are non-
destructive, eliminate the environmental variation associated with disease
resistance and can be evaluated for multiple traits simultaneously.
However, the use of molecular markers require the development of
breeding populations, which segregate for the trait of interest, and the trait
must be properly identified during marker identification. It is extremely
important that populations used for marker development be properly
identified. Mistakes made phenotyping plants used for marker development
will delay development and may cause potentially useful markers to be
missed. This is an important consideration since many of the traits that
would be most useful for marker development are traits that are highly
influenced by the environment or have some other high degree of variability
associated with them.
There are two important issues to consider regarding molecular
markers: time and cost. Although molecular breeding and the development
of molecular markers has great potential, it may take a significant amount
of time to properly develop the marker and thoroughly test the marker
in multiple populations. Along with this time is the expense of marker
development, which is significant. The potential for molecular markers to
save money is in their long-term utilization in combination with multiple
markers for a wide variety of traits; this will allow breeders to select for
multiple traits from large populations in a manner that is not currently
possible.
Another aspect of molecular breeding approaches includes the use
of genetically-modified organisms (GMO). Inserting a gene that does not
naturally occur in cucurbits can sometimes have dramatic effects on crop
performance. In fact, the second commercial GMO crop in the US was a
cucurbit crop, and variations of this GMO crop are still on the market today
(virus resistant squash), proving the success of this strategy. However,
genetically-modified cucurbits as well as other vegetables (other than
virus resistant squash) have thus far mostly been limited to research. The
increased use of grafting in cucurbit crops does present an avenue where
GMOs may impact this family of plants in the short-term. Rootstocks can
be genetically engineered to resist a host of biotic and abiotic stresses and
have the trait transferred to the scion through grafting rather than direct
transformation. It remains to be seen whether public opinion will influence
the development of GMO rootstocks.
3.4 Conclusion
Traditional genetics and plant breeding have made great strides in our
understanding and improvement of cucurbit crops. We have used classical

genetics to enhance our understanding of taxonomy and phylogenetic

relationships in the Cucurbitaceae; plant breeders have identified a
number of genes associated with commercially important traits and used
this information to create superior cultivars with greatly improved yield
and quality. However, previous advances were typically through adding
single traits with high heritability to adapted germplasm. Advancement
of some traits using traditional techniques is difficult and time consuming
since the complexity increases with each added trait. Molecular breeding
offers an avenue to overcome many of the problems associated with
traditional breeding and genetics. In fact, molecular breeding has already
made contributions to our understanding of cucurbit genetics, and has
been directly responsible for some of the improvements made in modern
cucurbit cultivars.
Molecular breeding offers avenues for crop improvement not otherwise
available to traditional approaches. Classical breeders have said, “Anything
is possible using traditional approaches; it is just that the world is not large
enough to hold the populations needed to find the variation required for
some traits.” Molecular breeding provides a tool to search for traits and
combinations of traits that are otherwise not feasible using traditional
approaches. As we move forward with molecular breeding in cucurbit
crops, it is important that we understand the need to maintain traditional
breeding programs, and that the skill set required for classical breeding is
not lost. Developing molecular markers requires traditional populations
with traits identified using traditional methods, at least in the initial stages.
Also, the time and expense of molecular marker development is significant,
and will often take time and money away from traditional breeding. If we
want to fully exploit the potential for molecular breeding, it is imperative
that we maintain a balance between molecular and traditional approaches.
Chapter 4 of this book delves into the advances made in cucurbit breeding
regardless of the breeding technique used.
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4
Breeding Squash and Pumpkins
J. Brent Loy
ABSTRACT
Within the genus Cucurbita (Cucurbitaceae) there are five domesticated
species, three of which, C. pepo, C. maxima, and C. moschata, are important
crop species grown world-wide. Although cultigens of all three species are
found in temperate and sub-tropical climates, C. maxima and C. pepo are
considered best adapted to temperate climates; whereas, many cultigens
of C. moschata are restricted to tropical and subtropical climates. Cucurbita
species have a vining habit of growth, and are monoecious with large
showy flowers that are bee pollinated. Horticulturally, domesticated
members of this genus are conveniently classified into three broad
groupings: (1) summer squash cultigens, the fruit of which is consumed
immature, about three to five days after fruit set; (2) winter squash, the
fruit of which is harvested when mature, about 50 to 60 days after fruit set,
but which may require additional storage to reach optimum sugar levels
for desirable eating quality; and (3) gourd and pumpkin cultigens that
are used mainly for ornamental purposes. Although Cucurbita cultigens
are considered highly outcrossed under natural conditions, they can be
highly inbred without readily apparent inbreeding depression. They are
often classified in breeding books along with other self-pollinating crops.
For this reason, the pedigree system of breeding has been widely and
successfully adopted by most cucurbit breeders. For wide, interspecific
crosses, the backcross system, together with selection, has also been
utilized together with the pedigree system. Most breeding efforts have
been for qualitative traits such as fruit appearance (size, color, shape),
bush or vine habit of growth, and resistance to a few major diseases.
Important quantitative traits are fruit size, seed size, and % dry matter
of flesh, the latter an important parameter of eating quality. Prior to 1980,
most varieties of squash and pumpkins were open-pollinated. However,
in North America there has been a plethora of new F1 hybrids introduced
into the seed trade during the past 30 years, with the majority of cultivars
having a bush or semi-bush habit of growth.
Department of Biological Sciences, University of New Hampshire, Durham, NH 03824;

e-mail: jbloy@unh.edu

4.1 Introduction
The genus Cucurbita within the family Cucurbitaceae comprises five
domesticated species, three of which, C. pepo L., C. maxima Duchesne, and
C. moschata Duchesne, represent economically important species cultivated
worldwide (Whitaker and Davis 1962; Robinson and Decker-Walters 1996).
The other two domesticated species, C. argyrosperma C. Huber and C. ficifolia
Bouché have more limited cultivation and use and will not be discussed
here. Squash, pumpkins, and to a lesser extent gourds, are economically
important crops, with world production estimated to be in excess of 20
million mtons grown on over 1.5 million hectares (FAOSTAT 2008). Decker-
Walters and Walters (2000) provide a comprehensive description of fruit of
both wild and domesticated forms of Cucurbita, along with an overview
of phytogeographic origins of the different species and general food and
ornamental uses. In North America in 2008, 859 thousand mtons of squash
and pumpkins were produced on 39.4 thousand hectares, and the farm value
was approximately 373 million U.S dollars (Statistics Canada 2008; USDA
2008). The agricultural statistical reporting does not separate ornamental
pumpkin production from that used for canning and does not distinguish
among winter and summer squash production.
There have been different interpretations on usage of the terms squash,
pumpkin, and gourd beginning with the time these crops were introduced
into Europe in the early part of the 16th century. In the first English printing
of Vilmorin-Andreieux’s “The Garden Vegetables” (Robinson 1885), all
members of the Cucurbita genus are described under the heading “Gourds,”
equivalent to the French name “courge,” and presumably, categories of the
different species followed the classification scheme proposed by Charles
Naudin. Although “marrows” clearly referred to fruit, mainly within
C. pepo, eaten immature, there was no clear distinction between pumpkins
and squash, except that pumpkins were generally mentioned as bearing
large fruit. In current North American usage “gourds” are defined as
various hard-rind forms of fruit within the Cucurbitaceae family, and in
the Cucurbita genus, most common within the species pepo (Bailey 1937).
“Pumpkin” typically refers to cultigens with round or oval fruit grown for
ornamental purposes, for pie processing, and less commonly for livestock
feed; whereas, “squash” is applied to any members of the three species
having culinary use. Summer squash includes cultivars in which the fruit
is consumed while immature and typically harvested within 3 to 5 days
after pollination, while fruit of winter squash is consumed when mature. A
squash can be defined as mature when its reproductive cycle is completed
in terms of embryo development (seed fill), which occurs about 55 days
after fruit set in temperate climates (Loy 2004). The three most important
classes of winter squash consumed in North America are acorn (C. pepo),

Breeding Squash and Pumpkins 95
buttercup/kabocha (C. maxima), and butternut (C. moschata) (Fig. 4-1A,B,C).

Summer squash are generally regarded as members of Cucurbita pepo, but
immature fruit of C. moschata and C. maxima cultigens have been used for
culinary purposes in some regions of South America and Asia.
Figure 4-1 Illustration of the three major squash types grown in North America: acorn (C.
pepo, A), kabocha/buttercup (C. maxima, B) and butternut (C. moschata, C).
4.2 Origin, Taxonomy and Early Distribution

All members of Cucurbita are endemic to the Western Hemisphere and
have been in cultivation for several thousand years. There is compelling
evidence that Cucurbita pepo, the most polymorphic of the three major
species, was first domesticated 9,000 to 10,000 calendar years ago (Smith
1997); however, there likely were multiple domestications of this species
in Meso and North America (Decker 1988). Current classification of C. pepo
favors evolution of two domesticated subspecies, C. pepo L. subsp. pepo. and
C. pepo subsp. ovifera (L.) D.S. Decker var. ovifera (L.) D.S. Decker (Decker
1988). The domesticated subspecies ovifera appears to have descended from
the wild species C. pepo subsp. ovifera var. texana (Scheele) D.S. Decker, to
which it has a close genetic affinity (Decker 1988; Decker-Walters et al. 2002).

A wild progenitor of C. pepo subsp. pepo, has not been found. The wild
species C. pepo subsp. ovifera var. texana is endemic to Texas, and another
closely related wild species, C. pepo subsp. fraterna (L.H. Bailey) Andres,
is native to northeastern Mexico. Domesticated forms of subspecies pepo
include ornamental pumpkins, zucchini, caserta, and vegetable marrow
summer squash, and a few ornamental gourds such as “Orange” and
“Miniature Ball”. Subspecies ovifera includes acorn, “Delicata”, and “Sweet
Dumpling” types of winter squash, yellow crookneck and straightneck
summer squash, and various gourds, including egg, pear and spoon gourds.
A third subspecies, C. pepo subsp. gumala Teppner, possibly endemic to
Guatemala, has been described by Teppner (2004). Fruit of this species are
characterized by a thick, hard, warty rind, and 10 main ribs with mostly
double interstitial ribbing. Teppner is of the opinion that cultivars of subsp.
gumala may be closely related to the original wild progenitors of subsp.
gumula and subsp. pepo.
Species diversity in C. moschata appears to be greatest in northern South
America (Nee 1990; Wessel-Beaver 2000; Andres 2004). Moreover, Dillehay
et al. (2007) have provided compelling evidence from radiocarbon dating
of macro plant remains of squash from a tropical dry forest region in the
Nanchoc Valley in Peru that domesticated C. moschata squash was being
used as far back as 10,000 years ago. This also agrees with the age (10,000
to 7,000 years BP) of phytoliths of C. moschata recovered from Ecuador and
Columbia, and believed, based on size, to represent domesticated species
(Piperno and Stothert 2003).
Cucurbita maxima is clearly a South American domesticate. There are few
archaeological sites documenting early use of C. maxima, but according to
Sauer (1993), the earliest archaeological remains of C. maxima are from the
coastal Viru Valley in Peru and are dated about 3800 BP. C. maxima subsp.
andreana (Naud.) Filov, native to Argentina and Uruguay, is considered to be
the wild progenitor of the domesticated subsp. maxima. The two subspecies
can readily hybridize and produce fertile offspring, and recent molecular
data support the view that subsp. andreana is the progenitor of domesticated
C. maxima (Sanjur et al. 2002).
4.3 General Botany

An extensive description of botanical characteristics of Cucurbita species
for taxonomic purposes is presented by Teppner (2004), and the overall
morphology of Cucurbita species is thoroughly reviewed by Whitaker and
Davis (1962). Squash and pumpkins are herbaceous annuals, characterized
by development of long vines or runners that may exceed 10 m in length
during a growing season. Leaves are large and cordate, with deep (C. pepo)
to shallow lobes (C. moschata and C. maxima). Long, compound tendrils

are produced from axillary meristems at leaf nodes, encouraging vertical

growth of Cucurbita plants when trellised. Many modern varieties of squash
have a compact or “bush” habit of growth, characterized by shortened
internodes, thicker stems, and sometimes thicker leaves (Broderick 1982).
All bush cultigens of summer squash and acorn winter squash, and some
vine cultigens of pumpkin and acorn squash, lack tendrils; whereas, all vine
cultivars of C. moschata, most vine cultigens of C. maxima and C. pepo, and
some, but not all bush cultigens of C. pepo pumpkins and C. maxima, have
tendrils (J.B. Loy unpubl. observations). Foliage of cucurbits is covered with
epidermal hairs or trichomes (Inamdar et al. 1990) that in some cultigens
of C. pepo can be harshly prickly and irritate the skin (Xiao and Loy 2007).
Foliage of C. maxima is generally less prickly than that of C. pepo, and foliage
texture in cultigens of C. moschata tends to be soft and hairy to the touch.
The root system of squash is extensive, characterized by development of
a strong tap root and several moderately shallow lateral roots that may
extend out beyond the aerial parts of the plant (Weaver and Bruner 1927).
Additional roots frequently develop at plant nodes laying over moist soil,
allowing plants to tap into water and nutrient reserves over a wide area.
Cucurbita species are monoecious. The bright yellow-orange flowers are
large and conspicuous, comprised of a companulate, 5-lobed corolla together
with a 5-lobed calyx, forming a basal perianth tube (Fig. 4-2). Usually
three, but occasionally four or five carpels, comprise the inferior ovary
of pistillate flowers, and each carpel develops a series of ovules borne on
placental ridges in each locule (Hayward 1938). A short, thick style ascends
Figure 4-2 Pistillate (left) and staminate (right) flowers of monoecious C. maxima.

from the perimeter of a nectary cup at the base of the perianth tube and
terminates in a three-lobed stigma. Staminate flowers are borne on long,
slender pedicels and have three stamens borne on single filaments that are
united by two tetrasporangiate and one bisporangiate anther (Hayward
1938; Kirkwood 1905; Fig. 4-2). Flower morphology differs among the
three species (Fig. 4-3). C. maxima is characterized by a long floral tube, and
edges of moderately lobed corollas are serrated. Flowers of C. pepo have
deep and sharply angled corolla lobes; whereas, in C. moschata, the floral
tube is shallow to moderate in length and the lobes tend to be rounded.
The nectaries in Cucurbita maxima are distinctly more fragrant than in the
other species. The fruit of Cucurbita is a multi-seeded berry. The peduncle
becomes highly lignified during the initial 40 days of fruit development
Figure 4-3 Flower morphology of C. maxima (left), C. pepo (middle) and C. moschata (right),
represented by pistillate flowers.
after pollination (Berg 2004), and the morphology of peduncles is a major

feature used to identify Cucurbita species. Fruits of C. maxima have a large
corky, non-ribbed peduncle (Fig. 4-4). In C. moschata, the peduncle usually
has five smoothly angular ridges, often flared slightly at the base, and is
very hard. In C. pepo subsp. pepo, peduncles are quite thick and often long,
but there is considerable variation in size of peduncles. They are highly
angular with deep furrows and typically five to eight ridges. On the other
hand, in C. pepo subsp. ovifera, the peduncles are thin and hard, usually
with five ridges and shallow furrows (Fig. 4-4).

Figure 4-4 Illustration of peduncle types in C. maxima (bottom laft), C. moschata (bottom right),
C. pepo subsp. ovifera (top laft), C. pepo subsp. pepo (top right). See text for explanation.
Flowers are usually borne singly in leaf axils. Staminate flowers

are produced near the crown of the plant; whereas, female or pistillate
flowers are produced distally to staminate flowers on the main stem and
lateral branches (Nitsch et al. 1952; Maynard et al. 1992). Once pistillate
flowering commences, solitary flowers are typically produced about every
4 to 6 nodes along stems, but two pistillate flowers occasionally occur on
successive nodes. Staminate flowering often occurs a few days before
pistillate flowering (Scott 1933; Nitsch et al. 1952; Decker 1986; Maynard
et al. 1992, 2002); however, in many modern bush and semi-bush hybrid
varieties, pistillate flowering precedes staminate flowering. Staminate
flowers produce prodigious amounts of large pollen grains, 120 to 200 µm
in diameter (Shridhar and Singh 1990; Nepi and Pacini 1993; Teppner 2004).
The primary pollinators of Cucurbita flowers in North America include honey
bee, Apis mellifera L., bumble bees, Bombus spp., and a species of squash
bee, Peponapis pruinosa Say (Shuler et al. 2005). The relative importance of
each of these pollinators varies with cultural practices, location and season
(Julier and Roulson 2009).

The pattern of flowering in summer squash is quite distinct from winter

squash and pumpkin cultivars, owing to an extreme bush phenotype
and selection for a proclivity of pistillate flowering. Typically, staminate
flowers are initiated only in leaf axils of the first 10 to 15 nodes, followed
by initiation of pistillate flowers on successive nodes with male flowers
dispersed every two to four nodes (Loy 2004). Even though staminate
flowers are differentiated at a lower node number than pistillate flowers,
pistillate flowering usually precedes staminate flowering, often by several
days. Zucchini cultivars initiate only one flower per node; whereas, yellow
straightneck and crookneck squash will often produce two flowers, and
occasionally three flowers, per node (Loy 2004).
4.4 Pollination Techniques

For hand-pollination, male and female flowers are typically tied off during
the day before anthesis with four inch Twist-ems ties available through
agricultural supply houses (Fig. 4-5). Pistillate flowers are retied following
pollination the next morning. In some varieties of C. pepo, the corolla is
somewhat rigid after opening, and the corollas of such flowers will often
rupture when retied. It is recommended that pollinators carry paper or mesh
poly bags to cover such flowers, so as to prevent contamination. Bumble
bees are often quite aggressive at penetrating small openings in squash
flowers and can contaminate pollinations. For identifying pollination set-
Figure 4-5 Pistillate flower (left) tied off with a Twist-ems tie one day prior to anthesis;
pistillate flower retied and tagged after pollination.

ups, vinyl colored flags on PVC stakes work well, and can be reused. One
color flag can be used for set-ups for self-pollinations, another color can
be used for female flowers set up for crosses, and an additional colored
flag can be placed next to fruit that have been pollinated. It is useful to
periodically walk through breeding plots to check for fruit set and remove
flags from unsuccessful pollinations. Pistillate flowers that have been self
or cross pollinated are conveniently labeled with ¾ x 9 inch polyethylene
bands (Sato Labeling Solutions, America, Inc.) placed around the pedicel
or the internode preceding the flower node. Biodegradable labeling tags
are available.
Squash pollen is not long lived, and typical recommendations are to
complete hand-pollinations by noon (Whitaker and Robinson 1986). Hayase
(1956b) reported that fruit set in C. maxima is greatest when pollinations
occur shortly after anthesis and progressively declines until mid-day. It has
been repeatedly observed that pollination success declines rapidly when
flowers begin to wilt or lose turgidity (JB Loy, pers. observation). In a study
reported by Nepi and Pacini (1993), wilting and flower closure in C. pepo
occurred by 11:30 to 12:30 hours, about six hours after anthesis, and this
was followed by rapid loss of pollen viability, presumably due to pollen
dehydration. In agreement with their results, it has been observed that
under overcast conditions with high humidity, flowers stay turgid much
longer, and it is possible to make successful pollinations much later in the
day (JB Loy, pers. observation).
There are also species and genotypic differences in duration of pollen
viability. Flowers of C. pepo will often begin to lose turgidity by 9:00 hours,
and flowers of certain groups of C. pepo, such as the small ornamental
gourds (C. pepo subsp. ovifera), generally wilt prior to those of ornamental
pumpkins (C. pepo subsp. pepo). In summer squash homozygous for the
glabrous gene (gl-2), flowers remain noticeably turgid longer than those on
non-glabrous genotypes, and successful pollinations have been obtained
on these genotypes beyond 12:30 hours on overcast days (JB Loy, unpubl.
results). Flowers of C. moschata stay turgid longer than those of C. pepo, and
closure of C. maxima flowers is usually last among the three species (JB Loy,
unpubl. observations). With judicious planning of planting schedules with
the different species of Cucurbita, it is possible to spread out pollinations
among the three species for more efficient use of labor. In addition, more
pollinations can be accomplished per day by taking advantage of differences
in length of pollen viability and flower closure among and within the three
species.
A further technical problem in pollinating squash is that of dichogamy,
in which flowers of one sex type reach anthesis prior to the other. For
example, early summer squash lines tend to exhibit pistillate flowering
as much as a week to 10 days before staminate flowering. In butternut

cultigens the ratio of staminate to pistillate flowers is typically low early in

the season, hampering pollination of single plant selections. One method
to partially alleviate this problem is to use bud-pollination. One day pre-
anthesis pistillate flowers will set quite well, and use of such flowers speeds
up the pollination process because such flowers only need to be tied on the
day of pollination. Hayase (1956a) reported that pollen germinating power
(pollen germination rate x pollen tube growth rate) was actually higher on
the evening before anthesis than during anthesis for all three major species
of squash. Under natural conditions, dehiscence of anther locules was
not complete until late in the evening of the day before anthesis (Hayase
1956a), but dehiscence could be accelerated by excising staminate flower
buds one or two days before anthesis and placing them in water at warm
temperatures. Therefore, it appears feasible, but not necessarily practical, to
use ½ day pre-anthesis staminate and pistillate flowers, thereby eliminating
the need to set up flowers the day before anthesis. Another method for
obtaining pollinations when appearance of staminate and pistillate flowers
is not in synchrony is to excise male flower buds the day before anthesis
and keep in cold storage (10oC) for up to one day following normal anthesis
(Hayase 1956a).
4.4.1 Fruit set

The percentage of fruit set in Cucurbita with hand pollinations is high, often
exceeding 90%. Nonetheless, extremes of weather can adversely affect fruit
set. Excessively warm days can cause female flower senescence and abortion
prior to anthesis (Wien et al. 2002); this can lead to delays in obtaining
adequate pollinations in breeding lines because it may take five or six days
for appearance of another pistillate flower on a plant. There appears to be
genotypic variation in the capacity of plants to produce female flowers
under high temperature (Wien et al. 2002; JB Loy, unpubl. observations).
Extended periods of rainy and overcast days also adversely affect fruit set
and can cause abortion of developing fruit. There is usually an elevated
incidence of fruit rot diseases during rainy and overcast periods of high
humidity, but low light intensity appears to play a role in increasing the
incidence of pistillate flower abortion as well. Suppression of female flower
production by low light intensity was confirmed in greenhouse studies in
which incident light levels were reduced to 20, 40 or 70% of normal levels
(Wien et al. 2002).
4.4.2 Harvesting and Seed Cleaning

Seed may appear fully developed by 35 to 40 days after pollination, but
seed fill is usually not completed until at least 55 days after pollination in

temperate climates (Vining and Loy 1998). Fruits harvested when immature
may require up to two to three months of storage to alleviate potential
seed dormancy problems (Young 1949). Seed development continues in
immature stored fruit, but seed fill may not be as complete as with intact fruit
(Vining and Loy 1998; Loy 2000, 2006). Well-filled seed has been obtained
from squash harvested as early as 18 days after pollination and stored for
6 or 7 weeks (J.B. Loy, unpubl. observations). Occasionally seed extracted
from mature fruits show dormancy (Odland 1937), which can usually be
broken by storing fruits for an additional period at room temperature prior
to extraction.
Seeds are easily removed from fruit of C. pepo and C. moschata, and
can be washed in a strainer. The moisture content of the embryos removed
from mature fruit is about 40 to 50% (Cui and Loy 2002). Seed should be
immediately dried on a screen in a forced air dryer at 30 to 35oC for 48 to 72
hours to about 12% moisture (Hawthorne and Pollard 1954). Seed extraction
from fruit of C. maxima is often difficult, especially when the seed cavity
is tightly filled with seed, and placental tissue often remains attached to
the micropylar end of the seed. Dried seed can be further cleaned, and
misshapen and unfilled seed can be removed. An air column separator is
useful for a final cleaning step, and aids in removing the clear, membrane-
like endocarp layer which adheres to seed.
4.5 Grouping of Cultivars within Species

Recognizable phenotypic classes or groupings of squash and pumpkin
cultivars within each species have evolved as a result of breeding efforts,
mainly over the past two centuries. The varietal classes suggested by
several authors (Castetter and Erwin 1927; Paris 1986, 1989; Whitaker and
Davis 1962) are similar and based primarily on fruit characteristics (size
and shape), on culinary use, and to some extent on historical precedence.
The groupings listed below (J.B. Loy, unpubl. results) are based on current
cultivar use in North America and practical grouping of squash for
breeding purposes, but follows somewhat closely the classifications used
by Whitaker and Davis (1962). Cultivar lists are extensive, so only some
representative types are included in the different groups. Variety updates
for North America are periodically listed in HortScience published by the
American Society for Horticultural Sciences. A fairly representative list of
winter squash and pumpkin cultivars in recent commercial use is provided
by Ferriol and Pico (2008) in an excellent review article of pumpkin and
winter squash.

4.5.1 Cucurbita pepo Variety Classes

Pumpkin (C. pepo subsp. pepo): mostly round to oval to slightly oblate
varieties, often with prominent ribbing. The rind of most fruit changes from
green to orange between 25 to 40 days after fruit set. There are now over
100 varieties representing this group, mostly F1 hybrids.
Summer squash:
Scallop (C. pepo subsp. ovifera): fruit usually consumed when immature; fruit
flattened with shallow ribbing and prominent, scalloped edges; immature
color varies from light green to yellow. It is one of the oldest forms of squash,
depicted in 1553 in the herbal of G. Őllinger (Teppner 2000).
Yellow Crookneck and Straightneck (C. pepo subsp. ovifera): Crooknecks
have a long, curved neck, a somewhat bulbous distal end containing the
seed cavity, and yellow or orange fruit at maturity. Older varieties, which
appeared in North American seed catalogs in the 1800s (Tapley et al. 1937)
are heavily warted with a hard rind at maturity; the stems, petioles and
the undersides of leaves are very spiny. All modern varieties have the bush
phenotype and some lack warts at maturity. The first straightneck variety,
Giant Straightneck, appeared in seed catalogs in 1896 (Tapley et al. 1937),
apparently a selection out of Giant Crookneck. Straightneck varieties have
largely replaced crooknecks in North American markets, but crooknecks
are still popular in the southern US.
Vegetable Marrows (C. pepo subsp. pepo): The marrow group is dominated
by bush varieties that produce oblong, green to cream-colored fruit eaten
immature. The long, cylindrical green zucchini squash are the most popular
type in this category. Also included in the category are the Middle Eastern
Cousa type with short, thick, light green fruit, a gray type similar to zucchini,
a striped caserta type, and the older cocozelle type with long striped fruit.
Paris (1986) placed the vegetable marrows, zucchini and cocozelle types
into separate groups on the basis of morphological differences and historical
records of use. In terms of genetic diversity within these groups, DNA
polymorphism supports this grouping (Paris et al. 2003). Nonetheless,
most modern varieties of summer squash within subspecies pepo have
converged through breeding efforts into a similar growth habit characteristic
of zucchini cultivars and differ most visibly only in fruit shape, color and
patterns of mottling or striping. Cultivars have also been bred with round
fruit.
Acorn and Related Culinary Types: This group is dominated by the popular
acorn varieties, characterized by a dark green rind, prominent ribbing or
furrows, and being round, oval or heart-shaped. However, there are now
several introductions that fall into the “Sweet Dumpling” class—0.8 to

1.5 kg fruit with more or less globular shape, shallow to deep ribs and a
distinct striping pattern, characterized by narrow green and wide white
stripes early in development changing to wide tan and narrow orange
stripes after storage. Another member of this group, more popular in the
past, is the “Delicata” type, characterized by oblong fruit about 8 to 10
inches long and 3 to 4 inches in diameter with the same striping pattern as
“Sweet Dumpling” squash.
Seed Pumpkins: In terms of fruit type and plant habit, seed pumpkins fall
into the same category as ornamental pumpkins (subspecies pepo). However,
seed pumpkins carry the hull-less seed trait which first appeared in oil seed
pumpkins grown in the Styrian region of Austria toward the turn of the
19th century (Teppner 2000). In these types, the seed coat is reduced to a
thin membranous covering of the seed resulting from reduced amounts of
lignin and cellulose in certain cell layers within the seed coat (Stuart and
Loy 1983), rendering the seed more efficient for oil extraction. In addition
to pumpkin seed oil, hull-less pumpkins seed are consumed as a snack,
and used in trail mixes, in crackers, in the confectionary trade, and also by
the pharmaceutical industry.
Spaghetti squash: Spaghetti squash is considered to be a member of the
marrow group in terms of origin and fruit type (Maynard et al. 2001). It
is a unique cucurbit in that the flesh separates into strands when cooked,
and has been marketed as a low calorie substitute for semolina products.
The flesh character has been ascribed to a recessive gene, sp (Mazurek and
Niemirowicz-Szczytt 1992). Bush cultivars and cultivars with orange flesh
(Paris et al. 1985) have been developed. Productivity and nutritional value
have also been evaluated (Beany et al. 2002).
4.5.2 Cucurbita maxima Variety Classes

Banana: large (6 to 12 kg) fruits elongated and somewhat tapering at both
ends; rind color pink or gray.
Hubbard: large (5 to 10 kg) fruits of general ovoid shape; fruit rind varies
from smooth to rough and warted (hard rind); rind color varies from green
to gray.
Golden Delicious: This group includes all of the orange processing varieties,
but is dominated by “Golden Delicious”. Varieties in this group are generally
ovoid in shape, weigh between 5 to 10 kg, and carry the Bmax gene (Shifriss
1989) for orange fruit coloration.
Show Pumpkins: Includes huge pumpkins and/or squash grown for show
at exhibitions, including many weighing over 500 kg.

Turban group: This group includes varieties with a button or protruding

ovary at the blossom end, and some cultivars, such as Turk’s Turban and
Rouge Vif D’Etampes, are grown primarily for ornamental use. This was not
a widely cultivated group at the time of Castetter and Erwins’s publication
in 1927, and the now popular “Buttercup” variety comprising this group
was introduced in 1932. Buttercup and related kabocha varieties having a
small blossom scar are now the most popular culinary class of this species
in North America with both labeled as “Buttercup” in supermarkets.
Kabocha: Small (1.5 to 2.5 kg), slightly oblate fruit, with either a dark green
or a mottled green rind, a small blossom scar and characterized by high dry
matter, deep orange flesh and exceptional eating quality. In North America
kabocha are usually marketed as buttercup squash, and for breeding
purposes, it is appropriate to place kabocha and buttercup squash in the
same group.
4.5.3 Cucurbita moschata Variety Classes

Cheese Group: Varieties with round to oval to slightly ovate 4 to 7 kg fruit
with smooth tan rind, with or without extensive ribbing, and having low
mesocarp dry matter (8 to 11%). Group includes processing pumpkins used
primarily for pie stock.
Crookneck or Cushaw: Large pumpkins with a long neck, a bulbous seed
cavity at the blossom end, and either tan or mottle green rind color. Fruit
show dimorphism for straightneck or crookneck (Pearson 1968). Tan
varieties are used for processing.
Bell-shape Group: This group represents one of the most popular squash
types grown for fresh market, and perhaps would be better referred today
as the “Butternut” Group after the original “Butternut” variety introduced
in 1936. Butternuts are characterized by a tan or buff rind color, a long, thick
neck, and a small bulbous seed cavity at the blossom end. The crookneck
dimorphic trait was bred out of “Butternut” with the introduction of
New Hampshire Baby Butternut in 1958. The “lack of dimorphism” trait
was introgressed into the still popular “Waltham Butternut” variety
released in 1970 by Dr. Robert Young of the Waltham Experiment Station
in Massachusetts. It is likely that many of the butternut cultivars grown
today have descended from “Waltham Butternut”. Most butternuts have
moderate to good eating quality and store well; the smooth rind and long,
thick neck lends itself to automated peeling and dicing for fresh and frozen
food processing.

Calabaza: Calabaza are ovoid squash, often with tan and green mottling,
and low to moderate dry matter content (Daniel et al. 1995; Maynard et
al. 2002), and are adapted to tropical regions. They are popular in parts of
Florida, the Caribbean basin, and Central and South America.
4.6 Interspecific Hybridization of Domesticated Cucurbita

Although Naudin (1856) attempted interspecific crosses as early as the mid
19th century, comprehensive research in North America on interspecific
crosses in the genus Cucurbita has largely been reported only during
the past 80 to 90 years. Early crosses were essentially made to define
compatibility and taxonomic barriers within the different Cucurbita species
(Erwin and Haber 1929; Castetter 1930; Van Eseltine 1936); whereas, later
interspecific crosses were directed at possibilities of transferring desirable
traits between species (Rhodes 1959; Wall 1961; Munger and Washek
1983) or on technologies to achieve such transfers (Wall 1954; Wall and
York 1960; Hayase 1961; Kwack and Fujieda 1987). Overall, there is little
cross-compatibility among the three major species of Cucurbita (Erwin
and Haber 1929), and tropical species are difficult to breed in temperate
climates because they flower only under short photoperiods. Nonetheless,
limited cross-compatibility between the domesticated species and some
wild species have afforded an important means for transferring disease
resistant traits into the domesticated species and in transferring some traits
between C. moschata and C. pepo (Munger and Washek 1983; Robinson and
Decker-Walters 1997; Andres and Robinson 2002; Cho et al. 2003; Oliveira
et al. 2003).
Wild species of Cucurbita are an important germplasm source for
introgressing disease resistance into domesticated species. Cucurbita
okeechobeensis (syn. C. martinezii) is a source of resistance to downy mildew
(Padley and Kabelka 2009), powdery mildew (PM) and cucumber mosaic
virus (Munger and Washek 1983). Cucurbita equadorensis, a relative of
C. maxima, is resistant to several virus diseases, as well as some fungal
diseases (Andres and Robinson 2002). Success with interspecific crosses is
difficult to predict, and numerous crosses are often required to obtain a few
viable seeds. Because most interspecific crosses yield nonviable seeds with
thin, misshapen embryos, it is often necessary to employ embryo culture
techniques to obtain viable plants (Wall 1954; Hayase 1961; Kwack and
Fujieda 1987). Most squash breeders are aware that wild species contain
relatively high contents of cucurbitacins in fruits and sometimes leaves.
Cucurbitacins are exceedingly bitter and the dominant Bi gene (Contardi
1939; Grebenščikov 1958; Herrington and Brown 1988) for bitterness can
be eliminated early in a backcross breeding program utilizing interspecific
crosses. The bitterness gene occasionally shows up in commercial seed

sources (Larry Hollar, Hollar Seeds; Rob Johnston, Johnny’s Selected Seeds;
pers. comm.), presumably due to mutational events.
Fertile F1 hybrids have been obtained from the cross of C. moschata x
C. martinezii (Contin and Munger 1977; Munger and Washek 1983; Cho et al.
2003), allowing for introgression of PMR and virus resistance into C. moschata
through subsequent backcrossing and selfing. Compatibility between C. pepo
and C. moschata is limited, and for the most part, researchers have relied on
the hope that two chosen parents will “nick.” Wall and York (1960) obtained
successful crosses between C. pepo (pistillate parent) and C. moschata using
Yankee Hybrid, an F1 hybrid between two yellow summer squash lines, as
the C. pepo parent. Cucurbita pepo is largely incompatible with C. martinezii
(Vaulx and Pitrat 1979), but by crossing C. martinezii with C. moschata, the
F1 hybrid has been used as a genetic bridge to transfer PMR and resistance
to cucumber mosaic virus (CMV) into C. pepo (Munger and Washek 1983;
Whitaker and Robinson 1986). Using embryo culture, Robinson (1997) was
able to recover F1 progeny from crosses of C. pepo to C. ecuadorensis. Through
a laborious series of backcrossing and screening, Robinson was eventually
successful in transferring zucchini yellow mosaic virus (ZYMV), papaya
ringspot virus (PRSV), and CMV resistance into C. pepo, with the subsequent
release of the variety “Whitaker”.
Crosses between C. maxima and C. pepo have been largely unsuccessful.
For example, Erwin and Haber (1929) performed nearly 2,000 reciprocal
crosses between different cultivars of C. pepo and C. maxima, resulting in
177 fruits and 23 fertile seeds. Twenty-one of the fertile seeds resulted from
crosses using Hubbard as either the male (10 seeds) or female (11 seeds)
parent. Using bud-pollination and embryo culture, Hayase (1961b) obtained
F1 hybrid plants with intermediate characters of C. pepo and C. maxima
parents. The F1 plants were gynoecious, but could be backcrossed to C. pepo.
Cucurbita maxima is, however, fully fertile with C. ecuadorensis, to which it is
closely related taxonomically (Sanjur et al. 2002). Genetic resistance to PRSV,
ZYMV, and watermelon mosaic virus (WMV) in C. ecuadorensis has been
transferred by backcrossing into C. maxima with the release of the varieties
“Redlands Trailblazer” and “Dulong QHI” (Herrington et al. 1991, 2001).
Gene transfer between C. maxima and C. moschata has been difficult.
Crosses between some cultigens of the two species yield neither seed nor
fruit. In other crosses, however, both fruit and ample, viable F1 seeds are
obtained (Erwin and Haber 1929; Hayase 1956; Robinson et al. 1978; Loy
and Uretsky 2010). The F1 hybrid progeny resulting from such crosses are
vigorous and fruit production is prolific, but the F1 hybrids are largely male-
sterile (Erwin and Haber 1929; Hayase 1956; Robinson et al. 1978). Japanese
breeders have produced interspecific F1 hybrids of C. maxima x C. moschata
on a commercial basis, most notably “Tetsukabuto”, a cross between
“Delicious” (C. maxima) x “Kurokawa No. 2” (C. moschata) (Robinson

and Decker-Walters 1996). “Tetsukabuto” was introduced into Brazil in

1960, and it is now the most popular cultivar in some states (Mendonça
et al. 2006). Cucurbita maxima varieties are commonly used as pollinators,
planted in intervening rows and occupying 10 to 20% of the cultivated area
(Mendonça et al. 2006). In addition to culinary use, C. maxima x C. moschata
hybrids are being used as rootstocks for melon grafts, an increasingly
important method of melon and watermelon production (Davis and King
2005/2006; Schultheis et al. 2008; Salehi-Mohammedi et al. 2009). Several
potentially beneficial advantages of C. maxima x C. moschata interspecific
hybrids warrant more breeding efforts: (1) the hybrids are seedless, which
may translate into increased biomass production of edible mesocarp tissue
and perhaps easier processing; (2) the F1 hybrids are earlier than the later
maturing C. moschata parents, thus potentially extending the latitude for
growing squash with favorable C. moschata traits; (3) the F1 hybrids may
display increased resistance to certain pests such as the squash vine borer
and powdery mildew as a result of the contribution of the C. moschata
genome, (4) the combination of a bush maxima parent with a more vining
moschata parent may result in a more desirable semi-bush growth habit, and
(5) it may be possible to combine certain maxima x moschata parents to obtain
interspecies hybrids with nutritionally beneficial carotenoid profiles.
The taxonomic differences between domesticated cultigens within
subspecies pepo and ovifera of C. pepo have been well described (Decker 1988;
Decker-Walters et al. 2002; Teppner 2004); however, except for noting that the
subspecies are cross-compatible, there has been relatively little description of
breeding outcomes when making subspecies crosses. Fruit size is larger and
peduncles are considerable thicker and more robust with more prominent
ribbing in C. pepo subsp. pepo than in subsp. ovifera. There is considerably
more variability in peduncle size within the pumpkin group than in the
entire ovifera subspecies, probably because this trait has been intensively
selected in ornamental pumpkins. Traits can be readily transferred between
the two subspecies, but because of substantial genetic differences between
them, two to three backcross generations may be needed to introgress a
trait of interest from one subspecies into a desirable genetic background
of the other subspecies. As an example, in transferring the glabrous trait
(Xiao and Loy 2007) from yellow straightneck squash (subspecies ovifera)
into zucchini (subspecies pepo), there was segregation for fruit length, fruit
taper, fruit color, peduncle size, petiole angle, petiole length, seed size, and
many other more subtle traits, some of which appear to be under complex
genetic control. In transferring traits between acorn squash, subspecies
ovifera, and pumpkin, subspecies pepo, there are differences in ribbing, fruit
size and shape, peduncle size, internode length, fruit color and color patterns
and flesh dry matter and texture. Moreover, some groups of traits appear
to be linked and inherited together, which can be either an advantage or

disadvantage depending upon whether they are linked to the trait being
transferred. There are numerous genes contributing to differences in fruit
coloration and patterns of fruit coloration (Paris and Brown 2005), and
variation in these traits may differ substantially in subspecies crosses,
depending upon the particular cultigens being crossed.
4.7 Germplasm Resources

Acquisition of suitable germplasm is one of the early and continuing
priorities in a breeding program. The primary source of germplasm for the
major commercial groups of squash and pumpkin are open-pollinated and
hybrid varieties in current use in the seed industry. The popular commercial
cultivars have suitable phenotypic traits for fruit size, shape and color and
plant growth habit, which are preferred in the current agricultural markets.
Also, as discussed above, alleles from wild species carrying resistance to
some of the major diseases, such as powdery mildew and viruses, have
been transferred into acceptable genotypes through various interspecific
hybridizations. Nonetheless, as new disease problems emerge, the wild
species most compatible with domesticated species, such as C. ecuadorensis,
C. andreana, C. lundelliana and C. okeechobeensis, may need to be evaluated,
along with existing cultivars and plant introduction (PI) germplasm, for
alleles conferring disease resistance. Although a majority of cultigens within
Cucurbita species show susceptibility to the major diseases within the genus
(Whitaker and Robinson 1986), screening of PI accessions has successfully
uncovered cultigens with intermediate levels of resistance to powdery
mildew (Krístková and Lebeda 1997), CMV (Lebeda and Krístková 1996a)
and WMV-2 (Krístková and Lebeda 2000), and several other virus diseases
(Provvidenti 1990). Information on global germplasm banks handling
Cucurbita species can be obtained from Bioversity International, now one
of 15 agricultural research centers supported by the Consultative Group
on International Agricultural Research (CGIAR). In the USA germplasm
repositories can be accessed through the National Germplasm System
(USDA-GRIN). Ferriol and Pico (2008) present an excellent review of
germplasm resources for Cucurbita.
4.8 Breeding and Selection for Specific Traits

Most breeding programs in squash and pumpkin focus on qualitative traits
such as fruit color, fruit color patterns, fruit size, fruit shape, growth habit
and disease resistance. Paris and Brown (2005) list 79 loci for phenotypic/
morphological traits in domesticated Cucurbita species, with only a few
reports of linked gene loci for such traits. Cucurbita species have 20 pairs
of small chromosomes which exhibit diploid pairing (Whitaker and Davis

1962), and progeny show no readily perceptible loss in vigor and fertility
with inbreeding. Given the focus on qualitative traits and considering the
reproductive characteristics in Cucurbita species, the pedigree system of
breeding has been widely adopted. When transferring only a few genes
into a desirable genotype from a wide or interspecific cross, the backcross
system is often used. The number of backcross generations needed prior
to selfing depends upon the genetic distance between parents used for
the initial cross, the size of the segregating backcross populations and the
degree of selection intensity for the phenotype desired.
Bush plants are considerably easier to manipulate than vine plants
because staminate and pistillate flowers are readily identified on individual
plants and higher plant populations can be grown per unit area. Of all the
cultivar groups, bush summer squash are easiest to breed because of their
exceedingly compact but open growth habit, together with the convenience
of being able to select for numerous immature fruit traits in segregating
progeny prior to making controlled pollinations.
Hull-less oil seed pumpkins (C. pepo subsp. pepo) are an important crop
in eastern Europe and China, but are not widely grown in North America.
Pumpkin seed oil will not likely become an important commodity in North
America, but the market for snackseeds, either alone or in trail mixes, and
in confectionary items, could expand. In breeding seed pumpkins, seed
yield is a major breeding focus, along with obtaining sufficient seed size
and uniformity (170 mg or greater), and good tip fill. Obtaining consistent
seed fill in high yielding cultigens is challenging because seed fill continues
until at least 55 days after pollination (DAP) in temperate climates, three
to four weeks after peak mesocarp dry matter is attained (Vining and Loy
1998), and 40 to 50 days after plants reach maximum leaf area (Loy 2004; Cui
2005). A major factor in achieving high yields has been developing inbred
lines and hybrids with relatively small fruit (1.0–1.5 kg) that partition a
large proportion (45 to 55% when expressed as kJ glucose energy per kg
fruit fresh weight) of photosynthate into seeds rather than mesocarp tissue
(Cui and Loy 2002; Loy 2004). A thorough review of the genetics, breeding,
and seed composition of oil seed pumpkins has recently been published
(Lelly et al. 2010).
There has been meager effort in breeding quantitative traits in squash
and pumpkin, and in the case of traits such as dry matter content of flesh,
seed size and fruit size which may exhibit quantitative inheritance (Singh
1949; Carle and Loy 1994), genetic manipulations of these traits can be done
in breeding cycles, such that no more than two or possibly three genes for
a particular trait are segregating in a breeding population.
Techniques for pollination, tagging fruit, and marking plants to be
pollinated were discussed above. Row and within row spacing varies
with the particular growth habit of the types of squash being bred, size

and number of fruit needed for evaluation, and the space available to the
breeder. Rows at least 3.0 m or more apart are preferable for vine genotypes.
To aid in pollinations, it is desirable to train vines in one direction. Related
genotypes are normally grown adjacent to one another, both for evaluation
in the same soil type and topography and for ease of making any necessary
crosses. Control lines of known performance for traits being selected should
be interspersed within breeding plots of segregating material.
Because of extensive space requirements, evaluation of elite germplasm
for possible commercial release can consume both time and resources
needed for breeding, especially in a mature breeding program. It is
frequently not possible to replicate evaluation plots involving dozens of
hybrid combinations, and in many instances, judgments concerning the
rating of pumpkin cultigens are based more on plant vigor and growth
habit and fruit appearance than on replicated yield records. Plot size for
comparing vining and semi-bush cultigens is often in the range of 10 to 15
plants spaced 1.0 m apart in rows 2.1 to 2.4 m apart. For summer squash, a
within-row spacing of 0.6 m and row spacing of 2.0 m is sufficient, and guard
rows are not needed because inter-row competition is minimal. Guard rows
for each cultigen are rarely used when evaluating winter squash or pumpkin
either because of space or seed limitations. It is therefore important to place
all cultigens with a similar growth habit (bush, semi-bush, or vine) in the
same block to minimize growth competition between adjacent cultigens.
Cultigens which look promising in initial non-replicated evaluations
can then be evaluated in replicated plots and in different locations in the
following years.
4.8.1 Plant Growth Habit

As in other vegetable crops, genetically determined attributes of growth
habit have been exploited by pumpkin and squash breeders, and indeed,
by early plant selectors as evidenced by the appearance of a bush (short
internode) form of scallop squash in an herbal dating to the mid 16th century
(Teppner 2000). The bush gene was selected and utilized in summer squash
at a very early period undoubtedly to provide easier access for continuous
harvesting of immature fruit, and perhaps because of the higher ratio of
pistillate to staminate flowers often associated with the bush habit of growth.
Bush plants in Cucurbita species are characterized by shortened internodes,
thicker stems, longer and more robust petioles, early flowering, and in many
cultigens, an absence of tendrils (Loy 2004). In winter squash, pumpkins
and gourds, the bush phenotype has gained widespread commercial use
only during the past three decades. The commercial adoption of bush and
semi-bush varieties can be attributed primarily to two factors: (1) increased
emphasis on F1 hybrid cultivars in the vegetable seed industry, and (2) earlier

maturation and amenability of bush cultivars to high density planting, rapid

leaf canopy closure, and more sustainable weed control. The technology
for hybrid seed production in squash was improved following reports
by Robinson et al. (1970) and Rudich et al. (1969, 1970) that monoecious
cucurbits could be converted to the gynoecious condition for an extended
period by spraying with appropriate concentrations of an ethylene-releasing
compound, 2- chloroethylphosphonic acid (ethephon). Ethephon-feminized
breeding lines can be interplanted with male pollinator lines for efficient
hybrid seed production. Bush lines are much easier to convert to gynoecy
than vine lines and generally set most fruit close to the crown of the plant
prior to late appearance of staminate flowers on ethephon-treated plants.
Either bush x bush or bush x vine hybrids can be produced via ethephon
treatment. In summer squash, bush x bush hybrids are used exclusively, but
the semi-bush phenotype appears to be more preferable for winter squash
and ornamental pumpkins because of a more suitable ratio of vegetative
growth to mature fruit load. Nonetheless, because breeders have been able
to modulate the ratio of vegetative growth to fruit load by selecting for later
reproductive development, some productive bush x bush hybrids of winter
squash and pumpkin have gained commercial acceptance.
In C. pepo, the bush habit of growth has been attributed to a single,
incompletely dominant gene (Shiffriss 1947); whereas, in C. maxima,
inheritance has been ascribed to two incompletely dominant genes (Singh
1949). Shiffriss labeled the incomplete dominance of the bush gene as a
“developmental reversal” of dominance because bush-vine heterozygotes
initially have short internodes, but revert to longer internodes later in
development. Nonetheless, internode lengths of heterozygous plants
usually remain intermediate to those in bush and vine genotypes (Denna
and Munger 1963). Hybrid plants obtained from an interspecific cross
between bush C. pepo and bush C. maxima exhibited short internode growth
suggesting that the two species share a common allele for the bush habit of
growth (Denna and Munger 1963). The bush allele (bu) in C. pepo has been
transferred to C. moschata (Munger 1990; Shiffriss 1990), and also, a dominant
bush mutant was recently discovered in C. moschata (Wu et al. 2007).
Expression of the bush phenotype is more complex and variable than
what is generally presented in the literature, and the short internode trait
conditioned by the bu locus appears to be influenced by an undetermined
number of modifying genes (Grebenščikov 1958). There are several major
features of the bush growth habit that must be considered by breeders,
including persistence of short internode growth during development,
pleiotropic effects on other phenotypic characteristics such as flowering
and branching habit, and fruit load in relation to vegetative growth. In
bush breeding lines of C. pepo, spanning yellow summer squash, zucchini
summer squash, acorn squash, gourds, and ornamental pumpkin, there

is considerable variability in expression of internode length (Table 4-2).

For instance, yellow straightneck summer squash retain extremely short
internodes throughout the plant life cycle, but modifying genes can still
modulate internode length as exhibited by YSN836 and YSN27A1221
(Table 4-2). In bush acorn squash, internode length is less than 2 cm for
12 to 16 nodes, but subsequent internodes become progressively longer,
reaching about 5 cm in absence of fruit set (Denna and Munger 1963;
Table 4-2). In ornamental pumpkin, on the other hand, typically only the
first 5 to 6 internodes are less than 2 cm, and mature internodes average
9 to 10 cm along the main stem when plants are trellised to a single stem
in the greenhouse. In vine plants only the first internode will typically be
less than 2 cm, and mature internodes average about 20 cm in length. In a
bush breeding line of C. moschata, only the first 6 internodes were less than
2 cm, and mature internodes were not appreciably shorter than the vine
cv. Waltham Butternut (Table 4-2).
Table 4-2 Successive number of internodes (IN) 2 cm or shorter in length beginning with the
first true leaf, and average length of mature internodes during exponential growth in bush
and vine cultigens of Cucurbita species. Data from greenhouse Spring 2010, Durham NH.
Variety Breeding Growthz IN numbery Mature INx
Species Classification line Phenotype (≤ 2 cm) length (cm)
C. pepo YSN squash NH836 extreme bush 28.2 (33.8) 1.2w
YSN squash NH27A122 extreme bush 25.3 (75.9) 3.0w
acorn NH459 bush 17.0 (17) 3.5
acorn NH1321 vine 4.3 (5.5) 9.2
pumpkin NH394-4-11 bush 5.0 (7.9) 10.3
pumpkin NH1141-3 bush 5.5 (7.9) 9.5
pumpkin NH432-1 bush 3.0 (5.3) 10.1
pumpkin NH314174 vine 1.0 (1.5) 20.9
pumpkin NH274-7 vine 1.0 (1.0) 20.5
C. maxima hubbard NH2556 digenic bush 12.7 (15.1) 12.4
kabocha NH5513 digenic bush 14.0 (19.5) 13.3
buttercup NH6320 digenic bush 11.6 (11.6) 11.1
kabocha NH46154 mono bush 5.8 (5.7) 11.7
kabocha NH5273 vine 3.0 (2.8) 15.7
hubbard Baby Blue vine 3.0 (3.2) 12.8
banana NH.BPB bush 25 4.7
C. moschata butternut Waltham vine 1.0 (0) 15.5
butternut HS221-7 bush 6.0 (6.0) 12.7
Z
Inheritance studies have not substantiated digenic inheritance nor which of the two putative
bush genes is present in monogenic bush maxima cultigens. Presumably one of the two genes
is allelic to the Bu allele in C. pepo.
y
Successive internodes 2 cm or less in length. Total stem length for internodes shorter than 2
cm is given in parenthesis.
x
Average of 3 mature internodes on 2 to 5 plants.
w
Average of all internodes, which were similar in length throughout development.

Selection for homozygous bush genotypes in C. maxima is more difficult

than in C. pepo. In the two gene model proposed by Singh (1949), both bush
genes were incompletely dominant and appeared to have additive effects,
such that Singh subdivided an F2 population segregating for growth habit
into five phenotypic classes—bush, bush tending to vine, bush-vining,
vining-bush, and vine. Under field conditions in a breeding program, it is
not practical to determine which plants carry a particular bush gene or a
particular combination of heterozygous alleles in a population segregating
for two incompletely dominant genes. To ascertain the action of bush
alleles it is necessary to determine internode lengths at different stages of
development and determine the transition period from short internodes
(those less than 2 cm) to mature internodes. Under greenhouse conditions
as illustrated in Table 4-2, putative digenic bush plants of C. maxima (lines
NH2656, NH3620 and NH551313) maintained short internodes for 12 to 14
nodes, similar to results reported previously (Denna and Munger 1963; Zack
and Loy 1981), but mature internodes of bush plants were not appreciably
shorter (11 to 13 cm) than those of vine plants (13 to 16 cm). Moreover,
mature bush internodes were about twice as long as those reported in the
two studies cited above, underscoring the strong influence of environmental
conditions, especially light intensity and light quality, on internode length
(Zack and Loy 1980). In plants homozygous for only one of the two bush
alleles in C. maxima, the initial expression of short internodes is very
transient and mature internode length is within the range of vine plants
(Table 4-2). There can be additional modifying genes in C. maxima exerting
an effect on the persistence of short internodes, similar to that observed in
summer squash (note Bush Pink Banana strain, Table 4-2).
F1 hybrids resulting from digenic bush x vine crosses in C. maxima
show less dominance toward expression of the bush phenotype than that
observed in C. pepo. Hybrid plants typically produce short internodes
(< 2.0 cm) for about 7 nodes, and then successive internode expansion is
quite rapid. By node 12 to 13, internode length approaches that of non-bush
plants (Denna and Munger 1963; Zack and Loy 1981). Cucurbita maxima
hybrids with a more persistent bush habit can be produced by crossing
a digenic bush line to a monogenic bush line such as exemplified in the
hybrid, “Autumn Cup”.
Under field conditions, the extent of lateral branching and the timing
of fruit set affect the overall vegetative phenotype of a plant, complicating
phenotypic analysis. Bush plants often flower and set fruit earlier than
vine plants. Early flowering may be a desirable trait for areas with a short
growing season, but early fruit set and high fruit loads on bush plants
suppresses vegetative growth, resulting in low dry matter accumulation in
mesocarp tissue (Loy 2004) and adversely affecting eating quality (Loy 2006).
In ornamental pumpkin, low dry matter in mesocarp tissue is correlated

with low dry matter and reduced integrity of peduncles (Berg 2004).
Branching habit appears to be under simple genetic control in Cucurbita,
but with some modifying genes (J.B. Loy, unpubl. results). In a small
population of acorn plants we observed a 3:1 segregation of plants with
a strong main stem and few laterals as compared to plants with multiple
branching habit. In numerous hybrid F1 plants resulting from crosses of
breeding lines with a dominant main stem to lines with multiple branching,
single stem is dominant (J.B. Loy, unpubl. observations). In summer squash,
there are clearly additional modifying genes that affect the intensity of a
strong single stem. In winter squash and pumpkin, unlike summer squash,
multiple branching habit is generally considered a desirable trait because
multiple growing points contribute to more rapid and uniform leaf canopy
development. Determinate growth habit, only recently reported in squash
(Kwack 1995), has not been utilized in breeding.
4.8.2 Flowering and Fruiting Habits

Flowering and fruiting patterns are a key attribute in breeding squash and
pumpkin. Important aspects of flowering are: (1) time of first appearance of
staminate and pistillate flowers, (2) ratio of staminate and pistillate flowers,
and (3) environmental effects on flowering patterns and fruit set. In early
maturing cultivars of summer squash, female flowering commences as
early as 30 to 40 days from seeding. Flowering of typical winter squash
and pumpkin cultivars of C. pepo and C. maxima begins about 6 to 8 weeks
from seeding. However, some of the more tropically-adapted cultigens of
C. moschata are daylength sensitive, and will flower only during short days
in temperate zones.
It is generally desirable from both a breeding and field performance
perspective to have simultaneous staminate and pistillate flowering.
In summer squash, early and prolific production of pistillate flowers is
associated with high yields, but on the negative side, early summer squash
cultivars will often produce pistillate flowers for several days prior to
appearance of staminate flowers. Early pistillate flowers will sometimes
produce parthenocarpic, mostly misshapen fruit. Such fruits negatively
impact allocation of photosynthates to the vegetative and reproductive
meristems. Moreover, when early fruits senesce, they can harbor various
soft rots which may adversely affect subsequent fruit sets.
In winter squash and pumpkin as well, it is not uncommon for modern
hybrids to initiate pistillate prior to staminate flowers. For most cultigens the
main concern with lack of staminate flowers is a delay in fruit set; however,
low pollen availability can also reduce seed yields. Pistillate flowering

usually precedes staminate flowering by 4 to 6 days in hull-less, F1 hybrid

seed pumpkins, yet seed yields per fruit are usually high (Loy 2004). One
plausible explanation is that fruit set under conditions of poor pollen loads
abort when later fruit set is accompanied by high pollen loads and greater
seed set as reported by Stephenson et al. (1988).
4.8.3 Vegetative Growth and Sink Strength

Sink strength in relation to vegetative growth and photosynthetic leaf area
is an important aspect of yield and fruit quality in squash and pumpkin.
The strength of sinks in squash is determined by the number and size of
each fruit, but may also be influenced by location of fruit on the plant and
time of development (Wien 1997). In addition to size and number of fruits,
sink capacity of an individual fruit is strongly influenced by its capacity to
store starch, the primary reserve carbohydrate of photosynthesis. Because
fruit size, fruit number and flesh dry matter are interrelated, breeders must
have a firm grounding of the relationship of these traits when breeding for
good eating quality in squash. High fresh weight yields are associated with
low flesh dry matter (Broderick 1982; Loy 2004, 2006). Good eating quality,
on the other hand, is associated with high dry matter, mostly in the form of
starch and sugars (Culpepper and Moon 1945; Harvey et al. 1997). Under
comparable growing conditions, cultivars with good eating quality should
not be expected to produce fresh weight yields as high as those varieties
with less acceptable eating quality. This presents a conundrum not only for
breeders and seed companies, but also for growers, because in absence of
quality standards, grower profits, particularly in wholesale markets, are
determined to a large extent by fresh weight yields. Mesocarp dry matter
is under multigenic control (Singh 1949), but the relative ratio of fruit load
to vegetative growth can affect dry matter accumulation (Loy 2004), and
thus confound estimation of potential dry matter content.
In large-fruited processing squash, it has been possible to breed a bush
plant ideotype that at appropriate spacing produces one fruit per plant, with
uniform fruit size and dry matter content (Broderick 1981). Producing one
fruit per plant results in uniform maturity and alleviates problems with
excessive production of immature fruit. Moreover, fruit size can be regulated
by adjusting planting density. Bush-vine processing hybrids possess these
same features, but appear to have wider adaptability than bush strains. Fruit
dry matter content in processing squash can be increased through selective
breeding, but large-fruited cultigens with higher dry matter invariably
exhibit lower fruit yields per unit area than lower dry matter cultigens (J.B.
Loy, unpubl. results).

4.8.4 Fruit Appearance

Fruit quality traits can be subdivided into two categories: (1) those traits
which affect external and internal appearance of fruit, and (2) those which
affect eating quality and nutrition. In ornamental cultigens, breeders focus
primarily on traits affecting external appearance. Important genetically
determined traits affecting external appearance are rind color, rind hardness,
degree of ribbing, size of blossom scar, size of peduncle, fruit shape and fruit
size. Internal appearance is largely limited to flesh thickness, flesh color and
uniformity of flesh color. In C. pepo the dominant allele Hr confers hard or
highly lignified rind (Mains 1950), and the dominant Wt allele conditions
warty fruit (Sinnott and Durham 1922; Paris et al. 2004), but only in the
presence of the Hr allele (Schaffer et al. 1986). In C. maxima, the dominant
allele, Hi, inhibits development of the hard rind trait (Herrington and Brown
1988). There is considerable genetic variability in the degree of ribbing,
fruit size and fruit shape within all three species, but scant genetic studies
describing gene action for these traits (Paris and Brown 2005). Degree of
ribbing and fruit shape appear to be under simple genetic control (one or
two genes) because they are easily manipulated in segregating populations.
Preference for degree of ribbing, fruit size and shape vary among the
different cultigen types, and occasionally, cultigens with new morphological
fruit characteristics evolve in popularity over the course of time.
4.8.4.1 Fruit Size

There is tremendous variation in fruit size within the three major
domesticated species of Cucurbita, ranging from 20 to 30 g egg gourds in
C. pepo subsp. ovifera to the humungous show pumpkins in C. maxima, some
of which weigh over 600 kg. Fruit size is a quantitative trait and large fruit
size is dominant over small fruit. Often, F1 hybrids will show overdominance
for fruit size, an expression of heterosis (Berenji 1986; Carle and Loy 1994).
In an F2 population of 450 plants generated from a cross of a breeding line
(NH29-13-5), with small fruit (0.61 kg ± SD 0.2) to a cultigen, PI285611,
with much larger fruit (2.64 kg ± SD 0.5), the smallest fruit recovered in
the segregating population weighed 1.13 kg, and the largest was 8.39 kg
(Carle and Loy 1994). Assuming that all genes conferring large fruit size
were dominant in the F2 population, one can infer that a minimum of four to
five recessive genes for small fruit size were segregating in the population.
Moreover, the range of fruit size in C. pepo is much wider than represented in
the above example. For breeding hybrid cultivars of ornamental pumpkin,
dominance for fruit size has been exploited by using small-fruited, bush
strains of pumpkins with high seed yields as female parents, crossed to
large-fruited vine strains.

4.8.4.2 Fruit Color

Fruit color traits are especially important attributes of squash and pumpkin.
The most detailed research on color inheritance has been in C. pepo by H.S.
Paris and colleagues. Because fruit color changes may occur at different
developmental stages (Paris and Nerson 1986), breeders have to be
cognizant of when certain color traits can be evaluated in cultigens and
segregating populations. In acorn squash, for example, dark green fruit
color is preferred to lighter hues, and stability of dark coloration during
storage is desirable. The dominant L1 and L2 alleles act in a complementary
fashion early in development to intensify green rind coloration (Paris and
Nerson 1986). The D gene, on the other hand, is expressed at intermediate
stages of fruit development, intensifies green fruit coloration, and is epistatic
to the l-1 and l-2 alleles conferring lighter green rind coloration (Paris and
Nerson 1986; Paris 1997). The D gene also confers dark green stem and
peduncle coloration, the latter being a desirable trait in acorn squash and
ornamental pumpkins. Also, in ornamental pumpkins the developmental
change from green to orange fruit occurs between 25 to 40 days after
pollination, and displays duplicate recessive epistasis for genes mo-1 and
mo-2 (Paris 1997). Both of these genes are present in most pumpkin cultigens,
but when making subspecies crosses in C. pepo, breeders have to be aware
of segregation at the Mo-1 and Mo-2 loci. The intensity and hues of orange
color appear to be modulated by several modifying genes, and also by the
“B” locus for precocious yellow fruit coloration (Shiffriss 1981). In summer
squash and pumpkin, the B allele normally has to be homozygous to be
expressed throughout the entire fruit, including partially into peduncle
and perianth tissues. In other genetic backgrounds, such as spoon gourd,
plants homozygous for the B gene display bicolor fruit coloration. A genetic
model invoking two incompletely dominant and additive modifying
genes, Ep-1 and Ep-2, for enlarging the area of yellow pigmentation, has
been proposed to account for differential expression of the B gene in spoon
gourds (Shiffriss and Paris 1981). The B gene has been incorporated into
several ornamental cultivars of C. pepo, in most cases to produce a bicolor
phenotype, but because it often has a pleiotropic effect on lowering starch
content (Schaffer et al. 1986), its use in edible cultivars has been largely
limited to bicolor cultivars heterozygous for the B allele.
In C. maxima fruit color varies from green cultivars of buttercup, kabocha
and hubbard types, to gray cultivars of Hubbard and Queensland Blue
types to pink and red kabocha and processing squash. There are various
shades of green, and Lopez-Anido et al. (2003) have proposed a three-gene
model to account for segregation of light and dark green fruit in zapallito
type (immature) C. maxima. Light orange to orange to red-orange coloration
in C. maxima is due to the effects of the Bmax allele and other unidentified

genes. In 1920, Lotsy described an Rd allele for fruit coloration in C. maxima

(quoted in Paris and Brown 2005), but perhaps the Rd allele is identical to
the Bmax allele. The Bmax allele has been incorporated into a few kabocha
cultivars and into processing squash because it eliminates problems with
green pigmentation in squash puree associated with cultigens with a green
rind. Gray or blue coloration is conferred by the bl allele, referred to as
incompletely recessive by Hutchins (1936). However, the bl gene apparently
affects the surface of the epidermis, perhaps via cuticular waxes, such that
all rind colors are muted, e.g., light green to light gray, dark green to dark
gray, orange to pink, and red-orange to dark pink (J.B. Loy, unpubl. data).
It seems that the expression of this allele is towards dominance and would
be more appropriately designated as incompletely dominant, and perhaps
also referred to as a color dilution (Cd) gene (J.B. Loy, unpubl. data).
In C. moschata, tan coloration dominates fruit color of butternut and
pie processing cultigens such as Large Cheese and Dickinson Field. Some
large crooknecks display green rind (Gr), dominant to tan (Robinson 1987).
There are various intensities of green among moschata cultigens, suggesting
genetic control similar to that in C. pepo and C. maxima. Mottled light and
dark green immature fruit color is dominant (Mldg) to uniform dark green
color (Cardosa et al. 1993). Both the B gene from C. pepo and the Bmax gene
from C. maxima have been transferred into C. moschata (Shiffriss 1989), and
they are non-allelic (Shriffriss 1991). The bicolor fruit phenotype has been
incorporated into at least one C. moschata cultivar (Boiteux et al. 2007).
4.8.5 Eating Quality and Nutrition

Starch and sugars are the dominant attributes of eating quality in squash
(Culpepper and Moon 1945; Daniel et al. 1995; Harvey et al. 1997). Starch
not only serves as the primary reserve carbohydrate for sugar conversion,
but also contributes textural properties to squash (Merrow and Hopp 1961;
Corrigan et al. 2001; Stevenson et al. 2005). Because sugars and starch often
comprise as much as 60 to 70% of the dry weight in high quality squash at
maturity (Phillips 1946; Hurst et al. 1995; Harvey et al. 1997), % dry matter
in squash, an easily measurable trait, correlates well with starch content
and eating quality. A minimum soluble solids level of 11% and dry matter
content of 20% is considered acceptable for good eating quality in kabocha
squash (Harvey et al. 1997). In butternut and acorn squash, somewhat
lower contents of dry matter (18 to 20%) are generally associated with good
eating quality in North America. Relative levels of sugars can be easily
estimated by freezing small (5 to 10 g) mesocarp samples and measuring %
soluble solids in liquid droplets squeezed onto a hand-held refractometer
from thawed samples. Some breeders have recommended taste panels to
evaluate squash cultigens for release (Murphy et al. 1966); however, given

the strong association of high soluble solids and starch levels to perceived
quality and the cost and time of group culinary evaluation, taste panels may
be an overly cumbersome and unnecessary addition to a squash breeding
program. On the other hand, once a breeder has identified a potentially
good cultigen, squash samples should be made available for evaluation
by other persons.
The stage of maturity of squash strongly influences evaluation of eating
quality and nutrition. Based on rind color alone, many squash cultivars
appear mature relatively early in development. Acorn squash, for example,
turn dark green and attain nearly full size within about two weeks after fruit
set. Tan rind color in butternut squash occurs by 35 to 40 days after fruit set.
Although peak dry matter is attained in all three species of squash by 30 to 35
DAP, acceptable soluble solids levels are usually not attained in acorn squash
until 50–55 DAP (Loy 2006), and in C. moschata, may not be attained until
several weeks of storage following harvest at 55 to 60 DAP (Noseworthy
and Loy 2008). Solids contents greater than 26% occur commonly in some
kabocha squash. Cooked squash with an over-abundance of starch and
low sugar has a dry flaky texture, and may score low in cooking tests. With
additional storage, accompanied by elevated sugar levels and some loss in
dry matter, texture and sweetness will become acceptable.
Carotenoids are an abundant dietary component of squash. Many
squash cultigens are not only a good source of β-carotene, important in
development and eye function, but also an excellent source of lutein,
which has an important photoprotective function in the macular region
of the retina (Azevedo-Meleiro and Rodriguez-Amaya 2007). Cultigens
with high carotenoid levels have been bred mostly using a visual scale
(Noseworthy and Loy 2008). However, accurate analysis of carotenoids
requires spectrophotometric determination of total carotenoids combined
with high pressure liquid chromatography (HPLC) to examine carotenoids
profiles. Increases in total carotenoid levels during fruit maturation and
subsequent storage correspond well with changes in soluble solids, so it
is appropriate to evaluate carotenoids when squash are near peak eating
quality (Noseworthy and Loy 2008).
There is considerable variability in percent dry matter of pericarp
tissue among cultigens, and even among fruits from the same plant. In
the classical paper of Culpepper and Moon (1945) in which 35 cultivars of
C. pepo, C. maxima and C. moschata were compared, percent solids varied
from as high as 15.7% for Table Queen to as low as 6.4% for King of the
Mammoths, a show pumpkin. Plant breeders have increased dry matter
content, and therefore eating quality, in some of the better cultivars. There
are now numerous cultivars of kabocha and buttercup squash with dry
matter consistently above 20%, and a few of the better C. pepo winter
squash cultigens average nearly 20%. The most popular C. moschata cultivar,

Waltham Butternut, often exhibits dry matter in the 17 to 21% range in New
England (J. Noseworthy and J.B. Loy, unpubl. results). The wide range in
dry matter in winter squash is undoubtedly under multigenic control; this,
together with seasonal, within plant, and plant to plant variability within
a cultigen, render inheritance studies of dry matter content a complex
genetic area to broach. Singh (1949) conducted a thorough genetic study
of solids content in C. maxima; however, the mean solids content (% DM)
of the two parental cultigens was exceedingly low (2.74 and 6.73%). His
results fit a two gene model and additive gene action. Observations have
been that for F1 hybrids to have high dry matter, both parents must have
high dry matter, and in F2 selections exhibiting high dry matter, progeny
in later generations retain the high dry matter trait. This suggests that high
dry matter is under control of either recessive or additive alleles (J.B. Loy,
unpubl. observation).
4.8.5.1 Quality Factors in Summer Squash

Because summer squash is harvested immature, about 3 to 5 days after
anthesis, sugar content is low and dry matter is only 5 to 6% (Lorenz 1949;
Aboul-Nasr et al. 2002). Size, shape, color and freedom from blemishes
caused by biotic and abiotic factors are the most important considerations
in assessing fruit quality. Zucchini is the most popular class of summer
squash in North America. Breeders have selected a fruit type that is long,
cylindrical and uniform in shape. Fruit are generally dark green, but there
are subtle differences in intensity of color and degree of light green stippling
among cultivars. There are also cultivars that have reduced presence of
trichomes on petioles and stems, reducing abrasion damage to fruit. Two
types of C. pepo spp. ovifera are unique to North America: yellow crookneck
squash, common in the southeastern US, and yellow straightneck squash,
more common in Central and northern US and Canada. Crookneck and
straightneck summer squash have tender skin when immature and are
very susceptible to bruising and scratches which eventually turn brown
with phenolic oxidation, leading to poor fruit appearance. In addition,
compound trichomes on petioles, stems and the abaxial veins of leaf blades
of this subspecies tend to be quite prickly, damaging fruit on windy days
and during harvesting operations, as well as irritating the skin of pickers. A
recessive glabrous gene (gl-2) has been reported in summer squash within
subspecies ovifera, which markedly reduces fruit damage (Xiao and Loy
2007). Glabrous cultivars are being introduced and may eventually lead
to improved fruit quality in supermarkets and other retail outlets. Other
than disease resistance and fruit shape and color, most breeding efforts
in summer squash have been aimed at developing productive and early
maturing cultigens with a thick, strong main stem, no lateral branching,

and long petioles that extend approximately perpendicular to the plant

axis. The latter morphological traits contribute to an open plant habit to
aid in harvesting. A more detailed presentation of specific traits in summer
squash, particularly those affecting color and patterns of color, is given by
Paris (2008).
4.9 Hybrid Cultivars and Hybrid Vigor

Although Cucurbita species are monoecious, bee-pollinated and show a high
degree of outcrossing under field conditions, they are often listed along
with self-pollinated species in books on breeding because they are easily
self-fertililized and early studies indicated that inbreeding depression was
either nonexistent or marginally detectible (Cummins and Jenkins 1928;
Haber 1928; Scott 1934). Although some studies have now shown evidence
of inbreeding depression in Cucurbita, both in domesticated (Chekalina 1976;
Cardoso 2004) and wild species (Johannsson et al. 2002; Hayes et al. 2005),
inbreeding depression is not readily apparent in breeding populations,
and therefore, not an issue in breeding Cucurbita. High parent heterosis
for growth and biomass yield would be expected to be small in Cucurbita
hybrids in comparison to crops that exhibit severe inbreeding depression.
Published references on heterosis in squash should be interpreted with
caution because of possible misinterpretation of data. For example, in bush x
vine hybrids F1 plants may display rates of leaf initiation similar to the vine
line, but larger leaves sometimes associated with the bush phenotype, and
therefore greater total leaf area. Bush, semi-bush and vine lines may have
substantially different spacing requirements for optimum yields, but this
discrepancy may be ignored in studies of heterosis. In winter squash, percent
dry matter of fruit tissue is negatively correlated with yield; therefore,
heterotic reports of fresh weight yield increases may be the result of lower
fruit dry matter in the F1 hybrid.
In spite of these pitfalls, hybrid vigor for a number of specific traits such
as early flowering, fruit size, fruit yield and seed yield has been substantiated
by a number of investigators. Furthermore, other assets of F1 hybrids, such
as uniformity, phenotypic stability, variety protection, and capacity to
combine favorable traits from two parents, have undoubtedly contributed
to the expanded adoption of F1 hybrids in squash and pumpkin. In 1983,
Pearson reported that about 56% of the summer squash seed and only 10%
of winter squash seed was of hybrid origin. Since that time, there has been a
huge increase in the number of hybrid winter squash and pumpkin cultivars
introduced, and they now dominate the commercial market in C. pepo and
C. maxima. In C. moschata, open-pollinated cultivars are still popular, and
there has been less adoption of hybrid cultivars. Few bush parental lines,
useful for hybrid seed production, have been developed in C. moschata.

Furthermore, open-pollinated cultigens resembling “Dickinson Field”,

which dominate the large acreage of C. moschata used for pie processing,
have low dry matter and extremely high yields. They are not likely to be
replaced by hybrid varieties in the future unless the hybrids have additional
attractive features such as multiple disease resistance. A comprehensive
review of hybrids and hybrid seed production in cucurbits was provided
by Robinson (1999).
A major heterotic effect in summer squash is earlier flowering, which
can impact total yields; therefore, it is not surprising that heterosis was first
documented in yellow summer squash (Curtis 1939). Other studies have
since documented heterotic yield increases in summer squash (Grebenščikov
1975; Amaya and Ortega 1996; Firpo et al. 1998; Ahmed et al. 2003). In a
study of 10 breeding lines crossed in a complete diallel, Firpo et al. (1998)
reported an average heterosis for early yield of 141%, for total yield of
35.2% and for leaf number at 30 days after seeding of 22% for the five best
combining lines. In summer squash, fresh weight yields are probably a
good parameter for heterotic effects on biomass yields because there is
not likely to be large differences in dry matter content of immature fruit,
and small differences would not be expected to affect perceived eating
quality. There have been attempts in C. pepo to extend genetic divergence in
potential crosses by intercrossing cultigens from different cultivar groups of
summer squash (Anido et al. 2004). In the preceding study, highly significant
heterosis was found only in combinations of crooknecks or straightnecks
with cocozelles.
In a comparison of 10 winter squash hybrids and inbred parents in
C. maxima, Hutchins and Croston (1941) found seven hybrids had higher
yields than the highest yielding parent, but they did not determine flesh dry
matter contents, so differences in dry biomass yields could not be assessed.
Hayase and Ueda (1956) likewise compared 15 hybrids and parents of
C. maxima for several traits. Most of the hybrids exhibited heterosis for fruit
numbers per plant, weight of fruit, and days to maturity. Most hybrids had
better eating quality than inbreds in terms of soluble solids and taste ratings,
but no data on % DM were obtained. In a study of nine inbred lines of
C. maxima and 49 F1 hybrids, Korzeniewska and Niemirowicz-Szczytt (1993)
evaluated heterosis for fruit yield, fruit number, fruit size, % dry matter, and
fruit biomass yield per plant. Heterosis was not observed in the majority
of hybrid combinations and was inconsistent between the two years of the
study, but heterosis was observed over both years for total fruit yield in
three hybrids and for fruit dry biomass per plant in three hybrids.
Heterosis has been substantiated in hull-less seeded pumpkins
(C. pepo), perhaps because the primary economic product is the seed
and not the whole fruit. Berenji (1986) compared several yield traits in
six inbred parents and 15 F1 hybrids created from a half diallel crossing

scheme. Seven out of 15 hybrids showed high parent heterosis for mean
fruit weight, 13 out of 15 were heterotic for seed weight per fruit, and all
hybrids showed heterosis for seed weight per plant. Cui and Loy (2002)
evaluated high parent heterosis for different components of seed yield in
two hybrids and four parental strains. Total fruit dry biomass per plot was
significantly higher in both hybrids than in parental strains. Seed yield per
plot was significantly higher in one of the two hybrids in comparison to
parental inbreds, and seed number per fruit was 24 and 36% higher in the
two hybrids as compared to the two highest parental lines.
Open-pollinated strains still dominate commercial acreage of C.
moschata cultigens, and there have been few detailed studies of heterosis
in this species (Robinson 1999).
4.10 Disease and Insect Resistance

4.10.1 Disease Resistance
Breeding for disease resistance is usually a strong priority in a breeding
program, particularly when breeders are dealing with cucurbit crops grown
in major agricultural regions where incidence of disease problems is likely
to be high. Pumpkins and squash are relatively tolerant of most soil borne
diseases. The major breeding emphasis has been on developing resistance
to numerous viral diseases, to powdery mildew, and to a few other fungal
diseases.
The primary viruses infecting squash and pumpkins from the aphid-
transmitted Potyviridae family are papaya ringspot virus (PRSV), watermelon
mosaic virus (WMV), and zucchini yellow mosaic virus (ZYMV). Cucumber
mosaic virus (CMV; Cucumoviridae) is a widespread aphid-transmitted
viral problem in squash with several alternate hosts. Squash mosaic virus
(SqMV; Comoviridae), vectored by beetles, is considered less of a problem in
Cucurbita. A summary of transgenic resistance to viral diseases in Cucurbita
is presented in the following section on “Contributions of Biotechnology.” A
comprehensive review of viral problems and use of biotechnology to achieve
resistance in cucurbits is provided by Gaba et al. (2004). As mentioned in the
section above on “Interspecies Hybridization,” natural resistance to CMV,
WMV, ZYMV and PRSV has been transferred from wild into domesticated
species (Munger and Washek 1983; Herrington et al. 1991, 2001; Robinson
1997). In general, germplasm within the domesticated species have not
provided many genes for viral resistance (Whitaker and Robinson 1986).
Nonetheless, some degree of resistance to ZYMV (Provvidenti 1997; Pachner
and Lelley 2004) and CMV (Zhou 1987) in C. moschata, to ZYMV (Paris and
Cohen 2000), CMV (Lebeda and Křístková 1996a) and WMV-2 (Křístková
and Lebeda 2000) in C. pepo and to CMV (Provvidenti 1982; Lebeda and

Křístková 1996a); PRSV (Provvidenti 1982) and WMV-2 (Provvidenti 1982;

Křístková and Lebeda 2000) in C. maxima have been reported.
Powdery mildew (PM) is the most widespread and devastating fungal
disease in Cucurbita. Two different organisms incite this disease, Podosphaera
xanthii (formerly Sphaerotheca fuliginea) and Golovinomyces cucurbitacearum
(formerly Erysiphe cichoracearum). The most commonly reported mildew
pathogen worldwide is P. xanthii (Zitter et al. 1996), but in some temperate
regions of the world, G. cucurbitacearum is dominant (Křístková et al. 1997).
Based on resistant differentials in Cucumis melo, most powdery mildew in
North America is incited by races 1 and 2 of P. xanthii; whereas, several other
prevalent races have been identified in other regions of the world (Pitrat et
al. 1998). In many temperate regions PM does not overwinter, and hence,
spores are spread by wind currents and infestations usually occur mid to
late in the growing season. Although various levels of PM resistance (PMR)
have been reported in cultigens of C. moschata (Adeniji and Coyne 1983;
Wessel-Beaver 1993) and C. pepo (Cohen et al. 1993; Lebeda and Krístková
1996b) and in C. lundelliana (Rhodes 1959, 1964), the source of resistance
used to date in all cultivars in North America is considered to be derived
from an initial interspecific transfer of resistance from C. okeechobeensis
into C. moschata, and hence, into C. pepo (Jahn et al. 2002). This resistance
is conferred by a single incompletely dominant gene, but the degree of
resistance may be modulated by modifying genes (Jahn et al. 2002). For
instance, among our breeding lines within C. pepo, homozygous resistance
is noticeably stronger in the acorn and yellow straightneck lines (subsp.
ovifera) than in the pumpkin lines (subsp. pepo). Our C. moschata PMR lines,
presumably homozygous for the same PMR allele, exhibit nearly complete
resistance to PM.
Crown, root and fruit rots, along with seedling damping off, caused
by Phytophthora capsici Leonian have been considered a sporadic problem
on squash and pumpkins (Zitter et al. 1996). Recently, however, several
major production areas of pumpkins and squash in North America have
been hit with epidemics of these diseases, resulting in a high incidence of
seedling death, leaf blight and fruit rot (Islam et al. 2005; Isakeit 2007). This
has stimulated breeding efforts to uncover genetic resistance. Padley and
Kabelka (2009) introgressed resistance to P. capsici crown rot into C. moschata
from two wild species, C. lundelliana and C. okeechobeenesis. Resistance
appears to be conditioned by three dominant genes.
Three additional pathogens often causing serious foliar blights and
fruit rots in pumpkin and squash are bacterial leaf spot rot [Xanthomonium
campestris (Pammel) Dowson pv. cucurbitae (Bryan) Dye], angular leaf spot
[Pseudomonos syringae van Hall pv. lachramans (Smith and Bryan) Young
et al.], and gummy stem blight [Didymella bryoniae (Auersw.) Rehm],
the anamorph of which, Phomo cucurbitacearum, produces dark pycnidia

characteristic of black rot. Gummy stem blight can overwinter on crop debris
and can be seed transmitted (Zitter et al. 1996). Specific cultivars resistant
to gummy stem blight (GSB) have not been released; however, Zhang et
al. (1995) screened 308 accessions for GSB resistance and reported seven
accessions of C. martinezii, two of C. moschata and three of C. Pepo, that
showed high resistance to the disease. Angular leaf spot is a widespread
bacterial disease of squash and pumpkin, often causing severe foliar
destruction but less fruit rot than bacterial leaf spot. In favorable weather
following a disease outbreak, new plant growth will often appear free of
the disease. Considerable variability in susceptibility of Cucurbita cultigens
to this disease on both foliage and fruit has been observed (J.B. Loy, pers.
observation). However, the reliance of natural field infestation for evaluation
of this disease has prevented a reliable ranking of cultivars for degree of
susceptibility (J.B. Loy, unpubl. results). Bacterial leaf spot often occurs
during weather episodes that favor angular leaf spot. This disease is very
subtle to detect because leaf symptoms, appearing as small necrotic spots
with a yellow halo, are often fairly obscure, and if lesions coalesce, may
look similar to angular leaf spot (Zitter et al. 1996). Initial symptoms on
fruit appear as tiny, corky tan dots, but many lesions expand into slightly
sunken tan spots, surrounded by a dark ring. Many of the lesions become
encapsulated with callose tissue at the fruit surface, but some eventually
penetrate into the flesh and seed cavity, causing severe fruit rot. Fruits of
zucchini and ornamental pumpkin (C. pepo subsp. pepo) are more susceptible
to both angular leaf spot and bacterial leaf spot than those of subspecies
ovifera. Oil seed pumpkins appear to be particularly susceptible. Tolerance
of fruit to this disease has been observed among ornamental pumpkins (J.B
Loy, unpubl. observations), and it appears that plants showing medium
resistance or tolerance to angular leaf spot are more tolerant to bacterial
leaf spot.
4.10.2 Insect Resistance

Research on breeding for insect resistance has been meager, perhaps because
reasonable insect control has been afforded in most cases by biological and
chemical control measures. Approaches for evaluating resistance to insects
in squash and pumpkin has been reviewed by Whitaker and Robinson
(1986). The major insect pests of squash and pumpkin in North America
are squash bug (Anasta tristis DeGeer), cucumber beetles (Diabrotica balteata
LeConte and Diabrotica undecimpunctata Mannerheim), squash vine borer
(Melittia cucurbitae Harris), aphid (chiefly green peach aphid, Myzus persicae
Sulzer), and in subtropical and tropical areas, the sweet potato white fly
(Bemisia tabaci Grennadius). White fly feeding induces a silver leaf disorder
in squash that reduces yields (Paris et al. 1987), and has been found to

vector squash leaf curl, a Begomovirus in the Geminivirus family. Cucumber

beetles can vector bacterial wilt and aphids can vector various viruses. It
has been demonstrated that cucurbitacins are an attractant for foraging of
the spotted cucumber beetle on squash (Chambliss and Jones 1966; Sharma
and Hall 1971), and cultivars differ in susceptibility (Wisemann et al. 1961).
However, no cultivars have been reported that give high resistance to
cucumber beetles. Resistance to the silvering disorder has been reported
in C. pepo (Cardoza et al. 1998) and C. moschata (Wessel-Beaver 1998).
Inheritance studies of resistance in C. pepo have been hampered by variation
in expressivity of resistance (Carle et al. 1998), but suggest that resistance to
feeding is complex. Resistance to squash leaf curl in C. pepo, conferred by a
single dominant gene, has been reported (Montes-García et al. 1998).
4.11 Breeding Applications of Biotechnology

The emergence of biotechnology as an asset to plant improvement in
Cucurbita has been slow as compared to crop plants having substantially
greater economic importance. Besides the relative paucity of molecular and
cellular geneticists working with squash, impediments to biotechnological
research on squash and pumpkins include a large space requirement
for growth, a long growing season, the necessity of performing hand-
pollinations, genomic differences among the domesticated species, and
difficulties in transferring traits through interspecific hybridization.
Nonetheless, molecular techniques have contributed greatly to our
understanding of the taxonomic relationships among species and the
genetic diversity among cultigens and among cultivar groups within species
(Decker-Walters et al. 2002; Sanjur et al. 2002; Ferriol et al. 2003a, b, 2004a, b;
Paris et al. 2003; Gong et al. 2008a, b). Since 2000, there has been considerable
progress in using molecular markers to map the cucurbit genome, and this
should greatly aid future breeding efforts using marker-assisted selection
and genetic engineering.
In the first attempt at mapping the genome of a Cucurbita species using
an F2 population derived from a cross of C. maxima x C. ecuadorensis, 11
isozyme loci were mapped to five linkage groups (Weeden and Robinson
1986). Using random amplified polymorphic DNA (RAPD) analysis of
backcross populations derived from a C. moschata x C. pepo cross, Lee (1995)
constructed the first molecular map with 28 markers in five linkage groups,
but no morphological markers. Brown and Meyers (2002) constructed the
first extensive Cucurbita linkage map using BC1 populations resulting from
an initial cross between a yellow straightneck inbred and a Nigerian local
landrace. Their map included 148 RAPD markers in 28 linkage groups,
and the map covered 1,954 cM, estimated to be about 75% of the genome.
More recently, Zraidi et al. (2007) constructed the first consensus map of

C. pepo using a combination of genetic markers including RAPD, amplified

fragment length polymorphism (AFLP), simple sequence repeat (SSR) and
morphological traits in two different populations. A total of 332 and 323
markers, respectively, mapped in the two populations, were spread over
21 linkage groups and estimated to cover 2,200 cM of the genome. The
C. pepo map was updated using SSR markers, expanding the map to 86%
genome coverage, representing 20 linkage groups and a map density of 2.9
cM (Gong et al. 2008b). Gong et al. (2008a) have also used 205 SSR markers
to construct a linkage map of C. moschata with a marker density of 7.6 cM.
Furthermore, they reported that 72 of the 76 common SSR markers between
C. pepo and C. moschata were located in homologous linkage groups, with
largely conserved orders and distances between markers.
The recent rapid progress in genome mapping in Cucurbita should
permit expanded use of marker-assisted selection by breeders. This should
be especially valuable for traits in which field selection is difficult, such as
with virus resistance, and for multigenic traits such as the bush growth habit
in C. maxima and % DM of mesocarp tissue in winter squash. Markers could
also reduce the size of field populations needed for combining various traits,
particularly in wide crosses. The Cucurbita map constructed by Brown and
Meyers (2002) included five morphological traits, but only two identified
by specific genes, and none were within 15 cM of a RAPD marker. Gong et
al. (2008b) reported four SSR markers closely linked to the h locus (hull-less
seed) and one SSR marker linked to the Bu locus (bush growth habit) in
C. pepo. In C. moschata, the Gr locus for green versus tan fruit coloration was
12.5 cM from an SSR marker (Gong et al. 2008a).
The successful application of genetic engineering to Cucurbita species
requires efficient and reliable methods for regeneration of plants by somatic
embryogenesis or organogenesis. Regeneration of somatic embryos from
tissue fragments in squash (C. pepo) cotyledons and hypocotyls was
reported as far back as 1972 (Jalaska). Continued expansion of regeneration
methods occurred in the 1980s and 1990s (Jalaska et al. 1986; Chee 1991,
1992; Gonsalves et al. 1995), laying the groundwork for genetic engineering
in squash, first accomplished in C. pepo (Fuchs et al. 1998). There continues
to be a steady stream of regeneration work, improving the efficiency of
regeneration (Kintzios et al. 2002) and developing somatic cell regeneration
methods for C. maxima (Lee et al. 2003) and C. moschata (Valdez-Melara et
al. 2009).
Transgenic squash cultivars incorporating single or multiple virus
coat protein (CP) genes were among the first genetically engineered crop
plants to be grown on a commercial scale. The first of these, the yellow
crookneck hybrid Freedom II, commercialized in 1995 (Fuchs et al. 1998),
was resistant to ZYMV and WMV-2. Lines expressing CP genes of CMV,
ZYMV and WMV have now been developed, and are resistant to these

three viruses (Fuchs and Gonsalves 2007). Once incorporated into breeding
lines, the CP gene constructs can be transferred to other breeding lines
and recombined with other CP genes to achieve multiple virus resistance.
Yield trials comparing cultivars with transgenic virus resistance to those
without resistance have generally shown favorable results under conditions
of natural virus pressure or with inoculated plants (Webb and Tyson 1997;
Fuchs et al. 1998; Schultheis and Walters 1998; Rowell et al. 1999). In 2005,
transgenic squash accounted for 12% of the summer squash acreage in the
US (Fuchs and Gonsalves 2007).
The development of in vitro techniques for producing haploid plantlets
via androgenesis and gynogenesis has been a significant development in
commercial breeding for rapid generation of homozygous lines. Among
cucurbits, this technology has been refined and utilized for cucumber and
melon breeding, but is still in its infancy in squash and pumpkin. Most
in vitro studies have focused on obtaining haploid plants from C. pepo,
using both ovule (Metwally et al. 1998a; Shalaby 2007) and anther culture
(Metwally et al. 1998b). More recently, pollen irradiation has been used to
induce haploid plants in C. pepo (Kurtar et al. 2002), C. maxima (Kurtar and
Balkaya 2010) and C. moschata (Kurtar et al. 2009). Further improvement is
needed in cultural techniques to improve the frequency of obtaining haploid
plants for wide scale adoption of androgenic and gynogenic techniques for
breeding purposes.
Acknowledgement
I wish to express my sincere thanks to Jacob B.Uretsky for his critical review
of this manuscript.
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5
Genetic Diversity Studies in
Cucurbits Using Molecular Tools
C. Esteras,1,* F. Nuez1,a and B. Picó1,b
ABSTRACT
Cucurbitaceae are among the largest and most diverse plant families.
These include several economically important cucurbits such as
watermelon (Citrullus lanatus), cucumber (Cucumis sativus), melon
(Cucumis melo) and squashes (Cucurbita spp.), but also minor crops
and wild species distributed worldwide. It is assumed to be of Asian
origin, but cucurbits have diversified around the world. In this chapter,
we describe studies that aim to know the extant genetic diversity
among cucurbits. Main tribes, comprising the most important genera
are first described, Benincaseae (Cucumis, Citrullus, Benincasa and
Lagenaria) and Cucurbiteae (Cucurbita). Most of the studies deal with
variability within the main crops of each genus, melon and cucumber
(Cucumis), watermelon (Citrullus), and pumpkins, C. pepo, C. moschata
and C. maxima (Cucurbita), but also studies dealing with minor crops
are included, African horned cucumber (Cucumis metuliferus), pickling
cucumber (Cucumis anguria), white gourd (Benincasa hispida), bottle
gourd (Lagenaria siceraria) and malabar melon (Cucurbita ficifolia). In
addition to the main tribes, we also review Joliffieae, Luffeae and
Sicyeae, including minor crops such as bitter melon (Momordica
charantia), chayote (Sechium edule) and sponge gourd (Luffa cylindrica),
respectively.
For each genus, a general view is provided on the taxonomy, distribution
and intra-genus relationships. For each species, we summarize the
information on taxonomy, morphological studies and molecular assays
using different marker systems. Reported works describe the variability
1
Instituto de Conservación y Mejora de la Agrodiversidada Valenciana (COMAV), Universitat
Politécnica de Valéncia, Camino de Vera 14, Valencia 46022, Spain.
a
e-mail: fnuez@btc.upv.es
b
e-mail: mpicosi@btc.upv.es
*Corresponding author: criesgo@upvnet.upv.es

Genetic Diversity Studies in Cucurbits Using Molecular Tools 141
of wild types, landraces or commercial cultivars in the centers of origin,

and primary and secondary diversification centers. The evolution of
the molecular analyses is shown. From those based on the protein
polymorphisms, through the use of chloroplastic and mitochondrial
DNA, to different types of DNA-based markers, dominant (ISSR, RAPD
and AFLP) and codominant and sequence-specific (SRAP, SCAR and
SSR), including the most recently used SNPs, coming from genomic
or ESTs sequencing projects. Knowing the amount and distribution
of genetic variability will be essential to conserve and classify all this
variation and to exploit it for cucurbits breeding.
Keywords: cucurbits, genetic diversity, molecular markers, phylogenetic
relationship, germplasm, geographical distribution, diversity centers
5.1 Introduction
The family Cucurbitaceae comprises some of the most economically
important crops. Cucurbits have been used by humans as food, containers,
musical instruments and as a source of medicine, for more than 12,000
years (Whitaker and Davis 1962; Brothwell and Brothwell 1969; Lira-Saade
1995).
Jeffrey (1990) and Robinson and Decker-Walters (1997) described 118
genera and about 825 species within the family. However, according to
the most recent classifications (Jeffrey 2005; Jeffrey and De Wilde 2006)
Cucurbitaceae comprises 130 genera, including about 800 species. The
family is divided into two subfamilies: Nhandiroboideae, also called
Zanonioideae (Jeffrey 1990), with 19 genera and about 60 species without
economic value; and Cucurbitoideae, with 111 genera and 740 species.
The subfamily Cucurbitoideae comprises some of the most important
tribes and genera within cucurbits: Benincaseae (Benincasa, Citrullus,
Coccinia, Lagenaria, Cucumeropsis, Cucumis), Luffeae (Luffa), Cucurbiteae
(Cucurbita), Sicyeae (Cyclanthera, Sechium), Joliffieae (Momordica, Telfairia),
Schizopeponeae (Schizopepon), Trichosantheae (Hodgsonia, Trichosanthes),
Bryonieae (Ecballium), Herpetospermae and Coniandreae (Jeffrey 1990;
Rubatzky and Yamaguchi 1997; Jeffrey 2005). There are some recent
phylogenetic studies of the family Cucurbitaceae using chloroplast DNA
sequences (cpDNA) (Kocyan et al. 2007). Molecular data weakly support the
traditional subfamilies Cucurbitoideae and Nhandiroboideae, and recover
most of the 11 tribes, but almost none of the subtribes. Within the subfamily
Cucurbitoideae, a clade comprising all the tribes but Joliffieae (polyphyletic)
was reported as a “fused stamen” clade due to their fused filaments or
connectives. In this clade several tribes clustered together in four groups:
Herpetospermae-Schizopeponeae group, Bryonieae group, Coniandreae-
Benincaseae-Cucurbiteae group and Luffeae-Sicyeae-Trichosantheae group

(Kocyan et al. 2007). The most important diagnostic characters for the
genera and tribes of Cucurbitaceae come from androecium and gynoecium
morphology (number of free styles, fusion of filaments and/or anthers),
tendril type, pollen structure and seed coat. Subfamily Nhandiroboideae
is characterized by free styles, small pollen grains with a striate exine and
branched tendrils with a sensitive basal part. Subfamily Cucurbitoideae
is characterized by having styles united in a single column, tectate to
semitectate pollen with a reticulate or echinate exine and simple, bifid or
multifid tendrils with non-spiralling basal part. These traits correlate quite
well with the phylogeny obtained with molecular data.
The genera Cucumis, Cucurbita and Citrullus include species (cucumber,
melon, watermelon and squash) that are among the most widely cultivated
crops worldwide. Apart from them, there are other notable cucurbits of local
or regional economic importance, such as Lagenaria, Momordica, Benincasa,
Luffa and Sechium (Lira Saade and Montes-Hernández 1994; Bates et al.
1995; Lira-Saade 1995).
Cucurbits have diversified around the world. A large amount of genetic
resources adapted to many different environmental and growing conditions
can be found in different areas. Knowing the extant genetic diversity among
cucurbits is important in order to optimize collection and conservation
programs and to facilitate the ongoing efforts by plant breeders worldwide
to improve melon, cucumber, watermelon and squash with traits from wild
relatives.
5.2 Tribe Benincaseae

5.2.1 Genus Cucumis
This genus comprises 33 species, including the most recent to be discovered,
C. canoxyi Thulin & A.N. Al-Gifri (Thulin and Al-Gifri 1994). Apart from
C. melo L. and C. sativus L., two species are also consumed: C. anguria L.
(West Indian gherkin) and C. metuliferus E. Meyer ex Naudin (African
horned cucumber or jelly melon). Others, such us C. dipsaceus Ehrenberg ex
Spach and C. myriocarpus Naudin, are used as ornamentals. The genus has
been traditionally conceived of as being an African genus divided into two
subgenera: cucumis and melo (Jeffrey 1980; Kirkbride 1993). The subgenus
cucumis (two species, C. sativus and C. hystrix Chakr.) is confined to Asia
and has chromosome numbers n = 12 and n = 7 (Chen et al. 1997a). Figure
5-1 shows the diversity centers of the main species within the genus. The
subgenus melo (30 species, including C. melo), with most of its species in
Africa, has a chromosome number n = 12 (Kirkbride 1993), although there

Figure 5-1 Distribution map of Cucumis spp. Principal diversity centers based on Kirkbride
(1993).
C. sativus L. and C. sativus L. var. hardwickii (Royle) Alef.
C. hystrix Chakravarty
C. myriocarpus Naudin ▲
C. africanus L. ▲
C. anguria L.
C. dipsaceus Ehrenberg ex Spach
C. zeyheri Sonder
C. ficifolius A. Richard
C. metuliferus E. Meyer ex Naudin
♦
C. melo L. ssp. Melo and C. melo L. ssp. agrestis (Naudin) Pangalo ♦
C. sagittatus Peyritsch
exists several tetraploid and hexaploid forms. Ashurmetov (1995) proposed

separating Cucumis into two genera, Cucumis and Melo, but it was not
broadly accepted.
Some molecular phylogenetic studies of Cucumis have been conducted
using seed storage proteins (Singh and Matta 2008), chloroplast restriction
sites (Perl-Treves and Galun 1985), nuclear isozymes (Perl-Treves et al. 1985),
nuclear ribosomal DNA from the internal transcribed spacer (ITS) region
along with nuclear simple sequence repeat (SSR) markers (Garcia-Mas et al.

2004) and chloroplast SSRs and sequence analysis (Chung et al. 2003, 2006).
One of the most recent studies, conducted by Renner et al. (2007), tested
the genetic relationships among Cucumis, combining cpDNA sequences
and nuclear ITS. This study extended the analysis performed in previous
studies considerably, using representatives of other potentially related
genera (Cucumella, Dicaelospermum, Mukia, Muellerargia, Myrmecosicyos,
and Oreosyce) included in the most recent morphology-based classification
of Cucurbitaceae (Jeffrey 2005). The Cucumis species relationships
found by these authors differ from those found in earlier studies. The
deepest divergence lies between the common ancestor of C. hirsutus and
C. humifructus and the stem lineage of the remainder of the genus. The
authors also concluded that the closest relative of Cucumis is Muellerargia,
and showed that the genera Cucumella, Dicaelospermum, Mukia, Myrmecosicyos
and Oreosyce are grouped among species of Cucumis. Similar results were
obtained by Ghebretinsae et al. (2007) and Schaefer (2007), who proposed
the expansion of the genus Cucumis to include these related genera. Renner
et al. (2007) extend the range of the genus throughout the Malesian region
and into Australia. According to this study, Cucumis comprises an old
Australian/Asian component. Cucumis sativus would have evolved within
this Australian/Asian clade. Cucumis melo is sister to this Australian/Asian
clade, rather than being close to African species as previously thought. In
fact, a more recent research, carried ourt by Sebastian et al. (2010), reports a
species from Australia (Cucumis picrocarpus) as the melon’s closest relative.
According to these studies (Renner et al. 2007; Sebastian et al. 2010), C. melo
might have originated in Asia and then arrived in Africa.
5.2.1.1 Cucumis Melo

5.2.1.1.1 Origin and Taxonomy
C. melo is one of the most important fruit vegetables cultivated in tropical
and temperate regions, as it is highly valued for the quality of its fruits.
Traditionally, C. melo is considered to be divided into two subspecies
according to ovary hairiness, subspecies melo (long hairs, distributed from
India to Europe and in America) and subspecies agrestis (short hairs, in
Eastern Asia from India to Japan and in Africa) (Jeffrey 1980). Today, the
species comprises wild, feral and cultivated varieties, including sweet
melons used for dessert and nonsweet ones consumed raw, pickled or
cooked.
The origin of C. melo remains unclear (Robinson and Decker Walters
1997; Yashiro et al. 2005; Lebeda et al. 2006). African origins have been
traditionally assumed, but, as previously mentioned, recent studies suggest
that C. melo could have originated somewhere in Asia and then reached

Africa from there (Renner et al. 2007). In fact, wild melons are very frequent
in East and West Africa, but also from Central Asia to India (Whitaker and
Bemis 1976; Staub et al. 1987; McCreight et al. 1993; McCreight and Staub
1993; Rubatzky and Yamaguchi 1997). It is also possible that in Africa and
Asia different domestication events took place (Bates and Robinson 1995),
the most extensive of which may have occurred in Asia (more edible
types). Independent domestication has also been proposed for the two
subspecies.
Melon has suffered an intense process of diversification, and today
shows great variation in morphological and physiological characters.
Primary and secondary centers of genetic diversity are located from eastern
Asia to the Mediterranean Sea (Afghanistan, Iran, Iraq, Saudi Arabia, Turkey,
China, Russia and India) (Robinson and Decker-Walters 1997; Akashi et al.
2002). Seeds of wild C. melo ssp. agrestis and possibly selected forms more
closely resembling currently cultivated C. melo could have been introduced
from Africa into the Middle East (Turkey, Iraq and Iran) and Asia (India,
China, and Japan) through land and sea commerce routes (Kajale 1979;
Walters 1989; Fujishita 1992). It seems that melon was cultivated in Iran
and China 3000 BC, in India 2000 BC, in Egypt 1500 BC and western Japan
as early as 100 BC (Walters 1989; Fujishita 1992; Decker-Walters 1999;
Stepansky et al. 1999; Karchi 2000; Luan et al. 2008). Melon spread through
Asia and subsequently to Europe (Roman-Greek periods) (Szabó et al.
2005). Three independent introductions to Europe (from the east: Russia,
Bulgaria, Hungary; from the south-east: Greece, Albania, Romania; from
the south: Italy) were hypothesized by Pitrat et al. (1999). Later, Columbus
introduced this crop to America, where it became popular and dispersed
quickly, producing a wide range of new cultivars.
C. melo is considered to be the most variable species in the genus
Cucumis (Kirkbride 1993; Bates and Robinson 1995). Great diversity in fruit
shape, size, color and taste exists among melons. This variability was first
classified as different species (C. melo, C. flexuosus, C. dudaim, C. callosus,
C. chate, C. conomon and C. momordica) that now are considered varieties
or morphotypes within the species. Naudin (1859) defined 9 “tribes” of
cultivated melons and one wild form. Many scientists have since added or
merged types (reviewed in Pitrat et al. 2000). Munger and Robinson (1991)
proposed a simplified version of Naudin´s taxonomy, which is still used
in many studies, with seven cultivar groups: agrestis Naud. (wild melon),
cantalupensis Naud. (cantaloupe or muskmelon; Middle East), inodorus
Naud (winter melons, honeydew, Cassaba; Middle East, southern Europe),
conomon Mak. (pickling melon, Chinese white cucumber; Asia), chito-dudaim
Naud (mango melon-queen’s pocket melon; Asia), flexuosus Naud. (snake
melon; Middle East), and momordica (Phoot or snap melon; Asia) (Robinson
and Decker-Walters 1997). More recently, Pitrat et al. (2000) described 16

botanical varieties: var. conomon, var. makuwa, var. chinensis, var. momordica,
var. acidulus (included in ssp. agrestis) and var. cantalupensis, var. reticulatus,
var. adana, var. chandalak, var. ameri, var. inodorus, var. flexuosus, var. chate,
var. tibish, var. dudaim, var. chito (included in ssp. melo). In later revisions,
Pitrat (2008) merged some groups. The description of the main botanical
groups is included in Table 5-1. Many of these botanical groups include
different cultivar-groups that are highly popular in different parts of the
world. Cantalupensis and inodorus are of commercial interest in the United
States, as well as in many European, Mediterranean and Asian countries
(McCreight et al. 1993), and include cultivars belonging to different market
classes. For example, while the popular market classes Charentais, Shipper,
Ogen and Galia are in the cantalupensis group, the inodorus group houses
an array of Cassaba market class types (e.g., Rochet, Piel de Sapo, Tendral,
Crensahw, Honeydew, Kirkagac and Yellow Canari).
5.2.1.1.2 Molecular Markers Used in Genetic Diversity Studies

Different types of molecular markers (see “Evolution of molecular markers”
in Chapter 6) have been used to assess genetic diversity in melon and to
study genetic relationships among commercial types and different botanical
groups. Isozymes were first utilized (Esquinas-Alcázar 1981; Perl-Treves et
al. 1985; Staub et al. 1997; Akashi et al. 2002), but soon DNA markers were
applied: restriction fragment lengh polymorphisms (RFLPs) (Neuhausen
1992; Silberstein et al. 1999), random amplified polymorphic DNA (RAPDs)
(García et al. 1998; Stepansky et al. 1999; Silberstein et al. 1999; Staub et al.
2000; Zhuang et al. 2004), amplified fragment lengh polymorphisms (AFLPs)
(Garcia-Mas et al. 2000), microsatellites or simple sequence repeats (SSRs)
(Katzir et al. 1996; Staub et al. 2000; Danin-Poleg et al. 2001; Mliki et al. 2001;
Monforte et al. 2003; Zhuang et al. 2004), internal simple sequence repeats
(ISSRs) (Stepansky et al. 1999) and the sequence and structural analysis of
the internal transcribed spacer (ITS) (Fantaccione et al. 2008). In general,
similar clustering is obtained with the different types of markers. An array
of selected RAPD markers and reference accessions has been frequently
employed to assess the genetic diversity of melon landraces and cultivars
from Europe, the USA and different centers of diversity (Staub et al. 2000).
Also, due to the reproducibility and the discriminating capacity of AFLPs,
and to the reliable, co-dominant and multi-allelic nature of SSRs, these
marker systems have been broadly used to define genetic relationships
among botanical groups and commercial market classes. Some recent
studies have focused on the development of SSR collections using genomic
microsatellite enriched libraries (Chiba et al. 2003; Ritschel et al. 2004), and
more recently using available collections of melon expressed sequence tags
(ESTs) (Kong et al. 2007; Fernández-Silva et al. 2008).

Table 5-1 Description of the main botanical groups in to which the species C. melo is divided, according to Pitrat (2008).
Variety Distribution Sex type Fruit (shape/ Flesh color Rind Sweetness Aroma Climateric
color)
CONOMON Eastern Asia Andromonoecious Elongated White Smooth, thin No No No

MAKUWA Eastern Asia Andromonoecious Flat-round-oval White Smooth, Yes Little Yes
White-yellow- thin, with
light green or without
sutures
CHINENSIS China, Korea Andromonoecious Pear-shaped Green- Smooth Medium Little Both types
(some Green with spots orange or no
hermaphrodite) aroma
MOMORDICA India Monoecious Flat-elongated White Smooth, Low Little Yes
slightly
ribbed, thin
ACIDULUS India Monoecious Oval-elliptic White Smooth No No No

Green-orange
with or without
spots
TIBISH Sudan Andromonoecious Small-oval White No No No

Dark green with
light green-
yellow stripes
Table 5-1 contd....

Table 5-1 contd....
148
Variety Distribution Sex type Fruit (shape/ Flesh color Rind Sweetness Aroma Climateric

color)
CHATE Mediterranean Monoecious (some Round-oval White-light Ribbed No No Yes
area and andromonoecious Green orange
Western Asia genotypes)
FLEXUOSUS Northern Monoecious Long or very long White-light Ribbed or No No Yes

(snakemelon) Africa to Green orange wrinkled
Turkey to Iraq
to India
CANTALUPENSIS Europe, Andromonoecious Flat-oval Orange Ribbed, Yes Yes Yes

(muskmelon) Western Asia, usually sometimes smooth
North and green sometimes
South America with warts
RETICULATUS Europe, Asia, Andromonoecious Round-oval Orange Netted Yes Yes Yes
North and (sometimes
South America ribbed)
AMERI Western and Andromonoecious Elongated-oval White-light Slightly Yes Little Yes
Central Asia Yellow-light orange netted
green

INODORUS Central Asia, Andromonoecious Round-elliptic White Often Yes No No
Mediterranean White-yellow- wrinkled,
area, North dark green ribbed or
and South (sometimes not
America spotted)
DUDAIM Central Asia Andromonoecious Small-round White Velvety No Strong Yes
Yellow with ochre

stripes
Color image of this table appears in the color plate section at the end of the book.

Melon has become a model within Cucurbits for genetic and genomic
studies (see Chapter 9). Different initiatives are generating sequence
collections (González et al. 2010, González-Ibeas et al. 2007, http://www.
melogen.upv.es; www.icugi.org) which are facilitating single nucleotide
polymorphism (SNP) detection. Morales et al. (2004) first detected SNPs
in melon using a small set of available ESTs. These authors indicated an
average frequency of 1 SNPs per 441 bp between two inodorus genotypes.
In a more complete study, screening 30,000 ESTs sequences from four
genotypes, 356 high-quality SNPs were found (González-Ibeas et al. 2007).
Some of them proved to study diversity in a set of melon accessions, giving
genetic relationships similar to that found with SSRs (Deleu et al. 2009).
In addition to genetic diversity studies, these markers have been used to
construct several melon maps (Baudracco-Arnas and Pitrat 1996; Wang et
al. 1997; Liou et al. 1998; Brotman et al. 2000; Oliver et al. 2001; Danin-Poleg
et al. 2002; Périn et al. 2002; Gonzalo et al. 2005; Deleu et al. 2009) that are
being merged within the International Cucurbit Genomics Initiative (ICuGI)
(http://www.icugi.org) (see “Molecular maps” in Chapter 6 and Chapter 9
on melon genetic maps) . The availability of increasing amounts of melon
sequences is allowing the application of new methods to study variability
in candidate genes of breeding interest (Nieto et al. 2007). In a recent study
reported by Esteras et al. (2009b) EcoTILLING techniques are being applied
to study polymorphisms in genes involved in quality and ripening processes
using a highly variable core collection of melons.
Molecular markers have been used to characterize elite melon
germplasm (commercial cultivars, hybrids and breeding lines, mainly
from the USA and European markets) mostly belonging to cantalupensis
(Charentais, Shipper, European, western and eastern USA types, Galia and
Ogen) and inodorus (Honey dew type and Cassaba Rochet, Piel de Sapo
and Yellow Canari types) types. Despite the fact that molecular analysis
discriminates between these market cultivars, the groupings were somewhat
ambiguous, most likely due to intogressions during plant breeding. They
also found a limited genetic diversity in some groups (Garcia et al. 1998;
Staub et al. 2000).
Much higher genetic diversity is reported when exotic germplasm (wild,
feral, landraces) from these and other botanic groups is also included. Most
of the molecular studies support quite well the division into two major
groups (ssp. melo and ssp. agrestis) (Staub et al. 1997; Silberstein et al. 1999;
Danin-Poleg et al. 2001; Monforte et al. 2003; Nakata et al. 2005; Deleu
et al. 2009; Esteras et al. 2009a). In general, higher molecular variability
(number of alleles and polymorphic loci) is reported in Central Africa and
India than in the extremes of melon distribution (the Mediterranean area
and China Sea). One of the most complete studies covering representatives
of most of the botanical groups was conducted by Stepansky et al. (1999).

They combined both phenotypic and molecular data (RAPDs and ISSRs),
analyzing accessions from 23 countries, including wild, feral and cultivated
forms representing the primary and secondary centers of diversity (Africa,
southern and western Asia and the Far East). Phenotyping was based on
a set of traits usually employed to define different botanical groups (seed
size, stem thickness, pubescence, sex type, ovary shape, ovary pubescence,
fruit shape and size, skin color, texture and design, splitting, abscission,
external aroma, flesh color, taste, sucrose, glucose and fructose and pH).
According to these traits, the subdivision into most of the varietal groups
persisted, indicating that the traditional classification is mainly based on
consistent and highly informative characters. Some traits with taxonomic
value show high variability. For example, wild types defined by small fruit
size, thought to be agrestis, can show ovaries with long or short hairs. Also
within the flexuosus types there exist both kinds of ovaries. The molecular
results did not substantially contradict the phenotype-based dendrogram.
The sweet-fruited cantalupensis and inodorus clustered together, in spite of
their ripening differences, and the nonsweet varieties agrestis, conomon and
momordica grouped together. There exist discrepancies in the classification
of some botanical groups. For example, dudaim and chito cultivars often are
grouped with agrestis types, although they have been reported to belong to
ssp. melo. The flexuosus types clustered closer to the non-sweet genotypes in
the phenotypic tree, and dispersed with the “dessert” ones in the molecular
tree. The non existence of reproductive barriers within the species makes
the crosses among the different cultivar groups possible, giving rise to a
continuous distribution-pattern of variation. Esteras et al. (2009a) found
similar results in a recent analysis with AFLPs of a melon core collection
of 212 accessions, representing all the genetic diversity of the species, with
dudaim and chito types being intermediate between ssp. melo and agrestis and
momordica and flexuosus types which, being highly variable, interspersed
among both groups.
5.2.1.1.3 Molecular Diversity of Melon Landraces in the Centers of

Origin and Diversity
Most of the molecular studies are focused on studying the genetic diversity
of landraces and cultivars in all the significant primary and secondary
centers of diversity.
Africa is a putative center of origin for melon, and so the study of
African germplasm is essential to know the extent of genetic diversity within
the species. Few studies have focused on African germplasm (Mliki et al.
2001; Akashi et al. 2006). Mliki et al. (2001), using RAPDs, clustered African
accessions into two groups according to their origin: northern (mostly from
Egypt, Tunisia, Libya, Morocco, Algeria, Ethiopia, Niger and Sierra Leone)

vs. central-southern Africa (Zimbabwe, Zambia, Mali, Senegal and South

Africa). Some accessions from Kenya, Senegal and Ghana grouped apart.
The relationship of African germplasm to accessions from secondary centers
of diversity suggests that major Indian introduction events originated from
southern Africa, whereas the Middle East was the beneficiary of North
African introductions (Staub et al. 2004; Sensoy et al. 2007). However,
other studies support that large- and small-seeded types found in India
(Akashi et al. 2002, 2006; Yashiro et al. 2005) were derived from northern
and southern Africa, respectively (Tanaka et al. 2007). Molecular results
support the polymorphic nature of the African germplasm.
The main center of diversity for melon and perhaps the origin of some of
the principal commercial types, such as cantalupensis or inodorus, is located
in the Near East and Central Asia (Jeffrey 1980). Groups like conomon,
makuwa, momordica and flexuosus also have an Asian origin. Many of the
studies aimed at elucidating the genetic structure of Asian melons focus
on these botanical groups, as they include most of the accessions used in
melon breeding as sources of resistance genes to different pests and diseases
(Akashi et al. 2002; Nakata et al. 2005; Yashiro et al. 2005; Tanaka et al. 2007).
Yashiro et al. (2005) selected a representative collection of Asian accessions
(from India, Myanmar, China, Korea and Japan). Using AFLPs they found
that East Asian melons (makuwa and conomon) grouped apart and showed
less variability than South Asian melons (especially those from India),
which supported previous results (Akashi et al. 2002). Tanaka et al. (2007)
confirmed these results. Using RAPDs these authors found a high genetic
diversity in India and a low variability in makuwa and conomon types.
The large genetic variation in India could be explained by the diverse
climatic conditions. India is divided into 21 agro-ecological regions and 131
agroclimatic subregions. This is the area of origin of var. momordica. This
botanic group presents most of the genes of resistance to diseases and pests,
as well as to abiotic stress used for breeding cultivated melons. Dhillon et
al. (2007, 2009) conducted the most complete morphological and molecular
characterization of snapmelon landraces using accessions collected in many
of the Indian agroclimatic subregions. A high level of variability was found
for certain morphological traits (fruit cracking, abscission, flesh texture,
acidity, sugar content, resistance to fungus and virus). The momordica
accessions analyzed showed high levels of genetic diversity and were not
closely related to melon accessions from other parts of the world, then
supporting an independent origin of this botanic group (Staub et al. 2004;
Dhillon et al. 2007, 2009). Moreover, regional differentiation among Indian
accessions was reported using a set of 16 SSRs (Dhillon et al. 2009). Most of
the accessions grouped according to agroclimatic subregions. Eastern Indian
snapmelon has unique traits, so it is important that more germplasm from
this region be sampled and preserved. These studies also included wild,

small-fruited types from India. These agrestis accessions showed a narrow

genetic diversity and cluster together with the momordica group. These data
confirm that India is a primary center of melon diversity.
There exists a clear divergence between momordica and East Asian
melons (conomon-makuwa). Oriental pickling melons are considered to be the
most ancient forms of domesticated melons in China. Different hypotheses
exist about where makuwa and conomon types were domesticated. Kitamura
(1950) proposed India from which point makuwa later established itself in
northern China and conomon in South China (Jeffrey 1980). Akashi et al.
(2002) suggested that these types derive from a small-seed type, which
adapted in East India. Recent molecular assessments of genetic diversity
of Chinese melons from diverse geographical origins and belonging to
different market classes [thick-skinned melon (netted and non-netted),
non-netted thin-skinned and vegetable types] also support this proposal.
Luan et al. (2008), using a previously defined standard RAPD marker array
(Staub et al. 2000), group Chinese cultivars in two groups. Results suggest
that certain melon types were introduced to western China via the Silk Road
(from the Middle East, Iran or Iraq), whereas oriental Asian melon types
(conomon and makuwa types) may have been introduced into China from
India. This study also reports stark molecular differences between Indian
and Chinese accessions and lends support to the occurrence of bottlenecks
and/or geographic or political isolation. Chinese accessions are therefore a
rich source of genetic diversity for plant improvement. Other studies relate
conomon types with African accessions (Nakata et al. 2005). These authors,
using RAPDs and SSRs, studied the variability of Japanese conomon. Cluster
analysis separated conomon accessions into two groups, one more closely
related to South African melons, and the other close to Japanese cultivars
belonging to cantalupensis and inodorus groups. The conomon-African clade
was separate from the rest of the examined genotypes, which might imply
an Asian origin of the African conomon-like accessions or an independent
domestication from similar ancestors. Some of the analyzed Japanese
market classes were relatively rich in genetic variation, more than the USA
or European accessions. Other studies, like that of Mo-suk et al. (1999) on
Korean diversity, also show the importance of the genetic diversity on the
Asian continent.
A recent work using RAPDs and morphological traits studies the genetic
diversity of melons in Myanmar (Yi et al. 2009). These authors indicated that
the genetic diversity of Indian melon is conserved in Myanmar. They found
that conomon and agrestis types from India group together and are apart
from sweet melons. They also found a high diversity for the conomon group,
equivalent to that of Indian melon populations, and higher than that of
conomon from East Asia. Genetic introgression among melon groups through

spontaneous hybridization is also indicated and considered important for

maintaining or increasing the genetic diversity in Myanmar.
Asia is not only rich in nonsweet melons but also in melons belonging
to the two most important commercial types. The Middle East (Iran, Iraq
and Turkey) exhibits one of the highest variabilities in cantalupensis, inodorus
and flexuosus types. This area has been reported as the origin of inodorus
types and is very important for the diffusion of these types to Europe. It is
thought that cantalupensis melons spread to Europe from the eastern part
of Turkey (Zhukovsky 1951; Günay 1993). Sensoy et al. (2007) used RAPD
to analyze a collection of local Turkish melon genotypes. Most nonsweet
melon types differed from sweet types. However, flexuosus types and some
momordica grouped with sweet genotypes. The diversity found was very
high, even higher than the examined African landraces. Distincion between
cantalupensis and inodorus cultivars was not possible. Intermediate forms
might have been formed between the inodorus and cantalupensis group due
to the ancient farming practices employed by some local small-scale melon
producers for centuries. Several melon genotypes grow together in many
regions of Turkey and introgression of genotypes occurs naturally. Some
types collected from the southeastern part of Turkey were related to some
conomon and momordica accessions. Turkey has some unique genotypes,
among them can be found several dudaim types or the inodorus kirkagac. Iran
also has a wide diversity of the inodorus group that has begun to be analyzed
(Kohpayegani 2004; Kohpayegani and Behbahani 2008). Kohpayegani and
Behbahani (2008) reported high variability in the Iranian melon, comparable
to that of Turkish melons and much higher than landraces from Europe. A
significant differentiation from reference inodorus has also been reported,
suggesting the singularity of this germplasm. The variability in this area,
including other countries of western Asia and eastern Europe (Iraq, Russia,
etc.) needs to be studied further, including the variability of types belonging
to other sweet and highly variable botanical groups such as ameri, adana or
chandalack. A recent study reports the distinction between the three classical
morphotypes of adana known in the Ukraine (Nimmakayala et al. 2009).
The analyzed genotypes represent a major non-US and non-western Europe
source of melon germplasm. In fact, the adana melons are considered to
have been introduced into Europe from Asia in the 15th century and to be
the ancestors of the cantalupensis group.
The Iberian Peninsula is considered to be a secondary diversification
center for melon and is a major world producer of both cantalupensis and
inodorus cultivars. The assessment of the genetic diversity of Spanish
cultivars, not only as reference types (Staub et al. 2000), has become of
a great interest. Typical Spanish types are Piel de Sapo, Tendral, Rochet,
Amarillo and Blanco melons (var. inodorus) as well as several landraces
belonging to var. flexuosus, which differ from types produced in other

European countries (Staub et al. 2000). Morphological variation surveys of

Spanish landraces and their market types have been carried out by several
authors such as Nuez et al. (1986, 1988), Gómez-Guillamón et al. (1985,
1995, 1998) and Costa et al. (1989). López-Sesé et al. (2002, 2003), using the
RAPD array reported by Staub et al. (2000) and SSR loci (Katzir et al. 1996;
Danin-Poleg et al. 2001) evaluated a complete set of Spanish accessions.
They found a high polymorphism between, but low within accessions.
While cluster analysis using fruit characteristics grouped accessions into
cultivars, RAPD-based genetic distance estimates did not provide consistent
accession groupings either by cultivar or geographic origin. The highest
level of polymorphism was detected among melons originating from the
central region of Spain, and in the Rochet cultivar, while accessions from the
Andalusia region and Green cultivars were comparatively less diverse. The
distinctive morphological characteristics among Spanish melon cultivars
(texture and specialized taste) have prevented the introgression of genes
from other germplasm of diverse origin, despite the lack of geographic
isolation, as has been confirmed by molecular analysis. Other studies have
evaluated traditional cultivars from different melon-producing regions in
order to fingerprint those traditional landraces (Escribano et al. 2008).
Genetic variability of landraces from other European countries has
also been reported. Fanourakis et al. (2000) and Staub et al. (2004) analyzed
Greek landraces, finding significant differences from Spanish melons.
The flexuosus types were the most variable and possessed affinities with
conomom types from western Asia. It is possible that some Spanish and Greek
flexuosus accessions may originate from or have ancestral relationships with
melons from western Asia. Lotti et al. (2005) analyzed Italian landraces,
including the typical Carosello (cucumber melon) belonging to the botanical
group chate, which is closer to flexuosus than to inodorus. This variety has
been suggested as the first cultivated melon in Africa, as it appeared on
Egyptian mural paintings from 2000–1500 BC. Italian accessions were widely
genetically different, and their clustering was related to their geographical
origin. The variability within Hungarian landraces has also been studied,
as Hungary is one the first European countries where melon cultivation
was reported (Szabó et al. 2008).
Most of the reported studies include accessions from Africa, Asia,
Europe and the USA as a set of reference commercial cultivars. However,
there are also North American wild populations of melons. These have
also been investigated in order to elucidate their origin (Decker-Walters et
al. 2002a). Whereas these populations were assumed to be escaped forms
of var. chito or var. dudaim, morphological-physiological and molecular
(RAPDs and SSRs) data reveal that enough distinctiveness exists for them to
be classified as ssp. agrestis var. texanus. This variety shows more similarity
to var. chito and to cultivars from eastern Asia. The possible introduction

to America from Asia is discussed, dating to pre-Columbian and post-

Columbian times. Diversity of melons in Australia has been studied less,
but this continent is also a center of complex morphological variation of
C. melo (Telford 1982).
5.2.1.2 Cucumis sativus

The species C. sativus and C. hystrix are found within Cucumis genus,
subgenus Cucumis. Fertile amphidiploids have been synthesized between
these two species, obtaining a species called C. hystivus (Chen et al. 1997a,
b; Chung et al. 2006). This fact along with chloroplast polymorphisms
observed among different Cucumis species, support the hypothesis that
C. hystrix is the progenitor of C. sativus. Both species may share a common
ancestral lineage as well (Chung et al. 2006). C. sativus houses several
botanical varieties, including the cultivated cucumber (var. sativus) and the
wild var. hardwickii (R.) Alef., cross-compatible with C. sativus and thought
to be a progenitor or a feral form of sativus (Horst and Lower 1978; Knerr et
al. 1989). It grows in the Himalayan foothills and has medicinal properties.
Var. hardwickii has been used for yield improvement in cultivated cucumber
due to its multiple fruiting and branching (Staub and Bacher 1997).
Cucumber, C. sativus var. sativus, is one of the largest crops in terms
of worldwide production value, mainly in East Asia. It is thought that
cucumber originated and was domesticated in Asia, likely on the Indian
subcontinent by 3000 BC, and was disseminated thanks to the Silk Road
and oceanic routes. China is considered a secondary center of diversity
(Leppik 1966; Meglic et al. 1996; Staub et al. 1999, 2008). Today this species
is less variable than melons, and cultivars are grouped basically into two
cultivar groups: those eaten fresh and those consumed as a processed
product (Staub et al. 2008).

Various marker systems have been applied to discriminate between
cucumber cultivars. The first isozyme-based studies analyzed US and
European cucumber germplasm (Staub et al. 1985; Knerr et al. 1989;
Staub and Meglic 1993), but the discrimination ability of these markers
was limited. Later, RFLPs were also assayed (Dijkhuizen et al. 1996),
detecting a low degree of variation, similar to that observed in melons by
Neuhausen (1992). More recent RAPD-based studies resulted in grouping
patterns consistent with accession origins, accepted dispersal routes and
discriminating morphological characters (i.e., sex expression and fruit

length to diameter ratio) (Horejsi and Staub 1999). RAPDs have also been
successful in discriminating elite accessions, but have detected limited
genetic diversity (Bernet et al. 2003; Duca et al. 2008; Onto et al. 2008).
Similar to what occurred in melons, several authors have made a big
effort to develop cucumber SSRs. Katzir et al. (1996) reported seven highly
polymorphic genomic SSRs in cucumber and melon. Later, Danin-Poleg et
al. (2001), Fazio et al. (2002) and Kong et al. (2006) increased the number of
microsatellites. SSR-enriched genomic libraries have also been developed
to increase SSR availability (Fukino et al. 2008; Watcharawongpaiboon and
Chunwongse 2008). Transferability to melon, bitter gourd, watermelon and
pumpkin has been assessed. These markers have been tested in diversity
studies using a different set of cucumber cultivars and have also been used
for constructing genetic maps (Park et al. 2000; Bradeen et al. 2001; Fazio
et al. 2003) (see “Molecular maps” in Chapter 6). EST-derived SSRs have
also been used (Kong et al. 2006). The draft genome sequence of Cucumis
sativus var. sativus was recently published. The availability of this sequence
will facilitate the high-throughput discovery of new markers, such as SNPs
(Huang et al. 2009).
In general, molecular studies report a low degree of genetic diversity
within C. sativus var. sativus compared to other cross-fertilized species of
the genus, such as melons (Dane 1976, 1983; Esquinas-Alcazar 1977; Knerr
et al. 1989; Dijkhuizen et al. 1996; Horejsi et al. 1999), but they also describe
higher levels of polymorphisms in var. hardwickii and consistently separate
both varieties (Dijkhuizen et al. 1996; Meglic et al. 1996; Horejsi et al. 1999).
Indeed, Staub et al. (2005) tried to identify a useful reference marker array
(from a set of 155 markers, SSRs and Sequence Characterized Amplified
Region (SCARs)) in order to distinguish very closely related varieties and
elite breeding lines, which is essential for variety protection. They found
difficulties in discriminating this genetic material suggesting that other
markers, such as SNPs are needed to better define these cultivars.
5.2.1.2.3 Molecular Diversity of Cucumber Landraces in the

Centers of Origin and Diversity
Several general studies characterized the genetic diversity of the cucumber
collection (about 1,000 accessions) maintained at the United States
National Plant Germplasm System (NPGS) using isozymes and RAPDs
(Meglic et al. 1996; Staub and Ivandic 2000). Meglic et al. (1996) found
that discrimination between the two varieties and within var. sativus was
possible. Accessions were grouped by continent or sub-continent, and a high
level of heterogeneity was detected in accessions belonging to var. hardwickii.
As expected, Staub and Ivandic (2000) found that NPGS accessions were
genetically more diverse than commercial varieties and presented a different

genetic structure. Accessions grown commercially exhibited a remarkably

narrow genetic base and thus could benefit from the introgression of exotic
genes present in the NPGS accessions.
The Asian gene pool is the most variable compared to the other pools
reported (the USA, Europe and Africa). Staub et al. (1997a, 1999) and Horejsi
and Staub (1999) described genetic diversity in the primary center of origin
(India) and secondary center of diversity (China). In these two works
the variability of cucumber collections from India and China collected in
independent expeditions was assayed. Differences in genetic variability
between the different expeditions reinforced the necessity of collecting
germplasm from the origin and diversity centers before they disappear.
Cases of genetic erosion were detected, as that of landraces of interest from
northern Rajasthan (India), which are tolerant to diverse environmental
stresses (Staub et al. 1997a). Chinese and Indian accessions were different
from each other and from all other groupings, including hardwickii. This
differentiation was also found in previous studies (Dijkhuizen et al. 1996)
and correlates with morphological observations. Differences between these
two countries are likely the result of the geographical and political isolation
of China. Molecular analysis weakly supports morphological differentiation
of cucumber cultigens from northern China (longer and larger fruits, thinner
skinned, warted, white-spined, slightly netted, yellow-to-brown epidermis
and more resistant to abiotic stresses) and southern China (black-spined,
netted and brown skinned). Chinese cultivars from northern and southern
regions are thought to have different origins. Southern germplasm has
been much more isolated due to the Himalayas and the social structure,
whereas the genetic diversity of northern China has taken advantage of the
Silk Road, which introduced types from India and eastern Europe. These
studies concluded that China and India represent the most diverse genetic
variation in this species.
Despite the fact that Africa is a continent where cucumber is not a
major crop, many cucumber landraces are traditionally cultivated on
small-farms. Thus, there exists a differentiated African genetic pool that
is of interest to analyze and maintain. Knerr et al. (1989) and Meglic et al.
(1996) described and analyzed African diversity in NPGS germplasm with
isozymes. They found differences among African cultigens and between
them and germplasm from other continents. A more recent study with
African accessions using RAPDs (Mliki et al. 2003) reports the existence
of three groups (one comprising genotypes from Egypt-Ethiopia-Libya,
separated from Kenyan and Algerian genotypes and the third with several
accessions from Egypt). The first group clearly differentiated from the other,
which was close to Chinese accessions, and therefore constitutes a different
source of variation. This could be of great interest for future breeding

programs, especially considering that Egyptian genotypes are known for

their resistance to several pathogens.
Although distinct genepools of cucumber have been reported in the
USA, Europe, Africa, and Asia, the genetic diversity within var. sativus
is limited. The wild relative var. hardwikii is an underexploited resource.
Until the collections performed by Bisht et al. in 2004, var. hardwickii was
poorly represented in genebanks. These authors reported the collection and
characterization of C. sativus var. hardwickii from different regions of India:
north-western Himalayas (widely and abundantly distributed), the Western
Ghats (fairly distributed), Eastern Ghats, Chhota Nagpur plateau and the
central plateau region at elevations from 800 to 1,700 m.s.a.l (sporadic
distribution). Some of the accessions collected were morphologically and
molecularly characterized, using RAPDs. A high level of diversity was
found, but grouping did not correlate to the geographical origin. This study
reported the existence of segregating populations and hybrids between var.
sativus and var. hardwickii in natural habitats, showing intermediate traits
(plant vigor, fruit skin color, fruit weight and fruit number) that are useful
for the improvement of commercial cucumbers.
5.2.1.3 Other Cucumis spp.

5.2.1.3.1 C. metuliferus
C. metuliferus E. Meyer ex Naudin, known as the African horned cucumber,
is mainly used for human consumption in Africa, where it originated.
However, most studies carried out on this species only have evaluated it for
breeding cucumber and melon because of its resistance to many important
pests such as root-knot nematode, powdery mildew and downy mildew.
Different crosses have been attempted to transfer resistances to C. sativus
(Nikolova et al. 2002; Walters and Wehner 2002) and C. melo (Beharav and
Cohen 1995), even using biotechnological tools like in vitro culture (Tabei
1997).
Evaluation and characterization of some C. metuliferus accessions has
been carried out to select for higher yield, disease resistance and plant
vigor (Marsh 1993) and also for fruit characteristics (size, color, firmness,
moisture content and chemical composition of protein, lipids, sugars, fibre,
organic acids and mineral elements) (Romero-Rodriguez et al. 1992; Krauze-
Baranowska and Cisowski 2001).
Helm and Hemleben (1997) studied relationships among some cucurbits
analyzing satellite DNAs. A new satellite from C. metuliferus was compared
to satellites from other species of the genus, having found the most similarity
with some satellite types from cucumber and melon. Results suggested
that an ancestral satellite type existed in a progenitor of all Cucumis spp.,

being modified during evolution. The pattern of satellite distribution was

in agreement with the taxonomy.
5.2.1.3.2 C. anguria
C. anguria L., commonly called “pickling cucumber”, has been considered
to have originated in America, but now it is supposed to come from tropical
Africa, having been introduced into the New World by African slaves
(reviewed by Baird and Thieret 1988). This species has been screened for
resistances to several diseases, like powdery and downy mildew (Nikolova
et al. 2002) and increased yield (Oliveira et al. 2009). Crosses between
C. anguria and C. anguria var. longaculeatus have been attempted to obtain
elite lines (Modolo and Costa 2003). Some phylogenetic studies on
Cucurbitaceae have also included this species, as well as C. myriocarpus
Naudin, to better represent genus Cucumis, but few accessions have been
assayed (Renner et al. 2007; Singh and Matta 2008). C. myriocarpus, which
has toxic fruits, has also been investigated for resistances, having been
reported as resistant to CVYV (Marco et al. 2003), and adequate mineral
ratios (Flyman and Afolayan 2007).
5.2.2 Genus Citrullus

The genus comprises four diploid (2n = 2x = 22) species with variable
degrees of cross-compatibility. C. lanatus ((Thunb.) Matsum & Nakai),
divided into var. citroides (citron) and var. lanatus (watermelon crop), and
C. rehmii De Winter, newly discovered by De Winter (1990), are annuals. The
remaining two are perennial: C. colocynthis L. Schrad. (colocynth or bitter
apple) and C. ecirrhosus Cogn. (Robinson and Decker-Walters 1997; Wehner
2008). The geographical distribution of these species is shown in Fig. 5-2.
Several works have established the phylogenetic relationships among
Citrullus spp. and related species. Results obtained with seed proteins and
allozymes were consistent with the characteristics of seed coat, grouped
separately from those previously classified as Citrullus spp. Acanthosicyos
naudinianus, from southern Africa, and Praecitrullus fistulosus, from India and
Pakistan (Navot and Zamir 1987). Other studies, using ISSRs and RAPDs,
suggested that P. fistulosus is a distant relative of Cucumis and Citrullus,
which is in agreement with the crossability barriers reported between
C. lanatus and C. colocynthis with P. fistulosus, as no viable seeds have been
achieved from the attempted crosses, thus making the transference of genes
of interest from these species to cultivated watermelon difficult (Levi et al.
2005).

Figure 5-2 Distribution map of Citrullus spp. Principal diversity centers based on Jeffrey
(2001).
C. colocynthis (L.) Schrad.
C. ecirrhosus Cogn.
C. lanatus ((Thunb.) Matsum & Nakai) var. lanatus ▲ and var. citroides ▲
C. rehmii De Winter ♦
Within Citrullus spp., isozyme studies indicate that C. ecirrhosus is

more closely related to C. lanatus than to C. colocynthis. Similar results
were reported using nuclear ITS, also placing C. rehmii closer to cultivated
watermelon than C. colocynthis and C. ecirrhosus (Jarret and Newman 2000).
Crossability and morphology traits, like foliage shape, also support these
findings. Genetic similarity between annual (C. lanatus and C. rehmii) and
between the perennial species (C. ecirrhosus and C. colocynthis) supports
the observation of Jobst et al. (1998) regarding the derivation of annual
forms from perennial ones. Dane (2002) and Dane et al. (2004) and Levi and
Thomas (2005), using PCR-RFLPs, studied the haplotypes of chloroplast
DNA in Citrullus species and close relatives. Most of the cpDNA regions
studied were invariant because the substitution rate found in this genus

is low compared to other species (Parducci and Szmidt 1999; Mohanty et

al. 2001; Davis et al. 2002; Yang et al. 2002). In fact, similar analyses with
mithocondrial DNA were abandoned due to lack of variant sites (Demesure
et al. 1995). Seven haplotypes within Citrullus were identified. C. lanatus
var. lanatus displayed only one haplotype, which suggests the occurrence
of genetic bottlenecks during domestication and low levels of outcrossing.
Higher variability was found in var. citroides. The presence of a similar
mutation pattern in citron and in the C. rehmii haplotype suggests that this
species may be the progenitor of citron melon.
C. ecirrhosus had a haplotype quite similar to C. lanatus, and might
be considered the ancestral species of watermelon (Dane and Liu 2007).
C. colocynthis was the most variable species according to Dane and Lang
(2004), showing five haplotypes associated with different geographic origins
(I: Mediterranean area, Morocco and Cyprus; II: Chad; III: Pakistan; IV:
Afghanistan; and V: Ethiopia), supporting previous reports which described
different races in this species (Yanev et al. 1999). Similar results were obtained
sequencing coding and non-coding cpDNA regions (Dane and Lang 2004).
C. lanatus var. lanatus, C. ecirrhosus and C. rehmii lacked molecular variability
and within the genus Citrullus two major clades separated C. colocynthis
from the other three species. In the latter clade, C. ecirrhosus and C. lanatus
appeared closer. The lack of strong crossing barriers between C. colocynthis
and watermelon (despite the wide genetic distance) make the gene flow
possible in order to enhance genetic diversity in this crop, marking this
species as the main source of genes of interest together with citron types.
Maggs-Kolling et al. (2000) analyzed chloroplast haplotypes in wild and
landraces of Citrullus spp. of Namibia. One accession from Swaziland and
another from South Africa are supposed to possess the ancestral chlorotype,
whereas accessions from South Africa, Botswana and Namibia present
the most recent chlorotype. This supports the Kalahari Desert as the area
of origin of var. citroides. The divergence found between var. lanatus and
var. citroides makes one suppose that they evolved independently from a
common ancestor, possibly C. ecirrhosus.
5.2.2.1.Citrullus lanatus
C. lanatus ((Thunb.) Matsum & Nakai) includes wild, cultivated and feral
forms. Fursa (1972) described three subspecies: ssp. vulgaris (divided into
var. vulgaris and var. cordophanus, including red sweet fruited cultivated
forms), ssp. lanatus (including tsamma types from the Kalahari Desert (var.
caffer), and the citron) and ssp. mucosospermus (including the egusi types
from West Africa). Recently, the species has been reclassified and divided

into two botanical varieties: var. lanatus (the cultivated forms, including
egusi types), distributed in tropical and subtropical regions worldwide, and
var. citroides (Bailey) Mansf. (including citron and tsamma types), which
grows in southern Africa.
Despite the numerous studies, the origin, distribution and domestication
of this species still remains unclear. Jeffrey (1967, 2001) and Zeven and
Zhukovsky (1975) proposed var. caffer as the ancestor of the species while
Navot and Zamir (1987) proposed the var. citroides. Other theories propose
C. colocynthis or, as previously noted, C. ecirrhosus (Dane and Liu 2007).
Primitive watermelons are supposed to have had nonsweet, bitter, white-
fleshed fruits, similar to those of citron or colocynth. Today it is generally
accepted that watermelon originated in Africa where it reaches maximum
diversity (DeCandolle 1883). The two putative ancestors, var. citroides and
C. colocynthis, can be found growing wild in Africa.
Different regions in Africa have been postulated as centers of origin
of this species: southern Africa, principally around the Kalahari Desert
(Meeuse 1962; Esquinas-Alcázar and Gulick 1983), Central Africa (Mallick
and Masui 1986) and northern Africa (Keay and Hepper 1985). In fact,
watermelon seeds (about 5,000 years old) recently discovered at an
archaeological site in southwest Libya (Wasylikowa and van der Veen 2004)
support northern Africa as the most probable domestication center. There
is evidence of watermelon cultivation in the Nile Valley by 2000 BC, when
southwest Africans did not yet practice farming (Zohary and Hopf 2000).
Colocynth seeds have been found at early sites in Egypt, Libya and the
Near East, indicating that they could have been used first. Some authors
assume that cultivation of watermelon began in ancient Egypt and India,
from where it spread to the Mediterranean area, Near East and Asia. The
Romans introduced this crop to Europe, and later the Muslims increased the
number of varieties on the continent. However, watermelon did not become
as popular there as it did in China where it arrived in the 10th–12th centuries.
Subsequently, watermelon reached America (17th century) (Rubatzky 2001;
Wehner 2008). Today, southern Africa and, to some extent, western Africa,
are considered primary centers of diversity. China constitutes a secondary
center of diversity, whereas a great variety of landraces and wild accessions
can also be found in other regions; India, where C. colocynthis grows wild,
the Middle East and countries in the Mediterrranean basin.
5.2.2.1 Molecular Markers Used in Genetic Diversity Studies

Cultivated watermelon (var. sativus) is morphologically highly variable,
mainly for fruit traits, size, shapes, flesh and rind colors and patterns (Ellul et
al. 2007; Wehner 2008). A very characteristic type is the well-known African
Egusi, commonly known in Nigeria and the Congo as wild watermelon,

whose fruits present bitterness and firm white flesh. Its seeds are coated
by an adherent layer of tissues. This type is used for its nutritional seeds
and for cattle consumption.
Despite this morphological variability, molecular variation is limited in
commercial cultivars (Jarret et al. 1997; Maggs-Kolling et al. 2000; Levi et al.
2004; Levi and Thomas 2005). This poor variability can be increased using
germplasm from the diversification areas. Germplasm of Citrullus from
China and South Africa is represented in genebank collections, but India,
south, southwest and tropical Africa and the southern areas of the former
USSR and Iran are still priorities for collection (Wehner 2008).
Molecular markers have been used to estimate genetic relatedness of
watermelon cultivars, and can be used to evaluate inbred lines for purity.
Studies with isozymes (Zamir et al. 1984; Navot and Zamir 1987; Biles et al.
1989; Walters et al. 1991) reflect a low degree of genetic diversity, and only a
few informative isozyme markers are available. Diversity within C. lanatus
(20 cultivated, 70 citron watermelon and reference types) has been recently
assessed by Dane and Liu (2007) by using PCR-RFLPs and sequencing of
cpDNA of several non-coding regions. RAPD markers were more efficient
at detecting genetic variation (Hashizume et al. 1993; Zhang et al. 1994). Lee
et al. (1996) used RAPDs with a representative collection of watermelon
cultivars, obtaining four groups. Molecular clusters correlated with fruit
quality traits such as sugar content. Moreover, Levi et al. (2001b) used this
marker system to characterize a collection of C. lanatus and C. colocynthis
exhibiting several disease resistances. They found three clusters, one with
watermelon cultivars, one with the C. lanatus var. citroides accessions, and
the third with C. colocynthis accessions. Levi and Thomas (2005) performed
their study using citoplasmatic markers, with which they also obtained
a clear differentiation of the accessions belonging to var. lanatus (the five
cultivars assayed), var. citroides and C. colocynthis as previously reported.
They found a closer relationship between citroides and colocynth vs lanatus
and colocynth. Levi et al. (2001a) found higher levels of variability in
C. colocynthis and var. citroides than in cultivated watermelon and differentiated
some watermelon accessions with introgressions from var. citroides. Similar
studies have been performed with ISSRs and AFLPs (Levi et al. 2004). This
study showed that AFLPs and ISSRs are highly informative and much more
efficient at differentiating between American heirloom cultivars with a
narrow genetic base. AFLPs were also successfully used to detect variability
among watermelon cultivars (Ke-peng et al. 2003). The polymorphism rate
detected with this kind of markers proved higher than with other previously
tested kinds, such as isozymes, and three groups were obtained among the
30 genotypes surveyed. Although low genetic diversity was found, classical
American ecotypes, breeding and selected lines and cultivars originating
from Japanese and Chinese pedigrees could be differentiated.

As in other cucurbits, both genomic SSRs and EST-SSRs have been

identified and used in genetic diversity studies (Jarret et al. 1997; Guerra-
Sanz 2002; Joobeur et al. 2006; Verma and Arya 2008). Jarret et al. (1997)
differentiated C. lanatus var. lanatus including egusi types, from wild and
cultivated citron (var. citroides) and C. colocynthis. The fact that most of the
alleles in var. lanatus are common in var. citroides supports the hypothesis
of citron as the wild progenitor. Joobeur et al. (2006) developed genomic
SSRs from a BAC library constructed for watermelon finding that 95% were
polymorphic, whereas Verma and Arya (2008) tested EST-SSRs in a set of
seven Indian genotypes showing 22% polymorphism. They found high
transferability to other genera of the family.
5.2.2.2 Molecular Diversity of Watermelon in Secondary Centers of

Diversity
The germplasm of secondary centers like northeast Brazil is also of interest.
Romao (2000) proposed that watermelon was introduced to this country
by African slaves about 300 years ago. A high level of diversity has been
reported in this area. The locally denominated “melancia de cavalo” had
been classified as C. colocynthis, but this author proposed adscribing it to
C. lanatus var. citroides. Similar morphological studies with commercial
types and landraces in Kenya have been carried out and have found low
diversity within the commercial ones (Gichimu et al. 2009) in comparison
with wild material and landraces. Namibia wild types and local landraces
have also been morphologically compared to commercial cultivars (Maggs-
Kölling et al. 2000). Indigenous classification into seed, cooking and fresh-
eating types was coherent with the clustering obtained, while commercial
types grouped separately. Diversity studies within this species in countries
like Turkey and Korea also demonstrated that high diversity exists there
for morphological traits despite their not being centers of origin (Huh et
al. 2008). Korean and Turkish accessions were easily separated. Korean
landraces were divided into two groups, whereas a continuous variation
was found in Turkish germplasm.
5.2.3 Genus Benincasa

Benincasa hispida (Thunb.) Cogn., known as ash gourd, white gourd, wax
gourd or white pumpkin, is the most important species of the genus.
In addition to their immature fruits, their young leaves and shoots are
consumed. It is considered to be one of the most polymorhic crops with
regard to fruit and certain phenotypic traits (Parkash et al. 2000; Singh 2002),
but few studies have been carried out in spite of its high nutritional value

and traditional medicinal properties. Its center of diversity is the Indo-China

region (Rubatazky and Yamaguchi 1999), where the crop is very popular.
RAPD and ISSR markers have been used to identify cultivars and
hybrids (Meng et al. 1996), to analyze genetic diversity (Sureja et al. 2006)
and to predict and develop hybrids with the highest heterosis value (Verma
et al. 2007). Morphological markers were used before to assess relationships
among B. hispida genotypes. Pandey et al. (2008), in a recent study using
RAPDs, concluded that accessions from northeastern India are quite
different from the other regions of the country.
5.2.4 Genus Lagenaria

The most important species is Lagenaria siceraria (Mol.) Stand., better known
as bottle gourd or calabash. It is an African crop grown for its fruit, which
can either be harvested young and used as a vegetable or harvested mature,
dried, and used as a bottle, utensil, or pipe. Japan, China and India are great
consumers of this fruit. Despite the lack of any early remains in Africa,
this continent is believed to be the origin of the species (Whitaker 1971).
Wild Lagenaria species are distributed in northern Africa. Morphological
analyses and archaeological data suggest that this species dispersed across
the ocean from Africa to Asia and America, where different domestication
events took place. Analyses using RAPD markers have been performed
to clarify its evolutionary history (Decker-Walters et al. 2001). Landraces,
cultivar accessions and a wild relative L. sphaerica were examined, revealing
that southern African germplasm is a divergent lineage from which some
cultivars have derived, and that American germplasm is distinct, but with
an African origin. According to their results, landraces from New Guinea
were not related to American germplasm as previously supposed, and
commercial cultivars have very different origins and genetic backgrounds.
Subsequent studies of diversity in this species have been carried out,
especially in countries where this crop is more important. For instance,
Kenyan landraces of L. siceraria and wild relatives L. sphaerica, L. abyssinica
and L. breviflora were morphologically characterized by Morimoto et al.
(2005), and it was found that more diversity exists within L. siceraria than
within the wild relatives supposedly due to human selection. In order to
have material resistant to diseases and pests, such as root-knot nematodes,
whiteflies, ZYMV and powdery mildew, recent researchers have examined
L. siceraria (Levi et al. 2009) and its relationship to other cucurbits. Moreover,
variability was detected within species with the accessions clustering into
two major groups. One cluster included accessions collected mostly in India
and a few collected in the Mediterranean region and in Northeast Africa.
The second cluster included accessions from southern Africa, North, Central
and South America, China, Indonesia and Cyprus.

5.3 Tribe Cucurbiteae

The genus Cucurbita comprises 20 species (2n = 2x = 40) according to the
most recent classification of the family (Jeffrey 2005). The most economically
important are C. pepo L., C. moschata Duchesne and C. maxima Duchesne. C.
argyrosperma C. Huber and C. ficifolia Bouché are also grown, but they have
a narrower distribution. Based on ecological adaptation, Cucurbita species
can be divided into two groups: the mesophytic annuals or short-lived
perennials with fibrous root systems, which includes the five cultivated
species mentioned above, and the xerophytic long-lived perennials with
fleshy storage roots. Among the latter group, C. foetidissima Kunth (buffalo
gourd) shows characteristics of interest to be domesticated (De Veaux
and Schultz 1985). Geographical distribution of the main Cucurbita spp. is
shown in Fig. 5-3.
The genus Cucurbita is native to America, from where the domesticated
species spread worldwide. Each cultivated species is thought to have
been domesticated independently from the others in distinct regions of
the continent, but all in the pre-Columbian era. The centers of origin for
these species are: C. maxima in southern South America, C. moschata in
the lowlands of southern Central America or northern South America,
C. pepo in northern Mexico or south-central USA, C. ficifolia probably in
the northern or Central South American highlands and C. argyrosperma in
southern Mexico.
A numerical study of taxonomic relationships in Cucurbita was
carried out by Bemis et al. (1970) using a set of 160 characters. They
found five groups of Cucurbita spp., one comprised species indigenous
to arid regions of the southwestern USA and northwestern Mexico
(C. digitata, C. palmata, C. californica, C. cylindrata and C. cordata) and the
second comprised tropical species (C. okeechobeensis, C. martinezii and
C. ludelliana); the third grouped mesophytic species (C. sororia, C. gracilior,
C. palmeri) and the cultivated C. argyrosperma; the fourth was constituted
by C. maxima and C. andreana; and the fifth by C. pepo and C. texana.
C. moschata, C. ficifolia, C. pedatifolia, C. foetidissima and C. ecuadorensis did
not join other species in clusters. Molecular systematic work on the genus
Cucurbita has also been carried out by various investigators by using
both nuclear and cytoplasmic markers. Most of these studies mainly
focus on the cultivated species and their wild relatives, many suggesting
that the crop plants are not derived from a common ancestor. Decker-
Walters et al. (1990), using isozymes, support a common ancestor for
C. moschata and C. pepo that is not shared by C. maxima. Wilson et al. (1992),
using cpRFLPs, separated C. ficifolia from the other cultivated species and
considered it basal to the mesophytic species in the genus. Jobst et al. (1998)
also indicate a polyphyletic origin of Cucurbita on the basis of nuclear ITS

Figure 5-3 Distribution map of Cucurbita spp. Principal diversity centers based on Lira-Saade
(1995) and Sanjur et al. (2002).
C. argyrosperma C. Huber
C. ficifolia Bouché
C. maxima Duchesne ▲
C. moschata Duchesne ▲
C. pepo L.
C. ecuadorensis
C. okeechobeensis (J.K. Small) L.H. Bailey
C. lundelliana L.H. Bailey ♦
C. digitata A. Gray, C. cylindrata L.H. Bailey and C. palmata S. Watson ♦
C. foetidissima Kunth ♦
sequences. They found extensive allele sharing among these species, which
led to an inconclusive phylogenetic analysis, suggesting a high frequency of
introgression during domestication or polyploidization events in the genus.
Analysis using ISSRs and microsatellite DNAs has also been reported (King
et al. 1995; Katzir et al. 2000a, b).One of the most relevant studies has been
that by Sanjur et al. (2002), using mtDNA, which suggests six independent
domestication events from distinct wild ancestors.

5.3.1.1 Cucurbita pepo

C. pepo is native to North America (Trumbull 1876; Erwin 1931; Whitaker
1947). It grows wild in northeastern Mexico and the southern, southeastern
and Central USA (Nee 1990). Its wild range most likely extends to the
northeastern USA (Petersen and Sidell 1996). In northern Mexico and the
eastern USA there is evidence of the presence of this primitive crop from over
4,000 years ago. Moreover, recent studies based on re-examination of early
domesticate assemblages from caves in Mexico indicate that this species is
the earliest domesticate (~8000 BC) in Meso-America (Smith 1997, 2005).
Taxonomically this species is divided into three subspecies according to
Decker-Walters et al. (2002b): ssp. pepo, ssp. ovifera (L.) D.S. Decker, which
comprises var. ovifera (L.) D.S. Decker, var. orkazana D.S. Decker-Walters and
var. texana (Scheele) D.S. Decker, and ssp. fraterna (L.H. Bailey) Andres. The
division of the cultivars of C. pepo into two distinct major lineages, ssp. pepo
and ssp. ovifera, is supported by many studies based on allozymatic, cpDNA,
mtDNA and nuclear markers (Wilson et al. 1992; Decker-walters et al. 1993;
Katzir et al. 2000a, b; Ferriol and Picó 2008; Paris 2008). Most authors have
also supported the theory of two independent domestication events for
these two subespecies (Decker 1985; Kirkpatrick and Wilson 1988; Wilson
et al. 1992; Decker-Walters et al. 1993, 2002b; Sanjur et al. 2002). Different
wild types have been proposed as the ancestor of cultivated C. pepo. Var.
texana might be the ancestor of ssp. ovifera domesticated types. Both species
are cross-compatible and share some morphological similarity. However,
allozymic patterns differed in several studies and the possibility of it being
a feral form instead of the ancestor has been also suggested (Kirkpatrick et
al. 1985; Decker 1988; Kirkpatrick and Wilson 1988; Nee 1990). The precursor
might be an individual similar to var. texana that has been wiped out.
The wild ssp. fraterna has also been suggested as the ancestor of ssp.
ovifera. Accessions of the ssp. fraterna and var. texana were shown by Wilson
et al. (1992) to be closely aligned with cultivars of ssp. ovifera, but no wild
taxon studied was considered a likely progenitor for ssp. pepo. In addition,
in Katzir et al. (2000a), with ISSRs and SSRs, var. fraterna clustered with the
specimens representing ssp. ovifera. var. orkazana is another wild form related
to the ssp. ovifera cultivars and has also been reported to be the ancestor of
var. ovifera after RAPD analysis (Decker-Walters et al. 2002b). These three
extant wild forms in this species are thought to have differentiated from
each other before domestication (Decker-Walters et al. 1993), although
they grouped together based on mtDNA comparisons (Sanjur et al. 2002).
According to their results, ssp. fraterna is the most probable predecessor
due to its higher genetic affinity.

Regarding the ancestor of ssp. pepo, some researchers think that it is

extinct while others maintain that it is unknown (Wilson et al. 1992; Decker-
Walters et al. 2002b). Sanjur et al. (2002) suggested that var. fraterna was
distributed in the past in small and nearly isolated populations, genetically
diverging. This situation explains that one of these populations, to date
uncollected, was the ancestor of ssp. pepo. Paris et al. (2003) found that
the accession Miniature Ball, previously placed within the orange gourd
group of ssp. pepo, possessed wild-type characteristics and yet shows
genetic affinity to a wide range of domesticated C. pepo, suggesting that it
may represent the wild ancestor preserved in cultivation. Teppner (2004)
reported a new subspecies, C. pepo ssp. gumala Teppner, which is cultivated
in Guatemala and Mexico. This subspecies might be the ancestor of ssp.
pepo according to this author.
5.3.1.1.2 Phenotypic Diversity Studies

C. pepo is likely the most polymorphic species with regard to fruit traits
(Naudin 1856). This species, along with other Cucurbita spp., has suffered
great diversification in America, Europe and Asia after Columbus arrived
to the Americas (Decker 1988). Edible cultivated types of this species have
been traditionally grouped in eight morphotypes according to fruit shape
(Paris 1986, 1989, 2001b, 2008): pumpkin, vegetable marrow, cocozelle
and zucchini which belong to ssp. pepo and scallop, acorn, crookneck and
straightneck, which belong to ssp. ovifera (Table 5-2). Most of them are
usually employed as summer squashes, except for pumpkin and acorn. The
subspecies pepo includes ornamental types with orange, round and smooth
or warty fruits and ssp. ovifera var. ovifera also includes ornamental types
with oviform and pyriform fruits. Many studies report the morphological
variation of the species mainly for traits of agronomic interest (quality traits
of fruits and seeds, resistance to pests and diseases). A greater variation in
ssp. pepo than in ssp. ovifera is often reported (Paris 1998, 2001a; Paris and
Nerson 1998; Lebeda et al. 1999; Danilchenko et al. 2000; Kristkova and
Lebeda 2000; Nerson et al. 2000; Younis et al. 2000).

Apart from phenotypic variation, molecular variation within the species
has been assessed with different markers. Isozymes, RFLPs, RAPDs, AFLPs,
SCARs, sequence-related amplified polymorphisms (SRAPs), ISSRs and
SSRs have been used to date to evaluate genetic diversity within C. pepo
(Ignart and Weeden 1984; Decker 1985; Kirkpatrick et al. 1985; Torres Ruiz
and Hemleben 1991; Lebeda et al. 1999; Stachel et al. 1998; Baranek et al.
2000; Decker-Walters et al. 2002b; Heikal et al. 2008). Most of the studies

Table 5-2 Description of the edible C. pepo morphotypes according to Paris (2001b, 2008).
Morphotype Distribution Fruit (shape/color) Rind Consumption

Pumpkin USA-Canada, Europe-Asia Spherical-ovalated fruits Lignified/tender Immature-
Minor, Mexico-Guatemala with or without ribs mature
Orange or yellow-green pattern Seeds

Vegetable Middle-East, northern Africa Short, tapered, cylindrical fruits Lignified Immature
marrow
Cocozelle Europe (Italy), Far East, Long, bulbous fruits Smooth Immature
Turkey, Yugoslavia Striped, light green non-striped, Female
ribbed or non ribbed flowers
Zucchini Worldwide Uniformly cylindrical fruits Tender Immature

Yellow and green types
Scallop Australia, North America Flat, scalloped fruits Lignified Immature

and Europe Yellow, white-green
Table 5-2 contd....

Table 5-2 contd....
172
Morphotype Distribution Fruit (shape/color) Rind Consumption

Acorn USA-Canada Furrowed, turbinate fruits non-lignified Mature
Green
Crookneck Southeastern USA Narrow necked fruits, thick, lignified Immature

usually curved and warted
Yellow
Straightneck USA, Europe Short-necked or constricted lignified Immature

fruits, usually warted
Yellow
Color image of this table appears in the color plate section at the end of the book.

report a high variability within the species. When different species are
compared, allelic diversity is greatest in C. pepo and C. moschata. Katzir
et al. (2000a), using SSRs and ISSRs grouped the cultigens of C. pepo ssp.
ovifera more-or-less according to fruit shape, whereas in C. pepo ssp. pepo,
subclustering differentiated the cocozelle group from the zucchini group.
Some of the SSR loci used for Cucurbita analysis were transferred from
Cucumis, as only a few microsatellites were available for Cucurbita. Paris
et al. (2003) attempted to apply cucumber and melon SSRs described
by Katzir et al. (1996) and Danin-Poleg et al. (2001) attempted to assess
diversity in C. pepo. A set of 102 Cucumis-SSR primers developed by Fazio
et al. (2002) were also proved in this species for mapping, but none turned
out to be polymorphic. Sixty Cucumis SSRs (gSSRs and EST-SSRs) were
tested in Cucurbita spp. accessions, but over 63% did not amplify in any
of them (Picó et al. 2005-2006). Transferability from Cucumis EST-SSRs
has also been assayed (Fernández-Silva et al. 2008), but the fact that these
markers are located in expressed regions of the genome did not increase the
transferability rate and only 5.4% were polymorphic. Therefore, SSRs are
not easily transferable between genera within Cucurbitaceae, and because
of this, a wide collection of SSRs has been developed recently using SSR-
enriched partial genomic libraries from C. pepo ssp. pepo and C. moschata.
These markers show high inter-species transferability and have been used
to construct the first published C. pepo map (Zraidi et al. 2007; Gong et al.
2008a, b). Stift et al. (2004) had already reported a better transferability rate
from C. pepo-SSRs to C. moschata, C. maxima and C. ecuadorensis and Gong
et al. (2008a) found with their new set of 500 SSRs (from SSR-enriched
partial genomic libraries) a higher percentage of C. pepo markers transferred
to C. ecuadorensis in comparison to C. moschata, which implies a closer
relatedness between C. pepo and C. ecuadorensis than between C. moschata
and C. ecuadorensis.
Two of the most complete studies performed to date in C. pepo are those
by Paris et al. (2003) and Ferriol et al. (2003a). Paris et al. (2003) assayed a set
of C. pepo accessions belonging to the three subspecies with three different
marker systems: AFLPs, ISSRs and SSRs, finding a high correlation between
them. Whereas previous studies describe high levels of variation in wild
genotypes (Decker-Walters et al. 2002b), Paris et al. (2003) found higher
variation among domesticated than among wild populations. In general,
their results were coherent with botanical and horticultural classification
and with other studies with allozymes (Ignart and Weeden 1984) and DNA
markers (Torres Ruiz and Hemleben 1991; Katzir et al. 2000a). Clustering
agreed with the division into ssp. pepo, ssp. ovifera (syn. texana) and ssp.
fraterna. Subspecies fraterna and ovifera appeared more closely related to each
other than to ssp. pepo. Results show that the cultivar-groups are genetically
quite distinct. In fact subclusters within ssp. ovifera were in accordance

with morphotypes, supporting six groups: acorn, crookneck, scallop,

straightneck, ovifera gourds and wild forms. Scallop group, thought to be
among the oldest, was closely related to the texana gourds, supporting the
theory of its having been developed directly from inedible fruit ancestors
by American natives. straightneck, the most recent group among ssp.
ovifera (Paris 2000), was consistently the most dissimilar to texana gourds,
and little phenotypic variation within the group was observed. Within
ssp. pepo, zucchini is the most recent (Paris 2000) and the most different
from the other morphotypes of its subspecies, showing limited variability.
Relationships within pumpkin accessions and between this group and
cocozelle and vegetable marrow were not as clearly defined, perhaps due
to the large amount of diversity found in these three groups.
Ferriol et al. (2003a), using SRAPs and AFLPs, focused on the diversity
of Spanish landraces, with representation from the different morphotypes
described in the species as well as several unclassified types. Spain was an
important country for Cucurbita diffusions and diversification in the Old
World as it acted as a bridge between Europe and America. Results were
similar to those reported by Paris et al. (2003). Both subspecies were also
clearly differentiated, pepo being more polymorphic. A more defined sub-
clustering according to morphotype within ssp. ovifera than wihin ssp. pepo
was observed with SRAP markers. In the ssp. ovifera cluster, crookneck and
straightneck grouped separately from scallop, whereas acorn was more
dispersed.
5.3.1.2 Cucurbita moschata

Mexico was initially proposed as the domestication center of this species
(Cutler and Whitaker 1967), however, subsequent studies at some
archeological sites also placed the oldest C. moschata remains in Ecuador
(5170 to 3780 BC) and on the Central Pacific coast of Panama (5000 BC),
while the oldest remains in the southwestern USA dated back 2,300 years
(Decker-Walters and Walters 2000; Piperno et al. 2000). Nowadays, South
America is considered to be the domestication center or a secondary center
of diversity because some landraces from Colombia, Panama and Bolivia
display primitive traits, such as dark seeds, small fruits, bitter flesh, lignified
and warty rind and indeterminate growing habits (Nee 1990; Wessel-Beaver
2000; Sanjur et al. 2002). Two independent domestication events in Mexico
and northern South America have also been proposed (Lira-Saade 1995;
Robinson and Decker-Walters 1997; Decker-Walters and Walters 2000).
After its domestication, C. moschata spread to the Caribbean islands and
subsequently to the rest of the world´s continents where it adapted to
different conditions (Piperno et al. 2000).

The wild ancestor of C. moschata still remains unknown. C. lundelliana,

located on the plains of the Yucatan Peninsula, was considered to be the
ancestor, but morphological, isozymatic and crossing studies do not support
it (Merrick 1990). According to ecological, morphological and molecular
studies using mtDNA, cpDNA and nuclear markers, C. moschata and
C. argyrosperma are closely related (Wilson et al. 1992; Jobst et al. 1998; Sanjur
et al. 2002). In fact, they were considered to be a single species until Pangalo
(1930) proposed C. argyrosperma (syn. C. mixta) as a different species. That
is the reason why a wild taxon of C. argyrosperma, the ssp. sororia, has also
been suggested as the ancestor of C. moschata, although some crossability
barriers and different isozymatic patterns exist between the species. Gene
flow between C. argyrosperma ssp. sororia growing wild near milpas, where C.
argyrosperma ssp. argyrosperma and C. moschata are cultivated, were reported
in Mexico by Montes-Hernández and Eguiarte (2002), which contributes to
increasing polymorphism levels. Wild gourds discovered in Bolivia, could
be key in the elucidation of the origin of this species (Decker-Walters and
Walters 2000).
5.3.1.2.2 Phenotypic Diversity Studies

C. moschata is the species of the genus Cucurbita for which the most
secondary centers of diversity have been described, with a large amount
of landraces with variable characteristics developed in different regions.
Filov (1966) classified over 20 varieties of C. moschata in several geographic
subspecies, which revealed the existence of important diversity centers in
Colombia, Mexico, Central America, the western USA, Florida, India and
Asia Minor and Japan. Despite the great variability of C. moschata landraces,
only a few types have been introduced in the commercial circuit, which
initially comprised three groups of cultivars: “Cheese” (variable in shape
with leather-colored rind), “Crookneck” (round end with a long straight or
curved neck) and “Bell” (bell-shaped to nearly cylindrical) (Castetter 1925;
Whitaker and Davis 1962; Robinson and Decker-Walters 1997). “Butternut”
was the first commercial cultivar, obtained from a Crookneck type in the
1930s (Mutschleer and Pearson 1987).
Landraces from different centers of diversity have been morphologically
and agronomically characterized. Chung et al. (1998) assessed accessions
from Korea, where flattened and round fruited types predominated. Wessel-
Beaver (2000) observed a high frequency of primitive traits in Colombian
landraces and Ríos et al. (1997) characterized Cuban landraces according
to their morphology and yield without pesticides and fertilizers. A great
morphological diversity in Spanish landraces has also been reported (Esteras
et al. 2008). Cultivars exhibiting bushy growth habit have been studied too
(Carle et al. 2000; Wu et al. 2007). This interesting agronomic trait (bushy-

growth) already known in modern cultivars of C. pepo and C. maxima, has

recently been evaluated in C. moschata, leading to the discovery of the gene
responsible for the inhibition of cell elongation. Some studies also describe
variation in quality traits such as the great variability in β-carotene content
observed in landraces from Zambia (Gwanama et al. 2002). Loche types, a
highly valued landrace from Peru, are also being characterized due to their
increasing interest in typical gastronomy. Variability for herbicide and pest
and disease resistance has also been assessed (Poe et al. 1988; Wessel-Beaver
1993; Maynard 2001).

Some of the molecular studies performed with C. moschata tried to establish
relationships to other Cucurbita spp. Isozyme analysis (Decker-Walters et
al. 1990; Puchalski and Robinson 1990) and comparisons of chloroplastic,
mithocondrial and nuclear ribosomal DNA (Wilson et al. 1992; Jobst et al.
1998; Sanjur et al. 2002) established the closer relatedness of this species
with C. argyrosperma. Great diversity within C. moschata, higher than in
C. maxima and similar to or higher than in C. pepo, has been reported
using allozymes, RAPDs and chloroplastic and nuclear ribosomal DNA
comparisons (Decker-Walters et al. 1990; Wilson et al. 1992, Jeon et al. 1994;
Jobst et al. 1998; Baranek et al. 2000).
Only a few studies have assessed infraspecific variation in this species,
most with dominant markers that do not need previous knowledge of
sequences. Youn and Chung (1998) and Gwanama et al. (2000) analyzed
genetic diversity in landraces from Africa and South Korea, revealing
groups consistent with agroclimatic origin and not with morphological
traits. One of the most remarkable works on C. moschata is that of Ferriol
et al. (2004a) using AFLPs and SRAPs, which studied both morphological
and genetic diversity within the species in a germplasm collection of
Spanish landraces and some American accessions. The variability found
was comparable to that reported for some secondary centers of diversity like
Korea, indicating that Spain maintains a great amount of diversity. In fact,
many landraces could not be classified in any of the horticultural groups of
commercial importance. A clear grouping according to geographical origin
was observed. A clear separation between the Spanish, Central American
and South American accessions was detected, the latter showing some
primitive traits. C. moschata landraces from the Canary Islands differed
molecularly from those of the peninsula, suggesting different germplasm
introductions from America or earlier/better adaptation of some types to
the more tropical climate of the islands (Ferriol et al. 2005).
AFLPs have also been used to study the genetic diversity of C. moschata
from Brazil for the establishment of a core collection (Ramos 2007). Until

recently few SSR markers have been developed for C. moschata. In this
genus, most molecular tools had been generated mainly for C. pepo or
transferred from more important species in the family such as Cucumis
melo. The inter-genus transferability was quite low, both for genomic and
EST-SSRs (Picó et al. 2005-2006; Watcharawongpaiboon and Chunwongse
2007; Fernández-Silva et al. 2008). Recently, Gong et al. (2008a) developed
a set of 500 SSRs from SSR-enriched partial genomic libraries of C. pepo
and C. moschata, reporting a high interspecific transferability between both
species. These newly developed SSRs have been used to construct the first
SSR-based map for C. moschata (Gong et al. 2008b). A high level of macrosynteny
was found comparing both C. pepo and C. moschata maps, revealing that
the transferability of markers may be more easily accomplished (see
Chapter 8).
5.3.1.3 Cucurbita maxima

The oldest archaeological remains that demonstrate C. maxima Duchesne
domestication dated back to 2000 BC and were found in coastal Peru. In
pre-Columbian times, primitive forms of C. maxima were already cultivated
in northeastern Argentina and Paraguay by the Guarani Indians as well as
in the Andean valleys (Ferriol 2003). Oliszeweski (2005) reported northern
Argentinian remains dating to between 200 and 500 AD. Nowadays, it is
generally accepted that C. maxima ssp. andreana is the wild ancestor of the
cultivated forms of the species. Subspecies andreana is endemic to South
America (Argentina, Uruguay, Bolivia and possibly Paraguay), growing
wild in temperate regions of Argentina and the plains of Bolivia, so these
areas can be included in the domestication center (Sanjur et al. 2002).
Initially, C. ecuadorensis, whose habitat is located in the coastal region of
Ecuador, was also thought to be the ancestor (Lira-Saade 1995).
C. maxima is divided into two subspecies: C. maxima Duch. ssp. maxima,
which includes the cultivated and ornamental types, and C. maxima Duch. ssp.
andreana (Naud) Filov, which only includes wild forms. Hybridization has been
reported between cultivated types and ssp. andreana, which has contributed
to increasing the genetic variation (Decker-Walters and Walters 2000).
Castetter (1925) classified the cultivated C. maxima types in six
horticultural groups (banana, delicious, hubbard, marrow, show, turban),
which have persisted until the present day (Whitaker and Davis 1962;
Decker-Walters and Walters 2000). However, many local cultivars and
landraces present different characteristics and can not be included in this
classification. A few studies have focused on these traditional cultivars,
adapted to very different conditions since this species spread worldwide

from America. Spain acted as a bridge between America and Europe after
the discovery of the continent, but other types first arrived in Australia,
Africa and Asia where they diversified (secondary centers) and were later
exported to Europe. However, it seems that commercial types were selected
in the USA from materials collected in South America.
5.3.1.3.2 Phenotypic Diversity Studies and Genetic Diversity

Studies with Molecular Markers
Most of the studies on genetic diversity in C. maxima aimed at establishing
genetic relationships with other Cucurbita species. Bemis et al. (1970)
reported the closer relationship between this species and the previously
denominated C. andreana, now ssp. andreana, employing biological,
geographical and ecological data. Similar results were obtained by means
of nucleic acid hybridization, isozyme and mitochondrial (Sanjur et al.
2002) and chloroplast DNA (Wilson et al. 1992) comparison. Moreover, the
genetic similarity between semi domesticated C. ecuadorensis and C. maxima
has been established (Goldberg et al. 1972; Puchalski and Robinson 1990;
Wilson et al. 1992; Sanjur et al. 2002).
Other studies carried out in C. maxima include, like in the other Cucurbita
spp., seed characterization (Joshi et al. 1993) and disease and herbicide
resistance assessment (Poe et al. 1988; Keinath and DuBose 2000; Kristkova
and Lebeda 2000).
Some molecular studies have been performed with enzymes, finding
no correspondence between morphological and molecular data (Decker-
Walters et al. 1990; Júnior 1999). Ferriol et al. (2003b, 2004b) employed
AFLPs, RAPDs and SBAPs to assess genetic diversity of Spanish landraces.
SBAPs markers grouped the accessions according to the type of use: human
consumption, cattle consumption and ornamental, which are related to
morphological and agronomical traits, while AFLPs grouped based on
geographical origin, indicating much more variability among the included
American landraces than among the Spanish ones, and supporting the idea
of a genetic bottleneck during the introduction into Europe. A recent study
about diversity of C. maxima in its area of origin (Esteras et al. 2009c) placed
the representative accessions of Spanish variability among the less variable
accessions from Peru and Ecuador. In this preliminary study performed
with AFLPs, the ssp. andreana accessions separated clearly from those of
ssp. maxima. Some Argentinian genotypes were intermediate between ssp.
andreana and the remainder of accessions from Bolivia, Ecuador, Paraguay,
Peru and Argentina, which supports Argentina as the center of origin. Apart
from Argentina, Ecuador also showed high diversity, indicating the necessity
of collecting germplasm in this area; in Bolivia, however, a low level of
variation was observed, which might be due to a genetic erosion process.

5.3.1.4 Other Cucurbita spp.

5.3.1.4.1 C. ficifolia
C. ficifolia Bouché, also called Malabar melon or Angora squash, is supposed
to have been originated and domesticated in the Andean region (Nee 1990;
Sanjur et al. 2002), although a Mesoamerican origin is not ruled out due
to existence of several indigenous names. This species is poorly diffused
outside the tropics due to its ecological requirements, and because of that
this crop presents little diversity. Fruits are very uniform, only varying in
color and size.
It is cultivated mainly for self-consumption. This is the reason why only
a few studies on this species have been carried out. Due to its cold resistance
C. ficifolia has been surveyed to improve yield in other crops like cucumber
by means of using as rootstock (Zhou et al. 2009). Disease resistances have
been described as well. Ivancic et al. (2004) evaluated hybrids C. ficifolia x
C. maxima for morpho-agronomic traits. Moreover, this species partially
crosses with other less important cucurbits like C. lundelliana, C. foetidissima
and C. pedatifolia. C. ficifolia has been included in several phylogenetic studies
to elucidate relationships among genera in the family (Kocyan et al. 2007)
or among species in the genus. The last one surveyed cultivars from four
Cucurbita species with AFLPs concluding that C. ficifolia and C. pepo had a
close relationship.
5.3.1.4.2 C. argyrosperma
C. argyrosperma C. Huber (syn. C. mixta Pang.) was first considered a
different species from C. moschata by Pangalo (1930). It is believed that its
domestication took place in southwestern and Central Mexico, since the
oldest archeological remains found in different caves of the region dated
back between 3085 and 115 BC (Merrick 1990; Smith 2005). The wild ancestor
of cultivated forms (ssp. argyrosperma) is supposed to be C. argyrosperma
ssp. sororia (L.H. Bailey) Merrick and Bates (Sanjur et al. 2002), which is
distributed from Mexico to Central America.
Decker-Walters and Walters (2000) described a low variability within
the species with few commercial cultivars existing. Due to the poor quality
of the flesh, many cultivars are only grown for their seeds and for cattle
consumption. Within ssp. argyrosperma (cultivated forms) three varieties
may be distinguished: var. argyrosperma (the more primitive one, commonly
striped with bright color), var. callicarpa (the most recent and variable one,
comprising most of the commercial cultivars and landraces) and stenosperma
(striped fruits, mainly used for its edible seeds).

Several studies have dealt with the traditional Mexican agroecosystem

called “milpa”. Montes-Hernández and Eguiarte (2002) analyzed genetic
structure in squashes grown in western Mexico, where C. moschata and C.
argyrosperma ssp. argyrosperma are cultivated within maize fields next to wild
populations of C. argyrosperma ssp. sororia. Results with allozymic markers
suggested a high exchange of genes among populations even if they were
several kilometers apart. This extent of high gene flow has increased the
squash genetic variability in this area. However, this diversity is considered
to be threatened in the near future due to the lack of young farmers who
want to maintain this agrosystem and its landraces (Montes-Hernández
et al. 2005). Morphological diversity in this kind of system has also been
studied in central-eastern Yucatan (Mexico). Canul et al. (2005) characterized
36 squash landraces (C. moschata and C. argyrosperma) using plant, fruit
and seed traits. Most of the quantitative variation was explained by seed
length, width and weight; days to female flowering; fleshy thickness and
fruit length and width; while qualitative variation was mainly explained
by leaf shape, fruit longitudinal shape and color intensity of leaf spots.
Both species separated clearly in the Principal Component Analysis (PCA),
C. moschata being more variable.
5.4 Tribe Joliffieae

The most important species in this genus is Momordica charantia L.,
known also as bitter melon or bitter gourd, which is widely cultivated in
India, China, Malaysia, Africa and South America. This crop possesses
comparatively high concentrations of ascorbic acid and iron and is used as
a traditional medicine as well. In fact, studies like the one by Krawinkel and
Keding (2006) report a high content in phenolics, polyphenolic compounds
and natural oxidants and antioxidants in some Indian varieties.
This country possesses great morphological diversity in sex expression,
growth habit, shape and size of the fruit, etc. (Robinson and Decker-Walters
1999; Behera et al. 2006b) that needs to be evaluated for possible uses in
breeding.
Marr et al. (2004) surveyed wild and cultivated populations of this
species using isozymes and morphological traits. Comparable phenotypic
variation was found in both kinds of accessions although isozyme
polymorphisms were higher in the wild ones. However, relatively few
polymorphic markers have been identified in bitter melon (Dey et al. 2006).
To date, the most complete study is that of by Behera et al. (2008), who
assessed the genetic diversity among 38 diverse bitter gourd accessions
from different Indian states using RAPD and ISSR markers. The percentage
of polymorphism detected was higher for ISSRs and a great difference was
observed beween var. charantia (domesticated genotypes) and var. muricata

(wild, free-living types). A more recent work (Kole et al. 2010) with AFLPs,
fruit traits and three healthy compounds (cucurbitacin-C, charantin and
plant insulin), reported a high genetic diversity among 22 genotypes from
six countries. The knowledge of the phytomedicinal compounds variation
in bitter gourd germplasm and the pathways and genes involved, makes
this species a model for the new phytomedomics era.
With regard to within-genus studies, the most recent one is a DNA
sequence phylogenetic research that includes 58 Momordica species (Schaefer
and Renner 2010). They conclude this genus is monophyletic and consists
of 11 well-supported clades.
5.5 Tribe Luffeae

The genus Luffa is a very popular vegetable in the tropics. Some types are
also used to make sponges or to extract fiber. Within this genus two species
are cultivated: L. aegyptiaca Mill. (syn. L. cylindrical M. Roem), known as
smooth gourd or sponge gourd, and L. acutangula Roxb, known as ridged
gourd or fluted loofah.
Three varieties are recognized within L. acutangula: var. acutangula (the
domesticated) and two wild varieties, var. amara (located in India) and var.
forskalii (Harms) Heiser & Schilling (located in Yemen) (Heiser and Schilling
1990). While DeCandolle (1959) suggested Asia as the domestication
center, Zeven and Zhukovsky (1975) and Heiser and Schilling (1990) have
supported India due to the fact that var. amara, which is more likely to be
the ancestor, is endemic there. One of the most recent studies on this topic
included landraces from China, Laos and Nepal (Marr et al. 2005). High
variability was described for fruits and seeds, although only one allozyme
locus proved to be polymorphic in this species out of the 29 loci assayed.
Nine of them showed different and fixed alleles in both L. ocutangula and
L. aegyptiaca, indicating that they are likely to be reproductively isolated,
although interspecific crosses allowing gene flow have been reported
between the two species.
The latest studies on Luffa acutangula related to its antioxidants, and
also its medicine and pesticide properties, and have increased breeders´
interest in this crop, especially in finding a higher yielding variety. Because
of this, the search for phenotypic and genetic diversity has become more
important.
Genetic diversity and relationships among Luffa spp. was studied by
Tolentino et al. (1997) analyzing total seed protein profile using SDS-PAGE
in a collection of 215 accessions. Great similarity was detected between
both species, the smooth luffa being less variable possibly because of its
major domestication. Recently, Hsieh et al. (2007) have evaluated diversity
among lines and cultivars of Luffa using RAPDs and morphological traits,

finding a clear separation between both two species. As previously reported,

L. acutangula displays more variability. Hoque and Rabbani (2009) studied
28 ridge gourd landraces collected from different parts of Bangladesh,
where many wild relatives are grown, using RAPDs and detected five
groups within the germplasm assessed. A significant level of variability
was found. In fact, the percentage of polymorphic loci was higher than that
found in other cucurbits like M. charantia (Dey et al. 2006), C. maxima (Ferriol
et al. 2003b) and C. melo (Garcia-Mas et al. 2000). Regarding phylogenetic
studies, several surveys have tried to elucidate the evolutionary history
of this species. L. acutangula together with L. aegyptiaca were placed in a
single clade apart from the remainder of the species of the genus according
to phenotypic variation (Heiser and Schilling 1990), although chloroplast
DNA markers data do not agree completely with this (Chung et al. 2003).
On the other hand, several studies on qualitative and quantitative
composition of triacylglycerols from seed oils of different cucurbits have
been carried out including both cultivated species of Luffa (Grondin et al.
2002).
5.6 Tribe Sicyeae

In the genus Sechium, the most important species is. Sechium edule (Jacq.)
Sw., (chayote). It is, like other minor cucurbits, a vegetable crop that is
important in low-income agriculture. However, commercial production
of chayote is important in several countries, such as Costa Rica, Mexico,
Brazil and Puerto Rico (Hord et al. 1997).
It is native to Central America, Mexico being the main center of
diversity (Lira-Saade 1996). According to morphological and biochemical
data, the wild ancestor of the cultivated forms is Sechium edule (Jacq.) Sw.
ssp. sylvestre, which is endemic to Mexico (Cross 2003; Cross et al. 2006).
In addition, significant genetic variation in fruit traits is present in Central
America (Engels 1983).
Some of the most recent studies include morphological and anatomical
characterization to better variety classification (Cadena-Iñiguez et al. 2008)
and utilization of isozyme markers (Abdelnour and Rocha 2008). Abdelnour
and Rocha (2008) reported a high degree of genetic diversity in a collection
of 42 accessions from Costa Rica and highlighted the usefulness of this
kind of markers in species without DNA quality markers and genomic
information. They reported the necessity of maintaining the threatened
diversity in this crop and the necessity of germplasm collections. It is
expected that more molecular markers for a better characterization will be
developed in the future.
Other countries also are making an effort to study and maintain
this diversity. In India, Sanwal et al. (2008) evaluated some indigenous

populations of S. edule, called chow-chow there, for morphological traits

such as number of fruits/plant, fruit yield/plant, total soluble solid content,
acidity and ascorbic acid content.
Acknowledgements
The authors acknowledges support from the INIA projects RTA2008-00035-
C02-02 and RF2008-00003-C02-02.
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6
Molecular Genetic Mapping and
Map-based Cloning
Yi-Hong Wang
ABSTRACT
Mapping of plant genomes has progressed rather rapidly in the
last two decades. The initial low density mapping with isozyme or
morphological markers has been replaced with high density mapping
using DNA markers. Such a trend is more visible within the last decade
when at least 24 maps have been created in cucurbits for melon,
cucumber, watermelon and Cucurbita ssp. Most maps were produced
for melon since 2004: 11, and six, three, and four were generated for
cucumber, watermelon and Cucurbita, respectively. In sync with this
trend, increasingly sophisticated DNA marker systems were also used:
from less abundant and radioactive RFLP to more abundant and user-
friendly SSR and SNP. The increased technical advance also facilitated
cloning of genes underlying agriculturally important traits such as
sex expression and disease resistance. The power of next generation
sequencing technology in uncovering more SSRs in the plant genome
has just been demonstrated in cucumber. This will no doubt increase
the speed of marker identification and mapping in cucurbits.
Keywords: cucurbit, genetic mapping, SSR, SNP, AFLP, RFLP, RAPD
6.1 Introduction
Genetic maps have been continuously developed for cucurbits in the last
two decades mostly using molecular markers. Maps are important for all
important agricultural crops because of two reasons. Firstly, these maps
can be used to track inheritance of traits of interest, be they single-gene
Department of Renewable Resources, University of Louisiana at Lafayette, Lafayette, LA

70504, USA; e-mail: yxw9887@louisiana.edu

controlled or quantitative. The latter is especially relevant because majority

of economically important traits are quantitative. And secondly, they are
essential to clone genes for which a phenotypic trait is only known by its
map position. For single-gene controlled phenotypic traits, the gene itself or
its associated markers can also serve as markers in molecular breeding.
Classic genetic maps developed before or during the early1990s in
cucurbits employed mostly isozyme or phenotypic markers (classical
markers). These classical genetic maps were first developed in cucumber
using morphological markers (Fanourakis and Simon 1987; Pierce and
Wehner 1990; Vakalounakis 1992), isozyme markers (Knerr and Staub 1992),
or both (Meglic and Staub 1996). These maps covered 168 (Fanourakis and
Simon 1987), 166 (Knerr and Staub 1992), 95 (Vakalounakis 1992), and 584
cM (Meglic and Staub 1996), respectively. Since the length of the cucumber
genome is 750–1,000 cM (Staub and Meglic 1993), these maps are far from
saturated.
Early studies with classical markers in other cucurbits were also
conducted. Esquinas (1981) surveyed variability of six isozymes in 125
melon cultivars and found 11 polymorphic loci. Perl-Treves et al. (1985)
reported 24% polymorphism between six melon cultivars with 29 enzyme
systems. Using 12 enzyme systems representing 19 loci, Zamir et al. (1984)
did not identify any polymorphism among 13 watermelon cultivars. Navot
and Zamir (1987) surveyed 26 isozyme loci in 550 watermelon accessions
and also found very little variation among cultivated watermelon varieties
although there was significant difference between cultivated and wild
watermelon species. The result was a classical watermelon genetic map with
isozyme markers spanning 354 cM over seven linkage groups (Navot et al.
1990). The first classical melon map was constructed by Pitrat (1991) that
consisted of eight linkage groups with 23 markers (disease resistance, flower
biology and vegetative characters). For squash, Weeden and Robinson (1986)
used isozyme markers to develop the first squash genetic map based on an
F2 population derived from a cross of C. maxima × C. ecuadorensis. The map
contained 11 isozyme loci distributed in five linkage groups.
However, phenotypic or isozyme markers alone could not cover the
whole genomes because of their low occurrence in the cucurbit genomes
and their unstable expression. In contrast, molecular markers are abundant
and environmentally neutral. They provide powerful tools for mapping
a phenotypic trait and for map-based cloning. Therefore, most cucurbit
genetic maps are generated with a high density of molecular markers. A
complete list of 36 cucurbit maps can be found in Table 6-1.

Table 6-1 Genetic maps of cucurbits using molecular markers.
Population Parents Marker type and Number Average Length of Mapping software Reference
number of linkage marker the map
groups distance (cM)
(cM)
Melon 2n = 2x = 24
99 RILs PI414723 (C. melo subsp. 386 SSRs, 76 SNPs, 12 2.672 1222 Harel-Beja et al. 2010
agrestis) × ‘Dulce’ (C. melo six INDELs and 200
subsp. melo) AFLPs
116 F2 Q 3-2-2 (Chinese accession) 155 SSRs, 9 ESTs, 7 12 6.4 1095 MAPMAKER 3.0 Cuevas et al. 2009
Molecular Genetic Mapping and Map-based Cloning 201

x Top Mark (C. melo var. SNPs
cantalupensis)
81 RILs USDA 846-1 (exotic 104 SSRs, 4 SNP, 7 EST, 12 4.6 1180 MAPMAKER 3.0 Cuevas et al. 2008
accession) x Top Mark (C. 140 RAPDs and/or
melo var. cantalupensis) AFLPs, 1 phenotypic
trait
93 RILs AR 5 (highly resistant to 157 SSRs, 7 SCARs, 3 20 877 MAPMAKER/ Fukino et al. 2008a
powdery mildew, MNSV phenotypic traits EXP3.0
and A. gossypii) × Harukei 3
(susceptible to all)
114 F2 4G21 (C. melo var. chinensis) 152 SRAP 12 13.67 2077.1 MAPMAKER/ Wang et al. 2008
× 3A832 (C. melo var. EXP3.0
saccherinus)
77 DHLs PI161375 (C. melo var. 46 additional SSRs on 12 4.2 1237 MAPMAKER 3.0 Fernandez-Silva et al.
conomon) × Pinyonet Piel de Gonzalo et al. (2005) 2008
Sapo (C. melo var. inodorus) map
81 RILs USDA 846-1 (exotic 49 SSRs, 116 RAPDs, 15 5.87 1116 MAPMAKER 3.0 Zalapa et al. 2007
accession) x Top Mark (C. 33 AFLPs and 1
melo var. cantalupensis) phenotypic trait
Table 6-1 contd....

Table 6-1 contd....
202

(cM)
77 DHLs PI161375 (C. melo var. 79 RFLPs, 90 SSRs, 3 12 7 1223 MAPMAKER/ Gonzalo et al. 2005
conomon) × Pinyonet Piel de SNPs, and Nsv EXP3.0
Sapo (C. melo var. inodorus)
93 F2 PI161375 (C. melo var. 16 additional SSRs on 12 4.3 1240 MAPMAKER/ Gonzalo et al. 2005
conomon) × Pinyonet Piel de Oliver et al. (2001) map EXP3.0
Sapo (C. melo var. inodorus)
DHL and PI161375 (C. melo var. 226 RFLPs, 97 SSRs, 12 3.11 1021 JoinMap 3.0 Gonzalo et al. 2005
F2 (merged conomon) × Pinyonet Piel de 3 SNPs, and the Nsv
map) Sapo (C. melo var. inodorus) locus
120 RILs Védrantais (Fom-1) × Isabell 165 AFLPs, 28 IMAs, 16 4.9 641 MAPMAKER 3.0 Perchepied et al. 2005
(Fom-1, Fom-2, and partial 7 SSRs, 2 SCARs, 1
resistance to Fusarium phenotypic trait
oxysporum fsp. melonis
race1.2)
Cucumber 2n = 2x = 14
77RILs Gy14 × PI 183967 995 SSRs 7 0.58 573 JoinMap Ren et al. 2009
113 F8 RILs PI197088-1 (highly resistant 120 SSRs and 6 SCARs 8 4.96 625.7 MAPMAKER/ Fukino et al. 2008b
to Podosphaera xanthii) × EXP3.0
Santou (susceptible)
130 F2 S94 (Northern China open- 116 SRAPs, 33 RAPDs, 7 5.9 1016 MAPMAKER/ Yuan et al. 2008b
field type) × S06 11 SSRs, 9 SCARs, 3 EXP3.0
(greenhouse European type) ISSRs, and 1 STS
224 RILs S94 (Northern China open- 206 SRAPs, 22 SSRs, 7 3.9 1005.9 MAPMAKER/ Yuan et al. 2008a
field type) × S06 25 SCARs, 1 STS and 3 EXP3.0
(greenhouse European type) phenotypic traits

112 F2 PW0832 (flood tolerant) × 30 ISSRs, 32 SRAPs 7 16 992.2 MAPMAKER/ Yeboah et al. 2007
PW0801 (flood sensitive) EXP3.0
138 F2 S06 (strong lateral 92 SRAPs 7 12.6 1164.2 MAPMAKER/ Wang et al. 2005
branching) × S52 (no lateral EXP3.0
branching)
Watermelon 2n = 2x = 22
98 testcross [Griffin 14113 (C. lanatus var. 71 AFLPs, 93 SRAPs, 19 5.8 1976 JoinMap 3.0 Levi et al. 2006
progeny citroide) × New Hampshire 14 SSRs, 151 RAPDs,
Midget (C. lanatus var. 30 ISSRs
lanatus)] × PI 386015 (C.
colocynthis)

117 F8 RILs 97103 (high total soluble 150 AFLPs 17 8.3 1240.2 Yi et al. 2004
solids content; C. lanatus
var. lanatus) × PI 296341
(Fusarium wilt resistant –
races 0, 1, and 2; C. lanatus
var. citroides)
117 F8 RILs 97103 (high total soluble 87 RAPDs, 13 ISSRs, 4 15 11.54 1027.5 Zhang et al. 2004
solids content; C. lanatus SCARs
var. lanatus) × PI 296341
(Fusarium wilt resistant –
races 0, 1, and 2; C. lanatus
var. citroides)
Cucurbita 2n = 2x = 40
94 F2 Waltham Butternut (WB) 205 SSRs and 2 27 7 1445.4 MAPMAKER/EXP Gong et al. 2008a
(from each × Nigerian Local (NL) and phenotypic traits 3.0 and JoinMap
mapping ZHOU (hull-less) × WB
population;
merged
map)
Table 6-1 contd....

Table 6-1 contd....
204

(cM)
92 F2 Lady Godiva (a US oil- 178 SSRs, 244 AFLPs, 20 2.9 1936 JoinMap Gong et al. 2008b
pumpkin; C. pepo subsp. 230 RAPDs, 5 SCARs,
pepo) × Bianco Friulano (an 2 phenotypic traits
Italian crookneck; C. pepo
subsp. ovifera)
92 F2 SZG1 (oil pumpkin; C. pepo 247 RAPDs, 82 AFLPs, 21 6.4 2140 MAPMAKER/ Zraidi et al. 2007
subsp. pepo) × True French 3 (major) EXP3.0
Resistant (zucchini; resistant SSRs, 1 phenotypic
to Zucchini Yellow Mosaic trait
Virus)
92 F2 Lady Godiva (a US oil- 196 RAPDs, 125 21 6.9 2234 MAPMAKER/ Zraidi et al. 2007
pumpkin; C. pepo subsp. AFLPs, 2 known genes (major) EXP3.0
pepo) × Bianco Friulano (an
Italian crookneck; C. pepo
subsp. ovifera)
Notes: 1. Only original maps using molecular markers published in refereed journals after 2004 are included. 2. Most maps were constructed with
MapMaker (Lander et al. 1987) for PC or Mac. Some used JoinMap (Stam 1993) and some used both. For example, Gonzalo et al. (2005) used JoinMap
3.0 to test marker segregation while using MapMaker 3.0 to construct the map.

6.2 Evolution of Molecular Markers

Early studies of molecular markers in cucurbits focused on a technique
that identifies specific regions of a genome using restriction fragment
length polymorphisms (RFLPs) developed in the early 1980s (Botstein et
al. 1980). Genomic DNA from individual samples is cleaved with DNA
sequence-specific restriction endonucleases, size fractionated in agarose gel
electrophoresis, and transferred to nylon membranes. The nylon membranes
are hybridized with labeled, low-copy-number genomic DNA clones or
cDNA clones (these are called probes). RFLPs specific to each sample are
identified by autoradiography. Shattuck-Eidens et al. (1990) and Neuhausen
(1992) evaluated RFLPs in melon and concluded that polymorphism at the
DNA level within melon is relatively low. This is evidenced by that fact
that only 53 of 162 tested probes differentiated at least one of seven melon
varieties (Neuhausen 1992). Nonetheless, 34 RFLPs were placed in the first
molecular map of melon (Baudracco-Arnas and Pitrat 1996). Similarly, 61
and 31 RFLPs were placed in the inter- and intra-specific maps of cucumber
(Kennard et al. 1994). In total, 13 of the 42 maps in Table 6-1 used RFLP
markers although it is not used in any of the Cucurbita maps.
Random amplified polymorphic DNA (RAPD) is the second molecular
marker system developed for genetic mapping especially for plants
(Williams et al. 1990). The technique used polymerase chain reaction (PCR)
to amplify arbitrary genomic regions with single random 10-base primers.
This allows amplification and identification of relatively short regions in
which the primer sequence exists as an inverted repeat (Williams et al.
1990). Since this is the easiest marker system to use, it has been employed
in 24 of the 42 maps including eight maps that either employed RAPDs
exclusively or RAPDs account for majority of the markers mapped (Table
6-1). But RAPD markers can not be easily transferred between populations
and a new genetic map must be prepared for each segregating population.
The technique is also inherently subject to reproducibility problems (Jones
et al. 1997), which significantly reduces its utility as a genetic marker.
A more efficient marker system named amplified fragment length
polymorphism (AFLP) was developed shortly after RAPD (Vos et al.
1995). AFLP involves three steps: (a) restriction of genomic DNA using
two restriction endonucleases (usually a rare cutter and a frequent cutter),
(b) ligation of specific double-stranded DNA adapters to the restriction
fragments to function as priming sites, (c) amplification of fragments using
two primers (one is radioactively labeled) complementary to the ligated
adapters, and (d) the resulting PCR products are separated on polyacrilamide
gel and exposed to the X-ray film. The primers usually include one or two
additional nucleotides at the 3′ end designed to selectively match genomic
DNA sequences flanked by the adapters in order to generate specific

fragment sets that perfectly match the adapters and adjacent nucleotide(s)
(Vos et al. 1995). Although it is a portable and efficient marker system and
has been used in merging maps (Périn et al. 2002), it is included in 16 of
the 42 maps presumably because of its technical sophistication. Three maps
were constructed mostly with AFLP markers (Wang et al. 1997; Park et al.
2000; Yi et al. 2004).
Both RAPD and AFLP can be amplified by PCR, facilitating rapid large-
scale genotyping. However, these markers are usually genotype-specific
and are dominant, which limit integration of independently constructed
genetic maps. Microsatellite or simple sequence repeat (SSR) markers, on
the other hand, are hypervariable, multiallelic, often codominant, evenly
distributed through the genome, highly reproducible and they can be used
as anchor points for molecular linkage group comparisons and map merging
(Danin-Poleg et al. 2000; Gonzalo et al. 2005). For these reasons, SSRs have
been used more frequently in the recently developed maps, including
four maps almost exclusively constructed with SSR markers (Fukino et al.
2008a, b; Gong et al. 2008a; Ren et al. 2009). This may explain that 21 of the
42 maps contain SSR markers. SSR markers can be designed from cucurbit
genomic (gSSRs; Katzir et al. 1996; Jarret et al. 1997; Danin-Poleg et al. 2001;
Fazio et al. 2002; Chiba et al. 2003; Fukino et al. 2008; Gong et al. 2008b; Ren
et al. 2009) or expressed sequence tags (EST, also denominated EST-SSRs)
sequences (Katzir et al. 1996; Fernandez- Silva et al. 2008). EST-SSRs have
several advantages: (i) development costs are relatively low; (ii) they are
related to genes, being functional markers that can be used as candidate
genes to study their association with phenotypic variation, (iii) the flanking
sequences are more likely to be conserved among close or distant species
than those derived from genomic sequences, making their use as markers
for comparative mapping easier (Katzir et al. 1996; Fernandez- Silva et
al. 2008). The use of next generation sequencing significantly speed up
SSR discovery in cucurbits. Cavagnaro et al. (2010) resequenced the Gy14
cucumber genome and detected a total of 112,073 perfect repeats and from
these developed 83,000 SSRs for mapping the cucumber genome (see also
Chapter 11).
The latest marker system is called single nucleotide polymorphisms
(SNPs), which are most abundant in the plant genome (Brookes 1999;
Deleu et al. 2009). For example, in a survey of a 15 kb melon sequence
between Piel de sapo and PI 161375, there is one SNP for every 441 bp on
average (Morales et al. 2004). This would make it an ideal marker system to
saturate a map. For example, a bovine genetic map has been created with
6,769 SNPs that mapped to 3,078 unique positions with average distance
of 1.01 cM between these positions (Arias et al. 2009). In plants, Sato et al.
(2009) mapped 1,717 EST sequences as SNP markers to create an ultra-high-
density barley genetic map. But because of the expense of high-throughput

technology for SNP discovery and detection, they have not been widely
used in cucurbits (Morales et al. 2004). However, four maps in Table 6-1
did employ SNP markers including the one in melon that used 115 SNPs
(Harel-Beja et al. 2010).
It is clear from Table 6-1 that genetic map construction typically requires
linkage analyses of hundreds of Mendelian loci (molecular markers and
phenotypic traits), using a relatively large mapping population that is in
linkage disequilibrium. It can be expensive and time-consuming especially
when the objective is to obtain a high-density map or to incorporate a large
number of markers into an existing map. Vision et al. (2000) proposed a two-
step strategy (called “bin mapping”) that uses a much smaller population
size to solve the problem. First, a mapping population of standard size
(60–250; see Table 6-1) is used to construct a saturated framework map,
and second, new markers are added to this map with lower resolution
using a selected subset of highly informative plants (the bin set) from the
mapping population. The objective is to lower the cost of genotyping new
markers with a minimal loss of resolution. The optimal bin set of a given
size has the maximum possible number of breakpoints evenly spaced
throughout the genome, resulting in a high number of small bins of uniform
size (Howad et al. 2005). Genetic mapping using this strategy have been
carried out in melon (Fernandez-Silva et al. 2008; Moreno et al. 2008). A
bin-map was constructed including 80 RFLPs, 212 SSRs, 3 SNPs and the
Nsv locus, distributed in 122 bins with an average bin length of 10.2 cM
and a maximum bin length of 33 cM. Map density was 4.2 cM/marker or
5.9 cM/SSR (Fernandez-Silva et al. 2008).
In addition to markers developed from genomic sequences, EST
sequences have been increasingly used in cucurbit genetic mapping. ESTs
are partial cDNA sequences from expressed genes. Since genetic mapping of
ESTs establishes locations of known genes, a high-density EST map provides
the foundation for map-based genome analysis such as map-based cloning,
gene tagging and comparative mapping. Fernandez-Silva et al. (2008)
developed 126 SSR markers from about 30,000 melon ESTs and applied these
in bin mapping of the melon genome. Similarly, Levi et al. (2008) identified
40 SSRs from 4,700 non-redundant watermelon fruit ESTs. On the other
hand, half of the markers (187 of 370) in the latest melon map developed
by Harel-Beja et al. (2010) are derived from melon fruit EST sequences. In
addition to EST-SSR markers, EST-derived sequence-tagged site markers
(eSTS) have been extensively explored in other plants. For example, Sato
et al. (2009) designed 10,366 eSTS primer sets based on analysis of 60,000
barley ESTs. Of the 10,366 sets, 7,700 amplified useful products, 3,975 of
these detected polymorphism between the mapping parents and 2,890
eSTS markers were mapped to the barley genome. Significant cucurbit EST
resources have been generated. There are 3,5547 melon ESTs, 7,757 cucumber

ESTs, 7,891 watermelon ESTs and 879 Cucurbita ESTs already deposited in
GenBank. The International Cucurbit Genomics Initiative (www.icugi.org)
aims to produce additional 100,000 melon ESTs. And effort to map ESTs has
already begun (Harel-Beja et al. 2010). It will be a matter of time before EST
mapping in cucurbits catches up those in other crop plants.
A marker system also utilizing EST is an adapted AFLP named cDNA-
AFLP. The technique is similar to the normal AFLP but uses double-stranded
cDNA derived from mRNA as a template instead of genomic DNA. The
obtained cDNA–AFLP fragments called transcriptome derived fragments
(TDFs; Bachem et al. 1996) target coding regions of the genome, which can
be used to construct a genetic map (Brugmans et al. 2002; Li et al. 2003;
Ritter et al. 2008). For example, the potato map created by Ritter et al.
(2008) contains nearly 700 TDFs. Since TDFs are expressed genes, a map so
created can help identify genes involved in, or controlling, various biological
processes ranging from development to responses to environmental cues.
But because TDFs represent mainly highly expressed house-keeping genes
(Ritter et al. 2008), polymorphisms detected by TDFs are due to genetically
different gene expression, not due to differential expression caused by
environmental/physiological factors (i.e., plants at slightly different
physiological stages or grown in slightly different microenvironments).
Based on results from cucumber and potato, the vast majority of absence/
presence polymorphisms in transcripts are caused by genomic sequence
polymorphisms (Brugmans et al. 2002; Bae et al. 2006). The technique may
also be useful for gene tagging using bulked segregant analysis (BSA;
Michelmore et al. 1991) in addition to the whole-genome mapping, which
has not been done in cucurbits.
All the marker systems except cDNA-AFLP discussed above have been
used in cucurbit genome mapping. If genetic maps are to be populated with
markers representing coding regions, then markers developed from ESTs
and cDNAs should be considered. Since a number of maps are already
using EST-based markers, it is time to consider cDNA-AFLP to explore its
utility in cucurbits. Additional marker system such as sequence related
amplified polymorphism (SRAP; Li and Quiros 2001), inter-microsatellite
amplification or inter-SSR (IMA or ISSR; Zietkiewicz et al. 1994) and
sequence characterized amplified regions (SCARs; Paran and Michelmore
1993) are used in six, 12 and nine maps, respectively (Table 6-1) and are not
discussed further in this chapter. A recent review of these marker systems
can be found in Semagn et al. (2006).
6.3 Molecular Maps

Cucurbit molecular maps have been developed in the last 20 years with
increased marker density and more sophisticated marker systems. Details

of all recent molecular maps are listed in Table 6-1. (see Ezura and Fukino
2009 and Wang et al. 2006 for earlier maps in cucurbits). Each map was
developed with a particular mapping population (backcross, F2 or its
derivative RILs/DHLs) from two chosen parental lines, which usually
possess two different sets of phenotypes. For example, one parent may
have a number of desirable agronomic traits but lacks resistance to multiple
diseases while the other parent is completely opposite. Maps created from
such parents are useful to identify markers linked to these traits, which may
eventually lead to cloning of the underlying genes. The cloned genes can
be used in a breeding program either as a tool in marker-assisted selection
(MAS; see Chapter 7) or through genetic engineering.
Tremendous efforts by various research groups have been devoted
to mapping of the cucurbit genomes, which are small and comparable
to that of rice in size (Arumuganathan and Earle 1991). In total, 9,324
markers including 80 phenotypic traits have been mapped to the cucurbit
genomes. Melon genome is the most mapped with 3,444 markers, followed
by cucumber, Cucurbita, and watermelon (Table 6-2). RAPD, AFLP, SSR
and RFLP account for over 85% of the mapped markers. While RAPD is
the predominant marker system used in watermelon and Cucurbita, AFLP
was predominantly used in melon and SSR was predominantly used in
cucumber. On the other hand, RFLP and SNP were mostly used in melon
and SRAP was mostly used in cucumber. It is possible that some of these
markers are redundant and were used in different maps so the number of
unique markers may be lower. In addition, the actual number of mapped
phenotypic traits may be higher because in a lot of cases a particular trait was
mapped using bulked segregant analysis (Michelmore et al. 1991), which
may not be included in the map of the whole genome. This is especially
true when the purpose of mapping was to positionally clone the gene
underlying the mapped trait, not the whole genome. For example, Fom-2
was not included in the map by Wang et al. (1997). Instead, it was further
mapped with additional markers using BSA (Wang et al. 2000), which
eventually led to cloning of the gene (Joobeur et al. 2004).
There is great interest to create a reference map for each cucurbit
species. Markers from such a map may be used to facilitate mapping/
cloning of new genes. This is especially useful if a map is high-density with
transferable markers such as SSR. Ren et al. (2009) tested 995 cucumber
SSRs mapped in the cucumber genome in other cucurbits. Among them,
49, 26 and 22% of the cucumber SSRs amplified PCR products in melon,
watermelon and pumpkin, respectively with polymorphism rate at 39.6, 46.5
and 54.8%. Obviously, these markers can be used to enhance the mapping
efforts in these cucurbits. An example of Cucurbita genetic map is shown
in Fig. 6-1.

Figure 6-1 contd....




Figure 6-1 A genetic map of Cucurbita pepo. The new map contains 659 loci: 178 SSR, 244
AFLP, 230 RAPD, five SCAR markers, and two morphological traits (h and B) (From Gong
et al. 2008b).

Table 6-2 Summary of cucurbit maps: number of markers used in cucurbit genome mapping*.
RAPD AFLP SSR RFLP SRAP ISSR SNP SCAR STS PT** Total
Melon 436 1,212 741 616 152 101 121 9 0 56 3,444
Cucumber 247 577 1,162 109 446 33 1 64 2 17 2,658
Watermelon 1,069 221 14 54 93 95 0 6 0 2 1,554
Cucurbita 821 451 386 0 0 0 0 5 0 5 1,668
Total 2,573 2,461 2,303 779 691 229 122 84 2 80 9,324
Note: *Isozyme marker is not included in the table. The numbers include those in earlier maps not listed in Table 6-3. **PT-phenotypic trait.

6.4 Map-based Cloning

One of the utilities of a genetic map is to clone a gene underlying a
phenotypic trait, which is known as map-based cloning. The first step for
this purpose is to identify a molecular marker that lies close to the gene of
interest. Markers in a genetic map that are close to the gene (several cM
away) can be used. The next step is to saturate the region with additional
markers that rarely show recombination with the gene. The size of mapping
population could increase to a large number of individuals. The next step
is to screen a large insert BAC library for genomic clones that hybridize to
the tightly linked markers. Once markers nearing or flanking the gene are
found located on the same clone, one can now determine where the gene
resides. Chromosome walking is used to delimit the gene. New markers
developed from the end sequences of the BAC clones are used to screen
additional segregating population (usually over 1,000 individuals). The goal
is to find a set of markers that co-segregate with/flank the gene of interest.
If two markers are known to flank the gene, DNA fragments between the
flanking markers are sequenced to identify candidate genes. New transgenic
plants are created by transforming with a single open-reading frame (ORF).
Once an ORF is shown to confer the phenotype, then the gene is considered
cloned and additional analysis is carried out.
The first gene in cucurbit cloned by map-based cloning approach is
melon Fom-2 (Joobeur et al. 2004), which confers resistance the races 0 and
1 of Fusarium oxysporum f.sp. melonis. To clone Fom-2, markers presented in
Wang et al. (2000) were used to fine-map the region with 159 RILs derived
from Védrantais × PI 161375. SSR154 (FM) and STS178 (AM) were found to
flank Fom-2 at 0.7 and 0.6 cM, respectively. Screening a melon BAC library
(Luo et al. 2001) with ACT/CAT1 marker (Wang et al. 2000) identified 23
BAC clones that belonged to the same contig. Two SSR markers (SSR138 and
SSR180) were derived from the BAC end sequences. SSR138 co-segregated
with Fom-2 while SSR180 was 0.2 cM away. Screening additional populations
with SSR154 and STS178 revealed 15 recombinant plants. Analysis of the
15 recombinants confirmed that Fom-2 was located between SSR180 and
STS178. No additional recombinants were found between SSR138 and Fom-2.
Chromosome walking was initiated with SSR154 and STS178 and SSR138
markers. All identified clones belonged to the same contig. STS411 and
SSR184 derived from end sequences of newly identified BAC clones were
found to flank Fom-2 at two and one recombination events, respectively.
Hybridization analysis indicated that STS411 and SSR184 were located on
two BAC clones that overlapped by 32 kb. Sequencing of the two BAC clones
identified all the markers used and additional markers were developed from
the sequenced BACs to further delimit Fom-2. The recombination event
located between Fom-2 and SSR184 was found within a 1.28 kb interval

between newly derived markers STS296 and SSR451. The two recombination
events found between STS411 and Fom-2 were confined to 5.5 kb between
STS411 and another new marker SSR430. Thus Fom-2 was assigned to 75
kb-size interval between STS411 and STS296. Three putative genes were
found in this 75 kb interval and only two were found to be complete. One
was similar to Arabidopsis AtCPSF73-II, which was primarily expressed
in flower tissue (Xu et al. 2004). The other was highly homologous to
previously characterized NBS-LRR class of resistance genes such as I2 in
tomato and was thus designed as Fom-2 (Joobeur et al. 2004).
Fom-2 was also cloned by Pitrat and colleagues (Pech et al. 2007) using
segregating populatins from Védrantais × PI 161375. A BAC contig was
built using clones identified from the MR-1 BAC library (Luo et al. 2001)
based on linked markers. Sequencing identified three candidate genes, one
of which shares high homology to the NBS-LRR class of resistance genes.
This gene is 3 kb in length and encodes an intronless protein of 1,073 amino
acids. Sequencing analysis also revealed variation in LRR region of the
gene between resistant MR-1 and susceptible Védrantais, AY and Durango
varieties (Pech et al. 2007).
The melon Vat gene is the second cloned cucurbit gene that mediates
resistance to the melon/cotton aphid Aphis gossypii. Two segregating
populations were used to clone the Vat gene (Pauquet et al. 2004). A
population of 200 RILs from Védrantais × PI 161375 was used to first fine-
map the region and another population of 6,000 backcross progeny from
(Védrantais × PI 161375) × Védrantais was screened for recombination
events within 1.7 cM delimited by markers flanking Vat to fine-map the
region further. Markers tightly linked to the gene were used to screen a
PI 161375 BAC library. Markers were generated from end sequences of
the identified BAC clones and fine-mapping of these markers using the
backcross population further delimited a physical interval that contains
a single gene of 5.9 kb. The gene has five exons and four introns and
encodes a protein which belongs to the coiled coil (CC)—NBS—LRR family.
Transferring an 11-kb genomic fragment carrying Vat and its own promoter
into the susceptible Védrantais confers to resistance to aphids (Pauquet et
al. 2004).
The recessive nsv gene, which confers complete resistance to melon
necrotic spot virus (MNSV), is another gene cloned by the map-based
cloning approach in cucurbits, except that the cloning process also took
advantage of microsynteny between melon and Arabidopsis. Using two
mapping population of 408 F2 (PI 161375 × Piel de sapo) and 2,727 BC1
([Védrantais × PI 161375] × PI 161375), nsv was mapped in a 3.2 cM region
flanked by CAPS markers M29 and M132 (Morales et al. 2005). Additional
markers were developed from BAC clones identified by linked markers
and one of these markers (52K20sp6) cosegregated with nsv in the mapping

populations. A single BAC clone of 100 kb was thus identified that covered
a genetic distance of 0.73 cM (Morales et al. 2005). Using microsynteny
between melon and Arabidopsis (van Leeuwen et al. 2003), markers linked
to nsv (including 1R3 and 1L3) were compared to the Arabidopsis sequence
by BLAST analysis, which identified a region in Arabidopsis chromosome
4 as the most probable nsv syntenic region (Nieto et al. 2006). 1R3 and 1L3
mapped within a 182-kb Arabidopsis genomic region located between genes
At4g17770 and At4g18100. Among the genes in the region was eIF4E at
position 18040. Degenerate PCR primers designed based on eIF4E sequences
from other species amplified a product of 1.9 kb which was sequenced and
was used to design CAPS marker (M-CmeIF4E) primers from the resistant
and susceptible parents. M-CmeIF4E cosegregated with nsv among more
than 3,000 progeny of the F2 and BC1. Full length Cm-eIF4E cDNA from
the homozygous dominant (Védrantais—susceptible) and homozygous
recessive (PI 161375—resistant) parents were sequenced. The cDNAs were
1,153 bp in length with a 5’-UTR of 122 bp, a coding region of 708 bp and
a 3’-UTR of 323 bp. Sequence comparison of Cm-eIF4E proteins from the
resistant (PI) and the susceptible (Ved) cultivars revealed a single amino acid
substitution at position 228. Védrantais carries a Histidine and PI 161375
carries a Leucine. A single nucleotide change lead to this amino acid change
in the protein. A molecular marker derived from the SNP cosegregated with
nsv in the mapping population of more than 3,000 segregating plants and
differentiated seven resistant genotypes from six susceptible ones (Nieto
et al. 2006).
The first non-resistance gene cloned by map-based cloning is the
gene andromonoecious (a), which together with gynoecious (g) governs sex
determination in melon. Monoecious (A_G_) and andromonoecious (aaG_)
plants bear male flowers on the main stem and female or hermaphrodite
flowers on axillary branches while gynoecious (AAgg) and hermaphrodite
(aagg) plants only produce female and hermaphrodite flowers (Kenigsbuch
and Cohen 1990). But these patterns can be modified by hormones such
as ethylene and environmental factors (Byers et al. 1972). To fine-map a,
Boualem et al. (2008) crossed a monoecious melon cultivar PI124112 (AAGG)
and an andromonoecious cultivar Védrantais (aaGG), and backcrossed
the resultant F 1 plants with Védrantais. To identify plants carrying
recombination events linked to a locus, DNA samples were extracted from
7,000 plants from the backcross population and analyzed with the a locus
flanking markers M64 and M47. The sexual phenotype was determined for
all the 235 recombinant plants. The fine-mapping and ensued chromosome
walking delimited the a locus to a single BAC clone that had seven genes.
The two closest flanking markers (L41 and R5) were used to identify a
14 kb region containing a single gene encoding a 1-aminocyclopropane-
1-carboylic acid synthase (ACS) designated CmACS-7. ACS catalyzes a

rate-limiting step in ethylene biosynthesis pathway. Mutation analysis

through TILLING (McCallum et al. 2000) confirmed the role of the gene in
sex determination in melon (Boualem et al. 2008).
Cloning of another sex determination gene (M/m) is underway in
cucumber. Li et al. (2008) identified eight markers linked to the M/m
locus. Among them, the two closely linked SRAP markers ME1EM26 and
ME1EM23 flank the gene while ME8SA7 cosegregated with it as mapped
by an F2 population of 167 progeny. A SCAR marker (S_ME1EM23) was
developed from BAC clones identified by the above markers. The gene has
been fine-mapped using a segregating population of 900 progeny (670 F2
individuals and 230 BC1 plants). It is now flanked by ME1EM26 at 5.4 cM
and ME1EM23 at 0.7 cM and cosegregates with ME8SA7 (Li et al. 2008).
All the four genes cloned are from melon. This reflects the fact that
melon has the most mapped genome, as described above although a high-
density map has been recently developed for cucumber (Ren et al. 2009)
and its genome has been sequenced (Huang et al. 2009). In terms of total
growing area and production, watermelon and cucumber are economically
more important. Research in these cucurbits may catch up with melon and
other crop plants hopefully with increased funding.
6.5 Concluding Remarks

Cucurbit mapping did not start in earnest until 1990s when genetic
mapping of other crop plants was already well established. New marker
technologies such as AFLP and SSR greatly facilitated cucurbit mapping in
recent years while SNP is just being adopted. High-density maps have now
been developed for all major cucurbits. It is expected that future efforts will
focus on comparative mapping of different cucurbit genomes, gene tagging
and cloning of more genes, especially those underlying quantitative traits.
The cloned or tagged genes may be used to develop markers for marker-
assisted selection or in direct gene transfer in breeding programs. The
cucurbit community should also think about posting genetic maps with
easily transferable markers such as RFLP (as probes) and SSR/ISSR/STS/
SCAR (as primers or primer sequences) online in the NCBI website where
other plant maps are available (http: //www.ncbi.nlm.nih.gov/genomes/
PLANTS/PlantList.html) or in the website of International Cucurbit
Genomics Initiative (ICuGI) (www.icugi.org) so that these maps can be
frequently updated and be available to other investigators. There are over
1,300 SSR and 779 RFLP markers used in cucurbit mapping (Table 6-2). Some
of these markers may be transferable to other cucurbits. Although reports
on such transferability among cucurbits is mixed (see Gong et al. 2008b),
Watcharawongpaiboon and Chunwongse (2008) tested 20 cucumber SSR
primer pairs and found 13 amplified products in melon, 11 in bitter gourd,

10 in watermelon and seven in pumpkin. In addition, there are more SSR

markers that have been developed but not included in cucurbit map yet
such as those reported by Levi et al. (2008).
Genetic mapping will be even more important when high-throughput
sequencing technology becomes more accessible. One such technology, the
Roche/454 Titanium platform, has been used to sequence the rice genome
(Rounsley et al. 2009) and a similar technology has been used to sequence
the cucumber genome (Huang et al. 2009). The technology produces a
large number sequencing reads which averaged 367 bp per read in rice.
Coupled with high-density genetic map and large-insert genomic library
already in place in cucurbits, this technology may help produce the first
sequenced cucurbit genome which will be tremendously valuable in genetic
improvement of this economically important gourd family.
Acknowledgement
I thank Hugo E. Cuevas for his contribution to Table 6-1.
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7
Mapping and Molecular Breeding
of Monogenic Traits
Yi-Hong Wang
ABSTRACT
Molecular breeding using tightly linked genetic markers has been
widely adopted. The new tool has offered the breeder unprecedented
advantages such as efficiency and time-saving. Like in other crop
plants, these markers have been extensively studied in cucurbits for
monogenic traits. During the last decade, markers linked to or genes
controlling 28 traits of agronomic importance have been identified
and are listed in this chapter. Based on germplasm characterization
results, markers more closely linked to target genes are more robust
in phenotype prediction. In an extreme case, a marker converted from
the a gene itself, which determines andromonoecy, correctly predicted
sex types of all 497 melon varieties. This further indicates that breeding
efficiency can be dramatically increased if tightly linked markers are
used to select for the underlying phenotypes.
Keywords: cucurbits, DNA markers, linkage, bulked segregant analysis,
trait
7.1 The Importance of Gene Targeting

Targeting or tagging of a gene underlying a particular trait of interest serves
two purposes. The first is that such a tagged gene can be cloned and its
function revealed through its role in the phenotype. This is map-based
cloning when it is initiated from tightly linked markers. Gene cloning helps
us understand how a particular trait is influenced by genes and how these
genes contribute to the development of the trait. The cloned gene can be
Department of Renewable Resources, University of Louisiana at Lafayette, Lafayette, LA,

USA; e-mail: yxw9887@louisiana.edu

introduced into other plants if trait transfer is so desired through either

direct gene transfer or molecular breeding. For example, the gene that
determines andromonoecy (a) in melon has been cloned (Boualem et al.
2008). Sex determination in melon is determined by the interplay of a and
gynoecious (g). Monoecious and andromonoecious plants have genotypes
of A_G_ and aaG_, respectively while AAgg is gynoecious and aagg is
hermaphrodite (Poole and Grimball 1939; Keningsbuch and Cohen 1990).
External factors, such as ethylene, can modify sex expression. Actually, the
cloned a gene encodes for a 1-aminocyclopropane-1-carboxylic acid sythase
(CmACS-7). The enzyme catalyzes the first committed and rate-limiting step
in ethylene biosynthesis in plants. The gene is specifically expressed in the
carpel primordia of female and hermaphrodite but not male flowers during
early stages of flower development. This leads to ethylene production,
which affects the development of the stamina in female flowers but is not
required for carpel development (Boualem et al. 2008).
The second purpose is that if a gene is tagged with tightly linked or
cosegregating DNA markers or if markers are developed from a cloned gene,
these markers can be potentially used in molecular breeding as a tool in
marker-assisted selection (MAS). MAS provides significant advantages to
traditional phenotypic screening because it directly selects for genotypes.
MAS procedure is rapid, effective, relatively inexpensive and is not limited
by environmental conditions (Robbins and Staub 2009; Behera et al. 2010),
since it can be performed off-season using DNA already isolated. And
just as important, it allows simultaneous screening for multiple traits if
markers linked to these traits are available. To demonstrate how powerful
this can be, we will again use the a gene as an example. When 497 melon
genotypes were typed for the a gene, all the 146 monoecious and three
gynoecious genotypes contained the dominant A57 allele while all 347
andromonoecious and hermaphrodite genotypes contained the recessive V57
allele (Boualem et al. 2008). This clearly indicates that by typing this locus
alone using DNA isolated from seedlings, one can easily determine whether
a particular plant is monoecious or andromonoecious without growing the
plants to the flowering stage. This is significant because the breeder can
now directly select for the monoecy genotype, which is an important goal
for melon breeding. Monoecious plants used in production of F1 hybrid,
which produces higher quality fruits, do not require the labor-intensive
hand emasculation (Périn et al. 2002).
MAS can also reduce ambiguity in phenotyping. In melon, resistance to
powdery mildew is controlled by two dominant genes in the variety PMAR
5, both of which are necessary for complete resistance (Fukino et al. 2004).
When lines are evaluated under some conditions, one resistance gene is
sufficient for complete resistance, and individuals with one resistance gene
cannot be distinguished from those with two (Fukino et al. 2008). This makes

Mapping and Molecular Breeding of Monogenic Traits 227
it impossible to pyramid both genes based on phenotype alone. But use of

linked markers will obviate this problem (Fukino et al. 2008). Because of
this, MAS can effectively reduce the cost of phenotyping and selection in
plant breeding. In cucumber, MAS has been shown to be very effective in
increasing multiple lateral branching and consequently fruit yield (Robbins
and Staub 2009). Due to these reasons, studies are being actively conducted
to identify markers linked to genes of interest and the more recent works
are summarized in Table 7-1.
7.2 Employment of BSA for Gene Tagging

Most monogenes or major quantitative trait loci (QTLs) can be tagged
using a technique called bulked segregant analysis (BSA) first developed
by Michelmore et al. (1991) to identify random amplified polymorphic
DNA (RAPD) markers linked to a disease resistance gene in lettuce. In
this technique, marker patterns from two DNA bulks, each comprising
DNA from individuals exhibiting the extreme phenotypes (i.e., high/low
or resistant/susceptible) of a particular trait in a segregating population
are compared. The two bulks have completely random genotype for most
of the genome other than the region around the gene controlling the trait
of interest. So the presence of polymorphism between the marker patterns
of the two bulks is expected only when they are genetically linked to the
underlying gene (Giovannoni et al. 1991). To construct the bulks, 10–20
individuals are commonly included in each bulk, mostly in the form of equal
amount of DNA. For example, Tezuka et al. (2009) constructed two resistant
and two susceptible bulks each with 12 plants in an effort to identify markers
linked to Fom-1, the gene that confers resistance to the melon Fusarium wilt
caused by the fungus Fusarium oxysporum f.sp. melonis race 2. The bulks were
screened with amplified fragment length polymorphism (AFLP) markers
detected by using ethidium bromide-stained polyacrylaminde gel. Two
markers were found to be tightly linked to Fom-1: one was at 0.5 cM and
the other was cosegregating with the gene in a population of 125 F2 plants
(Tezuka et al. 2009).
But AFLP technique could be too sensitive for BSA screening since it
might result in artefactual polymorphism. Also working on tagging Fom-1,
Brotman et al. (2005) used four resistant and four susceptible individual
samples for AFLP analysis, instead of constructing two bulks, and found
two closely linked markers out of 184 primer combinations that were
screened. Marker ATC/CAT497 is a 497 bp-fragment from the PI 414723
parent, while marker ACA/CAT90 is a 90-bp fragment amplified from the
Védrantais parent. The markers cosegregate at a distance of 1.7 cM from
the Fom-1 gene (Table 7-1). However, adjusting the number of plants in a
bulk may reduce the problem of artifacts (Tezuka et al. 2009).

228
Table 7-1 Cucurbit genes identified by molecular markers.
Gene Locus Parents Population Linked Marker/Distance (cM) Marker Type Reference

Melon
Gummy stem PI 420145 (resistant) × P1 136170 F2 E-TG/M-CTC200/2.0 AFLP Wolukau et al. 2009
blight (susceptible)
cmv1 PI 161375/SC12-1 (resistant to 171 F2 In a 2.2 cM interval between CMN61_44 SSR Essafi et al. 2009
cucumber mosaic virus) × Piel de AND CMN21_55
Sapo (susceptible)
Pm-1 [AF426-S × AF426-R(Pm-1)] × 143 BC1 M75/H35_155/4.9 AFLP Teixeira et al. 2008
AF426-S
powdery TGR-1551 (resistant to races 1, 295 F2 E38M82/dCAPS/5.7 AFLP/CAPS Yuste-Lisbona et
mildew 2, and 5 of Podosphaera xanthii) al. 2008
resistance × Bola de oro (susceptible to the
races)
powdery 1A151 (resistant) × Hengjin RRS F2/BC RAPD-S329/6.81 RAPD Wang et al. 2005
mildew (susceptible)
resistance
Prv1 [AF426prv1 (susceptible) × 197 BC1 EK190/0.5 AFLP Teixeira and
AF426Prv1 (resistant to papaya Camargo 2006
ringspot virus type W)] ×
AF426prv1
a [CM (monoecious) × CA2] × CA2 530 BC1 M3/5.5 (the gene has now been cloned) AFLP/SCAR Noguera et al. 2005
(andromonoecious)
Fom-1 P11 (fom-1) × MR-1 (Fom-1) 125 F2 C-MRGH13/0.8; S-MARGH9/0.6; STS/CAPS Tezuka et al. 2010
C-MARGH12/0.4; CAPS3/1.2
Fom-1 P11 (fom-1) × MR-1 (Fom-1) 125 F2 C-TCG/GGT-400/4.9; CAPS2/4.9; GTC/ AFLP/STS Tezuka et al. 2009
ATG-260/0.6; S-TAG/GCC-470/0
Fom-1 Charentais-Fom1 (Fom-1) × TGR- 116 F2 SB17645/3.5; SV01574/4.0 (both on the same RAPD/SCAR Oumouloud et al.
1551 side) 2008

Fom-1 Védrantais (Fom-1) × PI 95 RILs 62-CAPS/0.7 CAPS Brotman et al. 2005
161375
Fom-1 Védrantais (Fom-1) × PI 62 RILs ATC/CAT497 and ACA/CAT90/1.7; NBS1- AFLP/CAPS Brotman et al. 2005
414723 (Prv) CAPS/2.8; 62-CAPS/6.3
ms-3 ms-3 (male-sterile) × TAM Dulce 141 F2 OAM08.650/2.1 Park et al. 2004
(male-fertile)
Cucumber
Target leaf Q5 (resistant) × P57-1 241 F2 EST-SSR/2.9 SSR Wang et al. 2010
spot (susceptible)
de Gy-7 × H-19 CSWCTT14b/1.4; SSR13251/4.2 SSR Weng et al. 2010

ll Gy-7 × H-19 SSR02355/4.6; SSR03940/3.6 SSR Weng et al. 2010
Tu S06 × S52 247 F2 C_SC933/5.9; SSR16203/1.4; C_SC69/2.8; SSR, SCAR Zhang et al. 2010b
C_SC24/3.2
M/m S52 (monoecious; ffMM) × H34 1,067 F2/BC1 S_ME1EM26/5.4; S_ME8SA7/0.0; S_ SCAR Li et al. 2008
(hermaphrodite; FFmm) ME1EM23/0.7
M/m WI 1983G (gynoecious; MMFF) 96 F2 EACAMCAT_202/203/0.9; CsEIL1/0.0; AFLP Liu et al. 2008
× WI 1983H (hermaphrodite; EATGMCAA_380/1.6
mmFF)
wmv Qiupeng (resistant to WMV) × 120 RILs EACT/M-CTT/8 (Converted to SCAR) AFLP Zhang et al. 2009
European #8 (susceptible)
Anthracnose 66 (resistant to anthracnose) × 220 F2 E24M48251/2.727 AFLP Wang et al. 2007
resistance A18 (susceptible)
bi 9110Gt (bibi; bitterfree) × 03828 F2 TG/GCT150/6.43 (converted to SCAR AFLP Chi et al. 2007
(BiBi; bitter foliage) marker SC87)
Scab 9100 Gt (resistant to scab) × 9930 148 RILs SSR03084/0.7; SSR17631/1.6 SSR Zhang et al. 2010a
resistance (susceptible to scab)
Scab Q6 (resistant to scab) × Q12 145 F2 E20M64/4.83 (converted to SCAR) AFLP Zhang et al. 2006
resistance (susceptible to scab)
gynoecy 4401F (gynoecious) 4410F 80 F2 TG/CAC234/6.7 (converted to SCAR) AFLP Lou et al. 2007
(monoecious)
Table 7-1 contd....

Table 7-1 contd....
230
Gene Locus Parents Population Linked Marker/Distance (cM) Marker Type Reference

ZYMV-CH Qiupeng (resistant to ZYMV-CH) RILs E-ACG/M-CAG-182/5.0 AFLP Luo et al. 2006
× Euro #8 (susceptible)
Bt 931 (BtBt; bitter fruit) × 932(btbt; 129 F2 E23M66-101/5.0; E25M65-213/4.0 AFLP Gu et al. 2006
non-bitter fruit)
Powdery WIS2757 (resistant) × 19032 597 F2 SSR97-200/5.0 SSR Zhang et al. 2008
mildew (susceptible)
resistance
Powdery Q9 (resistant) × Q10 (susceptible) F2 P18M47-238/236/5.56 AFLP Zhang et al. 2004
mildew
resistance
Watermelon
zymv P1 595203 (ZYMV resistant) × 143 F2 ZYRP/8.0 SNP Harris et al. 2009
NUM (ZYMV susceptible)
flesh color PI 165002 (canary yellow) × PI 100 F2/BC1 LYCB-CAPS/0.0 SNP/CAPS Bang et al. 2007
593380 (red)
zymv-ch PI 595203 (resistant) × 98R 109 F2 AK13-644/8.0 RAPD Ma et al. 2006
(susceptible)
Cucurbita
Squash Zuc76 (resistant) × Black Beauty 93 F2 and 30 M121/3.3 SSR Kabelka and
silverleaf (susceptible) BC1 Young 2010
dwarf S2 (dwarf) × Mingri 306 F2 S1225-548/2.29 RAPD/SCAR Li et al. 2007
cmv Ko1 (resistant) × K02 (susceptible) 76 F2 S4391400/7.1 RAPD Zhao et al. 2007
*Fom-1 and Prv are tightly linked (~2 cM) to each other (Brotman et al. 2005). A list of genes tagged with molecular markers before 2004 can be found
in Wang et al. (2006). Only tightly linked markers are included in the table.

BSA can also be used to identify markers linked to a QTL although not
reported yet in cucurbits. Kanagaraj et al. (2010) used 11 drought-tolerant
and 12 drought-susceptible rice recombinant inbred lines (RILs) to construct
the two bulks. The parents were first screened for polymorphism using 1,206
rice simple sequence repeat (SSR) markers. Out of 134 SSR polymorphic
primers between parents, three primers showed polymorphism between
bulks. These three primers co-segregated among the individual RI lines
constituting the respective bulks. The genomic regions flanked by these
markers have been reported to be associated with several drought resistance
component traits (Kanagaraj et al. 2010). In sugarbeet, root elongation rate
controlled by QTLs was tagged with three AFLP markers using BSA on an F2
population generated from a cross between a high and a low root elongation
parent (Stevanato et al. 2010). The technique can be used in cucurbits to
identify markers linked to important quantitative traits.
7.3 Germplasm Characterization

DNA markers derived from cloned genes tend to be most robust across
diverse germplasms. We have seen this earlier when genotyping a gene in
the 497 melon accessions and two alleles perfectly divided all accessions
according to their sex types (Boualem et al. 2008). When the cloned recessive
nsv gene, which confers complete resistance to melon necrotic spot virus
(MNSV), a perfect association of a single nucleotide polymorphism
(SNP) in the gene was also found between seven MNSV-resistant and six
MNSV–susceptible melon accessions (Nieto et al. 2006). In watermelon, a
key gene, lycopene β-cyclase (LCYB) was found to determine canary yellow
and red flesh color of watermelon. An SNP marker developed from this
gene showed a perfect association with fruit color among 182 germplasm
accessions (Bang et al. 2007).
For most other traits, the underlying genes are not yet cloned.
Markers tightly linked to these genes can still be effectively used to predict
phenotypes. In cucumber, an AFLP marker was found to be 2.7 cM distant
from the anthracnose resistance gene (Wang et al. 2007). When this marker
was converted to a sequence characterized amplified region (SCAR) marker
SCEM178/172 (with primers 5’-CGT TTA CTT CTC TCC CAT TTC-3’ and
5’-TTG AGC GGA GTA GGA GAC-3’) and used to genotype 40 different
cucumber varieties with known anthracnose phenotypes, it correctly
identified disease phenotypes of in 39 of the 40 varieties, suggesting that the
marker is tightly linked to the resistance gene (Li et al. 2008). Zhang et al.
(2010a) tested SSR03084 and SSR17631, linked to cucumber scab resistance
at 0.7 and 1.4 cM, respectively, on 59 diverse inbred lines and hybrids, and
the accuracy rate for the two markers was 98.3%. In watermelon, a SCAR
marker was found to differentiate between 12 susceptible and 7 resistant

varieties against Fusarium wilt (Lin et al. 2009). Another dominant SCAR
marker developed from an AFLP marker linked to a gynoecy gene at 6.7
cM was tested on 21 genotypes with known sex phenotypes. The marker
correctly identified 14 of 15 gynoecious genotypes but it misidentified
one monoecious genotype as gynoecious (Lou et al. 2007). When two
markers linked to Tu (warty fruit) locus at 2.8 (C_SC69) and 3.2 (C_SC24)
cM were tested on 28 warty fruit (TuTu) and 34 non-warty fruit (tutu)
cucumber germplasms, C_SC24 and C_SC69 could correctly predict the
fruit phenotype of 55 and 58, respectively (Zhang et al. 2010b).
It is clear from these examples that MAS is a very effective tool for
selection of plants with desired phenotype. This is the reason that MAS
has been adopted by breeding companies to release commercial varieties
sooner and at lower cost although one does not see it in the literature. For
example, Harris Moran stated in its website (http: //www.harrismoran.
com/products/biotechstatement.htm) that “Harris Moran employs many
advanced techniques to assist and improve the classic plant breeding
work that is the core of our business. One example is the use of Molecular
Markers to allow the identification of specific genes and plant traits. Our
breeders use markers to assist in selection of new plant lines with important
characteristics, our plant pathologists use markers to identify plant diseases
and our quality assurance folks use markers to help insure the seed you
buy is true to type—truly versatile and valuable technology”. Syngenta
also stated in its 2009 Annual Review that “modern technologies such as
marker-assisted breeding enable Syngenta scientists to identify genetic traits
that relate to certain plant characteristics. Using DNA testing, plants with
the desired traits can be identified at an early stage of growth, enabling
much faster development of varieties with enhanced flavor, color, nutrition
and agronomic performance” (Syngenta 2009). The seed company Pioneer
also uses MAS extensively in its commercial variety development program
(Cahill and Schmid 2004). In their experience, MAS is most efficient in early
generation single plant selections.
7.4 Limitations of MAS

Marker genotypes may not be an accurate predictor of the phenotype if
the underlying genetics of the trait is not very well understood. Fom-1
is a case in point (Zink and Gubler 1985; Risser 1987; Danin-Poleg et al.
1999; Oumouloud et al. 2010). When 43 fixed lines and 27 commercial F1
hybrids were screened for their Fom-1 genotype with tightly linked markers,
prediction of the phenotype depends on the varieties tested (Tezuka et al.
2010). For var. cantalupensis, C-TCG/GGT-400 (5.2 cM from Fom-1) was

suitable for the Fom-1 genotype prediction. The Fom-1 genotype in var.
chinensis, conomon and makuwa seemed to be predicted accurately by CAPS2
(5.2 cM), NBS1-CAPS, C-MRGH12 (0.4 cM) and 62-CAPS (1.2 cM). CAPS2
was identical in all cultivars and lines excluding MR-1 as also reported in
their earlier study (Tezuka et al. 2009). The Fom-1 genotype in var. reticulatus
could be predicted accurately by C-TCG/GGT-400, C-MRGH12 and the
cosegregating S-TAG/GCC-470 (Tezuka et al. 2009, 2010). What is interesting
is that C-TCG/GGT-400 and CAPS2 are both CAPS markers derived from
the same AFLP marker TCG/GGT-400. Yet C-TCG/GGT-400 could predict
the resistant phenotype in var. reticulatus and var. cantalupensis but CAPS2
could only do so in var. chinensis, conomon and makuwa (Tezuka et al. 2009,
2010). Similar results were reported in two other studies (Brotman et al. 2005;
Oumouloud et al. 2008), i.e., markers linked to Fom-1 do not always predict
the resistance phenotype in all varieties, only in certain variety groups as
described above. However, one can always combine all the markers to select
the right resistant genotype in MAS.
These results may revive the issue of Fom-3, another gene conferring
resistance to F. oxysporum f.sp. melonis races 0 and 2 in the cultivar “Perlita
FR” (Zink and Gubler 1985). In “Tortuga”, a Spanish cantalupensis accession,
resistance to races 0 and 2 of F. oxysporum f.sp. melonis is found to be
conferred by two independent genes: one dominant and the other recessive.
The dominant gene was shown to be Fom-1 but the recessive gene was
suggested to be named fom-4 (Oumouloud et al. 2010). It should be noted
that both “Perlita FR” and “Tortuga” belong to var. cantalupensis. Obviously,
this is a very intriguing finding and more investigation is warranted to
resolve the issue.
Despite this shortcoming, MAS remains to be an effective breeding tool
for selection of qualitatitive traits controlled by single genes or quantitative
traits controlled by major genes. Genomics and genome-wide study in other
crop plants have shown in the last two years that more markers can result
from the next generation sequencing. By resequencing 517 rice landraces,
Huang et al. (2010) identified 3.6 million SNPs and found markers associated
with 14 agronomic traits of rice. These markers no doubt will be an asset for
MAS. In soybean, Lam et al. (2010) resequenced 17 wild and 14 cultivated
soybeans and concluded that marker-assisted breeding of soybean will
be less challenging than map-based cloning. Not surprisingly, the new
sequencing technology has already been used in cucurbits. Cavagnaro et
al. (2010) resequenced the Gy14 cucumber genome and detected a total
of 112,073 perfect repeats and from these developed 83,000 SSRs. Such a
large number of markers will speed up marker discovery for MAS in the
immediate future.

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8
Genome Mapping and QTL
Analysis in Cucurbits
Hugo E. Cuevas,1,* Jack E. Staub2 and Juan E. Zalapa3
ABSTRACT
The cucurbits are among the most culturally and commercially
important plant species cultivated worldwide. The considerable genetic
diversity within the major cultivated species, such as melon (Cucumis
melo L.), cucumber (Cucumis sativus L.), watermelon (Citrullus lanatus)
and squash (Cucurbita moschata), provides a reservoir of genes for the
development of new cultivars in breeding programs. The development
and use of genomic tools for the genetic improvement of cucurbits
has been one of the major objectives of research programs across the
world. In fact, 34 linkage maps and more than 400 genes or quantitative
trait loci (QTLs) associated with economic important traits have been
indentified in cucurbits during the last 15 years. Subsequently, an
extensive number of publications related to genome mapping and QTL
analysis are dispersed in the literature. The objective of this chapter
is to summarize and illustrate the importance of DNA marker and
genomic analysis (e.g., mapping, QTL detection, and map-based gene
cloning) in the study of diseases, yield and fruit quality components
in cucurbit crop species. Moreover, it integrated independent research
results in order to improve the utility of genomic analysis in cucurbit
breeding programs.
1
Plant Genome Mapping Laboratory, Center for Applied Genetic Technologies, 111 Riverbend
Road, Athens, GA 30602, USA; e-mail: hcuevas@uga.edu
2
USDA-ARS, Forage and Range Research Laboratory, Utah State University, Logan, UT
84322-6300, USA.
3
USDA-ARS, Madison WI; Dept. Horticulture, 1575 Linden Drive, Madison, WI 53706,
USA.

Genome Mapping and QTL Analysis in Cucurbits 239
8.1 Introduction
The globally ubiquitous, cross-pollinated family Cucurbitaceae includes
825 species representing 118 principally tropical genera of which some
have economic importance (Jeffrey 1990). For instance, species in the genera
Cucumis (cucumber; C. sativus var. sativus L; melon; Cucumis melo L.),
Citrullus [watermelon; C. lanatus (Thumb.) Matsum & Nakai], and Cucurbita
[squash (C. pepo L. and C. moschata Duchesne)] provide an array of market
types that are grown worldwide (~17.9 x 107 metric tons of marketable
product; FAO 2004). In fact, taken collectively species of the Cucurbitaceae
are among the most culturally and commercially important plant species
(Nayar and More 1998).
The origin and domestication of cucurbit crop species differ dramatically.
For example, while squash was domesticated in America more than 8,000
years ago (Smith 1997), cucumber, melon and watermelon originated in
the Near East, India, and North Africa (Kirkbride 1993, Robinson and
Decker-Walters 1997). Genetic improvement of cucurbits relies on the
incorporation of economically important alleles from genetically diverse
exotic germplasm. Consequently, the documentation (i.e., taxonomy,
nutrition, and culinary attributes) and conservation (i.e., collection and
curation) of unique accessions (e.g., Cucumis hystrix Char.; Chen and Staub
1997) is essential. Recently, the intra- and interspecific variation in Cucumis
(Staub et al. 1997, 1999, 2000, 2002, 2004; Horejsi and Staub 1999; Mliki et
al. 2001; Akashi et al. 2002; López-Sensé et al. 2002, 2003; Nakata et al. 2005;
Sensoy et al. 2007; Luan et al. 2008), Cucurbita (Merrick 1991; Paris et al.
2003), and Citrullus (Levi et al. 2001a, b) germplasm has been described and
summarized (Lebeda et al. 2007).
Contemporary/modern cucurbit domestication efforts since 19th
century primarily involved farmer-selection of landraces. However, genetic
diversity in “commercial” germplasm has more recently (after 19th century)
been augmented by the strategic introduction of novel traits (e.g., disease
resistance and plant architecture) housed in exotic germplasm (Lebeda
et al. 2007). Such initial hybridization with elite lines was followed by
intensive selection efforts primarily focused on improving the fruit yield
and quality traits. Recent increases in yield and quality in many cucurbit
crop species have, in fact, resulted from complementary improvements in
cultural practices, increased pest and disease resistances, and modifications
of plant architecture and sex expression (e.g., increased gynoecy). While
traditional breeding has been successful, it is clear that in some cucurbit
species, this process might be accelerated (i.e., increased effectiveness and
efficiency) by the application genomic tools including marker assisted
selection (MAS) (Fazio and Staub 2003b; Fan et al. 2006; Robbins and Staub
2009). Fully implemented molecular marker technologies, such as use of

tightly linked marker/trait associations (< 5 cM), can permit plant selection
without exposure to environmental (abiotic) or pathogen (biotic) challenges
(Helentjaris et al. 1986).
A wide range of DNA markers have been employed in cucurbits for
diversity analysis (Lebeda et al. 2007), genetic map construction [melon
(Baudraco-Arnas and Pitrat 1996; Wang et al. 1997; Liou et al. 1998; Oliver
et al. 2001; Danin-Poleg et al. 2002; Perin et al. 2002; Monforte et al. 2004;
Gonzalo et al. 2005; Perchepied et al. 2005a, b; Zalapa et al. 2007; Fernández-
Silva et al. 2008; Cuevas et al. 2008, 2009; Fukino et al. 2008), cucumber
(Kennard et al. 1994; Serquen et al. 1997a; Park et al. 2000; Bradeen et al.
2001; Fazio et al. 2003a; Yaun et al. 2008; Ren et al. 2009), squash (Brown and
Myers 2002; Zraide et al. 2007; Gong et al. 2008a, b), and watermelon (Levi
et al. 2001a, 2002, 2006; Hashizume et al. 2003; Zhang et al. 2004)] and MAS
(Fazio and Staub 2003b; Fan et al. 2006; Robbins and Staub 2009). Marker
analysis technologies in cucurbits have employed restriction fragment
length polymorphism (RFLP), random amplified polymorphic DNAs
(RAPD), simple sequence repeats (SSR), inter-simple sequence repeats
(ISSR), amplified fragment length polymorphism (AFLP), sequence-related
amplified polymorphisms (SRAP), sequence characterized amplified regions
(SCAR), cleaved amplified polymorphic sequence (CAPS), and single
nucleotide polymorphism (SNP). The joint analysis of marker genotyping
and trait phenotyping enables the detection and location of loci controlling
quantitative traits (QTL) (Asíns 2002). The importance of DNA marker (e.g.,
genotyping) and genomic (e.g., mapping, QTL detection, and map-based
gene cloning) analysis in major cucurbit crop species (i.e., melon, cucumber,
watermelon, and squash) are presented and discussed herein.
8.2 Cucumis melo L.

8.2.1 Introduction
Melon (C. melo L.; 2n = 2x = 24) has been subdivided into seven cultivar
groups (i.e., Flexuosus, Conomon, Cantalupensis, Inodorus, Chito, Dudaim,
and Momordica) based on phenotypic variation in plant architecture
(e.g., leaf, stature, and branching habit) and fruit characteristics (Pitrat
2008). Genetic variability assessments (genotypic and phenotypic) of elite
breeding germplasm and exotic accessions have been conducted by various
researchers using different methodologies (Staub et al. 2000, 2004; Mliki et
al. 2001; Akashi et al. 2002; López-Sesé et al. 2002, 2003; Nakata et al. 2005;
Sensoy et al. 2007; Luan et al. 2008). While marker-based phylogenetic
analysis established a closer relationship between group Cantelupensis
and Indorus accessions than previously thought, group Conomon, Chito,
Dudaim, and Momordica germplasm share comparatively few genetic

affinities (Stepansky et al. 1999). Moreover, specific, geographically-based,

primary gene pools have been identified in melon (Lebeda et al. 2007).
Indeed, exotic and domesticated African melon accessions are different
from Europe and US market classes (secondary centers of diversity) (Mliki
et al. 2001). Likewise, Spanish and Greek (secondary centers for diversity),
and Turkish (primary center for diversity) melon landraces possess unique
genetic variation and differ from other “reference” accessions examined
(López-Sesé et al. 2002; Staub et al. 2004; Sensoy et al. 2007). In contrast,
an array of diverse Asian melon accessions examined possessed genetic
affinities with either African accessions or US and European cultivars
depending on geographic origin (Nakata et al. 2005). A recent assessment
of Chinese melon accessions (secondary center of diversity) found that
they differed dramatically from a diverse array of group Cantalupensis
and Inodorus accessions examined (Luan et al. 2008). In fact, Chinese
germplasm share genetic affinities to Indian and African accessions, and
differ from Japanese, European, and US germplasm. These results indicate
that a rich source of genetic diversity exists in primary and secondary centers
of diversity and that diversity is and will continue to be critical for plant
improvement and the exploitation of complex traits.
More than 27 million metric tons of melons were produced in 2004
worldwide, where China, Turkey, Iran, the US, and Spain were the major
economic markets (FAO 2004). Consequently, many international institutions
have devoted resources to melon research for example US Department of
Agriculture-Agricultural Research Service (USDA-ARS); The Institute
of Research and Technology in Agriculture (IRTA, Spain); The Chinese
Academy of Agricultural Sciences (CAAS), as well as in France [The National
Institute of Agronomic Research (INRA)], Japan [The National Institute of
Vegetable and Tea Science (NIVTS)], Korea [Korea Research Institute of
Bioscience and Biotechnology (KRIBB)], India [Indian Agricultural Research
Institute (IARI)], and Israel [the Agriculture Research Organization (ARO)].
Many of these institutions investigate the genetics underlying economically
important traits, and develop enhanced germplasm. In the last 15 years,
research efforts have resulted in the construction of 16 linkage maps derived
from 14 mapping populations (Baudraco-Arnas and Pitrat 1996; Wang et
al. 1997; Liou et al. 1998; Oliver et al. 2001; Danin-Poleg et al. 2002; Perin et
al. 2002; Silberstein et al. 2003; Monforte et al. 2004; Perchepied et al. 2005a,
b; Zalapa et al. 2007; Fukino et al. 2008; Cuevas et al. 2009; Table 6-1) and
the identification of 379 genes or QTLs that control disease resistance, fruit
quality and yield components (Dogimont et al. 2000; Oliver et al. 2001;
Perin et al. 2002; Pitrat 2002; Monforte et al. 2004; Perchepied et al. 2005a, b;
Zalapa et al. 2007; Eduardo et al. 2007; Fukino et al. 2008; Paris et al. 2008;
Cuevas et al. 2008, 2009; Obando et al. 2008, 2009; Table 8-1).

242
Table 8-1 A summary of traits and their associated quantitative trait loci (QTL) in melon (Cucumis melo L.).
Traita QTL R2 (%)b Parent Populationc Reference

Yield components Primary branch number 6 05.0–18.0 ‘USDA 841-1’ x ‘Top Mark’ RIL Zalapa et al. 2007
Earliness 9 06.0–41.0 ‘Piel de Sapo’ x PI 161375 DHL, F2 Monforte et al. 2004
Fruit maturity 3 08.0–35.0 ‘Q 3-2-2’ x ‘Top Mark’ F2-3 Cuevas et al. 2009
Percent of maturity fruit 5 07.0–13.0 ‘USDA 841-1’ x ‘Top Mark’ RIL Zalapa et al. 2007
Maturity index 11 n/a ‘Piel de Sapo’ x PI 161375 NIL Obando et al. 2008
Fruit weight 6 08.0–34.0 ‘Piel de Sapo’ x PI 161375 DHL, F2 Monforte et al. 2004
14 n/a ‘Piel de Sapo’ x PI 161375 NIL Eduardo et al. 2007
Fruit weight/plot 12 06.0–20.0 ‘USDA 841-1’ x ‘Top Mark’ RIL Zalapa et al. 2007
Average weight fruit 5 07.0–43.0 ‘USDA 841-1’ x ‘Top Mark’ RIL Zalapa et al. 2007
Fruit number 9 04.0–21.0 ‘USDA 841-1’ x ‘Top Mark’ RIL Zalapa et al. 2007
Fruit diameter 13 n/a ‘Piel de Sapo’ x PI 161375 NIL Eduardo et al. 2007
6 06.0–28.0 ‘USDA 841-1’ x ‘Top Mark’ RIL Paris et al. 2008
Fruit length 13 10.0–29.0 ‘Piel de Sapo’ x PI 161375 NIL Eduardo et al. 2007
Fruit area 5 n/a ‘Piel de Sapo’ x PI 161375 NIL Obando et al. 2008
Fruit shape 5 n/a ‘Védrantais’ x PI 161375 RIL Perin et al. 2002
8 08.0–33.0 ‘Piel de Sapo’ x PI 161375 DHL, F2 Monforte et al. 2004
16 11.0–52.0 ‘Piel de Sapo’ x PI 161375 NIL Eduardo et al. 2007
Fruit quality components Fruit netting 5 10.0–18.0 ‘USDA 841-1’ x ‘Top Mark’ RIL Paris et al. 2008
7 n/a ‘Piel de Sapo’ x PI 161375 NIL Obando et al. 2008
Fruit hardness 4 n/a ‘Piel de Sapo’ x PI 161375 NIL Obando et al. 2008
Ovary shape 6 n/a ‘Piel de Sapo’ x PI 161375 NIL Eduardo et al. 2007
Skin color and ground spot color 25 n/a ‘Piel de Sapo’ x PI 161375 NIL Obando et al. 2008
Organic acids 21 n/a ‘Piel de Sapo’ x PI 161375 NIL Obando et al. 2009
Sugar content 30 n/a ‘Piel de Sapo’ x PI 161375 NIL Obando et al. 2009
Soluble solid content 5 07.0–30.0 ‘Piel de Sapo’ x PI 161375 DHL, F2 Monforte et al. 2004
19 n/a ‘Piel de Sapo’ x PI 161375 NIL Eduardo et al. 2007

Seed cell diameter 6 07.0–24.0 ‘USDA 841-1’ x ‘Top Mark’ RIL Paris et al. 2008
Seed cell diameter: fruit diameter 7 07.0–17.0 ‘USDA 841-1’ x ‘Top Mark’ RIL Paris et al. 2008
ratio
Fruit flesh proportion 14 n/a ‘Piel de Sapo’ x PI 161375 NIL Obando et al. 2008
Mesocarp pressure 5 07.0–20.0 ‘USDA 841-1’ x ‘Top Mark’ RIL Paris et al. 2008
Flesh color 16 n/a ‘Piel de Sapo’ x PI 161375 NIL Obando et al. 2008
Beta-carotene-associated flesh 3 04.0–50.0 ‘Q 3-2-2’ x ‘Top Mark’ F2-3 Cuevas et al. 2009
color
Beta carotene 8 08.0–32.0 ‘USDA 841-1’ x ‘Top Mark’ RIL Cuevas et al. 2008
Juice color 10 n/a ‘Piel de Sapo’ x PI 161375 NIL Obando et al. 2008
Extractable juice 8 n/a ‘Piel de Sapo’ x PI 161375 NIL Obando et al. 2008

Juice pH and acidity 4 n/a ‘Piel de Sapo’ x PI 161375 NIL Obando et al. 2008
Dry matter (flesh tissue) 7 n/a ‘Piel de Sapo’ x PI 161375 NIL Obando et al. 2008
Diseases Resistance Resistance to Fusarium 9 05.0–36.0 ‘Védrantais’ x ‘Isabella’ RIL Perchepied et al.
oxysporum sp. melonis 2005a, Brotman et
al. 2005
Resistance to Pseudoperonospora 11 05.0–38.0 ‘Védrantais’ x PI 124112 RIL Perchepied et al.
cubensis 2005b
Resistance to Podosphaera 2 12.0–46.0 ‘AR 5’ x ‘Harukei 3’ RIL Perchepied et al.
xanthii 2005a, Fukino et al.
2008
Resistance to Melon necrotic spot 1 n/a ‘Piel de Sapo’ x PI 161375 F2 Oliver et al. 2001
virus
Resistance to Cucumber mosaic 7 12.0–79.0 ‘Védrantais’ x PI 161375 RIL Dogimont et al.
virus 2000
a
Trait name according to their published designation.
b
Percentage of phenotypic variation explain by QTL association.
c
Population type used to construct linkage maps, where F2, RIL, NIL, DHL, and F2-3 refer to F2 population, recombinant inbred lines, nearly isogenic
lines, double haploid lines, and F3 family analysis, respectively.

8.2.2 Genetics Maps and Mapping Populations

The genome size of melon has been estimated to be between 450 to 500
Mbp (~900 to 1,200 cM), about three times that of Arabidopsis thaliana (L.)
Heynh. (Arumuganathan and Earle 1991). In 1984, M. Pitrat (INRA) initiated
the systematic characterization of linkage relationships among phenotypic
traits and developed the first melon linkage map (Pitrat 1984). A relatively
narrow genetic base in melon (i.e., low DNA polymorphism level), however,
has hampered progress towards the development of highly saturated
maps (i.e., mean marker map intervals between 1–5 cM). Persistence in
the development of unique mapping populations and continued efforts
in marker development has resulted in the construction of marker-based
(SNP, SSR, AFLP, RFLP, and RAPD) melon maps that include economically
important traits (Baudraco-Arnas and Pitrat 1996; Wang et al. 1997; Liou
et al. 1998; Oliver et al. 2001; Danin-Perin et al. 2002; Poleg et al. 2002;
Silberstein et al. 2003; Monforte et al. 2004; Perchepied et al. 2005a, b; Zalapa
et al. 2007; Fukino et al. 2008; Cuevas et al. 2008, 2009; Table 6-1).
Initial melon linkage maps were developed using F2 and BC1 populations
(Baudraco-Arnas and Pitrat 1996, Wang et al. 1997, Liou et al. 1998, Oliver
et al. 2001; Danin-Poleg et al. 2002). However, these maps were not well
suited for QTL analysis of yield and quality components since extensive,
replicated, multi-location evaluations could not be easily performed. More
recently, the development of immortalized mapping populations [e.g.,
double haploid lines (DHL) and recombinant inbred lines (RIL)] have
provided the structure needed for rigorous QTL analysis and assessment
of marker/trait associations for plant improvement [RIL (Perin et al. 2002;
Perchepied et al. 2005a, b; Zalapa et al. 2007; Cuevas et al. 2008; Fukino et
al. 2008; Paris et al. 2008) and DHL (Monforte et al. 2004)].
The research group at INRA (France) developed two RIL populations
derived from the cross “Védrantais” x PI 161375, and “Védrantais” x PI
414723 (Perin et al. 2002). These mapping populations were utilized to
construct two genetic maps possessing 481 and 318 markers, respectively.
These maps were later integrated to produce a 668-point composite map
spanning 1,654 cM on 12 linkage groups, which included nine previously
described functional genes, and 23 morphological traits (Perin et al. 2002).
This research group then developed two additional RIL populations derived
from the cross “Védrantais” x “Isabella”, and “Vendrantis” x PI 124112,
allowing for the mapping of 133 and 510 additional genetic markers,
respectively (Perchepied et al. 2005a, b). Most of these markers were
dominant AFLPs (~95%), which are not directly amenable to comparative
mapping (i.e., map colinearity analysis) when maps have been created using
different parental stocks (Staub et al. 1996a, b).

Researchers at IRTA (Spain) have also contributed significantly to the

development of moderately saturated melon maps. Initially, a set of 77 DHL
derived from the cross “Shongwan Charmi” PI 161375 x “Piel de Sapo”
cultivar was utilized to construct a 107-point map (82 RFLPs and 25 SSRs)
spanning 994 cM over 12 linkage groups (Monforte et al. 2004). Subsequently,
the saturation of this map was increased to 327 markers (226 RFLPs, 97 SSRs,
and 3 SNPs; Gonzalo et al. 2005). Codominant markers that were broadly
distributed across this latter genetic map provided an appropriate framework
(i.e., common anchor points) for comparative mapping. More recently, a bin
mapping approach was employed to further increase SSR saturation in this
framework map (Fernández-Silva et al. 2008). This strategy improved map
construction efficiency by reducing mapping population size [i.e., using the
most recombinant individuals (7–14) in the mapping population to place new
markers] (Vision et al. 2000; Howad et al. 2005). This map was characterized
by partitioning the melon genome map into 122 bins, where the average bin
length was 10.2 cM. Such partitioning allowed for the placement of 121 new
SSRs (125% increase), resulting in a bin-based reference map consisting of
80 RFLP, 212 SSR, and 3 SNP markers (Fig. 8-1).
The recent dramatic increase in the number of published SSR melon
markers (> 550; Chiba et al. 2003; Ritchel et al. 2004; Gonzalo et al. 2005;
Fukino et al. 2007; Kong et al. 2007; Fernández-Silva et al. 2008), and
genomics resources [e.g., expressed sequence tag (EST) libraries; cucurbit
genomics resource (http: //www.icugi.org)] afford an opportunity for the
construction of a unique SSR-based genetic maps. To this end, the NIVTS
(Japan) constructed a RIL-based genetic map derived from a cross between
line “AR 5” (USDA, ARS) and “Harukei 3” using 157 SSR and 7 SNP
markers (Fukino et al. 2008). Since 43 of these markers were common with
previously published maps, comparative mapping is now possible using
these marker loci as anchor points.
The melon USDA-ARS breeding programs at Salinas, California and
Madison, Wisconsin USA have jointly developed a RIL population derived
from a cross of “USDA 846-1” x “Top Mark” (Group Cantalupensis). Initially,
a genetic map was constructed using 114 RAPDs, 32 AFLPs, and 35 SSRs,
which spanned 1,032 cM with a mean marker interval of 5.7 cM on 15
linkage groups (Zalapa et al. 2007). This map proved useful in identifying
and mapping QTLs controlling yield and quality components in melon
(Zalapa et al. 2007; Paris et al. 2008). Subsequently, this RIL-based map was
further saturated by the addition of 104 SSR and 11 SNP markers (Cuevas et
al. 2008), and then merged with an F2-based map (“Q 3-2-2” x “Top Mark”;
154 SSRs and 15 SNPs) to construct a consensus map possessing 172 SSR
markers and 13 SNP markers (Cuevas et al. 2009). This map has potential
utility for comparative mapping and synteny analysis since 120 markers are
common with previously published maps (Gonzalo et al. 2005; Fernández-
Silva et al. 2008; Fukino et al. 2008).

Figure 8-1 A bin map [framework map (FM)] of Cucumis melo obtained by genotyping 14
selected doubled haploid lines (DHL). The vertical bars represent linkage groups (LG) that
are characterized by bins defined by the genotype of the selected DHL. Markers in bold were
used for the estimation of bin size. Genetics distance (GD) is shown on the left side of LG,
indicating the postion of the last marker included in the bin according to the FM. Markers
in italic define “new bins”, and the hypothetical position of the last marker in each bin is
indicated by a dashed horizontal line within the LG bar without the GD specification. Figure
adapted with kind permission from Springer Science and Business Media: Fernández-Silva
et al. 2008, Theor Appl Genet 118: 139–150.

The investment of resources by many research institutions (above) has

led to the development of moderately saturated genetic maps in melon
(Perin et al. 2002; Fernández-Silva et al. 2008; Fukino et al. 2008; Cuevas
et al. 2009). Those possessing common codominant markers (e.g., SSR)
have been proposed for map merging experiments (Gonzalo et al. 2005). A
single merged map developed from such individual moderately saturated
maps would lead to map integration (synteny analysis), an appraisal and
validation of mapping efficiency (e.g., RIL vs. DHL, and comparisons of
maps derived from different parental stocks; Liu 1998), and an evaluation
of QTL position and an assessment of their effects across differing genetic
backgrounds (Staub 2007). To this end, the International Cucurbit Genomic
Initiative (ICuGI) has just completed the construction of a “Melon Reference
Map” by merging melon maps possessing common SSRs identified and
mapped by several international laboratories using different mapping
parents and populations (A. Monforte, pers. comm. 2009). In this merged
map, the nomenclature of Perin et al. (2002) will be used to designate linkage
groups (LG; LG I-XII), to facilitate comparisons among various individual
maps (i.e., marker synteny analysis) and previously published QTL, and
provide a framework for further QTL dissection and gene cloning.
8.2.3 Genetic Mapping of Quantitative Traits in Melon

The development of genetic linkage maps has provided tools for the
molecular analysis of economically important traits in melon (Perin et al.
2002; Pitrat 2002; Monforte et al. 2004; Zalapa et al. 2007; Fukino et al. 2008).
Since QTL mapping in melon was initiated by Dogimont et al. (2000) as part
of their ongoing studies of disease resistance, more than 300 QTLs have been
identified that are associated mostly with morphological traits (e.g., yield
and quality components). The majority of this research has been performed
by international research institutions that have employed different parental
stocks (e.g., Groups Inodorus and Cantalupensis; Table 8-1). The currently
identified marker-QTL associations form the basis for gene cloning and
germplasm improvement through MAS, and are thus detailed below.
8.2.3.1 Yield Components

Several yield-related QTLs have been recently identified and mapped in
melon by Monforte et al. (2004), Eduardo et al. (2007), Zalapa et al. (2007),
Obando et al. (2008), and Cuevas et al. (2009). Monforte et al. (2004; Group
Inodorus) studied the inheritance of earliness and fruit weight by evaluating
DHLs in multiple environments. While nine QTLs associated with earliness
were detected, only six were considered major QTLs (LOD > 5.0 or R2 >

20%). Likewise, six QTLs controlling fruit weight were detected at the same
level of stringency, where three were considered to contribute major effects.
Subsequently, four fruit weight (fw) QTLs (i.e., fw5.1, fw5.2, fw12.1, and
fw12.2) were confirmed by Eduardo et al. (2007) using near-isogenic lines
(NILs) in the same genetic background. Twelve additional QTLs for fruit
weight and 11 genomic regions associated with maturity were subsequently
identified employing these NILs (Obando et al. 2008).
The genetics of plant architecture in melon was studied by Zalapa et al.
(2007; Group Cantalupensis) using RILs evaluated at two contrasting US
environments (Wisconsin and California). Thirty-seven QTLs associated
with primary branch number (PB = 6), percent of maturity fruit (PMF =
5), fruit weight/plot (FW = 12), average weight fruit (AWF = 5), and fruit
numbers (FN = 9) were detected at both the locations. Although, genotype x
environment interactions (G x E) were identified for those traits, 16 QTLs (PB
= 4, PMF = 1, FW = 4, AWF = 2, and FN = 5) were stable across the growing
environments examined. More recently, two major (R2 > 20%) and one minor
(R2 > 8%) QTLs associated with fruit maturity were detected using F2-3
family analysis, where progeny were derived from Chinese (early flowering,
smooth-skinned epidermis, horticultural group undetermined) and US
Western Shipping (late flowering, netted epidermis, Group Cantalupensis)
parents (Cuevas et al. 2009).
8.2.3.2 Fruit Quality Components

Melon cultivars display enormous genetic diversity with respect to fruit
quality traits, including fruit shape, interior (mesocarp and endocarp) color,
epidermis texture and architecture (e.g., smoothness, vein tracks, ribbing,
etc.), sugar concentrations (soluble and insoluble), aroma, and pH (Liu et al.
2004; Obando et al. 2008, 2009). Consequently, the genetics and physiology
of fruit quality in melon has been intensively studied (Table 8-1).
The expression of several fruit quality QTLs has been found to be
consistent across growing environments, which portends their potential for
gene cloning and deployment in MAS. In fact, QTLs in the central region of
LG I (i.e., associated with SSR markers TJ27, CMCCA145, and CMCT505),
the distal region of LG II [containing andromonoecious gene (a)], and a
large portion (~1/2) of LG XI (i.e., interval bounded by SSR CMTC160 and
CMGA104) have been found to be associated with fruit shape in different
studies (Perin et al. 2002; Monforte et al. 2004; Eduardo et al. 2007; Paris et
al. 2008). Moreover, although the heritability of this trait is comparatively
higher than other fruit quality traits (e.g., soluble solids content), genotype
x environment (G x E) interactions have been detected (Eduardo et al.
2007; Paris et al. 2008). Moreover, correlations between fruit shape and

fruit diameter are dependent on the growing environment (Eduardo et al.

2007; Paris et al. 2008).
Genomic regions associated with soluble solids content (SSC) have
been identified (i.e., marker/trait associations) in LGI (SSR TJ27), LGII
(SSR CMGA108), LG VI (SSR CMTC123), LGVII (SSR interval CMAGN75
to TJ38), LG IX (SSR interval CMTCN1to CMATN22) and LG X (SSR
CMGA172) by Monforte et al. (2004), Eduardo et al. (2007), and Paris
et al. (2008). This trait is dramatically affected by growing environment
and is not correlated with other recently examined quality traits. Soluble
solids content involves complex metabolic processes, especially sugar
accumulation that is associated (i.e., regulated by) with sugar metabolism
and fruit development. Recently, Obando et al. (2009) evaluated a panel
of NILs constructed in a Group Inodorus genetic background (Eduardo et
al. 2005) using high performance liquid chromatography (HPLC), which
permits the quantification and analysis of different types of sugars (i.e.,
content of glucose, fructose and sucrose). Results showed that previously
genomic regions associated with SSC were related to different types of
sugars. For instance, the LG I region is associated with fructose content,
while the LG IX region is associated with glucose, fructose, total sugar,
and sucrose equivalents (i.e., sweetness). Likewise, the LG X region is
associated with sucrose, total sugars, and sucrose equivalent. In fact, 62
QTLs associated with SSC and sugar content have been detected under
differing experimental conditions, where the large majority are location
specific (Monforte et al. 2004; Eduardo et al. 2007; Paris et al. 2008; Obando
et al. 2009; Table 8-1).
The inheritance of mesocarp/endocarp fruit color has been difficult
to interpret in melon, and color intensity and hue depend on genetic
background (e.g., Groups Cantalupensis vs. Inodorus; Cuevas et al. 2008,
2009). Although, green flesh (gf; Hughes 1948) and white flesh (wf; Imam
et al. 1972) genes have been mapped to LG VIII (Perin et al. 2002; Monforte
et al. 2004) and LG IX (Fukino et al. 2008), respectively, the expression of
these loci do not explain all of the color variation (i.e., intensity and hue)
observed in melon. Initially, an epistatic interaction model (i.e., gf and wf)
was described by Clayberg (1992), where an F2 population derived from
a cross between the Casaba cultivar “Goldeb Beauty” (Group Inodorous;
white flesh color) and a green flesh experimental line (Group Inodorous)
segregated into 12: 3: 1 (orange:white:green) ratio. Subsequently, Monforte
et al. (2004) confirmed the recessive inheritance of green flesh and identified
three QTLs associated with orange flesh color, suggesting that orange and
green flesh segregation is independent. However, posterior analysis using
NILs derived from the same cross could not confirm the action of such QTLs
(Eduardo et al. 2007). More recently, an RIL-based QTL analysis of intensity
and hue of orange flesh color (Group Cantalupensis; Cuevas et al. 2008)

identified eight QTLs, where the QTL in LG VI (SSR CMTC123) was colinear
with a previously reported fruit color QTL (ofc12.1; Monforte et al. 2004).
Moreover, QTL analysis of F3 progeny derived from the cross between line
“Q 3-2-2” (Chinese; white flesh) and “Top Mark” (US Western Shipping;
Group Cantalupensis; orange flesh) identified QTLs colinear with gf and
wf and with the previously identified QTLs (Cuevas et al. 2008) mapped to
LG VI (Cuevas et al. 2009). The discrepancies between these studies (i.e.,
Monforte et al. 2004 and Cuevas et al. 2008, 2009) are likely due to differences
in parental constitution and the cross-specific genetic control (i.e., allelic
constitution) conditioning the flesh-colored phenotype.
The morphological diversity present in melon fruits have allowed for
QTL analysis of other important quality traits, such as seed cavity size, dry
matter content, the color of extracted mesocarp juice and pH, organic acid
components, mesocarp flesh rigidity (i.e., pressure to compress), ovary
shape, fruit hardness (pressure to compress), and netting (Table 8-1). The
lack of consistency (i.e., data repeatability) for many fruit quality traits
has contributed to the incomplete genetic dissection (i.e., gene cloning) of
QTL. For instance, the expression of each of the above traits is dramatically
affected by environment and epistasis and, thus, their inheritance is complex
[i.e., multi-genic (> 4) highly interactive QTL].
8.2.3.3 Disease Resistance

The discovery of economically important genes that condition resistance
to pathogens and pests has fostered considerable interest among plant
breeders and geneticists. Genetic studies have primarily focused on four
major diseases: 1) resistance to Fusarium wilt (Fusarium oxysporum f. sp.
melonis); 2) downy mildew [Pseudoperonospora cubensis (Berk. and Curtis)
Rostovzev]; 3) powdery mildew [Podosphaera xanthii (formerly Sphaerotheca
fuliginea Schlech ex Fr. Poll.) and Golovinomyces cichoracearum (syn. Erysiphe
cichoracearum DC. ex Merat)], and; 4) virus resistance (e.g., melon necrotic
spot virus, zucchini yellow mosaic virus, papaya ring spot virus, and
cucumber mosaic virus).
One of the most economically important diseases of melon is Fusarium
wilt (Martyn and Gordon 1996). Wilt resistance is under the control of two
single dominant genes, Fom-1 and Fom-2, which confer resistance to races 0
and 2, and races 0 and 1, respectively (Wang et al. 2000). Molecular markers
linked to Fom-2 have been identified, characterized, and mapped by Zheng
et al. (1999), Wang et al. (2000), Brotman et al. (2002), and Joobeur et al.
(2004). During the process of gene cloning, Joobeur et al. (2004) identified
two makers (STS 411 and SSR 430) 6 kb apart that flanked a gene controlling
wilt resistance (R; likely Fom-2). This R-gene encodes a protein that contains
leucine-rich repeat (LRR) and nucleotide binding site (NBS) domains, and is

located on the distal region of LG XI. The gene conferring resistance to races
0 and 1 (Fom-1) is linked to the gene conferring resistance to papaya ring
spot virus (Brotman et al. 2002). The most closely linked markers (NBS1-
CAPS and 62-CAPS) are within 1.7 and 1.4 cM of the gene, respectively. This
genomic region is located at the terminal region of LG IX, near SSR marker
CMTC47. Nevertheless, Fusarium race 1.2 has overcome the resistance
provided by these genes (Fom-1 and Fom-2). Although, partial resistance to
race 1.2 has been identified (Risser and Rode 1973), it is under polygenetic
control (Perchepied et al. 2005a). Nine QTLs have been identified that
explain a significant portion of the phenotypic variation associated with
resistance to race 1.2 (LOD > 3.0; R2 = 41–67%), and seven of these QTLs
demonstrate digenic epistatic interactions, indicating that this resistance
has a complex genetic basis (Perchepied et al. 2005a).
During early experimentation, the inheritance of resistance to downy
mildew in melon was found to be partially dominant and under control
of many genes (Cohen et al. 1985; Kenigsbuch and Cohen 1989, 1992a;
Epinat and Pitrat 1994a, b). The identification of trait/marker associations
(i.e., via QTL analyses) using RILs derived from PI 124112 (resistance) and
“Védrantais” (susceptible) has identified one major QTL (pcXII.1; LOD > 5)
located in LG XII that explains from 12 to 38% of the resistance to P. cubensis
(Perchepied et al. 2005b). In addition, 10 minor QTL (LOD > 3.0; R2 = 5–20%)
variable express themselves under differing experimental conditions,
suggesting that this resistance is dramatically affected by environmental
conditions and epistasis.
Resistance to powdery mildew in melon has been studied (Kenigsbuch
and Cohen 1992b; Epinat et al. 1993; Pitrat et al. 1998; McCreight 2003), and
major genes and QTLs have been identified (Perin et al. 2002; Perchepied
et al. 2005b; Fukino et al. 2008). Although two genes for powdery mildew
resistance, Pm-x, and Pm-w, have been located in LG II, and V, respectively
(Perin et al. 2002), the two major QTLs controlling resistance, PmV.I and
PmXII.I, are located on LG V and XII, respectively (Perchepied et al. 2005b).
While QTL PmV.I (LOD > 12.0) explains from 33 to 89% of the observed
phenotypic variance associated with resistance to P. xanthii races 1, 2, and
3, PmXII.I (LOD > 10.0) explains from 25 to 93% of the phenotypic variance
associated with resistance to P. xanthii races 1, 2, 5, and G. cichoracearum
race 1. In addition, interactions between PmV.I and PmXII.I explain for 68
and 80% of the variance of resistance to P. xanthii races 1 and 2, respectively
(Perchepied et al. 2005b). More recently, Fukino et al. (2008) identified two
major QTLs (LOD > 5; R2 = 12–46%) on LG II (SSR interval CMBR008-
CMBR120) and LG XII (SSR interval CMN01_38 & CMBR150), which confer
resistance to P. xanthii races 1 and N1. However, due to the lack of sufficient
“anchor” markers among these genetic maps (i.e., Perchepied et al. 2005b

and Fukino et al. 2008), it is currently impossible to confirm whether QTL

located on LG XII of these maps are syntenic.
Several virus resistance genes have been mapped and cloned in melon
(Daning-Poleg et al. 2002; Perin et al. 2002; Brotman et al. 2005; Morales et
al. 2005; Nieto et al. 2006). The zucchini yellow mosaic virus (Zym) and the
necrotic spot virus (nsv) genes were mapped to LG II and XII, respectively,
using a RIL population constructed in a Charentais market type genetic
background (Perin et al. 2002). The gene, Prv, controlling resistance to
papaya ring spot virus has also been mapped using the same RIL population
and is linked to the Fom-1 gene conditioning Fusarium wilt resistance
(Brotman et al. 2005) on LG IX. In parallel, a bulk segregant analysis (BSA)
employing individuals from a BC1 population derived from a Group-
Cantalupensis (syn. Reticulatus) identified that Zym was tightly linked to the
SSR marker, CMAG36 (Daning-Poleg et al. 2002). More recently, BAC-end
sequence analysis identified a SNP marker (52K20sp6) that co-segregated
with the gene conferring nsv resistance in BC1 (Group Cantalupensis,
Charentais type) and F2 (Group Inodorus) populations (Morales et al. 2005).
Similarly, using NILs developed from Group Inodorus genetic background
(Eduardo et al. 2007), Essafi et al. (2009) mapped resistance to cucumber
mosaic virus (cmv-1) to LG XII in an interval between SSR markers CMN61_
44 and CMN21_55.
8.3 Cucumis sativus L.

8.3.1 Introduction
Cucumber (Cucumis sativus L.; 2n = 2x = 14) has been of culinary importance
to humans for millennia (Robinson and Decker-Walters 1997). Five major
types of cucumber are cultivated worldwide including American processing
and fresh market types, the Dutch gherkin and greenhouse types, the
German Schalgurken type, the Middle-Eastern Beit Alpha type, and
the Oriental trellis (burpless) type (Staub et al. 2008). It is the fifth-most
grown vegetable crop worldwide behind tomatoes (Solanum lycopersicum
L.), watermelon, cabbage (Brassica oleracea L.), and onion (Allium cepa L.)
(FAOSTAT 2004).
The initial domestication of cucumber is thought to have occurred
in India about 3,000 years ago (Lower and Edwards 1986). The primary
gene pool consists of two interfertile botanical varieties, the domesticated
C. sativus var. sativus L. and the wild or feral type, C. sativus var. hardwickii
(R.) Alef. Genetic diversity within and between these two varieties is very
low (3–12%; Dijkhuizen et al. 1996; Horejsi and Staub 1999) when compared
with other Cucumis species [e.g., melon (10–25%); Staub et al. 2000; López-
Sesé et al. 2003].

The identification and exploitation of genetic variation is critical in crops

having a narrow genetic base (Hoisington et al. 1999). This is particularly
true of cucumber where, because of the comparatively low genetic variability
among elite lines, wide inter- and intra-species crosses must be made to
improve yield and reduce vulnerability to pest and pathogens (Lebeda et al.
2007). India is the likely center of origin and a primary center of diversity for
cucumber, where considerable geographically-based differences in genetic
diversity exist between North and South India (Staub et al. 1997). In fact,
northern and southern Chinese cucumber cultivars likely have different
origins; therefore, represent distinct, potentially important germplasm
pools for plant improvement programs (Staub et al. 1999). Curiously
and singularly unique to the Cucurbitaceae, exists a Cucumis species,
C. hystrix Char. (2n = 2x = 24; origin southern China) that is sparingly
cross-compatible (i.e., via embryo rescue and chomosome doubling) with
C. sativus and affords an opportunity for enhancement of the C. sativus gene
pool and provides a potential bridge between C. sativus and C. melo (Chen
and Staub 1997, 2002 and 2003).

The genome size of cucumber is smaller than that of melon (243.5 Mbp),
being about two times that of A. thaliana (Koo et al. 2005; Huang et al. 2009).
Cucumber linkage maps have been constructed using phenotypic data
(Fanaourakis and Simon 1987; Pierce and Wehner 1990; Vokalounakis 1992),
isoenzymes alone and in combination with morphological traits (Knerr and
Staub 1992; Meglic and Staub 1996), and with molecular markers (e.g., SNP,
SSR, SCAR, RAPD, and AFLP) (Kennard et al. 1994; Serquen et al. 1997;
Horejsi et al. 2000; Park et al. 2000; Bradeen et al. 2001; Fazio et al. 2003a;
Yuan et al. 2008; Ren et al. 2009; Table 6-1).
The first two molecular marker maps in cucumber were developed by
Kennard et al. (1994) using F2 populations derived from a cross between the
elite inbred line “Gy14” (a gynoecious, US processing type) and PI 432860
(a monoecious, long-fruited slicing cucumber from China; designated
narrow-based) (RFLP backbone; 58 markers spanning 766 cM over 10 LGs,
mean marker interval = 13.2 cM), and from a cross between “Gy14” and PI
183967 (C. sativus var. hardwickii, a monoecious small-fruited wild or feral
form; designated wide-based) (RAPD backbone; 70 markers spanning 480
cM over 10 LGs; mean marker interval = 6.9 cM). Likewise, Serquen et
al. (1997a) employed 77 RAPD markers and three morphological traits to
construct a map spanning nine LGs (600 cM; mean marker interval = 7.5
cM) using an F2 population from the narrow cross between the determinate,
gynoecious, standard leaf size, line “G421” (synom. “Gy-7”) and the
indeterminate, monoecious, little leaf size line “H-19”. Park et al. (2000)

then used an RIL population derived from the cross of “TMG1” and “ST8”
[resistance and susceptible to Zucchini yellow mosaic virus (ZYMV) and
papaya ringspot virus (PRSV-W), respectively], to generate a 353-point
map (RAPD, RFLP, AFLP, and loci conditioning virus resistances) defining
12 LGs with a mean marker interval of 4.2 cM. Information from these
maps was subsequently used in map merging experiments to create two
consensus maps (narrow and wide-base maps) by Bradeen et al. (2001)
using common AFLP anchor markers. The narrow-based consensus map
was constituted by merging the maps of Kennard et al. (1994), Serquen et al.
(1997a) and others previously published maps constructed with isozymes
and morphological characters (Fanourakis and Simon 1987; Pierce and
Wehner 1990; Meglic and Staub 1996; Horejsi et al. 2000). This map included
255 markers distributed over 10 LGs having a total length of 538.6 cM and
an average of 2.1 cM between markers. The wide-based consensus map
(C. sativus x C. sativus var. hardwickii) was constituted by merging wide-based
map of Kennard et al. (1994) with the maps of Pierce and Wehner (1990),
Knerr and Staub (1992), and Meglic and Staub (1996). This map included
197 markers that were distributed over 15 LG having a total length of 450.1
cM with an average of 2.3 cM between markers.
The recently dramatic increase in genomic resources [e.g., expressed
sequence tag (EST) libraries] and the development of new types of molecular
makers (e.g., SSR, SNP, CAPS, SCAR, and SRAP) have contributed to the
development of moderately saturated maps. The first map possessing
seven LGs (the theoretical haploid chromosome number of cucumber)
was constructed by Fazio et al. (2003a) using RILs derived from a cross
between ‘G421” and “H19”. This map possessed 128 molecular markers
(14 SSRs, 24 SCARs, 27 AFLPs, 62 RAPDs, and one SNP), three morphological
markers, and spanned 706 cM with a mean marker interval of 5.6 cM.
Subsequently, a 173-point linkage map (116 SRAPs, 33 RAPDs, 11 SSRs, 9
SCARs, 3 ISSRs, and 1 STS) was constructed using an F2 population derived
from the indeterminate line “S94”’ (northern China open-field type) and
the gynoecious indeterminate Chinese line “S06” (European greenhouse
types) (Yuan et al. 2008). This map spanned seven LG having a total length of
1,016 cM with a mean marker interval of 5.9 cM. More recently, a saturated
SSR-based cucumber map was assembled by Ren et al. (2009) using 77 RILs
derived from the wide-based cross between “Gy14” (C. sativus) x PI 183967
(C. sativus var. hardwickii) (Kennard et al. 1994), and 130 RILs derived from
the intraspecific (C. sativus) cross between line “9930” and “9110 Gt”. Nine
hundred sixty-six (966) novel (previously unmapped) SSR markers (Ren
et al. 2009) and 29 SSR markers previously reported (Danin-Poleg et al.
2000; Fazio et al. 2002; Kong et al. 2007) were utilized to construct a map
possessing seven LGs spanning 573 cM with a mean marker interval of 0.58
cM, that defined ~680 recombinant breakpoints.

8.3.3 Genetic Mapping of Quantitative Traits in Cucumber

Yield and quality are a major focus of cucumber improvement and consist
of many extensively reviewed, interrelated traits that are often the focus
of the cucumber breeder (Lower and Edwards 1986; Tatlioglu 1993;
Staub et al. 2008). These quantitatively and qualitatively inherited traits
range from disease resistance to plant and fruit architecture and habit.
Therefore, breeding objectives often focus on high fruit yield and quality
(i.e., appearance, taste, and nutrition) (Yuan et al. 2008). The development
of genetic linkage maps has provided tools for the molecular analysis
of important characteristics in cucumber including fruit quality, disease
resistance, and yield components (Serquen et al. 1997a; Park et al. 2000;
Dijkhuizen and Staub 2003; Fazio et al. 2003a; Sun et al. 2006c; Yaun et al.
2008; Table 8-2). The marker-QTL associations identified in these studies
form the foundation for cucumber improvement through MAS (Staub et
al. 2008).
8.3.3.1 Yield Components

Yield has been a focus of cucumber breeders for over 50 years (Staub et al.
2008). However, measurement of cucumber yield in the US is often difficult
because fruits are harvested before they reach physiological maturity (see
yield measurement as reviewed by Wehner 1989). Recently, breeding for
yield has focused on the genetic and physiological control of sex expression
(e.g, gynoecious or monoecious), modification of plant architecture (e.g.,
multiple lateral branches), and earliness (refer to the time of the first
harvested) (Trebitsh et al. 1997; Fazio et al. 2003a; Nam et al. 2006).
Sex expression in cucumber is determined by three major loci and
hormonal control as it relates to growing conditions (F, M, and A; Galun
1959, 1961; Shifriss 1961; Kubicki 1969). The F locus influences the degree
of femaleness (FF>Ff>ff), while the M locus determines whether flowers
are unisexual (M_) or bisexual (mm). The A locus conditions increased male
tendency if a plant is homozygous recessive aa and ff.
Genetic control and environmental variation of sex expression is
mediated through changes in plant hormonal levels (Staub et al. 2008).
Current theory holds that sex expression in cucumber is regulated by a
balance between ethylene, auxins, absissic acid (ABA), and gibberellins (GA;
Galun 1959; Roy and Saran 1990). While ethylene is considered the primary
hormone affecting femaleness (Byers et al. 1972), gibberellins regulate male
sex expression (Atsmon et al. 1968; Rudich et al. 1972a, b).
While the three-gene model (F, M, and A) describes the basic regulation
of sex types, a plant’s phenotype is also influenced by modifying genes
and environmental factors (Serquen et al. 1997a, b; Staub et al. 2008). The

256
Table 8-2 A summary of traits and their associated quantitative trait loci (QTL) in cucumber (Cucumis sativus L.).
Traita QTL R2 (%) Parents Populationb Reference

Sex expression 6 06.0–67.0 ‘Gy7’ x ‘H19’ F2-3 Serquen et al. 1997a
Female nodes on mainstem 3 05.0–16.0 ‘Gy7’ x ‘H19’ RIL Fazio et al. 2003a
Female node on primary lateral branches 4 05.0–06.0 ‘Gy7’ x ‘H19’ RIL Fazio et al. 2003a
Yield components
Earliness 4 03.0–08.0 ‘Gy7’ x ‘H19’ RIL Fazio et al. 2003a

Mainsteam length 5 08.0–45.0 ‘Gy7’ x ‘H19’ F2-3 Serquen et al. 1997a
Lateral branch number 4 11.0–40.0 ‘Gy7’ x ‘H19’ F2-3 Serquen et al. 1997a
13 02.0–32.0 ‘Gy7’ x ‘H19’ RIL Fazio et al. 2003a
Days to anthesis 3 08.0–13.0 ‘Gy7’ x ‘H19’ F2-3 Serquen et al. 1997a
Fruit number 4 06.0–20.0 ‘Gy7’ x ‘H19’ F2-3 Serquen et al. 1997a
Cumulative fruit per plant 7 03.0–22.0 ‘Gy7’ x ‘H19’ RIL Fazio et al. 2003a
Fruit weight 4 09.0–40.0 ‘Gy7’ x ‘H19’ F2-3 Serquen et al. 1997a
4 04.0–14.0 ‘S94’ x ‘S06’ F2-3 Yuan et al. 2008
Fruit length 1 21.0–31.0 ‘Gy7’ x ‘H19’ F2-3 Serquen et al. 1997a
Fruit quality components
6 06.0–23.0 ‘S94’ x ‘S06’ F2-3 Yuan et al. 2008

Fruit diameter 4 09.0–22.0 ‘Gy7’ x ‘H19’ F2-3 Serquen et al. 1997a
3 05.0–15.0 ‘S94’ x ‘S06’ F2-3 Yuan et al. 2008
Ratio length: diameter 2 14.0–15.0 ‘Gy7’ x ‘H19’ F2-3 Serquen et al. 1997a
6 04.0–22.0 ‘S94’ x ‘S06’ F2-3 Yuan et al. 2008
Fruit-stalk length 4 06.0–30.0 ‘S94’ x ‘S06’ F2-3 Yuan et al. 2008
Fruit length/stalk ratio 6 07.0–15.0 ‘S94’ x ‘S06’ F2-3 Yuan et al. 2008
Fruit pedicel length 3 06.0–31.0 ‘S94’ x ‘S06’ F2-3 Yuan et al. 2008
Seed cavity diameter 3 08.0–14.0 ‘S94’ x ‘S06’ F2-3 Yuan et al. 2008
Fruit flesh thickness 3 07.0–20.0 ‘S94’ x ‘S06’ F2-3 Yuan et al. 2008
Parthenocarpy 9 08.2–25.5 ‘2A’ x ‘Gy8’ F2-3 Sun et al. 2006c

Resistance Resistance to papaya ringspot virus 1 - ‘Straight 8’ x‘ Taichung Mou RIL Park et al. 2000
Diseases
Gua’
Resistance to zucchini yellow mosaic virus 1 - ‘Straight 8’ x ‘Taichung Mou RIL Park et al. 2000
Gua’
a
Trait name according to their published designation.
b
Percentage of phenotypic variation explain by QTL association.
c
Population type used in linkage map construction, where F2-3 and RIL refer to F3 family analysis and recombinant inbred lines, respectively.

existence of sex modifying genes is supported by the observation that inbred

gynoecious plants differ in their level of gynoecy and their capacity to confer
femaleness in F1 hybrids (Kubicki 1969; Zhang et al. 1992).
Interactions between F, M, and A produce the basic sex types in
cucumber and are important in the identification of QTL having major
effects on plant phenotype (Staub et al. 2008). The first QTL sex expression
analysis was performed by Serquen et al. (1997a) using a F2-3 assessment
(US processing cucumber), where the population was fixed for the M and
A genes (i.e., alleles segregating only at F locus). This analysis identified
four location-specific QTLs (LOD = 2.7–29.8) and two location-independent
QTLs (LOD = 3.4 and 5.1), which accounted for over 85% of the observed
phenotypic variation. Moreover, two major location-specific QTLs (LOD =
28.3 and 29.8; R2 > 67%) were detected within the genome region associated
with the F locus. An RIL-based QTL study was then performed using an RIL
population derived from the F3 populations of Serquen et al. (1997a). In that
study, three QTLs were detected for the number of female nodes on the main
stem (Fazio et al. 2003a). These genomic regions accounted for 31% (LOD
> 5.0) of the phenotypic variance, of which 16.4% was attributed to a QTL
associated with the F locus. Although a large portion of the genetics of sex
expression is controlled by the F locus, it is clear that there are modifying
genes having relatively small effects involved in the expression of gynoecy
(Staub et al. 2008).
Lateral branch number (multiple lateral branched, MLB) is positively
correlated (r = 0.58 to 0.42) with the number of fruit per plant in elite US
processing cucumber and wide-based populations (Fredrick and Staub 1989;
Cramer and Wehner 1998, 1999, 2000; Kupper and Staub 1998; Fazio 2001).
Moreover, narrow-sense heritability estimates for MLB range from 0.4 to
0.8 depending on growing environment and population structure (Wehner
et al. 1987; Serquen et al. 1997b). It is not surprising then that four QTLs
affecting MLB were identified by F2-3 family analysis that explained 48% to
66% (LOD = 3.3–10.4) of the observed phenotypic variance depending upon
growing environment (Serquen et al. 1997a). Subsequently, 13 QTLs (LOD =
2.7–32.9) were identified by an RIL-based analysis (advanced lines derived
from F3 populations of Serquen et al. 1997a) when lines were evaluated in
three environments (Fazio et al. 2003a). However, only five QTLs were
detected in at least two of the three locations with a combined R2 of 37%
to 55% (LOD = 2.7–32.9) depending on location. Although QTL colinearity
analysis in these populations is limited by the lack of common markers,
one major QTL in each mapping population [R2 = 32% (LOD = 32.9) and
40% (LOD = 29.8); Fazio et al. 2003a and Serquen et al. 1997a, respectively]
was located near the little leaf locus (ll).

The number of MLB are often positively correlated (r = 0.58 to 0.42)

with number of fruit per plant (FNP) in processing cucumber in different
growing environments (Fazio 2001). Consequently, the genomic locations
of three of the 13 QTLs associated with FNP overlap with QTLs associated
with MLB (see information above) in certain commercial US cucumber
populations (Fazio et al. 2003a). The combined effects of these 13 QTLs
explain 72.6% and 41.9% (LOD = 2.7–7.2) of the phenotypic variance for
FNP at the first and second harvest, respectively.
Earliness and stable gynoecious sex expression are important
components of yield in processing cucumber, especially in once-over
machine harvest operations (Staub et al. 2008). Several minor QTLs (R2
< 13%; LOD = 2.7–7.1) associated with earliness and yield components have
been identified by the analysis of F2-3 family and RIL populations derived
from the same parents (Serquen et al. 1997a; Fazio et al. 2003a). For instance,
QTL analysis for days to anthesis (earliness) revealed two QTLs in close
proximity to each other explaining 13% (LOD = 3.4–3.9) of the phenotypic
variance common in two environments, and a third QTL of smaller
magnitude (R2 = 8.1%; LOD = 3.1) in another environment (Serquen et al.
1997a). Subsequently, Fazio et al. (2003a) identified four QTLs for earliness,
which, in combination explained 20.7% (LOD = 2.7–7.1) of the phenotypic
variance. Furthermore, yield component QTLs detected in several diverse
cucumber populations (i.e., wide- and narrow-based) have been found to
be colinear (Sun et al. 2006c). These results indicate that, in cucumber, QTLs
identified in one population may have utility (i.e., in MAS) in another. This
phenomenon is likely associated with the narrow genetic base of cucumber
and the paucity of marker variation between cucumber germplasm sources
(especially elite commercial lines).
8.3.3.2 Fruit Quality

Processing cucumbers in the US are graded based on their size, and, thus,
fruit length: diameter (L: D) determines marketable yield (Staub et al. 2008).
The QTL analyses performed on fruit L: D to date suggest that a few stable
QTLs (perhaps 2–5), and their interaction with environmental factors play
a role in trait expression (Serquen et al. 1997a; Fazio et al. 2003a). In initial
studies, two QTLs linked to the F and ll loci regions, respectively, were
detected that explained 28% (LOD = 3.1 and 3.3) of the observed variation
for L:D (Serquen et al. 1997a). The magnitude and effect of these QTLs
were subsequently confirmed by Fazio et al. (2003a; R2 = 9.7–11.6%, LOD
= 3.6–6.0). Although Fazio et al. (2003a) identified 12 QTLs associated with
L:D (R2 = 2.7–11.6%, LOD = 2.6–8.6), only five were location-independent

(R2 = 3.0–11.6%, LOD =2.6–8.6), including the F and ll genomic regions.

Recently, Yuan et al. (2008) identified six QTLs associated with L:D in
greenhouse-grown cucumbers (R2 = 4.1–21.7%, LOD = 4.1–14.3), however,
only one QTL was location-independent (LOD > 6.6, R2 = 14–22%). The
development of more powerful mapping populations and inbred lines (e.g.,
near-isogenic lines) should allow for the identification of additional fruit
quality QTLs and eventually the complete dissection of fruit quality traits
in cucumber. This will be more straightforward for greenhouse-grown than
field-grown market types given the large environmental effects observed
in the open-field.
Parthenocarpy (seedless fruit) is an important quality-related trait in
cucumber, where higher quality is typically observed in seedless when
compared to their seeded counterparts. It is clear that parthenocarpy is
genetically controlled, but historically there has been little agreement
regarding the number of genes and type of gene action involved in
the expression of this trait (Staub et al. 2008). The results of de Ponti
(1975), El-Shawaf and Baker (1981), and Sun et al. (2006a, b) indicate that
parthenocarpy is quantitatively inherited in this species. In fact, 10 QTLs
have been detected that provide genetic control of parthenocarpy in a
narrow cross in US processing cucumber (“2A” × “Gy8”; Sun et al. 2006c),
where three QTLs map to the same genomic regions as QTLs controlling
fruit yield (Fazio et al. 2003a).
8.3.3.3 Diseases Resistance

The genetic control of resistance to scab (Ccu; Cladosporium cucumerinum Ellis
& Arthur), downy mildew [dm; Pseudoperonospora cubensis (Berk & Curt)
Rostow], bacterial wilt [Bw; Erwinia tracheiphila (Smith) Bergey, Harrison,
Breed, Hammer, and Huntoon], angular leaf spot (psl; Pseudomonas syringae
pv. lachrymans (Smith and Bryan) Young, Dye and Wilkie), anthracnose
(Ar, cla; Colletotrichum lagenarium), target leaf spot (Cca; Corynespora
cassiicola), and Fusarium wilt (Foc; Fusarium oxysporum f.sp. cucumerinum)
is conditioned by few genes (Robinson et al. 1976; Pierce and Wehner
1990; Meglic and Staub 1996; Zitter et al. 1996). In contrast, the genetics of
resistance to powdery mildew (Podosphaera xanthii; pm-1, -2, -3, pm-h), green
mottle mosaic virus (GMMV), gummy stem blight [GSB; Didymella bryoniae
(Auersw.) Rehm], belly rot (Rhizoctonia solani Kuehm), cottony leak (Pythium
spp.), Phytophthora rot (Phytophthora capsici Leo.), and gray mold [Rhizopus
stolonifer (Ehrenb.: Fr) Vuill] is complex (Staub et al. 2008). Likewise, the
genetic control of resistance to viruses is, in many instances, not well defined
and likely complex. For instance, the inheritance of resistance to cucumber

mosaic virus (CMV) and watermelon mosaic virus (WMV) are highly
quantitative, while resistance to papaya ring spot virus (PRSV-W), zucchini
mosaic virus (ZYMV), and Moroccan watermelon mosaic virus (MWMV)
are control by relatively few genes (Wai and Grumet 1995; Kabelka et al.
1997; Kabelka and Grumet 1997; Grumet et al. 2000). Moreover, only a few
of these resistances have been mapped to define associated QTL (Kennard
et al. 1994; Grumet et al. 2000; Horesji et al. 2000; Park et al. 2000; Bradeen
et al. 2001).
Resistance genes to downy mildew (dm) and scab (Ccu) were initially
mapped using F3 families derived from wide and narrow-based crosses in
greenhouse evaluations (Kennard et al. 1994). These two genes segregate
independently, and were located on different linkage groups. More recently,
Horesji et al. (2000) evaluated two narrow-based F3 populations [“WI
1983G” × “Straight 8” (Pop1), and “Zudm1” × “Straight 8” (Pop2)] in five
open-field and greenhouse environments in North America and Europe
to identify RAPD markers linked to dm. Bulked segregant analysis (BSA)
of susceptible and resistance families identified five markers (OPAS5-800,
BC526-1000, BC519-1100, OPG14-800 and OPX15-1100) linked to dm in
Pop1 and Pop 2. Subsequent map merging experiments (Bradeen et al.
2001) positioned the Ccu gene close to RFLP marker CMTC51 (~1 cM) in
LG A, and the dm gene within an 1 cM region flanked by AFLP (E11/M62-
F-170) and RAPD (BC526-1000) markers in LG C. Given their relatively
close linkage associations with resistance genes, these markers will likely
be exploitable in MAS.
Inheritance studies have demonstrated close linkage association among
potyvirus resistance genes (e.g., zymv, PRSV-W, wmv, MWMV) (Wai et al.
1997; Grumet et al. 2000; Park et al. 2000). For instance, studies by Wai et
al. (1997) demonstrated that zymv and PRSV-W co-segregate in populations
(F2 and backcross analysis) derived from inbred lines “TMG1” (resistance)
and “WI-2757” (susceptible). A subsequent linkage analysis using RILs
derived from “TMG1” and “Straight 8” (susceptible) confirmed that zymv
and PRSV-W were tightly linked (2.2 cM), and located in the distal region
of an LG (LG Q; Park et al. 2000). In addition, one AFLP marker (E15/M47-
F-197) co-segregated with zymv gene. Moreover, co-segregation among
four potyvirus resistance genes (zymv, PRSV-W, Wmv, and MWMV) was
detected during the evaluation of F2 and backcross populations derived
from three resistance sources (“TMG1”, “Dina-1” and “Surinan”) (Grumet
et al. 2000). These results suggest that multiple potyvirus resistance in
cucumber may be due to either the action of different alleles at a single
potyvirus resistance gene or multiple resistances conferred by a tightly
linked cluster of resistance genes.

8.4 Other Cucurbits

8.4.1 Introduction
Other genera of the Cucurbitaceae, such as Citrullus and Cucurbita, are
economically important and represent important sources of nutrition
worldwide, especially in developing countries (Lebeda et al. 2007). The
considerable genetic diversity within Citrullus and Cucurbita provides
a reservoir of genes for the development of new cultivars in breeding
programs. Nevertheless, the development and use of genomic tools for
the genetic improvement of Citrullus and Cucurbita presently lags behind
that of Cucumis species and most other vegetable crop species (Brown and
Myers 2002; Lebeda et al. 2007).
While Cucurbita species have a New World origin (Smith 1997), Citrullus
species originated from wild species in Africa (Dane and Liu 2007). Cucurbita
species are presumed to have been domesticated independently, and the
wild ancestor is known for each species, except for C. moschata (Merrick
1995). In contrast, the domestication of the watermelon is still unclear, where
two possible wild ancestors have been hypothesized: C. colocynthis (L.)
Shard. and C. lanatus var. citroides (Bailey) Mansf. (Dane and Liu 2007).
The genera Cucurbita, commonly referred to as squashes, pumpkins,
and gourds, include five domesticated species; C. pepo L., C. moschata
Duchense, C. maxima Duchense, C. ficifolia Bouché, and C. argyrosperma
Huber (Robinson and Decker-Walters 1997). All Cucurbita possess a basic
chromosome number of 2n = 2x = 40, and C. pepo, C. moshcata and C. maxima
are the economically most important. Although these domesticated species
are reproductively isolated, crosses among them can be made with difficulty
(Merrick 1995).
Watermelons (Citrullus lanatus) are grown throughout the world in areas
where a long, warm growing season prevails (Lebeda et al. 2007). The genus
contains four species universally with a common chromosome number
of 2n = 2x = 22 (Shimotsuma 1963). The cultivated species in this genus is
C. lanatus var. lanatus (Thub) Matsun & Nakai, where genes from cross-
compatible exotic taxa are often introgressed during plant improvement
(Lebeda et al. 2007). Morphological variation is high such that fruits vary
in size and shape, rind and seed color, exocarp, mesocarp and endocarp
color, hue, and intensity. However, cultivated watermelon has a relatively
narrow genetic base (i.e., by molecular marker assessment) and thus the
judicial use of biotechnologies (e.g., mutagenesis, diversity analysis, and
MAS) could be beneficial for plant improvement (Levi et al. 2000; Lebeda
et al. 2007).


The first linkage map in Cucurbita was constructed using an interspecific
cross between C. pepo and C. moshata (Brown and Myers 2002). This map
contained 148 RAPD loci and three morphological marker loci, distributed
among 28 LGs. The total length of the map was 1,954 cM, covering
approximately 75% of the Cucurbita genome. More recently, two F2 mapping
populations derived from intra-subspecific (C. pepo subsp. pepo; oil seed
pumpkin x zucchini) and an inter-subspecific (C. pepo subsp. pepo and
subsp. ovifera; oil seed pumpkin x crookneck, respectively) matings were
used to construct more saturated maps in Cucurbita (Zraide et al. 2007;
Table 6-1). These maps employed RAPD, AFLP, SSR, and morphological
markers, possessing 332 (intra-subspecific cross) and 323 (inter-subspecific
cross) markers distributed over 21 LGs with an average length of 2,200 cM.
Alignment of these maps using 62 common markers (RAPD and AFLP)
distributed across 14 LGs indicated that, in most cases, marker order was
conserved across linkage maps.
The recent development of more than 500 SSRs for use in genomic
analysis of Cucurbita has improved linkage map saturation (Gong et al.
2008a). For instance, the inter-subspecific C. pepo map of Zraide et al. (2007)
was enhanced by the addition of novel SSR (178) and ALFP (105) markers.
This moderately saturated map (659 loci; 178 SSRs, 244 AFLPs, 230 RAPDs,
5 SCARs and 2 morphological traits) spanned over 1,936 cM across 20 LGs
with an average marker interval of 2.9 cM. In addition to this map, two
F2 populations derived from a cross between “Waltham Butternut” and
“Nigerian local” (Map 8.1), and “ZHOU” and “Waltham Butternut” (Map
8.2) were recently used to construct two SSR-based linkage maps (Gong et
al. 2008b). These linkage maps contained 182 (Map 8.1) and 93 (Map 8.2) SSR
markers, and shared 62 common markers that allowed for their subsequent
integration through map merging experiments. This integrated C. moschata
map included 205 SRR markers spanning over 1,445 cM across 27 LGs,
where the mean marker interval was ~7 cM. Seventy-two SSR markers
resident on the integrated C. moschata map (Gong et al. 2008b) were also
common with the C. pepo map (Gong et al. 2008a), where marker orders
and marker map distances were conserved demonstrating a high level of
macro-synteny between these species.
As is the case with Cucurbita, linkage maps and genome studies in Citrullus
(i.e., watermelon) are also in their infancy. The first three maps constructed
in Citrullus employing RAPD markers were relatively unsaturated (i.e., 58,
26, and 13 loci; Hashizume et al. 1996; Hawkins et al. 2001). Subsequently, a
map was constructed containing 155 RAPD markers and one SCAR marker
(Levi et al. 2001c). Construction of this map employed a BC1 population
[(PI 296341 x “New Hampshire Midget”) x “New Hampshire Midget”] and

spanned over 1,295 cM across 17 LGs, where the mean marker interval was
8.3 cM. A testcross population [(PI 14113 x “New Hampshire Midget”) x PI
386015] was also used by Levi et al. (2002) to construct a map spanning over
1,166 cM, where 141 RAPD, 27 ISSR and one SCAR marker were distributed
across 25 LGs with a mean marker interval of 7.9 cM. In an effort to construct
a useable map comprised largely of codominant markers, Levi et al. (2006;
Table 6-1) then added 71 AFLP, 93 SRAP and 14 SSR markers to the map of
Levi et al. (2001c) which yielded a 347- point map spanning over 1,976 cM
across 19 LGs, where the mean marker interval was 5.7 cM. More recently, an
RIL-based watermelon map (elite inbred line “97103” x PI 296341) consisting
of 104 loci (87 RAPDs, 13 ISSRs, and 4 SCARs) was constructed (Zhang et
al. 2004). This map spanned over 1,028 cM across 15 LGs, where the mean
marker interval was 9.9 cM.
Initially, it has been difficult in watermelon to obtain genetic maps with
an LG number equivalent to its haploid chromosome number (n = x = 11).
This was achieved by Hashizume et al. (2003) who employed F2 and BC1
mapping populations derived from the same parents [“H-7” elite inbred
line x “SA-1” (African wild accession)] to construct a moderately saturated
map that defined 11 LGs. While the F2 mapping population was genotyped
with 554 loci (477 RAPD, 53 RFLP, 23 ISSR, and one isozyme marker), 240
selected markers (facilitating genome coverage given F2 map placement)
were used to create a BC1-based map. These F2 and BC1 maps spanned over
2,384 cM (mean marker interval = 4.3 cM) and 1,729 cM (mean marker
interval = 7.2 cM), respectively. Given the marker saturation obtained in
such maps, additional inbreeding might be appropriate leading to the
construction of immortalized mapping populations [i.e., RIL (F2-based)
and NIL (BC1-based)].
8.4.3 Genetic Mapping of Quantitative Traits

With the exception of melon and cucumber, the analysis of quantitative
traits in cucurbit crops has focused on the morphological evaluation of traits
not related to yield. For instance, Brown and Myers (2002) evaluated leaf
mottle, precocious yellow fruit, mature fruit color intensity and peduncle
color in Cucurbita (simply inherited), and fruit shape and leaf indentation
(quantitatively inherited). However, to date, only one QTL analysis has
been performed in watermelon (Hashizume et al. 2003), where one QTL
for hardiness of the rind (fruit exocarp) (R2 = 25.8%, LOD = 2.76), one QTL
for sugar content (BRIX) (R2 = 19%, LOD = 2.61), one QTL for yellow flesh
color (R2 = 55.2%, LOD = 11.03) and two QTLs of red flesh color (R2 = 35%,
LOD > 5) were detected in a backcross population.

8.5 Map-based Cloning in Cucurbits

A comprehensive understanding of the molecular basis of quantitative
inheritance in most if not all crops, including cucurbits, is lacking because
factors (i.e., genes/genomic regions) that control and regulate phenotypic
variation at the systems level (i.e., biochemical pathways) have only been
isolated in a few cases (Salvi and Tuberosa 2005). This fact is important to
plant improvement programs since a complete description of quantitative
trait architecture (i.e., gene action, epistasis) is not possible until the genetic
control defined by QTL can be associated with specific genes (i.e., isolated
by gene cloning) (Mackay 2001). For example, in cucurbits, only sparingly
few genes [e.g., Fom-2, Vat, nsv, andromonoecious (a) and gynoecious (g)]
have been cloned, and such genes control trait expression that is simply
inherited (e.g., Fusarium wilt, melon/cotton aphid, melon necrotic spot
virus, sex expression) (Joobeur et al. 2004; Pauquet et al. 2004; Nieto et al.
2006; Boualem et al. 2008; Martin et al. 2009).
8.5.1 Gene Identification

The Fom-2 gene conferring resistance to melon Fusarium wilt (causal
agent: Fusarium oxysporum f.sp. melonis) was the first gene to be cloned
in a cucurbit species via map-based methodologies (Joobeur et al. 2004).
This was a tedious process, where initially two co-dominant markers that
co-segregated with Fom-2 (Wang et al. 2000) were used to screen a melon
BAC-library to identify clones linked to the gene (Luo et al. 2001). A specific
BAC-end sequence was then employed to develop eight SSR and seven STS
markers, which were in turn used to fine-map the Fom-2 region (Joobeur et
al. 2004). A comparative analysis of 662 highly inbred individuals derived
from a previously characterized RIL population ( “Védrantais” x PI 161375;
Perin et al. 2002) was used to determine that Fom-2 resided in a 75 kb map
interval that was flanked by two specific sequence markers (STS411 and
STS296; Joobeur et al. 2004). Sequence analysis of the region predicted that
Fom-2 in melon encodes an NBS-LRR type R protein of the non-TIR family.
This gene is clearly polymorphic in this species since 25 of the 541 amino
acids assessed differed between the resistance and susceptible individuals
examined. An evaluation of these markers (STS411 and STS296) in 45 elite
breeding lines was not capable of differentiating among resistant and
susceptible lines, suggesting that a higher than predicted allelic diversity is
present at this locus. Additional analysis of putative resistance-associated
allelic sequences may allow for the development of more specific markers
for use in MAS of Fom-2 resistant genotypes.

In parallel to the cloning of Fom-2, Pauquet et al. (2004) cloned the

resistance gene in melon, Vat, which confers resistance to the melon/
cotton aphid Aphis gossypii Glover. The Vat resistance gene embodies a 6
kb long sequence (5 exons and 4 introns) that encodes a predicted 1,473-
amino acid protein with a coiled domain (CC), a nucleotide binding site
(NBS), and leucine-rich repeats (LRR). This resistance gene also confers
resistance to CMV and potyviruses such as ZYMV and PRSV when they are
transmitted by A. gossypii (Lecoq and Pitrat 1980). Moreover, Dogimont et
al. (2007) demonstrated that Pm-W, which confers resistance to the fungus
Podosphaera xanthii (causal agent of powdery mildew), in fact, possesses a
parallel allelic constitution with the Vat gene. Recently, the allelic variation
at the Vat/Pm-W locus was evaluated using a geographically diverse array
of 31 melon accessions exhibiting resistance to A. gossypii (Dogimont et al.
2008). The results of that analysis suggest that the number of repeats (varied
from two to five) of a conserved motif of 65 amino acids within the LRR
domain play a major role in the specific recognition of aphid type and/or
powdery mildew race as associated with resistance.
The isolation of the nsv gene, the only known source of resistance to
MNSV, resulted in the identification of a resistance-associated translation
initiation factor 4E (eIF4E) (Nieto et al. 2006). The characterization of nsv
occurred as a result of the construction of a high-resolution physical map
of a genomic region on LG XII using more than 3,000 offspring and nine
co-dominant markers that flanked the nsv region. Such mapping allowed
for the identification of a SNP (marker 52K20sp6) that co-segregated with
the nsv locus and a 100 kb BAC spanning a genetic distance of 0.73 cM
that physically contained the resistance gene (Morales et al. 2005). BAC-
sequence analysis and microsynteny analysis with A. thaliana identified
the melon eukaryotic translation initiation factor 4E (Cm-eIFE4E) as an nsv
candidate resistance gene (Nieto et al. 2006). It was then determined that a
single amino acid change at position 228 of the protein Cm-eIFE4E resulted
in MNSV resistance.
Sex determination in melon is governed by the interplay of the
andromonoecious (a) and gynoecious (g) genes (Poole and Grimball 1939;
Kenigsbuch and Cohen 1990). For instance, monoecious (AAGG) and
andromonoecious (aaGG) individuals bear male flowers on the main stem,
and female or hermaphrodite flowers on axillary branches, respectively.
While, gynoecious (AAgg) and hermaphrodite (aagg) individuals only bear
female and hermaphrodite flowers, respectively. The andromonoecious (a)
gene was isolated through the evaluation of 7,000 BC1 individuals derived
from the cross of monoecious and andromonoecious cultivars PI 124112 and
“Védrantais”, respectively (Boualem et al. 2008). Initially, a locus flanking
markers (AFLP M64 and M47; LG II) were employed to identify recombinant
individuals and screen melon BAC libraries. Subsequently, two BAC

sequences derived markers (L41 and R5) were used to delimit the region to
14 kbp containing a gene encoding for a 1-aminocyclopropane-1carboxylic
acid synthase (ACS), designated as CmACS-7. Genomic sequence analysis
of PI 124112 and “Védrantais” identified a single missense mutation in
one conserved region of the protein, which affects the enzymatic activity,
and in turn, the development of male organs. A similar strategy, along
with 12,660 BC1 individuals derived from a cross of PI 124112 and the
gynoecious cultivar “Gynadou”, was implemented to clone the gynoecious
(g) gene (Martin et al. 2009). The analysis located the g locus within a 1.4
kbp no-coding region containing a heavily methylated DNA transposon
element of the hAT family. Subsequent analysis found that this methylation
extends to one neighboring transcription factor gene [C2H2 zinc-finger of
the WIP protein subfamily (designated as CmWIP1)]. Functional analysis
determined that expression of CmWIP1 leads to carpel abortion, resulting
in the development of unisexual male flowers in melon.
8.5.2 Candidate Gene Analysis

Candidate gene analysis of complex resistance in cucurbits has not always
been straightforward. For instance, the fine dissection of the factors
associated with the presumably complex resistance to CMV in melon
(cmv-1; Perin et al. 2002) was initially attempted using near-isogenic lines
(NILs) (Essafi et al. 2009). This genetic analysis demonstrated that a single
gene, cmv-1, conferred host resistance to several CMV strains. Additional
mapping delimited cmv-1 to a 2.2 cM genomic region on LG XII, between
SSR markers CMN61_44 and CMN21_55. However, subsequent candidate
gene analysis determined that 10 previously identified translation initiation
factors associated with virus resistance in eukaryotes [Hordeum vulgare L.
(Stein et al. 2005); Oryza sativa (Albar et al. 2006)] were not present in this
interval.
Candidate gene analysis, however, has been successfully employed
in the analysis of carotenoids (i.e., mesocarp/endocarp fruit color) in
cucurbits. For instance, Bang et al. (2007) identified that lycopene β-cyclase
(LCYB), a critical gene in the carotenoid biosynthetic pathway, may control
the canary yellow and red flesh interior fruit color differences observed in
watermelon. Sequence comparisons among melon germplasm possessing
such color differences identified an SNP that introduces an amino acid
replacement in an evolutionarily conserved protein-coding region. In fact,
one-to-one co-segregation was observed between this SNP and flesh color
in 221 individuals segregating in an F2 and BC1 population. Moreover, this
association (SNP and color phenotype) was also observed in a diverse set
of 29 watermelon commercial cultivars.

In melon, the orange gene (Or; Lu et al. 2006) was mapped to LG IX and
aligned with a major QTL interval associated with the genetic control of
flesh color (Fig. 8-2; Cuevas et al. 2009). Syntenic relationships between this
and other melon maps (Perin et al. 2002; Fukino et al. 2008) indicates that
the Or gene maps to the same genomic region as wf, which conditions flesh
color in melon (Imam et al. 1972). The Or gene regulates the synthesis of
an DnaJ Cysteine-rich-domain protein (i.e., chaperon protein). This protein
is involved in the differentiation of proplastid and/or other non-colored
plastid into chromoplast plastids, which then provides a “metabolic sink”
for carotenoid accumulation (Lu et al. 2006). Such casual associations
must be subjected to rigorous confirmation (i.e., functional analysis and/
or genetic transformation). If confirmed, knowledge of such associations
can improve our knowledge and understanding of the gene-regulated
physiological processes in cucurbits.
LG IX LG IX Thresho LOD
Fukino et al. 2008 F2-3 population ld LOD = LOD =
0.0 CMN53_68 0.0 CMTC47/ CMN22_47
-carotene in
13.1 CMN53_68a -carotene in
16.3 CMN04_19
18.4 ECM150
21.0 ECM66
28.6 CMN04_19
34.2 wf
37.1 ECM180
40.3 Or
57.7 CMN53_72 57.7 CMN53_72A
66.0 CMATN22 66.0 CMATN22
70.7 CMMS35_05
Figure 8-2 Colinearity and synteny relationships in Linkage Group IX between melon (Cucumis
melo L.) maps constructed using recombinant inbred lines derived from a cross between “AR
5” (orange-fleshed) x “Harukei 3” (green-fleshed) [Fukino et al. 2008; mapping of white flesh
color (wf; Imam et al. 1972)], and an F2-3 population derived from a cross between Chinese line
“Q 3-2-2” (white-fleshed) and “Top Mark” (orange-fleshed). The latter was used to map Or
gene (Lu et al. 2006) and QTL-associated with β-carotene (i.e., orange flesh) in melon. Figure
adapted with kind permission from Springer Science and Business Media: Cuevas et al. 2009,

8.6 Marker-assisted Selection (MAS) in Cucurbits

The primary utility of genetic maps and QTL studies in plant improvement
is their deployment in MAS and breeding (Lebeda et al. 2007). The QTL
reported in various crop species, however, have not been rigorously
exploited in breeding programs (Bernardo 2008). The utility of QTL
mapping information for use in MAS depends largely on the complexity of
the target trait, and the inherent repeatability of marker/trait associations
(i.e., genotype x environment and epistatic interactions). Theoretically,
when relatively few QTLs (3–5) with substantial effects (R2 > 0.20) operate
to control a trait(s), significant gain per year might be predicted using
MAS. In cucumber, yield may be increased by altering plant architecture to
produce unique early flowering (days to flower; DTF), female (gynoecious;
GYN), highly branched (multiple lateral branching; MLB), long-fruited
(length:diameter ratio; L:D) cultivars with diverse plant statures. These
traits are controlled by relatively few genes (3–7), and genetic analyses have
identified QTLs that explain significant portions of the observed variation
(see above).
Unsaturated and saturated genetic maps have been constructed for
several cucurbit species (Park et al. 2000; Bradeen et al. 2001; Perin et al.
2002; Fazio and Staub 2003; Monforte et al. 2004; Levy et al. 2006; Zalapa
et al. 2007; Gong et al. 2008a, b; Fukino et al. 2008; Cuevas et al. 2009),
and QTL analyses of yield and quality components have been reported in
Cucumis species (Fazio et al. 2003a; Monforte et al. 2004; Zalapa et al. 2007;
Cuevas et al. 2008; Paris et al. 2008). However, extensive use of maps for
QTL analysis and MAS in most cucurbit species has not been widespread.
In cucumber, however, MAS has been evaluated and results indicate that
its use could improve selection efficiency (Fazio and Staub 2003b; Fan et
al. 2006; Robbins and Staub 2009). For instance, Fazio and Staub (2003b)
compared three breeding schemes in an effort to increase multiple lateral
branches (MLB; i.e., yield component); 1) phenotypic selection under open
field conditions (PHE); 2) random intermating without selection (RAN), and;
3) MAS, employing five QTL-markers (R2 = 3.2–19.2%). After three cycles of
selection, no significant difference was detected between means of PHE and
MAS, however, both were significantly higher than RAN. Since the three
cycles of MAS could occur in one year, while phenotypic selection required
three years to be completed, MAS increased overall breeding efficiency.
Fan et al. (2006) constructed a base population by intermating four
unique but complementary cucumber lines, that was subsequently subjected
to three cycles (C1–C3) of phenotypic mass selection (PHE) for DTF, GYN,
MLB, and L:D. In tandem, after two cycles of PHE, five C2 progeny (C2S)
were selected based on their allelic constitution at 15 QTLs associated with
these traits (see Fazio and Staub 2003a for genotype selection strategies).

Subsequently, two cycles of marker-assisted backcrossing [F1(i.e., C2S x

C2S), and BC1 (i.e., F1 x C2S)] for these traits were employed to produce
lines for comparative analysis of gain from selection by PHE and MAS.
Similar gains from selection were detected as a result of PHE and MAS for
MLB (~0.3 branches/cycle) and L: D (~0.1 unit increase/cycle). However,
while the percentage of female flowers of plants (GYN) was increased by
MAS (5.6 to 9.8% per cycle), no genetic gain was realized during PHE. In
this case, MAS operated to fix favorable alleles that were not exploited
by phenotypic selection in this population, indicating that MAS could be
applied for altering plant architecture in cucumber to improve its yield
potential during line development.
MAS might also be used in population development during plant
improvement. To assess the efficacy of MAS for population development,
Robbins and Staub (2009) intermated four cucumber inbred lines, and then
maternal bulks were used to create four base populations for recurrent mass
selection. Each of these populations then underwent three cycles of PHE
(open-field evaluations), MAS (genotyping at 18 QTLs), and RAN. MAS
and PHE were practiced for yield by selecting for MLB, GYN, DTF, and L:
D [i.e., yield-component traits that are quantitatively inherited (2–6 QTLs
per trait)]. Both MAS and PHE provided improvements in all traits under
selection in at least one population, except for DTF, which did not respond
to MAS. Generally, PHE was most effective for GYN, DTF, and L:D, while
MAS was most effective for multiple lateral branching and provided the
only increase in yield (fruit per plant).
When potentially valuable QTLs (LOD > 4; R2 > 5%; Staub et al. 2008)
are identified, their effects can be theoretically pyramided during breeding
to develop improved germplasm (Bernardo 2008). This introgression
process, however, is often affected by the genetic background of the elite
germplasm and QTL interactions (i.e., epistasis) that can mitigate expected
genetic gains through MAS (Staub et al. 2008). For instance, Robbins et
al. (2008) utilized molecular genotyping to create two sets (standard- and
little-leaf types) of cucumber inbred backcross lines [IBL; (BC2S4)] possessing
various numbers of QTLs that promote lateral branching. These lines were
evaluated under differing open-field conditions (i.e., plant spacing), and it
was observed that as the number of QTLs increased among inbred lines,
the number of branches did not generally change in the little-leaf lines, and
decreased in the standard-leaf lines. These results demonstrated that branch
development is determined by QTL allelic constitution, genetic background
and growing environment (i.e., plant spacing). Similar results have been
reported for grain yield in maize, suggesting that an understanding of QTL
x genetic background interactions is critical for the successful introgression
of complex traits (Blanc et al. 2006).

Where genetic diversity in a crop species is relatively low (e.g.,

cucumber, see above), MAS might be used to increase allelic diversity in
plant improvement programs. Delannay (2009) used MAS successfully to
increase genetic diversity in Beit Alpha, US Processing, and European Long
market type cucumber populations. Initially, marker diversity analysis was
used to identify the most genetically diverse accessions in a broad array
of commercial cultigens in each market type when compared to exotic
germplasm. This analysis identified elite Beit Alpha, US Processing, and
European Long lines that were then crossed to PI 285606, C. hystivus (Chen et
al. 2002), and PI 432858 accessions, respectively. These resulting F1 progeny
were subsequently backcrossed twice to their respective elite parents, after
marker genotyping (22 SSRs and 7 SCARs) and selection (most heterozygous
individuals) in each generation. These selections were then self-pollinated
to produce inbred backcross lines (IBLs; BC2S3). Phenotypic and genetic
analyses indicated that the IBL created possessed considerable inter-line
morphological and genotypic diversity that differed appreciably from
parental lines. Thus, these broad-based IBLs will likely be useful in genetic
studies (e.g., QTL analysis and epistasis quantification) and enhancing the
genetic diversity of commercial cucumber.
The relatively strong marker/trait associations (i.e., tight linkage)
identified in melon (see above) might also be exploited effectively using
MAS. For instance, the QTLs associated with fruit flesh color in cucurbits
(Bang et al. 2007; watermelon and Cuevas et al. 2009; melon) could be
used to identify plants in the first true-leaf stage that would potentially
bear fruit with the desired flesh color. These “selected” plants could be
transplanted to the open field, further selected for economically important
traits (i.e., disease resistance and yield and quality components) during the
growing season, and then phenotyped for interior fruit color at the end of
the season. This strategy would optimize field and human resources. In the
case of breeding for disease resistance, such a strategy (MAS followed by
open-field evaluation) has the potential for increasing selection efficiency
by reducing the selection of false positives (i.e., problems associated with
artificial inoculation). For instance, Wang et al. (2000) and Burger (2003)
screened genotypes of melon for resistance to Fusarium wilt using two
markers linked to the resistance gene, Fom-2. Marker/trait associations
were confirmed when high correlations (r = 0.82) were observed between
marker genotypes and disease resistance phenotypes in 69 geographically
diverse melon accessions. The application of MAS for resistance genes
in melon populations, however, requires an understanding of the allelic
diversity present at the desired locus (Joobeur et al. 2004) and the rate and
type of mutagenesis (i.e., evolution) related to resistance genes (Hulbert
et al. 2001).

8.7 Comparative Genomics in Cucurbits

Comparative genomics can characterize similarities and differences in
structure and function of hereditary information across taxa (Paterson et
al. 2000). Linkage map development and QTL identification has permitted
opportunities for genomic comparisons within and across species, genera,
and higher taxa. Furthermore, comparative genome analysis can define
the colinearity among genomes of related species (Gales and Devos 1998),
which, in turn, can aid in the isolation and dissection of agronomically
important genes (Schmidt 2002).
Comparative genome analysis of cucurbit species is in its infancy.
However, several synteny analyses in cucumber and melon have been
performed (Park et al. 2004; Staub et al. 2007; Al-Faifi et al. 2008; Meyer et
al. 2008; Huang et al. 2009). For instance, Staub et al. (2007) assessed the
degree of colinearity among three C. sativus maps: 1) US processing line
“2A” x “Gy8”; C. sativus var. sativus; 7% polymorphism; 2) US processing
line “Gy7” x “H-19”; C. sativus. var. sativus; 8% to 12% polymorphism, and;
3) a broad-based C. sativus var. sativus x C. sativus var. hardwickii (R.) Alef.
(“Gy14” x PI 183967); 12% polymorphism. Common makers (RAPD, SCAR,
SSR, RFLP, and AFLP) were identified in seven LGs, providing evidence
for synteny. These common markers were used as anchor markers for map
position comparisons of yield component QTLs. The relative order of anchor
markers in each of six linkage groups (LG 1, 2, and 4–7) that had two or
more anchor markers within each group was colinear. It was also determined
that there exist commonalities in the position of some yield component
QTLs in LG 1 and 4 of the maps examined. The general synteny among
these maps indicates that identification and mapping of additional anchor
markers would lead to successful map merging to increase cucumber map
saturation that might be exploited during cucumber breeding.
Park et al. (2004) used a 5 kb genomic region linked to the zym locus in
cucumber to hybridize melon BAC clones and assemble a 180 kb contig for
microsynteny analysis of those species. One molecular marker developed
from a melon BAC contig mapped close to the zym locus in cucumber
demonstrating a syntenic relationship between melon and cucumber. A
similar strategy was implemented to identify melon markers linked to the
Psm, a locus that controls sorting of paternally transmitted mitochondrial
DNA in cucumber (Al-Faifi et al. 2008). In this experiment, the Psm genomic
region was initially mapped to LG IV of melon, which in turn, was used
to identify additional markers linked to the Psm locus. Subsequently, LG
IV melon marker analyses located three markers (HS_07-C10, CMTC168,
and MC60) ordered at 8.3, 12.3, and 17.3 cM, respectively, from the Psm
locus in cucumber. A microsynteny analysis of orthologous regions of
the cucumber and melon genomes possessing the eukaryotic initiation

factors 4E (eIF4E) and eIF(iso)4E (i.e., genes associated with resistance to

portyvirues in plants) (Stein et al. 2005; Nieto et al. 2006), demonstrated a
high level of microsynteny and sequence similarity (Meyer et al. 2008). The
34 kb genomic region close to eIF4E in cucumber and melon contains three
putative genes in the same orientation with the same size and number of
exons. This was also the case for the 41 kb eIF(iso)4E region, which contains
five putative genes. Moreover, sequence similarity among these genes is
> 95% for coding regions, > 80% for introns, and > 70% for surrounding
sequencing. Similarly, the homology between watermelon and cucumber,
and between watermelon and melon is 78 and 84%, respectively (Pasha and
Sen 1998). Moreover, comparisons of melon and watermelon genetics maps
over the whole cucumber genome sequence revealed that there has been
no substantial chromosome rearrangement among these cultivars (Fig. 8-3;
Huang et al. 2009). For instance, cucumber chromosome 7 corresponds to
melon chromosome 1 and watermelon group 7. This considerable degree
of microsynteny and sequence similarity might be exploited for the cloning
and functional analysis of orthologous genes in cucurbit species.
Figure 8-3 Comparative analysis of the melon (Cucumis melo L.; n = x = 12) and watermelon
(Citrullus lanatus (Thumb.) Matsum & Nakai; n = x = 11) with the cucumber (Cucumis sativus
L.; n = x = 7) sequence map. The watermelon genetic maps employed in the analysis are
organized into 18 linkage groups. Figure adapted by permission from Macmillan Publishers
Ltd: Nature Genetics; Huang et al. 2009.
Microsynteny analysis between C. melo and A. thaliana has also been

performed to show gene order conservation and orientation (Leeuwen
et al. 2003). In such comparative analyses, a 117-kb melon BAC sequence
possessed two distinct genomic regions; one containing a cluster of three
resistance genes and other containing 11 putative functional genes. The latter
region is similar to two regions previously defined on chromosomes 3 (At3g)
and 5 (At5g) of the A. thaliana genome (Leeuwen et al. 2003). Likewise, six
of these melon BAC genes were found to be homologous to five Arabidopsis
genes, having identical gene order and orientation as that described for
A. thaliana. Furthermore, the exon structure of A. thaliana and that of melon
genes was found to be similar. Although the melon BAC resistance gene
cluster examined was not found to be syntenic with that of Arabidopsis,

their “clustering pattern” was similar to that of A. thaliana resistance genes

(Leeuwen et al. 2005).
Recently, two melon genome regions were compared to three model
plants genomes [A. thaliana, Medicago trucatula (Gaertn), and Populus
trichocarpa (Torr & Gray)] using two complete melon BAC sequences
(Deleu et al. 2007). The BAC’s examined contained homologous resistance
genes that are located in genomic regions on melon LG IV and LG XI. The
genomic region on LG XI (128 kb) was found to be syntenic with three
Arabidopsis (At1g, At2g, and At4g), two Populus (Pt_XI and scaffold Pt_204)
and one Medicago (Mt_4) genomic regions (Fig. 8-4). Additionally, this
genomic region in melon contains 13 putative genes, of which seven are
homologous to and six possess the same order and orientation as genes
found in Populus. Likewise, a genomic region on LG IV [215 kb; including
a previous overlapping BAC sequence described by Leeuwen et al. (2003)]
Figure 8-4 Overview of microsynteny between melon (Cucumis melo L.) BAC 1-21-10 and
regions in the Arabidopsis thaliana, Medicago truncatula, and Populus trichocarpa (not drawn
to scale). Genes are represented by arrows where gene name, number or ID given above or
below the arrow. Homologous genes are illustrated with the same color and indicated by
narrow connecting lines of the corresponding color. Arrows representing genes that have
one or more ESTs are designate with a spot. Genes without homologs are given in black.
Transposable elements are delineated in gray and indicated by Tn. At1g, At2g, At4g referrers
to A. thaliana chromosomes 1, 2 and 4, respectively. Cm11 referrers to C. melo Linkage Group
11, Pt_XI referrers to Populus trichocarpa Linkage Group XI, and Pt_204 referrers to Populus
unmapped scaffold 204. Mt4 referrers to M. truncatula chromosome 4, consists of BAC
clones AC137837, AC153460 and AC144608, and * = End of scaffold. Figure adapted with
kind permission from Springer Science and Business Media: Deleu et al. (2007), Mol Genet
Genomics 278: 611–622.

was syntenic to three Arabidopsis (At1g, At3g, and At5g), two Populus (Pt_XIII
and the scaffold Pt_70), and two Medicago (Mt1 and Mt7) genomic regions.
As was the case with the melon genomic region on LG XI, the greatest
synteny was detected in two Populus genomic regions. In fact, the highest
microsynteny values [53.8 (Pt_XI) and 40.9 (Pt_204)%, LG IV; 54.2 (Pt_XIII),
and 59.6 (Pt_70)%, LG XI] were obtained with Populus genome. Although,
the comparative analysis of additional melon sequences are necessary to
verify this putatively high degree of synteny, data currently suggest that the
Populus genome may have utility for genomic analysis (i.e., gene position
and candidate gene) in the Cucurbitaceae.
8.8 Further Perspective on Cucurbit Genomics

The genetic resources (i.e., technologies, sequence information, and genetic
stocks) in cucurbits related to genomics have increased dramatically in the
last five years to revolutionize genomic analyses. These resources include
BAC or fosmid libraries (Luo et al. 2001; Nam et al. 2005; Jobbeur et al.
2006; Meyer et al. 2008), more than 1,000 published cucurbit SSRs (Fazio et
al. 2002; Chiba et al. 2003; Ritchel et al. 2004; Gonzalo et al. 2005; Kong et
al. 2007; Fukino et al. 2007; Gong et al. 2008a), and immortalized mapping
populations (Park et al. 2000; Perin et al. 2002; Fazio et al. 2003a; Monforte
et al. 2004; Zhang et al. 2004; Eduardo et al. 2005; Zalapa et al. 2007; Ren et
al. 2009). Moreover, the decline in sequencing costs associated with novel
and new high-throughput technologies will allow for the sequencing and
synteny analyses of several cucurbit genomes in the near future (e.g.,
cucumber; Huang et al. 2009). Such comparative genomic information about
chromosome organization is of high value, especially in closely related taxa
(e.g., Cucumis and Citrullus species) (Paterson et al. 2000).
The International Cucurbit Genomics Initiative (ICuGI) has created
an EST library, which includes 16,128 unigenes for melon, 81,401 and
4,719 unigenes for cucumber and watermelon, respectively (accessed
November 2009; http: //www.icugi.org). In addition, unigene sequences
resident in this database have been used to develop 1,530, 1,679, and 257
SSR markers for melon, cucumber and watermelon, respectively. The
development of useful markers in cucurbits (especially cucumber, melon,
and watermelon) should not be underestimated. In a recently constructed
highly saturated map in cucumber (995 mapped SSRs; Ren et al. 2009),
595 SSR markers amplify in at least one of the cucurbit species surveyed
(i.e., melon, watermelon and squash). In fact, 115 of these SSRs amplify in
melon, watermelon, and squash, and as such should be considered for use
in colinearity/synteny analyses.

The genomic analysis of cucurbit species might also take advantage of

association mapping (syn. linkage disequilibrium mapping) approaches
(Risch and Merikangas 1996; Nordborg and Tavare 2002). This approach offers
three advantages over traditional linkage analysis: 1) increased mapping
resolution; 2) increased efficiency (reduced resources), and; 3) the ability to
identify large numbers of alleles (Yu and Buckler 2006). Association mapping
harnesses the genetic diversity of natural populations to resolve complex
trait variation (Zhu et al. 2008) and can be applied to cucurbits possessing
abundant diversity that has been characterized by phylogenetic analysis
of molecular and morphological markers (Lebeda et al. 2007). Since whole
genome-association scans of cucurbit species currently cannot be conducted
due to limited genome information, candidate gene association mapping is an
appropriate strategy to dissect complex trait (i.e., gene isolation). Candidate
gene association mapping exploits the results of genetic, biochemical, and/
or physiology studies in model and non-model plants species (Mackay 2001).
Moreover, nested association mapping (NAM; Yu et al. 2008; Buckler et al.
2009), a design that simultaneously exploits the advantages of both linkage
analysis and association mapping could be integrated into cucurbit mapping
efforts. This strategy involves the selection of a diverse “founder panel”,
follow by the development of a large set of related mapping progenies (e.g.,
RIL). The choice of germplasm is critical to the success of association analysis
and NAM (Yu et al. 2006, 2008), and, thus, the assembly and characterization
of cucurbit-specific diversity panels suitable for association mapping and
NAM will be important during plant improvement.
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9
Genomic and Functional Genomic
Resources of Melon
Zhangjun Fei1,2,* and Yang Liu1,a
ABSTRACT
Melon is an economically important vegetable crop and belongs to the
Cucurbitaceae family, which includes several other important crops
such as watermelon, cucumber, pumpkin and squash. It has served
as a model system for fruit ripening and sex determination studies. In
recent years, significant progress has been made in melon structural
and functional genomics. Several BAC libraries, a physical map, and
a number of high-density genetic linkage maps have been constructed
for melon and currently its genome is being sequenced by the Spanish
Genomics Initiative using a whole-genome shotgun strategy with the
Roche 454 GS FLX Titanium system. A large collection of ESTs consisting
of approximately ~130,000 ESTs derived from various tissues and
genotypes was created and is publicly available at the International
Cucurbit Genomics Initiative (ICuGI) website (http: //www.icugi.org).
Availability of these ESTs allowed the design of melon microarrays for
transcriptome analysis and the identification of SSR/SNP markers for
breeding programs. Other functional genomic resources and studies in
melon, including proteomics and metabolomics researches and melon
mutant libraries, are also discussed and summarized in this chapter.
Keywords: melon, genome sequencing, expressed sequence tags,
transcriptome profiling, molecular breeding
1
Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, NY 14853, USA.
2
USDA-ARS, Robert W. Holley Center for Agriculture and Health, Ithaca, NY 14853, USA.
a
Current address: College of Medicine, Texas A&M Health Science Center, Temple, TX 76504,
USA; e-mail: lyang@medicine.tamhsc.edu
Corresponding author: zf25@cornell.edu

Genomic and Functional Genomic Resources of Melon 287
9.1 Introduction
Melon (cucumis melo L.) belongs to the family Cucurbitaceae, which includes
several other important crops such as watermelon, cucumber, pumpkin
and squash. Melon is one of the most intensively studied species in terms
of fruit ripening and sex determination and is becoming an increasingly
important economic vegetable crop. During 2003–2005, the average global
production of melon reached 60.4 billion pounds, ranking at the 16th
position and sharing 1.3% of the world production of fruits and vegetables
(FAOStat database, 05/2006). In addition, melon is also one of the favorite
fruits for dessert and salad uses because of its unique flavor and aroma.
The average per capita consumption of melon in the US has been increasing
consecutively each decade since the 1960s. During the past few years
(2000–2006), the estimated average US per capita consumption exceeded
12 pounds per year, an 8% rise from 1990–1999 (Vegetables and Melons
outlook/VGS-320/April 19, 2007; Economic Research Service, USDA).
Compared with other major crops in the cucurbit family, melon is
one of the species producing fruits with the greatest amount of genetic
and morphological diversity. Melon includes a wide variety of cultivars
producing fruit deferring in many traits including fruit shape (round,
flat, elongated), size (from 50 g to 15 kg), flesh color (orange, light green,
white), sweetness (high or low sugar content), aroma volatiles and fruit
texture (Nunez-Palenius et al. 2008). In addition, melon fruits also have
significant variations in ripening behavior. Melon fruits can be categorized
as either climacteric or non-climacteric types based on their ripening
related respiration rate and ethylene evolution profiles. Usually fruit
in C. melo var. cantalupensis Naud and C. melo var. reticulatus Naud are
considered as climacteric types, including Cantaloup, Vedrantaise, Noy
Yizre’el and Dulce cultivars. Fruits from C. melo var. inodorus Naud are
generally classified as non-climacteric types, including for example Tam
Dew and Rochet cultivars. In addition to ripening physiology, climacteric
and non-climacteric melons also differ in additional phenotypes. Most of
the climacteric melons have orange flesh, high aroma and quick softening
upon ripening, while non-climacteric melons usually display pale-green
flesh, low level of aroma and slow softening resulting in typically longer
shelf-life than climacteric varieties. It is interesting to note that climacteric
and non-climacteric varieties exist in the same species implying that in at
least the case of melon, these differences are more likely the result of genetic
differences in ethylene synthesis or response and probably do not reflect
major differences in primary ripening programs (Giovannoni 2007).
Extensive molecular and genetic studies have been carried out in
order to better understand regulatory mechanisms underlying these traits
with the aim to improve melon fruit quality and to extend storage time.

Traditional breeding has produced numerous cultivars with enhanced fruit

traits in melon, especially disease resistance and environmental tolerance.
Traditional breeding, while highly effective, is generally slow and limited
by many factors including sexual compatibility and saturation of genetic
potential (Niemirowiczszczytt and Kubicki 1979; Pitrat et al. 1999; Nunez-
Palenius et al. 2008). Biotechnological methods have been applied to
generate transgenic melon in order to produce varieties with more desirable
agronomic traits, particularly to reduce softening and extend shelf-life.
Several lines of transgenic melon with suppression of the expression of an
ethylene synthesis gene, ACC oxidase, using antisense strategies have been
generated for climacteric melon varieties (Ayub et al. 1996; Bauchot et al.
1998; Flores et al. 2002; Nunez-Palenius et al. 2007). Reduction of ethylene
production in transgenic melon resulted in reduced fruit softening, aroma
volatile production and rind yellowing while carotenoid content and sugar
accumulation were not affected. Further analysis showed that shelf life
was extended from days to a few weeks and 68–85% of total volatiles were
reduced in the transgenic melons which greatly decreased the final quality of
the resulting fruit (Bauchot et al. 1998; Flores et al. 2002). While the outcome
of this effort did not yield marketable transgenic fruit, it did demonstrate the
primary role of ethylene in regulating important postharvest characteristics
with additional demonstration of an ethylene requirement for the critical
quality factor of ripe fruit aroma.
Melon, as well as cucumber, have long served as the primary model
system for sex determination studies due to its diverse floral sex types
(Tanurdzic and Banks 2004). Sex determination in flowering plants is a
fundamental developmental process of great economical importance. Sex
determination occurs by the selective arrest of either the male stamen or
female carpel during development (Kater et al. 2001). Ethylene is highly
correlated with the femaleness in cucurbit species and has been regarded
as the primary sex determination factor. In melon, sex expression is mainly
determined by two genes, andromonoecious (a) and gynoecious (g). Melon
plants with different alleles at these two loci display a wide range of
floral sex types, including hermaphrodites (aagg), which bear only bisexual
flowers; monoecious (A-G-), which bear both pistilate and staminate flowers;
andromonoecious (aaG-), which bear both hermaphrodite and staminate
flowers; and gynoecious (A-gg), which bear only pistilate flowers (Poole
and Grimball 1939; Kenigsbuch and Cohen 1990). Both A gene and G
gene have been cloned recently. A gene encodes 1-aminocyclopropane-1-
carboxylic acid synthase, a key enzyme in ethylene biosynthesis pathway
that represses stamen development in female flowers (Boualem et al. 2008).
G gene encodes a C2H2 zinc-finger transcription factor of the WIP protein
subfamily and can indirectly represses the expression of the A gene to allow
stamen development (Martin et al. 2009).

Despite significant progress in melon molecular and genetic studies,

genomic and functional genomic resources in melon have been very
limited. This situation has changed during the last couple of years due
to the advances in next-generation sequencing technologies. Recently
considerable efforts have been made to generate genomics and functional
genomics resources and tools for melon species with a primary aim to
develop a complete catalog of genes in this economically important crop,
including sequencing of melon genome and transcriptome, investigation
of transcriptome and metabolome dynamics under diverse conditions,
creation of high-density genetic and physical maps, and development of
comprehensive and saturated mutant populations. All of these are powerful
tools for forward genetic dissection of the genetic basis of biologically
interesting phenotypes.
9.2 Sequencing of Melon Genome

Melon is a diploid species that contains 2n = 24 chromosomes. The genome
size of melon is around 450–500 Mb (Arumuganathan and Earle 1991),
which is close to rice (430 Mb) and about four times of Arabidopsis (125
Mb). Currently genome sequences of more than 10 plant species, including
Arabidopsis (Arabidopsis Genome Initiative 2000), rice (International Rice
Genome Sequencing Project 2005), poplar (Tuskan et al. 2006), grape
(Jaillon et al. 2007), papaya (Ming et al. 2009), sorghum (Paterson et al.
2009), cucumber (Huang et al. 2009), maize (Schnable et al. 2009), soybean
(Schmutz et al. 2010), Brachypodium (International Brachypodium Initiative
2010), apple (Velasco et al. 2010), and castor bean (Chan et al. 2010), have
been published, while genome sequencing of several other plant species,
including Medicago truncatula (http: //www.medicago.org/genome),
tomato (http: //solgenomics.net/genomes/Solanum_lycopersicum),
potato (http: //www.potatogenome.net), peach (http: //www.rosaceae.
org/peach/genome), and watermelon (see Chapter 10) is completed or
nearly complete. It is worth noting that among the Cucurbitaceae family, the
genome of cucumber is published and the genome sequence of watermelon
will be available in the near future. Sequencing of the melon genome will not
only enable the discovery of genes and molecular markers associated with
diverse agronomic traits creating new opportunities for crop improvement,
but also provide an excellent opportunity for comparative genomics to
unveil distinct and common aspects of cucurbit plant evolution.
Currently the melon genome is being sequenced by the Spanish
Genomics Initiative (MELONOMICS). The estimated 450 Mb genome has
been sequenced using a whole-genome shotgun strategy with the Roche
454 GS FLX Titanium system. A combination of single and paired-end reads
derived from 3, 8 and 20 kb fragments have been performed. In total, more

than 24 million reads representing 17.6× of the melon genome have been
sequenced and assembled. Additionally, 0.05× of BAC end sequences have
been included in the assembly. The current melon assembly contains 367
Mb (81.5%) of the genome sequence, with an N50 scaffold size of 5.2 Mb
and an N90 index of 77. Anchoring the assembled genome scaffolds onto
melon chromosomes based on genetic and physical maps and annotating
the genome are currently underway (Garcia-Mas, pers. comm.).
9.3 Melon Genetic Maps

Genetic maps constructed based on molecular markers can serve a number
of purposes in basic and applied research. They are a key tool for cloning
genes or QTLs of interest by chromosome walking and for plant breeding
program through marker-assisted selection (MAS). In addition, genetic
maps can serve as the basis of physical map construction. Both genetic
and physical maps are critical in whole-genome sequencing projects as
combined with the fluorescent in situ hybridization (FISH) technology,
they provide an essential tool to anchor genome sequences onto individual
chromosomes. To date a total of around 20 melon genetic maps have
been constructed and several genes and QTLs of interest, most of which
confer resistances to different pathogens and pests or are related to fruit
quality and yield, were placed on these maps (see Chapter 8). Early
melon genetic maps mainly used markers of restriction fragment length
polymorphism (RFLP), amplified fragment length polymorphism (AFLP),
and random amplified polymorphic DNA (RAPD) and these maps are far
from saturation. RFLP, AFLP and RAPD makers have proved to be very
valuable in mapping genomes of various species; however these types of
markers are not user-friendly as they are either labor intensive to generate,
low rate of polymorphisms in melon (Shattuck-Eidens et al. 1990), or not
readily transferable to other populations (Ezura and Fukino 2009). With
the accumulation of sequence information (mainly expressed sequence
tags (ESTs)) during the past several years, markers of simple sequence
repeats (SSRs) and single nucleotide polymorphisms (SNPs) are being
more and more widely used in construction of melon genetic maps. These
markers have the following advantages: they are hypervariable, multiallelic,
codominant, locus-specific, and evenly distributed throughout the genome
(Ezura and Fukino 2009), and for markers derived from ESTs, they are
directly linked to expressed genes. Recently a high-throughput marker
discovery approach, which used an oligo-nucleotide microarray designed
based on melon EST sequences to identify single-feature polymorphisms
(SFPs), was reported in melon (Ophir et al. 2010). A total of 6,184 putative
SFPs between the parents of the mapping population, PI414723 and Dulce,
were identified. Sequencing of a subset of these SFPs in both parents

indicated that 79% of them could be validated and most of the SFPs (81%)
contained SNPs. The usefulness of these SFPs was further confirmed by
testing them in parents of another mapping population, PI161375 and “Piel
de Sapo” (Ophir et al. 2010). This approach provides an efficient way to
discover markers on a large scale for melon, which further facilitates genetic
mapping and molecular-assisted breeding.
Genetic maps in melon have been generated using different mapping
populations and different types of molecular markers. Nevertheless there
is no unique consensus map with the same name for the linkage groups.
In 2005, the International Cucurbit Genomics Initiative (ICuGI; http: //
www.icugi.org) was founded and one of its main goals was to develop a
consensus melon genetic map by merging the existing melon genetic maps
using SSRs as anchor markers. The consensus map is now available at ICuGI
website (http: //www.icugi.org). The map contains a total of 1,244 markers,
including 544 SSRs, 223 SNPs, 235 RFLPs, 109 AFLPs, 92 RAPDs, 18 ISSRs,
7 INDELs, 9 morphological traits, and 7 other markers. The map has 12
linkage groups and a total length of 1,449.1 cM, with an average distance
of 1.16 cM between points (ICuGI unpublished; http: //www.icugi.org).
This consensus map is significantly more saturated than any of the melon
genetic maps published so far and expected to play a significant role in
melon whole-genome sequencing and melon breeding programs.
9.4 Melon BAC Libraries and Physical Maps

Bacterial artificial chromosome (BAC) libraries are a critical tool for
plant genomic studies. It can be used for physical mapping, positional
cloning, genome sequencing, as well as comparative genomics to identify
microsynteny between different species. Several BAC libraries have been
constructed in melon (Luo et al. 2001; van Leeuwen et al. 2003; Boualem
et al. 2008; Martin et al. 2009). Two BAC libraries, a HindIII library and an
EcoRI library, were constructed for the multidisease-resistant line of melon
MR-1. The HindIII library contains about 68,000 clones and 95.6% of the
clones contain inserts with an average length of ~118 kb, providing 15.4×
coverage of the entire melon genome, while the EcoRI library contains
about 85,000 clones and 96% of the clones contain inserts with an average
length of ~114 kb, providing 18.7× coverage of the genome (Luo et al. 2001).
The two MR-1 BAC libraries are currently available through Clemson
University Genomics Institute (CUGI) BAC/EST Resource Center (http:
//www.genome.clemson.edu). The two libraries were used to map-based
clone FOM-2, an R gene which confers resistance to race 0 and 1 of soil-
borne fungus Fusarium oxysporum f.sp. melonis (Joobeur et al. 2004). A
BamHI BAC library was constructed for double-haploid melon line “PIT92”
(“PI161375” x “Piel de Sapo T111”). The library contains ~23,000 clones,

among which 80% are estimated to be non-empty clones with an average

insert size of 138 kb, representing 5.7× genome coverage (van Leeuwen et
al. 2003; Gonzalez et al. 2010). Full sequencing of a 117 kb BAC from this
library indicated a significant degree of microsynteny between genomes of
melon and Arabidopsis (van Leeuwen et al. 2003). The BamHI BAC library
was used to clone the nsv gene which confers resistance to an uncapped
and non-polyadenylated RNA virus in melon and encodes a melon
eukaryotic translation initiation factor 4E (Nieto et al. 2006). During the
process of map-based cloning andromonoecious (a) and gynoecious (g), two
major sex determination genes in melon, three additional BAC libraries
were constructed (Boualem et al. 2008; Martin et al. 2009). The first library,
a HindIII library, was constructed from the monoecious PI124112 cultivar.
The library contains ~93,700 clones with an average length of ~100 kb,
representing 20× genome equivalents (Boualem et al. 2008). The second
library, an EcoRI library, was constructed from the andromonoecious
Vedrantais cultivar. The library contains ~120,000 clones with an average
length of ~100 kb, representing 26× genome equivalents (Boualem et al.
2008). The third library was constructed from the gynoecious cultivar
Gynadou. The library consists of 50,000 clones and represents the haploid
melon genome at least 10 times over (Martin et al. 2009).
Recently a physical map of the melon genome was generated
(Gonzalez et al. 2010). The map was constructed using the BamHI BAC
library generated by van Leeuwen et al. (2003). The 23,040 BAC clones
from the library were fingerprinted using the high-information-content
fingerprinting (HICF) method with five-enzyme (BamHI, EcoRI, NdeI, XbaI,
and HaeIII) digestion and SNaPshot labeling. A total of 14,484 clones with
high quality fingerprints were obtained and their restriction patterns were
analyzed with FingerPrinted Contigs (FPC) program to obtain BAC contigs.
The resulting physical map consists of 1,355 contigs and 441 singletons, with
an estimated physical length of 407 Mb (0.9× coverage of the genome). The
size of the longest contig is 3.2 Mb and the average length of the contigs is
about 300 kb. Furthermore, 845 BAC clones could be anchored to the melon
genetic map using a set of 178 genetics markers (100 RFLPs, 76 SNPs and 2
SSRs). This anchoring determined the genetic position of 183 physical map
contigs/singletons, which represent 55 Mb (12%) of the melon genome. The
melon FPC database is available for download at http: //melonomics.upv.
es/static/files/public/physical_map/.
During the last several years, we have evidenced the huge expansion
of melon genomic and genetic resources. The melon genetic and physical
maps, as well as BAC libraries, will play significant roles in assembling and
refining the draft genome that will be available in the near future, which
will further facilitate the identification of genes/loci of interest and the
melon breeding program.

9.5 Functional Genomics of Melon

Although complete genome sequences can provide a wealth of information
on gene structure and its physical position on the genome and give us new
insights into areas such as genome content, architecture and organization,
they do not tell us the expression of genes under different conditions
and developmental stages and how genes work together to comprise
functioning cells and organisms. The development of a complex organism
and its interaction with the environment are mostly related to dynamic
changes of gene activities, which lead to downstream changes of protein
activity and metabolite accumulation, and ultimately phenotype changes.
Investigating the dynamics of transcriptome, proteome, metabolome,
and/or phenome and their interactions and relationships is the basis of
functional genomics.
9.5.1 Melon ESTs

In recent years rapid progress has been made in the area of functional
genomics, both theoretically and technologically. In melon, most functional
genomics studies focused on transcriptome and its dynamics. Expressed
sequenced tags (ESTs), typically created by single-pass and partially
sequencing randomly isolated gene transcripts that have been converted into
cDNA and cloned (Adams et al. 1991), directly represent the transcriptome
or transcribed portions of the genome. ESTs have been successfully applied
to accelerating gene discovery including gene family expansion, elucidating
phylogenetic relationships, facilitating the construction of physical and
genetic maps, inferring intron-exon boundaries and identifying alternative
spliced and polyadenylated transcripts, and facilitating large-scale gene
expression analysis (reviewed in Rudd 2003). Moreover, ESTs can provide
valuable transcribed sequence information which can serve as the basis for
high-throughput expression analysis via microarray technologies.
Prior to the establishment of ICuGI in 2005, only several thousand ESTs
were available in public domains. One of the major goals of ICuGI was to
sequence ~100,000 ESTs from different melon genotypes and tissues. In
the meantime, the Spanish Melon Genomics Project (MELOGEN) reported
the generation of ~30,000 ESTs from eight normalized cDNA libraries
prepared from tissues of fruits, roots, leaves, pathogen-infected roots and
cotyledons derived from four different genotypes: agrestis pat81, “Piel de
Sapo” Pinyonet torpedo, cantaloupe C-35, and “Piel de Sapo” T-111 (Table
9-1; Gonzalez-Ibeas et al. 2007). These ESTs were assembled into 16,637
unigenes, among which 6,023 were contigs and 10,614 were singletons. A
total of 1,052 potential SSRs and 356 SNPs were identified from this EST
collection (Gonzalez-Ibeas et al. 2007). Omid et al. (2007) also reported

~1,800 ESTs from phloem-sap of melon cultivar Hales Best Jumbo (Table
9-1). Comparison of these ESTs to those from other tissues allowed the
identification of a set of phloem-sap specific genes, which were mainly
associated with biotic stimulus, response to stress, and metal-ion binding
(Omid et al. 2007). Recently ICuGI released ~94,000 ESTs (Table 9-1; http: //
www.icugi.org), which represents a significant addition to the current melon
EST and functional genomic resources. These ESTs were generated from
fruits, flowers, and callus of four different genotypes: Dulce, Vedrantais,
PI161375, and “Piel de Sapo” T111, as well as melon necrotic spot virus
(MNSV)-infected leaf, root, and cotyledon of “Piel de Sapo” T111; more
than three-fourths of these ESTs were derived from full-length cDNA clones
(Table 9-1). To date, a total of 129,067 melon ESTs have been generated (Table
9-1). All these ESTs, together with 173 mRNA sequences from GenBank,
were assembled into 24,444 unigenes with an average length of 776.7 bp,
comprising 11,653 contigs with an average length of 972 bp and 12,791
singletons of 598.7 bp. The melon unigenes were extensively annotated by
comparing them to different kinds of sequence databases and by assigning
them gene ontology (GO) terms. The sequences and annotations of all
melon ESTs and unigenes are currently available at ICuGI website (http:
//www.icugi.org) in a searchable manner. Functional classification of the
melon unigenes according to a set of plant-specific GO slims, which are
a list of high-level GO terms providing a broad overview of the ontology
content (http: //www.geneontology.org/GO.slims.shtml), is shown in Fig.
9-1. A number of genes that are potentially involved in fruit and flower
development, fruit ripening, and responses to different biotic/abiotic
stresses can be identified from the melon EST collection (Fig. 9-1; ICuGI
unpublished). As shown in Table 9-1, melon ESTs were generated from
more than 10 different genotypes which show high genetic diversity, thus
SNPs were expected to be enriched in the EST collection. We were able to
discover a total of 3,073 high-confidence SNPs, among which were 1,972
transitions, 976 transversions, and 125 single-base indels. Furthermore, over
4,000 SSRs were also identified from the melon EST collection (ICuGI, http:
//www.icugi.org). These SNPs and SSRs are potential valuable markers
for melon breeding programs and part of them have been used to construct
high density genetic maps (Deleu et al. 2009; Harel-Beja et al. 2010).
9.5.2 Melon Transcriptome Profiling

Gene expression can be measured one gene at a time using traditional
methods such as RNA gel-blots and reverse transcriptase PCR. These
methods limit the number of transcripts that can be analyzed simultaneously.
Microarray technologies allow global detection of thousands of genes at one
time and have been the dominant gene expression analysis tool throughout

regulation of gene expression, epigenetic 97
growth 222
cell dif f erentiation 630
secondary metabolic process 603
cellular homeostasis 251
protein metabolic process 1525
cell growth 344
cellular component organization 2003
photosy nthesis 252
response to extracellular stimulus 201
cellular process 5423
f lower dev elopment 347
pollination 104
abscission 21
ripening 78
post-embry onic dev elopment 747
embry onic dev elopment 484
response to endogenous stimulus 1273
anatomical structure morphogenesis 818
response to abiotic stimulus 1534
response to biotic stimulus 844
tropism 76
response to external stimulus 468
biosy nthetic process 2421
catabolic process 2155
cell death 547
metabolic process 2885
multicellular organismal dev elopment 1445
signal transduction 1729
cell communication 111
cell cy cle 638
response to stress 3054
transport 2400
lipid metabolic process 1020
cellular amino acid and deriv ativ e metabolic process 942
protein modif ication process 1824
translation 870
transcription 1746
DNA metabolic process 634
nucleobase, nucleoside, nucleotide and nucleic acid metabolic process 1275
generation of precursor metabolites and energy 595
carbohy drate metabolic process 1052
reproduction 795
0 1000 2000 3000 4000 5000 6000
Figure 9-1 Functional classification of unigenes derived from a collection of ~130,000 melon
ESTs.
the last decade. Currently there are two microarray platforms available for
melon. The first is a cDNA array, which was designed based on the early
collection of melon ESTs. The array contains 3,068 unique cDNA clones
with each clone printed in triplicate on the array, as well as 12 negative
controls. The array is available through the ICuGI website (http: //www.
icugi.org). The second melon array is an oligo-nucleotide array constructed
using the Nimblegen Maskless Array Synthesis technology and currently
is commercially available at the Nimblegen Company. The array was

296
Table 9-1 Melon EST libraries.
library author cultivar tissue No. ESTs

SFTP2a Clepet & ICuGI Piel de Sapo T-111 mixture of fruits in four developmental stages 3,769
VFTP2a Clepet & ICuGI Vedrantais mixture of fruits in four developmental stages 3,770
DFTP2a Clepet & ICuGI Dulce mixture of fruits in four developmental stages 3,619
PFTP2a Clepet & ICuGI PI161375 mixture of fruits in four developmental stages 13,220
SFLP2a Clepet & ICuGI Piel de Sapo T-111 mixture of flowers in three developmental stages 320
VFLP2a Clepet & ICuGI Vedrantais mixture of flowers in three developmental stages 19,899
DFLP2a Clepet & ICuGI Dulce mixture of flowers in three developmental stages 3,537
PFLP2a Clepet & ICuGI PI161375 mixture of flowers in three developmental stages 3,610
CM-TEa Joobeur & ICuGI Piel de Sapo T-111 callus 5,700
CM-VEa Joobeur & ICuGI Vedrantais callus 5,467
CM-DEa Joobeur & ICuGI Dulce callus 5,485
CM-PEa Joobeur & ICuGI PI161375 callus 5,527
MNFG2a Aranda & Clepet & ICUGI Piel de Sapo T-111 melon necrotic spot virus (MNSV) infected leaf 8,071
MNRP2a Aranda & Clepet & ICUGI Piel de Sapo T-111 melon necrotic spot virus (MNSV) infected root 8,250
MNCP2a Aranda & Clepet & ICUGI Piel de Sapo T-111 melon necrotic spot virus (MNSV) infected cotyledon 3,512
A Gonzalez-Ibeas et al. 2008 agrestis pat81 healthy root 3,598
AI Gonzalez-Ibeas et al. 2008 agrestis pat81 root infected with M. cannonballus 3,190
PS Gonzalez-Ibeas et al. 2008 Piel de Sapo Pinyonet torpedo healthy root 3,315
PSI Gonzalez-Ibeas et al. 2008 Piel de Sapo Pinyonet torpedo root infected with M. cannonballus 3,442
HS Gonzalez-Ibeas et al. 2008 cantaloupe C-35 healthy leaf 2,925
CI Gonzalez-Ibeas et al. 2008 cantaloupe C-35 cotyledon infected with Cucumber Mosaic Virus (CMV) 5,468
15d Gonzalez-Ibeas et al. 2008 Piel de Sapo T-111 fruit at 15 days after pollination (DAP) 3,450
46d Gonzalez-Ibeas et al. 2008 Piel de Sapo T-111 fruit at 46 days after pollination 3,364
PF Omid et al. 2007 Hales Best Jumbo phloem sap stem collected between the 4th leaf and the 1,072
6th leaf from plants bearing fruits
PUN Omid et al. 2007 Hales Best Jumbo phloem sap stem collected between the 4th leaf and the 714
6th leaf from plants not bearing fruits

NYMF Katzir & Giovannoni Noy Yizre’el a mixture of mature green and mature yellow fruits (35 378
DAP and 37 DAP)
NYYF Katzir & Giovannoni Noy Yizre’el a mixture of fruits at 0, 1, 3, 12, and 25 DAP 459
DMF Katzir & Giovannoni Dulce mature fruit 969
TDMF Katzir & Giovannoni Tam Dew mature fruit 953
mc_p MELOGEN PI161375 seedling 728
mc_fi MELOGEN Piel de Sapo T-111 fruit at 15 DAP 104
f MELOGEN Piel de Sapo T-111 fruit at 20 DAP 208
SSH Katzir & Giovannoni various genotypes mainly fruits (17 subtraction libraries) 892
others - - - 82

Total - - - 1,29,067
a
Full length cDNA libraries.

designed based on the EST collection reported in Gonzalez-Ibeas et al.

(2007) and covered 17,510 melon unigenes with each unigene represented
by an average of 11 60-mer probes (Mascarell-Creus et al. 2009). Both
melon array platforms have not been widely used. Samuel (2008) used the
cDNA arrays to investigate transcriptional differences between the near-
isogenic aphid susceptible PMR 5 (Vat-) and aphid resistant AR 5 (Vat+)
melon plants in response to aphid (Aphis gossypii) infestation. Significant
expression changes of 50 genes (14 up and 36 down) in susceptible plants
and 27 (17 up and 10 down) in resistant plants upon aphid feeding were
able to be identified. Several genes responding to aphid feeding in both
susceptible and resistant lines, which might contribute to the basal level
of responses, as well as genes specifically regulated in either susceptible
or resistant lines, which might contribute to the different resistances of the
two lines, were identified (Samuel 2008). Zhang et al. (2009) compared the
transcriptomes of mature fruits from two melon varieties, flavor No 3, a
sour taste flesh melon, and cantaloupe, using the cDNA arrays. A total of
251 and 224 genes were found to be up- and down-regulated in flavor No
3, respectively. Among these differentially expressed genes, a citric synthase
was remarkably up-regulated in flavor No. 3, which could be a major factor
causing its sour taste (Zhang et al. 2009). We also used melon cDNA arrays
to investigate the differences of transcript levels during fruit development
and ripening between climacteric and non-climacteric melon cultivars.
The climacteric cultivar, Dulce, has high levels of aroma volatiles, orange
flesh and a relatively quick softening process during ripening, while the
ripening of the non-climacteric cultivar, Rochet, is accompanied by low
aroma, pale-greenish flesh and relatively slow softening. A total 183, 302,
293, and 480 genes showed differential expression between Dulce and
Rochet at immature unripe, mature, ripe and over-ripe stages, respectively.
By focusing the analysis on the expression patterns of genes that may
participate in biological pathways related to fruit quality traits, we were able
to identify specific differences that were consistent with the variable fruit
traits between these two varieties including: fruit softening, aroma, flavor
and carotenoid biosynthesis. We found that the quick softening phenotype of
Dulce during ripening was mainly caused by the concomitant up-regulation
of genes involved in cell wall degradation. Multiple regulatory mechanisms
may contribute to the orange color (β-carotene) of Dulce flesh but their gene
targets are clear in that transcriptional regulation of 1-deoxy-D-xylulose-5-
phosphate-reductoisomerase (DXR) and phytoene desaturase (PDS) appears
to be highly consistent with the carotenoid accumulation profiles of Dulce
versus Rochet. Aroma variation between Dulce and Rochet is likely due to
the differential expression of alcohol acyltransferases (AATs). In addition, as
expected, a number of genes involved in ethylene biosynthesis and signal
transduction showed significantly higher expression in the climacteric

cultivar (Dulce) during fruit ripening (Liu, Fei, Katzir and Giovannoni
unpubl.data). Mascarell-Creus et al. (2009) reported using the melon
Nimblegen oligo-nucleotide array to characterize global gene expression
profiles during fruit ripening of a non-climacteric melon, “Piel de Sapo”
T111, and in response to viral and fungal infections in melon roots and
cotyledons. They found that fruit ripening of “Piel de Sapo” T111 involved
down-regulation of ethylene biosynthesis genes and differential regulation
of sugar metabolism and cell wall-loosening enzymes, while in cultivar
agrestis Pat 81, responses of roots to fungi Monosporascus cannonballus
involved down-regulation of signal transduction pathways and cell wall
and cytoskeleton rearrangements, and in cultivar “Piel de Sapo” tendril,
cotyledons infected with cucumber mosaic virus (CMV) induced structural
cell-cycle deregulation (Mascarell-Creus et al. 2009).
It has been proved that the occurrence of ESTs from large unbiased
(non-normalized and non-subtracted) cDNA libraries represents the
relative expression of genes from which the ESTs are derived. Using ESTs
as an approach of expression profiling has been reported in several plant
species including tomato (Fei et al. 2004), grape (da Silva et al. 2005),
and apple (Park et al. 2006). As shown in Table 9-1, the current melon
EST collection includes ESTs derived from unbiased libraries (especially
those by ICuGI) of different tissues, development stages, and conditions.
These ESTs provided a valuable and rich source for identification of tissue
specific genes, highly expressed genes, and differentially expressed genes.
However, the overall effort and cost of EST sequencing are major hurdles
of this approach. This situation has changed during the past several years
due to rapid advances of next-generation sequencing technologies, led by
Illumina/Solexa and Roche/454 sequencing by synthesis technologies,
which can generate million to 10s of millions short tags (40–400 bp)
efficiently and cost effectively. In addition, these technologies do not require
labor intensive cloning effort and the cost can be reduced significantly.
RNA-seq, an emerging technology for large-scale gene expression analysis
through sequencing the whole RNA population using high-throughput
sequencing technologies (Wang et al. 2009), is expected to eventually phase
out microarrays for transcriptomic studies. Microarray is a hybridization-
based technology, thus it is prone to suffer from hybridization artifacts.
Cross-hybridization of closely related members in the same gene family
or short stretches of nucleotide homologies has been observed in cDNA
and oligo-nucleotide arrays (Kachalo et al. 2002), respectively. Stable
probe secondary structures (Southern et al. 1999) may interfere with
array hybridization. In addition, problems such as high background (e.g.,
nonspecific hybridization) and limited dynamic range (e.g., nonlinear and
saturable hybridization kinetics) have been well documented (Velculescu
and Kinzler 2007). RNA-seq approaches bypass the longstanding technical

problems inherent to microarrays and offer several additional advantages.

First, they allow direct enumeration of transcript molecules, providing the
statistical rigor of digital quantification. Additionally, digital expression data
are absolute. This is an important advantage over microarrays as data can
be directly compared across different experiments and laboratories without
the need for extensive internal controls or other experimental manipulation.
Finally, such approaches provide open systems that allow detection of
previously uncharacterized transcripts, as well as rare transcripts. This
facet allows profiling of transcripts in organisms where the compendium
of genes has not been fully characterized (Velculescu and Kinzler 2007).
With the full genome sequences available in the near future and advances in
sequencing technologies, we expect more and more transcriptome analysis
using RNA-seq approaches in melon, as evidenced in other model species
(Wang et al. 2009).
In the past few years, small RNAs (sRNAs) have been found to act as
key regulators of cellular processes. They regulate gene expression by acting
either on DNA to guide sequence elimination and chromatin remodeling
or on RNA to guide cleavage and translation repression (Vaucheret 2006).
Advances in high-throughput sequencing technologies have greatly
accelerated the discovery and characterization of new classes of sRNAs
including miRNA, ta-siRNA and nat-siRNA, as well as identification of
their novel regulatory roles in diverse biological processes. sRNAs have
been generated and characterized in many plant species including pumpkin,
a member in the Cucurbitaceae family (http: //smallrna.udel.edu/).
Unfortunately, to date no sRNAs have been reported in melon.
9.5.3 Melon Proteomics and Metabolomics

High-throughput analysis of differential gene expression through tag
or microarray approaches has proved to be a powerful tool for gaining
information about a certain biological process at the whole-genome scale.
However, some discrepancies between mRNA and its corresponding protein
levels can exist. Thus coupling transcriptome with proteome studies will
help us to identify post-transcriptional events and lead to better knowledge
of gene networks. Extensive proteomics studies have been reported for
other cucurbit species, such as in pumpkin to study phloem physiology
and long distance signaling system (Lin et al. 2009; Zhang et al. 2010), and
in cucumber to investigate salt stress responses (Du et al. 2010) and plant-
fungus interactions (Segarra et al. 2007). However, no reports have been
found for proteomics studies in melon. With the rapid accumulation of
melon genomic and functional genomic resources and advances of high-
throughput technologies, we foresee that proteomic analyses of melon

under various conditions and during different developmental stages will

get extensive attention.
During the last decade, analyses of mRNA at the whole-genome level
have proven central to most functional genomics initiatives. Recently,
metabolite profiling has emerged as an additional layer of phenotypic
information to more fully inform gene functional interpretation and
has the potential not only to provide deeper insight into complex
regulatory processes but also to determine biochemical and downstream
phenotypes directly (Fiehn et al. 2000). Currently more and more studies
in plants focus on metabolome analysis; however in melon this kind of
analysis is very limited. A metabolomic approach using 1H NMR and gas
chromatography−electrospray ionization time-of-flight mass spectrometry
(GC-EI-TOFMS) profiling was employed to assess both the concentration
and spatial localization of the main primary metabolites in three cultivars
of melon flesh fruits (Biais et al. 2009). It highlighted a number of metabolite
concentration differences among the cultivars and discovered a number of
metabolite gradients from the epicarp to the inner mesocarp (Biais et al. 2009).
Further investigation of the spatial localization of a number of metabolites
and the energy status changes in melon fruit using a metabolomics approach
combining primary metabolites with adenine nucleotide quantification
revealed concentration gradients for some sugars, amino acids, organic
acids and ethanol. The adenine nucleotide measurements in the fruit showed
that, consistent with the alanine and ethanol gradient, the ATP/ADP ratio
decreased drastically from the periphery to the center of the fruit. These
changes in metabolite concentrations reflected a decrease in the biosynthetic
processes (accumulation of sucrose), an inhibition of the TCA cycle
(accumulation of organic acids) and an activation of anaerobic metabolism
(diversion of pyruvate to ethanol and alanine) by low oxygen tension (Biais
et al. 2010). Recently, the META-PHOR (metabolomics for plants, health
and outreach; http: //www.meta-phor.eu/) project was launched. This is
an EU project with one of its objectives being to generate knowledge on
nutrition, health and quality-related metabolites present in three important
European crops: broccoli, melon and rice and enhance our understanding
on how this is influenced by physiology, genetics and environment (http:
//www.meta-phor.eu). Metabolomic analysis of mature fruits of various
melon cultivars has been performed (Allwood et al. 2009).
9.5.4 Melon Phenome

To study functions of interested genes and elucidate molecular mechanisms
of target traits, a comprehensive and saturated mutant population is
indispensable. In tomato, mutants have been collected over several
decades and are catalogued in the C.M. Rick Tomato Genetics Resource

Center (http: //tgrc.ucdavis.edu/); while in melon this effort is lacking.

A large melon (cultivar Noy Yizre’el) mutant library containing a total of
3,000 M2 families was developed through ethyl methane sulfonate (EMS)
mutagenesis (Tadmor et al. 2007). Phenotypic analyses of these mutants
revealed newly induced variation, mostly governed by single recessive
mutations, which affect different plant organs including cotyledon, leaves,
flowers, and fruits at different growth stages. Another EMS-induced mutant
library of melon (cultivar “Piel de Sapo” M62–113) was constructed by the
Melogen project. A total of 5,000 M2 mutant families were collected for this
library (Puigdomènech et al. 2007). Japanese groups also constructed an
EMS-induced mutant library for melon cultivar Harukei 3, which consisted
of 600 M2 families (Ezura and Fukino 2009). In addition, an EMS-induced
mutant library, which contained 3000 M2 families, was generated for melon
monoecious plants and used to confirm the functional roles of two major
sex determination genes, andromonoecious (a) and gynoecious (g) (Boualem
et al. 2008; Martin et al. 2009). All these mutant libraries can serve as the
material basis for targeting induced local lesions in the genomes (TILLING)
platform. EcoTILLING is a technology that is similar to TILLING, except that
its objective is to uncover natural genetic variations as opposed to induced
mutations. In melon, EcoTILLING was employed to screen for natural allelic
variation for resistance to melon necrotic spot virus (MNSV) in various
Cucumis accessions. High conservation of eIF4E, a translation initiation
factor, was found among 113 accessions evaluated. Six polymorphisms
were identified; however, only one site produced an amino acid change
that correlated with disease resistance (Nieto et al. 2007).
The mutant libraries and collections of various accessions of melon,
combined with TILLING or EcoTILLING platforms, provide a valuable
tool for melon functional genomics and crop improvement.
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10
Watermelon
Amnon Levi,1,* W. Patrick Wechter,1 Judy. A. Thies,1 Kai-Shu Ling,1
Umesh K. Reddy,2 Yong XU,3 Shaogui Guo3 and Xingping Zhang 4
ABSTRACT
This chapter elucidates the challenges in genetics, breeding, and
genomics of watermelon, dealing with crop origin and major diseases
and pests. It also delineates challenges in enhancing watermelon
cultivars for disease and pest resistance, genetic mapping and the
identification of disease resistance genes. It describes the recent
advances in genomics of watermelon and the construction of a physical
map and genetic maps for watermelon. At present, about 80% of the
watermelon (Citrullus lanatus var. lanatus) genome has been sequenced
and assembled using the advanced 454 and Solexa technologies. There
are two major genomic projects for watermelon. The first is in China
using the elite Chinese watermelon line 97103 for whole genome
sequencing. The second sequencing project is in the USA, using the
heirloom cultivar Charleston Gary genome for sequencing. Alongwith
the ongoing genomic project, there is a great need to phyenotype
and identify genes associated with disease or pest resistance in wild
watermelon and incorporate these genes into watermelon cultivars
without compromising fruit quality. The genomic and genetic studies
described here should be a useful platform for further studies and gene
discovery in watermelon.
Keywords: Citrullus, genomics, genetic map, resistance, genome
sequencing
1
USDA-ARS, U.S. Vegetable Laboratory, 2700 Savannah Highway, Charleston, SC, 29414,
USA.
2
Department of Biology, West Virginia State University, Institute, WV 25112, USA.
3
National Engineering Research Center for Vegetables Banjing, 100097 Beijing, PO Box 2443,
PR China.
4
Syngenta Seeds, 21435 Road 98, Woodland, CA 95695, USA.
*Corresponding author: Amnon-Levi@usda.ars.gov

10.1 Crop Origin and History

Watermelon is an important vegetable crop accounting for 2% of the
world area devoted to vegetable production (FAO 1995). It belongs to the
xerophitic genus Citrullus Schrad. ex Eckl. et Zeyh. that thrives in the Old
World tropics (Singh 1990). The center of diversity and possibly the center
of origin of Citrullus is southern Africa (Rubatzky 2001; Dane and Lang
2004). There, diverse populations of the Citrullus spp. grow freely (Jarret et
al. 1997; Mujaju et al. 2010; B. De Winter, pers. comm.). The genus Citrullus
comprises four known diploid (n = 11) species. Among these species is the
annual Citrullus lanatus (Thunb.) Matsum et Nakai, which is considered the
most important Citrullus species, grows in tropical and subtropical climates
throughout the world, and is a native of the dry sandy areas of southern
Africa (Bates and Robinson 1995). The C. lanatus species includes the
C. lanatus subsp. citroides [L.H. Bailey], the watermelon that is considered by
some botanists as a group of ancient cultigens derived from the “tsamma”
melon in southern Africa, and is also known as the Citron melon. The
tsamma melon is adapted to semidry conditions and is an essential source
of water and nutrients for animals, which results in the dissemination of
the watermelon seeds throughout the African deserts. It is for this reason
Citroides watermelon is also known as the cow melon.
In contrast to watermelon cultivars that usually have red, sweet
flesh, the citron watermelon has white or green flesh, and a wide range of
flavors and sugar content. An additional subspecies is the C. lanatus subsp.
mucospermum that thrive in the Kalahari Desert and is known as the “egusi”
type watermelon (also called “Ibara”), having unique large seeds rich in
fat and proteins. According to Jeffrey (2001), C. lanatus subsp. vulgaris
(Schrad. ex Eckl. et Zeyh.) Fursa is the desert watermelon group that gave
rise to the red, sweet cultivated watermelon. The cultivated watermelon,
which is grown and consumed by people throughout the world, has been
designated as C. lanatus var. lanatus (Schrad. ex Eckl. et Zeyh.) (Whitaker
and Davis 1962; Whitaker and Bemis 1976; Burkill 1985; Jarret et al. 1997).
Other known species are the annual Citrullus ecirrhosus Cogn. that have
bitter-tasting fruit (Meeuse 1962) and Citrullus rehmii De Winter that have
pink and olive green, spotted, mandarin sized, non-edible fruits (De Winter
1990; Singh 1990; Bates and Robinson 1995) and thrives in the Namibian
desert (Meeuse 1962; Jarret et al. 1997). Another distinct species is the bitter
Citrullus colocynthis (L.) Schrad. (syn.: bitter apple) that populates the deserts
of northern Africa, south-western Asia, and the Mediterranean (Zamir et
al. 1984; Burkill 1985; Navot and Zamir 1987; Jarret et al. 1997).
The history of watermelon has not been sufficiently investigated (Jeffrey
2001), and taxonomic classification of various Citrullus types collected in
the world has yet to be validated, as indicated for C. rehmii De Winter

Watermelon 311
(Robinson and Decker-Walters 1997; Jarret and Newman 2000). Several

studies used DNA markers including random amplified polymorphic DNA
(RAPD), inter-simple sequence repeat (ISSR), amplified fragment length
polymorphism (AFLP), and chloroplast or mitochondrial markers for
examining the phylogenetic relationships among the Citrullus species and
subspecies (Levi et al. 2001c, 2004; Dane and Lang 2004; Dane et al. 2004;
Levi and Thomas 2005; Nimmakayala et al. 2010). Overall, these studies
are in consensus, showing closer genetic relationships among the C. lanatus
subspecies and greater distance with desert species C. colocynthis (Figs. 10-1
and 10-2). A recent study using sequence-related amplified polymorphism
(SRAP) and expressed sequence tag-simple sequence repeat (EST-SSR)
markers showed that the watermelon (Citrullus spp.) is not closely related
to the round melon Praecitrullus fistulosus (Stocks) Pangalo that has been
cultivated in Asia since ancient times and has been considered a close
relative of watermelon (Levi et al. 2010). That study showed that P. fistulosus
is more closely related to Benincasa hispida (Thunb.) Cogn.
Citrullus lanatus subsp. citroides
Citrullus lanatus subsp. vulgaris
Citrullus lanatus subsp. citroides
Citrullus colocynthis
Figure 10-1 A dendogram showing genetic relationships among 31 Citrullus spp. based on
the neighbor-joining (NJ) method (Saitou and Nei 1987) using amplified fragment length
polymorphism (AFLP) and simple sequence repeat (SSR) fragments. Numbers shown at
different nodes represent percentage confidence limits obtained in the bootstrap analysis.

1 2 3 4 5 6 7 8 9 10 11 12
p8/Dra1
–1.8 Kb
Figure 10-2 Autoradiogram (chloroplast DNA P8 fragment to DNA digested with restriction
enzyme DraI), showing close affinity of the chloroplast genome of C. colocynthis (PI 386016, PI
388770, and PI 432337; lanes 10–12) to those of C. lanatus subsp. citroides (PI 271778, PI 296341,
and PI 500332; lanes 7–9), but not to those of Citrullus lanatus subsp. lanatus accessions (PI
162667, PI 169289, and PI 185635; lanes 4–6) or watermelon cultivars ( “Allsweet”, “Charleston
Gray” and “Black Diamond”; lanes 1–3).
Watermelon has been cultivated in Africa and in the Middle East for
thousands of years, and in China before 900 AD, and was brought from
Africa to the New World in the 1500s. Although watermelon was first
domesticated near the center of origin in Africa and is popular in most African
countries, Asia is the leading continent in production and consumption of
this important vegetable crop. Over 75% of the world watermelons are
produced in Asia. China is the leading producer, growing 76 million tons
of watermelon per year (about 73% of the total world production), while
the United States produces 3.8 million tons of watermelon per year (about
2.5% of total world production) (http://www.fao.org). Watermelon is
grown in 44 states in the US, Florida, Georgia, California, and Texas,
having long warm growing seasons are the major producing states. In the
US, watermelon production has increased from 1.2 M tons in 1980 to 3.8 M
tons in 2009, with an annual farm value of $470 million (US Department of
Agriculture, Agricultural Statistics, 2009). In recent years, there has been
an increase in consumer demand for seedless watermelons and production
of seedless watermelon has increased significantly. Today, over 80% of the
watermelons produced in the US are triploid seedless watermelons (US
National Watermelon Promotion Board; www.watermelon.org ).
Watermelon needs high temperatures and intense light to promote
flowering, and fruit set and development. Watermelon plants have trailing
vines with branched tendrils at each node, and their leaves are divided
into three or four pairs of lobes. In the field, the vines of certain wild
type accessions can reach up to 8–9 meter long. However, there are dwarf
watermelon accessions with short and/or less-branched stems controlled
by two recessive genes (dw-1 and dw-2). Roots of wild type watermelons
that grow in the African deserts are extensive, penetrating deep into the soil
layers. However, in commercial fields, with drip irrigation, most of the root
system of watermelon cultivars resides in the upper soil surface.
Watermelon has small flowers (about 0.5–0.6” and 0.6–1.0” in diameter in
C. colocynthis and C. lanatus, respectively) compared with other major cucurbit
crops, including Benincasa hispida (Thunb.) Cogn, Lagenaria siceraria (Mol.)

Watermelon 313
Standl. and Cucurbita spp.(Levi et al. 2010). Like most cucurbit species, the
flowers of watermelon have five yellow petals. Most watermelon cultivars are
monoecious, having separate male (staminate) and female (pistillate) flowers
(that are typically formed in this order) on the same plant. Many of the wild
type watermelon accessions are andromonoecious, producing both male and
perfect (hermaphroditic) flowers on the same plant. The female flowers have
an inferior ovary, and the size and shape of the ovary is correlated with final
fruit size and shape. In many cultivars, the male flowers are positioned at
most nodes while the female or perfect flowers are positioned at every seventh
node. The male:female flower ratio of typical watermelon cultivars ranges
from 3:1 (Extra Early Sugar Baby) to 7:1 (Black Diamond) to 11:1 (Charleston
Gray) and to 15:1 (C. lanatus subsp. citroides PI 500331). Watermelon plants
start to form flower at the 5–7th internodes, at about 4 weeks after planting.
Ethylene is known to play a critical role in floral sex determination of cucurbit
species. Ethylene promotes female flowers in cucumber and melon. However,
in watermelon it is possible that ethylene promotes the formation of male
flowers (Salman-Minkov et al. 2008).
The phylogenetic relationships among Citrullus species and subspecies
were examined using isozymes (Zamir et al. 1984; Navot and Zamir 1987),
genomic DNA markers (Jarret et al. 1997; Levi et al. 2001a, b) and organelle
DNA markers (Dane and Lang 2004; Levi and Thomas 2005). These genetic
studies (Navot and Zamir 1987; Levi et al. 2001a, b) indicated that although
possessing a wide phenotypic diversity, a narrow genetic base exists
among watermelon cultivars. In these studies, most isozymes produced
monomorphic patterns among cultivars (Zamir et al. 1984; Biles et al. 1989).
RAPD analysis produced low polymorphism, but provided informative
markers that could distinguish among watermelon cultivars that share a
narrow genetic base (Hashizume et al. 1993; Zhang et al. 2004). This narrow
genetic base is a result of many years of cultivation and continuous selection
for a set of desirable qualities, suitable to the needs of growers, shippers,
and consumers.
Over 314 American heirloom cultivars are maintained by the USDA, ARS,
Regional Plant Introduction Station, Griffin, Georgia and are considered an
essential germplasm resource for watermelon breeding programs. Among
them are “Allsweet”, “Au-Producer”, “Charleston Gray”, “Crimson Sweet”,
“Jubilee”, and “Peacock”. These heirloom cultivars are grown throughout
the world and are widely used as parents for many F1 hybrid lines. There
is incomplete information regarding the ancestries of many American
watermelon cultivars developed during the 19th and early 20th centuries.
For a long time, the identification of watermelon cultivars relied mainly
on fruit characteristics. Today, molecular markers are extremely useful in
determining genetic relatedness and genetic purity of cultivars. Overall, the
watermelon cultivars do not show any inbreeding depression, and inbred

lines can be developed without difficulty through self-pollination. However,

due to the narrow genetic diversity, there is little or no heterosis among
watermelon cultivars. Still, F1 hybrids are considered valuable by breeders
and growers because they combine a variety of qualities, including disease
resistance and fruit quality donated by their parents. During the last decade,
consumer demand for seedless watermelons has increased considerably.
Consequently, production practices for seedless watermelon have improved
significantly. Today, most triploid seedless watermelon cultivars are F1
hybrids, resulting from cross-pollinating a tetraploid female parent with
a diploid male parent. This practice expands the genetic diversity among
watermelon cultivars through crosses with different Citrullus species and
subspecies. Selection for enhanced breeding lines could be useful for
producing F1 hybrids that are heterozygous in many gene loci and as a
result they are more vigorous, exhibiting significant heterosis.
A large number of watermelon heirloom cultivars have been developed
in the US in the last 100 years. These cultivars represent a wide range of fruit
phenotypes, including fruit size (ice box, small, medium, large, or giant),
shape (round, oval, blocky, or elongated), rind colors (white, gray, light green,
green, dark green), and rind pattern (dappled, narrow, medium, or wide
stripes, light solid, or dark solid). The flesh of watermelon fruits can be white,
light green, yellow, orange, or red in color. Most wild watermelon types have
white, light green or yellow flesh, while most watermelon cultivars have red
flesh. Genetic analysis using RAPD markers revealed low genetic diversity
among 46 heirloom cultivars, indicating the need to enhance watermelon
cultivars with genes from the wild type watermelon (Levi et al. 2010).
In contrast with the narrow genetic base among watermelon cultivars,
wide genetic diversity exists among wild watermelon and Citrullus species.
These species are considered viable sources for disease and pest resistance
genes that could be useful in enhancing watermelon cultivars (Whitaker
and Davis 1962; Jarret et al. 1997; Levi et al. 2002; Ling et al. 2009; Harris
et al. 2009a, 2009b). The National Plant Germplasm System (NPGS) of the
United States Department of Agriculture (USDA), Agricultural Research
Service (ARS) maintains a large number of wild Citrullus spp. accessions
that were collected from different parts of the world (majority from Africa).
In addition, the USDA, ARS maintains a large number of heirloom cultivars
that represent the different germplasm and the core of watermelon cultivars.
There are over 1,800 US Plant Introduction (PI) accessions that are kept at
the Regional Plant Introduction Station, Griffin, Georgia. Of these, about
1,640 PIs belong to C. lanatus subsp. lanatus, 134 PIs belong to C. lanatus
subsp. citroides, and 40 PIs belong C. colocynthis. These wild watermelon PIs
should be a useful source for disease or pest resistance, as has been shown
in a recent study examining nematode resistance among watermelon PIs
(Fig. 10-3) (Thies and Levi 2007).

Watermelon 315
Figure 10-3 Root systems of Citrullus colocynthis, C. lanatus subsp. lanatus “Charleston Gray”
heavily infected with peanut root-knot nematode (RKN), Meloidogyne arenaria race 1, versus
C. lanatus subsp. citroides showing resistance to RKN.
10.2 Watermelon Fruit

The watermelon fruit contains mostly water (93%) and carbohydrates
(6%), and diminutive amounts (~1%) of protein, fat, and minerals. Still, it is
known to have numerous health benefits. It is a viable source of potassium,
magnesium, health-promoting amino acids (citrulline, arginine, and
glutathione), and vitamins A, B1, B6, and C (Rimando and Perkins-Veazie
2005), and is an excellent source of the antioxidant carotenoids, lycopene
and β-carotene. Similar to the tomato, watermelon has a wide range of
fruit color mutations associated with carotenoid biosynthesis. However, in
contrast to the tomato, very little is known about carotenoid biosynthesis
in watermelon fruits (Tadmor et al. 2004, 2005; Levi et al. 2006; Wechter et
al. 2008). Watermelon cultivars with red flesh have high lycopene content
(2,300–7,200 µg/100 g) compared to those having orange flesh. Like in the
tomato, the watermelon fruits accumulate large quantities of lycopene in their
fleshy mesocarp tissue. The lycopene has strong reactive-oxygen scavenging
abilities (Stahl and Sies 1996; Agarwal and Rao 2000), which is thought to
promote human health (Tadmor et al. 2004, 2005), and may reduce the risk of
prostate or lung cancer and coronary heart disease if consumed on a regular
basis (Giovannucci et al. 2002; Fraser and Bramley 2004; Perkins-Veazie et al.
2006). Watermelon is also a natural source of the carotenoids, prolycopene
and violaxanthin, as well as of the cardiovascular health-promoting amino
acids citrulline, arginine, and glutathione (Rimando and Perkins-Veazie 2005).
Watermelon varieties produce widely varying amounts of these carotenoids
or amino acids due to mutations in genes controlling their biosynthesis
(Tadmor et al. 2004, 2005). Bang et al. (2007) developed a cleaved amplified

polymorphic sequences (CAPS) marker associated with the lycopene β-cyclase

(LCYB), a key gene in the carotenoid pathway that causes watermelon
to accumulate lycopene and other downstream carotenoids. This CAPS
marker proved useful in distinguishing between red and canary yellow
flesh watermelon (Bang et al. 2007). The nutritional and health benefits of
carotenoids have been recognized by producers and consumer groups, and
there is great interest in the watermelon industry to enhance the nutritional
qualities of watermelon.
Like fruits of most plant species (Seymour and McGlasson 1993),
ripening watermelons undergo changes in pigment accumulation, flavor
and aromatic volatiles, conversion of starch to sugars, and increased
susceptibility to post-harvest pathogens (Karakurt and Huber 2003; Levi et
al. 2006; Wechter et al. 2008). In this respect, there are differences between
seeded and seedless watermelon fruits. For example, seeded watermelons
may have shorter shelf-life than seedless watermelons due to faster
degradation of the tissue surrounding the seeds. Seedless watermelons
may have different sugar and aromatic compound contents than seeded
watermelons of similar genetic background. Identifying the genes that
control watermelon fruit quality and analyzing their differential expression
in seeded versus seedless watermelon will be useful in enhancing fruit
quality, nutritional values, and shelf-life of seedless watermelons, to make
them suitable to consumer needs.
10.3 Watermelon Seeds

Watermelon seeds are diverse in size, shape, texture and color, and are rich
in fat and protein. The egusi-type watermelon that is widely cultivated in
Nigeria has the most unique seeds. In the egusi watermelon the ovary wall
tissue surrounding the seeds (pericarp) is fleshy, and the seeds are thick and
edible and have high protein and carbohydrate content. Watermelon seed
size varies greatly. The edible seed watermelon growing in China has seed
size as big as 9 seeds per gram and the “tomato seed” watermelon mutant
produce seed as small as 200 seed per gram (Zhang 1996a). Watermelon seed
typically do not have dormancy; however, the seed of citron watermelon
germination improves after 6 months storage. The seed coat of Citrullus
colocynthis is very hard and usually does not germinate without seed
scarification.
10.4 Fruit Genomics

The high-throughput sequencing of cDNA clones (libraries) has produced
extensive genomic databases and large numbers of ESTs for various plant
species. Levi et al. (2006) isolated RNA from watermelon fruits at early,

Watermelon 317
maturing, and ripe stages (Davis et al. 2006), and constructed normalized
cDNA libraries that were subtracted by hybridization with leaf cDNA. Eight
thousand eight hundred cDNA clones of the watermelon flesh subtraction
library were sequenced and ESTs associated with fruit setting, development,
and ripening were identified. These 8,800 ESTs were assembled into 4,770
EST-unigenes. These EST-unigenes can be found at the International Cucurbit
Genomics Initiative (ICuGI) website (http://www.icugi.org). About 29%
of the 4,770 watermelon EST-unigenes had no detectable homologous gene
sequences to any other genomes or protein sequences in GenBank (Fig. 10-4)
(Levi et al. 2006; Wechter et al. 2008). These results indicate that watermelon
may offer a large number of unique genes and metabolites that may not exist
in other plant species. Also, about 20% of the EST-unigenes correspond to
genes with unknown function, whereas about 50% correspond to genes with
known function in other plant species. These “EST-unigenes” are mainly
Figure 10-4 Classification of 880 EST-unigenes Illini Red watermelon fruit based on homology
of 880 EST-unigenes to gene sequences in other plant species.
associated with metabolism, membrane transport, cytoskeleton synthesis

and structure, cell wall formation and cell division, signal transduction,
nucleic acid binding and transcription factors, defense and stress response,
and secondary metabolism. These EST-unigenes provide an expanded pool
of genes associated with fruit development. These EST-unigenes have been
useful for the development of EST-SSR markers that were used in saturating
the genetic map of watermelon based on a recombinant inbred line (RIL)

population derived from a cross between PI 296341-FR and the elite Chinese
breeding line 97103 (Xu et al. 2010). Also, the expression of 832 EST-unigenes
was examined using microarray and quantitative real-time PCR approach
to elucidate the genetic flow of events associated with fruit development
and ripening in watermelon fruit (Wechter et al. 2008). The microarray
analysis identified 335 unique ESTs that are differentially modulated by
at least two-fold in watermelon fruit during the early, ripening, or mature
stage when compared to leaf. Primers for the differentially expressed genes
have been developed and are being used to determine their association with
fruit quality. This microarray study also elucidated the role of ethylene in
fruit development in watermelon and in non-climacteric fruits (Salman-
Minkov et al. 2008), showing that ethylene levels are highest during the
early development stages and decrease during maturation and ripening of
the watermelon fruit. These genomic and metabolomic profiles provided
valuable information on genes and metabolites that affect watermelon fruit
quality (Wechter et al. 2008).
In the US, watermelon is a major specialty crop, which provides
an important source of income for farmers and a vital lifeline to the
surrounding rural communities in the southern states. However, in recent
years, watermelon growers in the US have faced new challenges related to
heightened competition with vegetables from Central and South America;
increased production costs due to the significant increase in the cost of oil
and its byproducts; and increased disease, pest, and environmental pressures
(urbanization and reduction in agricultural land, poor soil conditions,
increased salinity and poor water quality, and intense use of pesticides and
other chemicals), that reduce watermelon yield and quality.
10.5 Major Diseases and Pests of Watermelon

As a result of a narrow genetic base, the watermelon cultivars are vulnerable
to a large number of pests and diseases (phytopathogens) caused by viruses,
bacteria, fungi, oomycetes, and insects. In recent years the watermelon crop
has suffered significant losses from soil-borne diseases such as Fusarium wilt
(Dane et al. 1998), gummy stem blight (Gusmini et al. 2005), and root-knot
nematode (Thies and Levi 2003, 2007). With the loss of the soil fumigant
methyl bromide, there is serious concern among growers that soil-borne
pathogens and plant-parasitic nematodes will cause severe crop losses.
Fusarium wilt [caused by Fusarium oxysporum Schlechtend.:Fr. f. sp.
niveum (E.F. Sm.) W.C. Snyder & H.N. Hans] (Fon) is a major disease of
watermelon (Netzer and Martyn1989). In 1991 over 60% of the watermelon
fields in the Southeast US suffered from Fusarium wilt (Zhang and Rhodes
1993). Three pathogenic races (0, 1, and 2) of Fon are documented (Netzer
and Martyn 1989). Various commercial cultivars contain mild resistance to

Watermelon 319
Fon race 0 and 1, but not to race 2. Fon is a seed-borne pathogen and can
rapidly spread in watermelon production areas. An RIL population (Zhang
et al. 2004) derived from a cross between the Fon race 2 resistant PI 296341-
FR (C. lanatus var. citroides) and the high quality inbred watermelon line
97103 (C. lanatus var. lanatus) has been used in genetic studies to identify
markers associated with Fusarium wilt resistance. Xu et al. (2010) have
constructed a suppression substractive hybridization (SSH) library from
root tissues of PI296341-FR that was infected with Fusarium wilt race
1. They sequenced approximately 3,895 cDNA clones. Their sequence
analysis identified a large number of the EST-unigenes that are putatively
associated with the disease-defense response. They analyzed the gene
expression of the root tissue infected by F. oxysporum race 1, using 32
Agilent 8 × 15K microarray chips designed on the watermelon ESTs and
public databases. This gene expression study identified 12 putative genes
in the Glycosphingolipid metabolic pathway that may be associated with
resistance to F. oxysporum race 1.
Root-knot nematodes (RKN) are among the most important pests of
cucurbit crops worldwide. Three primary species, Meloidogyne incognita,
M. arenaria, and M. javanica, cause substantial damage to watermelon
throughout the southern US (Thomason and McKinney 1959; Winstead
and Riggs 1959; Sumner and Johnson 1973; Thies 1996). The RKN invade
watermelon roots, inducing root galls that damage the vascular system and
interfere with the uptake of water and translocation of minerals, resulting in
stunted plants that produce poor or no yields (Williamson and Hussey 1996).
Root-knot nematodes also increase the occurrence and severity of Fusarium
wilt in watermelon and could reduce resistance in Fusarium wilt-resistant
varieties (Sumner and Johnson 1973). The RKN have been controlled
in watermelon by pre-plant fumigation with methyl bromide or other
nematicide treatments. The exclusion of methyl bromide from pre-plant soil
fumigation is projected to result in annual yield losses of at least 15 to 20% for
watermelon in Georgia and Florida (Lynch and Carpenter 1999). Thies and
Levi (2003, 2007) screened a watermelon germplasm and identified several
C. lanatus subsp. citroides PIs that contain resistance to root-knot nematodes
(Fig. 10-3). These C. lanatus subsp. citroides PIs are being used in breeding
programs and in developing rootstocks for grafting experiments in fields
infected with root-knot nematodes (Thies et al. 2010). Watermelon has also
suffered major losses from aphid and whitefly-transmitted viruses. The
aphid-transmitted potyviruses, including ZYMV, WMV, PRSV-Watermelon
(W), and cucumber mosaic virus (CMV) are considered the most important
viruses of watermelon (Provvidenti 1996; Lecoq et al. 1998). Recently, the
whitefly transmitted squash vein yellowing virus (SqVYV) was identified
as the causal agent of watermelon vine decline (WVD) that has caused
severe damage to the watermelon industry in Southwest Florida, Indiana,

and South Carolina (Adkins et al. 2007; Egel and Adkins 2007; Kousik et al.
2009). These viral diseases are difficult to control due to their transmission
by insect vectors (aphid or whitefly). Several mechanisms for resistance
to potyviruses in cucurbit species were elucidated in recent studies (Ling
et al. 2009). Recently, Ling et al. (2009) and Harris et al. (2009b) identified
markers associated with ZYMV resistance in the C. lanatus var. lanatus PI
595203. The markers are on the “eIF4e” gene sequence, which is known to
be one of the eukaryotic elongation factor genes that are associated with
resistance to poyviruses in plants (Ling et al. 2009). These markers (Fig.
10-5) are being used by seed company breeders to incorporate ZYMV
resistance into watermelon. However, our experiments indicated that the
marker is not useful for incorporating resistance to all ZYMV strains, and
in addition to the “eIF4e” gene a modifier gene(s) might be associated with
the resistance (A. Levi unpubl. data).
a) Line: P N F1 F2
Phenotype: R S S R S R S S S S S M
Genotype:
N
P
Figure 10-5 A restriction fragment length polymorphism generated using CAPS-1 marker for
the eukaryotic elongation factor “eIF4E” gene sequence on randomly selected F2 progenies
along with F1 and parental materials (P: PI 595203, N: New Hampshire Midget). Phenotype
on virus susceptibility was determined through seedling inoculation with ZYMV-FL (R:
resistance, S: susceptible). Genotype was determined through comparisons to RFLP patterns
in parents, P: PI 595203 (106 bp); N: New Hampshire Midget (131 bp). M is 1 kb plus molecular
weight marker.
Whitefly is a virulent pest that has become problematic in the last

two decades and has a wide variety of hosts. Whiteflies transmit several
important viruses to cucurbit crops, including the cucumber vein yellowing
virus (CVYV) that caused severe damage in watermelon fields in Spain
(Hong et al. 1995), and the cucurbit leaf crumple virus (CuLCrV) that infects
commercial watermelon in California and Arizona (Guzman et al. 2000).
Spraying with insecticides is the primary control strategy for whiteflies
(USDA 2000). However, these same insecticides also kill the whitefly
parasitoids and predators, ensuing unmanageable infestation of this pest in

Watermelon 321
watermelon fields. Furthermore, recent studies indicated that the B-biotype

whitefly has developed resistance to several insecticides, including a number
of pesticides that are currently registered on cucurbits. Watermelon cultivars
resistant to whiteflies should be the paramount strategy for controlling this
pest. Resistance to whitefly was identified among the desert watermelon
C. colocynthis (Simmons and Levi 2002). Presently, a genetic study is being
conducted (Simmons and Levi unpubl.data) to identify and map the gene(s)
conferring whitefly resistance in C. colocynthis with the intent to incorporate
the resistance into watermelon cultivars while excluding the alleles that
adversely affect fruit quality (Levi et al. 2006).
10.6 Genetic Studies and Genetic Mapping

Although a number of genetic studies were conducted in watermelon (Levi et
al. 2001a, 2002, 2006; Wechter et al. 2008), only limited genetic data are available
for genes that could confer disease and pest resistance in watermelon (Zhang
et al. 2004; Ling et al. 2009; Harris et al. 2009a, b). In addition, there is little
genetic information with respect to genes controlling the unique physiological
and metabolic properties related to rapid fruit development, metabolism,
and formation of secondary metabolites (carotenoids) in watermelon fruit
(Levi et al. 2006; Wechter et al. 2008). Several small linkage maps have been
constructed in watermelon based on isozymes (Navot and Zamir 1986; Navot
et al. 1990), RAPD and ISSR (Hashizume et al. 1996, 2003; Hawkins et al. 2001;
Levi et al. 2001, 2002; Zhang et al. 2004), and AFLP and SRAP (Levi et al. 2006)
markers. However, the most dense and useful map based on a large number
of EST-SSR (Levi et al. 2009) and SSR markers have been constructed by Xu
et al. (2010) (Fig. 10-6) . This map was constructed using an RIL population
(F2S8) derived from a cross between the Fusarium wilt resistant PI 296341
(C. lanatus subsp. citroides) and the elite Chinese line 97103 (C. lanatus var.
lanatus). In genetic mapping experiments, DNA markers including RAPDs
or ISSRs produced low polymorphism among watermelon cultivars. On
the other hand, in F2 populations derived from a wide cross between
C. lanatus subsp. citroides PI 296341 and watermelon cultivar (C. lanatus
subsp. lanatus), a large number of markers did not exhibit the expected
Mendelian segregation, but instead indicated skewed segregation. Most
of the skewed markers were clustered in their respective linkage group
(Levi et al. 2006). This preferential segregation for most markers could be
the result of the wide genetic distance between the watermelon cultivar
(C. lanatus subsp. lanatus) and the wild Citrullus lanatus subsp. citroides (Levi
et al. 2001a, b). This wide genetic distance is due to changes in chromosomal
structure that could lead to unequal transmission of chromosomal regions
during meiosis (meiotic drive) that result in preferential (non-Mendelian)
genetic composition of a population. Skewed segregation resulting from

2 3 4 5
1
0 WMN73C12 WMN11H07 SSR20354 WSSH387 WMN42A02 WMU799
WMN74D10 WSSH765 0 Bac_SSR33 0 WSSH970 0 WMG00426
0 wep33 4 WMG00362 WMG00327
7 wep32 3 WMG00222 WMG00653 5 WMN54F06 WMN04G10 WMN04F04 WSSH163
WMG00654 WMG00007 WMU567 wep30 SSR17818 WMN07G11 WMG01643 WMF00056
9 wep22 13 9 6 WMG01099
11 WMG01836 WMG01835 WMG01156 WMG01426 WMU617 WSSH1194 WSSH1004
11 WMG01634 WMG00863 15 Bac_SSR8 WSSH1015 WSSH1196 WMI00092 WMG00955
16 WSSH984 8 WMG00769 WMG01557
18 WMG00034 WMF00190 WMG00864 16 WMG00131 WMG00207 WMG00208
18 WMG01413 12 WMG00124 WMF00087
WMG00291 SSR10055 12 WMG00344
WMG00627 WMG00751 19 wep18 16 WMG00326 WMG00316 WMG00482
20 SSR10042 WMF00091 WMG00652 WMF00041
WMG01166 WMG01617 WMG01324 WMG00958 WMN03E10 19 SSR15737 10
20 WMG00658 WMG00455
25 WMG01168 WMG01379 WMG01417 WSSH497 20 WSSH410
WMG01303 WMG00949 22 WMN36H04 24 WMN30B12 WMI00015
28 SSR01859 13 WMG01433 WMG00651
30 WMI00072 14 WMG00003 WMG01281 24 SSR04992 WSSH572 30 WMG00102
WMG01568 WMG01911 25 wep16 wep48 32 WMG01592 WMG00354 WMG00971
35 WMG01114 WSSH531 WMG00464 WMG01374
37 WMG00255 WMG00166 WMG01912 WMF00243 WMG00676 WMG01691 36 WMN30E07 WSSH399 14
WMF00244 WMG01534 WMG01212 WMG00911 37 WMU880 WMF00038 WMG01752
WSSH322 WMN22H02 26 WMF00184
44 WMI00043 WMI00086 WMG00805 WMF00020 WMG01521 WMF00110
wep3 18 WMG00419
WMG00602 WMG00845 18 WMG00183 WMG00085 WMF00070 WMI00046 WMG01034 WMG00933
WMG01451 WMG00566 WMI00054 WMG00998 WMG01183 WMG01427 21 WMG01455 WMG00854
45 WMG01041 WMG01404 21 22 WMG01163
WMG01576 WMG01771 WMG00681 WMG01553 28 WMG01458 WMG00453 WMG00564 WSSH1133
38 24 WMG01686
SSR01534 SSR07284 22 WMI00013 WMG00454 WMF00129 WMG00808
WMG00328 WMG01096 29 WMG01276 WMG00807 WMG00898 25 WMG01068 WMG01067
50 WMG01487 WMG00230 WSSH752 WSSH321
WMG00846 27 WMF00144 WMG01097 WMI00018 WMI00017 WMG01144 WMG00589 27
WMG01393 WMG00765 WMG01076 WMG00660 WMI00091 MCPI_14
52 WMG01852 WMG00985 30 31 WMI00021
53 WMG00582 WMG01351 31 WMG00314 WMG01602 WMG01623 WMG01047 45 WMG00405
34 WMG00221 WMG01454 48 WMI00069 38 WMI00076
54 WMU226 wep65 WSSH1125
WMG00071 WMG01369 WMG00009 WMF00101 WMG00486 WMG00485 53 WMG00458
35 31 WMG01151 WMG01685 WMG01192 WMG00410 41 WSSH1113 WSSH1199
WMG00708 WMG00281 WMG00010
45 MCPI_15 MCPI_11 WMG01199 54 WMG00457 WMG01750 WMU328 WMN39F06
WMF00009 WMG00546
WMG01126 WMG01758 46 WSSH740 wep62 32 WMG01728 WMG01708
56 WMG00080 WMG01743 47 WMG00058 WMG00114 WMG00874 WMG01383
WMG01007 WMG01601 49 WSSH786 WMG00226 WMG01335
WMG01032 WMG00018 52 WMU656 33 WMG00873 WMG00038
WMG00948 WMG00515 55 WMN08D09 EIF4e_SSR12 WMG01296
WSSH820 56 WMN23G09 WMG00337 EIF4e_SSR7
58 WMU149
61 WMG00621
WMG00201 WMG00029 6
68 WSSH872 63
WMG00084 WMG00813 WMG00028
WMG01119 WMG01113 WMU157 WSSH313 SSR20083 WMU597
67 WSSH573 0
WMG00126 WMG01577 WSSH892
WMF00096 WMG01346 73 WMU316 WSSH14 WSSH15
78 WMN09D12 6 WSSH398
WMG00480 WMG00795
70 MCPI_47 MCPI_27 11 WMN16F06
WMG01139 WMG01362
WMG00554 WMG00288 MCPI_29 MCPI_13 12 WMU512
81
WMG00667 WMG00668 WSSH705 SSR04385 15 WSSH807 WMN77H08
WMG01357 WMG00462 MCPI_07 19 WMG01309 WMF00048
WMF00002 WMG00040 20 WMG00133
71 MCPI_41 WMG00246 WMG01776 WMI00090
WMG00592 WMG00365 WMI00020 WMI00031
72 24 WMG01790 WMG01718
WMG00976
75 WMG01107 WMG01358 WMG00287
76 WMG01824 WMG00647 WMG00544
79 WMG00076 9
80 WMG00075 10
wep24 WSSH891
SSR11439 WMU400 WSSH698 wep64
84 WMU297 MCPI_21 0 SSR20063 SSR33278 0 SSR12763 WMU307
WMN23E12 wep19 3 WMI00067
WMN17F06 wep74 1 WMN74G08 7 SSR21065
87 3 WMN55B08 11 WMG00269
WMU995
13 WMG01478
91 SSR15203 8 5 WMU525 WMN73E02
16 WMG01136
92 WMG00228 WMG00165
WMG00383 WMG00013
7 13
wep63 SSR11741
SSR00048 23 WMU227 WMN54E05
99 WMG00014 WMI00050 WMG00369 WMG00049 16 WMG01133 26 wep14
0 22 WSSH422 MCPI_37 MCPI_46
104 WMG00801 WMI00087 WMI00034 WMG00050 27
107 WMG01709 0 WMG01397 WMG00434 4 WMG00329 WMG00265 26 WMG00212 WMG00567 MCPI_25 MCPI_10
WMG00304 WMG00324 6 WMF00044 WMG00969 WMG00483 29 WMG00079
WMG01507 WMG01859 SSR11343 SF2SR10 29 WMG00635 WMI00029 WMG00712 WMG01407
2 31 WMG00711
WMG00345 WMN24D04 WMU398 WMG00019
3 WMI00055 WMF00097 WMG00373 WMU529 WMG00016 34 WMG01881
5 WMG01045 WMG00374 WMI00041 WMG01523 WMG00959 36 WSSH405 WMG01035
7 WMI00048 WMG01714 WMG01491 WMG00926 WMG00883 38 WSSH307 WMG00903
WMG01005 WMG01436 WMG01461 WMG00037 WMG00884 WMG01887 WMG00618 WMG00932
8 WMG01103 WMG01883 WMG00609 WMG00571 WMG00851 WMG01722 40 WMG00844 WMG01039
WMG01701 WMI00084 12 WMG00333 WMG01314 WMG01178
WMG01292 WMG00610
WMG00353 WMG01639 WMG01001 WMG00886 30 WMG00508 WMG01512 41 WMG00686
10 43 WMG00819
WMG01094 WMG01724 WMG01287 WMG00522 WMG01387 WMG00593
11 WMF00003 WMI00060 WMG00528 WMG01209 WMG00688 WMG01731 46 WMG01442
14 WMG00612 WMG01589 WMG00620 WMG00861 WMG01256 49 WSSH306
15 WMG00611 WMG01147 WMG01698 WMG00826 WMG00507 MCPI_33 50 WSSH1200
18 WMG01149 WMG00371 WMU97 WSSH799 WMU56 WMU155_2 WMG00257
51
27 WSSH1110 WMG00252 WMG00992 WMG01838 WMG00671 WMG00435
31 wep79 SSR07236 WMG00991 WMG00726 WMI00093 WSSH889 WMG00867 WMG00850
33 55 WMG00262
38 wep59 WMG01402 WMF00251 MCPI_23
WMN12E07 SSR14498 19 WMF00071 WMF00250 36 WMG00001 56 SSR02008 WMN20D05
39 WSSH917 WSSH192 SSR20270
WMG00738 WSSH837 38 WMN07D02 SSR22259
WMU3 41 wep50 57 WMU155_1 WMN09C06
21 WMN09B07 WSSH434 43 WMG00068 WSSH341 WSSH711
48 WMG00030
11 12 13 14
WMG01161 WMG00090
0 WSSH1136 WMG01636
WMI00078 WMG00134 0 0 SSR13292 MCPI_26 0 Fo_SSR23 Fo_SSR13
2 wep55 WMN44B03 WSSH791
4 WMI00080 10 WMU758 SSR00842 10
WSSH118 WSSH1151 Fo_SSR44 Fo_SSR16
Fo_SSR26 Fo_SSR12
15
11 WMU548 MCPI_24 WMU430 3
5 WMI00081 WMN65D12 WSSH1174 MCPI_09 Fo_SSR27 Fo_SSR29
WMN19D04 SSR23148 14 WMU626 15 WMN12A04 WMN28B09
6 16 SSR07461 WMU134 Fo_SSR36 Fo_SSR37 0
WMI00065 21 WSSH28 WMN37G11 WMN20A02 WMN76F12
9 C31 C80 20 WMN67H04 WSSH331 SSR20704
22 WMN18B09 SSR02237 WMN29C04 WSSH1201 9 wep13 WMN02H02
25 WMU503 SF12SR5 WMN15E04 5 SSR05195 SSR15802
SSR23265 14 Fo_SSR6 WSSH195
28 WMN41G05 WMN19F06 17 WSSH326
WSSH214 WSSH215
20 wep21
Figure 10-6 Genetic linkage map for watermelon based on 103 RILs derived from a cross
between the Fusarium wilt resistant PI 296341-FR and high quality Chinese watermelon line
97103. Most of the markers in this linkage map represent SSRs and EST-SSRs that exist in
EST-unigenes of watermelon (Levi et al. 2006).
meiotic drive has been reported in a number of crop species, particularly

in mapping populations derived from interspecific or intergeneric crosses
(Zamir and Tadmor 1986; Durham et al. 1992). Meiotic drive is prevalent in
crop plants, and plays a dominant part in plant genome evolution (Buckler et
al. 1999). Thus, in these population types it may not be sufficient to determine
the genetic mode of inheritance of a trait through phenotypic observations,
but by mapping gene loci and determining their segregation pattern.
Overall, in the genetic mapping experiments RAPD, ISSR, and SSR
primers produced low polymorphisms while SRAP primer produced higher
polymorphism among watermelon heirloom cultivars (Levi et al. 2001c,
2004, 2006). Also, AFLP primers produced higher polymorphism. However,
mapping experiments indicated that most of the AFLP markers were clustered

Watermelon 323
on several linkage groups and did not cover all regions of the watermelon
genome (Levi et al. 2006). Xu et al. (2010) have constructed an extensive
map, based on 555 SSR and EST-SSR markers that have proven quite useful
as a watermelon genome representation. This map consists of 11 linkage
groups with a total coverage of 739 cM and the average distance between
markers of 1.33 cM. Many markers are still required for the construction
of a dense map that can be used extensively in watermelon breeding
programs and to isolate genes that control fruit quality or confer resistance
to diseases and pests. Recently, they implemented a simple procedure for
developing and using a new type of PCR primers, named “high frequency
oligonucleotides—targeting active genes (HFO-TAG)”. These primers proved
useful in producing polymorphic markers in watermelon cultivars and in
genetic mapping of watermelon (Levi et al. 2010). The HFO-TAG primers
are constructed by first using a “practical extraction and report language
(Perl)” script to identify short oligonucleotides (8-, 9-, and 10-base) that exist
in high frequency in a 4,700 EST-unigene watermelon fruit library (Levi
et al. 2006; Wechter et al. 2008). This computer-based screening yielded
3,200 oligonucleotides that exist 32 to 335 times in the 4,700 EST-unigenes
constructed for watermelon. Of these, 192 HFO-TAG primers (present 51-269
times in the 4,700 EST-unigenes) were used to amplify DNA from closely
related watermelon cultivars. The average number of DNA fragments
produced by a single HFO-TAG primer among the watermelon cultivars was
considerably higher than the number of fragments produced by inter-simple
sequence repeat (ISSR) or randomly amplified polymorphic DNA (RAPD)
primers. Also, the HFO-TAG primers produced considerably more fragments
than the ISSR or RAPD primers from a watermelon cDNA library that was
used as a template. These results suggest that the HFO-TAG primers should
be more specific in targeting active gene loci. Xu et al. (2010) have sequenced
the genome of the elite Chinese watermelon line 97103. Additionally, they
produced extensive EST data for watermelon. A sequencing project for the
genome of the heirloom cultivar Charleston Gray has been conducted by Levi
et al. (2011). These extensive EST data should be useful for developing HFO-
TAG primers that can be utilized in genetic mapping and targeting of gene loci
of watermelon. Several linkage maps were constructed for watermelon using
a BC1 population (Levi et al. 2001c), a testcross population (Levi et al. 2002,
2006), an F2 population (Hashizume et al. 2003) and a recombinant inbred
line (RIL) population (Zhang et al. 2004; Xu et al. 2010). The genetic maps
and mapping populations (Levi et al. 2006) have been useful for mapping
the eukaryotic elongation factor “eIF4E” gene linked to ZYMV resistance in
watermelon and for identification of markers linked to this resistance (Ling
et al. 2009; Harris et al. 2010a). Also these maps have been used for genetic
mapping of NBS-LRR genes (possible resistance-gene analogs) that we
recently identified in watermelon (Harris et al. 2009b).

10.7 Identifying Nucleotide-Binding Site–Leucine-Rich Repeat

(NBS-LRR) Families in Watermelon
NBS-LRR genes have been shown to be involved in pathogen sensing and
host defense by binding to the pathogen proteins or by conformational
alterations in their amino-terminal and LRR domains, promoting the
exchange of ADP for ATP on the NBS domain and activation of an unknown
mechanism that generate pathogen resistance (DeYoung and Innes 2006).
The NBS domain includes the P-loop, kinase-2 motif, kinase-3a motif as
well as conserved blocks of unknown function, RNBS-A, RNBS-C, GLPL,
RNBS-D and MHD (De Young and Innes 2006). Many of these R-gene
candidate (RGC) sequences have been linked to gene loci that confer
resistance to pathogens (Deng et al. 2000). Furthermore, these RGC appear to
be clustered within plant genomes (Brotman et al. 2002). One hundred and
forty-nine NBS-LRR-type genes have been found in the Arabidopsis genome,
the vast majority of which have no definite function (Belkhadir et al. 2004a,
2004b). The precise mechanism by which the NBS-LRR proteins detect a
pathogen is not known. However, both direct and indirect contact with
pathogen avirulence factors is possibly involved (Jia et al. 2000). Identifying
and mapping of NBS-LRR genes might be critical in enhancing disease and
pest resistances in watermelon. To date, nine NBS-LRR gene analogs were
identified among Citrullus PIs and mapped on the genetic linkage map for
watermelon (Harris et al. 2009b). Three of these resistance gene analogs
are clustered in one linkage group, indicating the possibility that resistance
gene islands may exist in the watermelon genome (Harris et al. 2010). The
watermelon genome sequencing projects will facilitate the identification and
mapping of additional R-genes, as well as the subsequent transfer of these
genes or islands from wild accessions into watermelon cultivars. Indeed,
there are efforts to use the genome sequence data generated for watermelon
and identify and map additional resistance genes on the genetic linkage
maps constructed for watermelon (Xu et al. 2010).
10.8 Genomics of Watermelon

Xu et al. (2010) initiated the sequencing of the watermelon genome
(International Watermelon Genomics Initiative). To accomplish this
sequencing project, Solexa’s Sequencing-By-Synthesis technology was
applied in Watermelon Whole Genome Sequencing (WWGS). Watermelon
line 97103, which is the paternal parent of JingXin No.1, was sequenced.
Until now, a total of 49.6 Gb high-quality base pairs of 97103 have been
generated, which is about 115.42 fold coverage of the genome. K-mer depth
distribution of the sequenced reads was used to estimate the genome size
of watermelon. The estimated genome size is 421.75 Mb. Additional 16

Watermelon 325
watermelon lines with different fruit qualities and resistance to diseases and
pests were selected and used for shallow sequencing and SNP discovery
(Tables 10-1 and 10-2; Fig. 10-7). As in previous studies using DNA markers
(Jarret et al. 1997; Levi et al. 2001b, 2001c), a large number of SNPs was
discovered among the C. lanatus var. lanatus (watermelon cultivars) versus
the wild type C. lanatus var. citroides (cow watermelon) while lower genetic
diversity existed within the subspecies.
A special assembling platform was constructed for the Solexa sequence
data assembly. The total length of the assembled genome sequence was
360.3 Mb, which is about 83.8% of the genome and 16.2% smaller than the
estimated genome size 430 Mb. The size of scaffold N50 is 2.51 Mb and
Figure 10-7 The genetic diversity estimation based on number of single nucleotide
polymorphisms (SNPs) among 17 sequenced genomes.

Table 10-1 Seventeen watermelon genotypes that were sequenced and of which genomes
were assembled and used for SNP discovery.
No. Name Species Special traits

GS1 97103 C. lanatus var. lanatus East-Asia type
GS2 RZ-900 C. lanatus var. lanatus unknown
GS3 RZ-901 C. lanatus var. lanatus unknown
GS4 Sugarlee C. lanatus var. lanatus Resistant to anthracnose race 1 &
Fusarium wilt race 0 & 1
GS5 JX-2 C. lanatus var. lanatus Resistant to Fusarium wilt races 0 and 1.
GS6 JLM C. lanatus var. lanatus Small size, yellow flesh
GS7 JXF C. lanatus var. lanatus Small size, high sugar
GS8 XHBFGM C. lanatus var. lanatus Gynoecious lines
GS9 Calhoun Gray C. lanatus var. lanatus Resistant to Fusarium wilt races 0 and 1.
GS10 Black Diamond C. lanatus var. lanatus Susceptible to Fusarium wilt and
anthracnose
GS11 PI296341-FR C.lanatus var. citroides Resistant to races 0, 1, and 2 of Fusarium
wilt
GS12 PI595203 C. lanatus var. lanatus Resistant to ZYMV, PRSV & WMV
GS13 PI386019 C. colocynthis Resistant to Powdery mildew, & B. tabaci
GS14 PI482271 C. lanatus var. lanatus Resistant to Powdery mildew race 1
GS15 PI482303 C.lanatus var. citroides Nematodes resistance
GS16 PI482276 C.lanatus var. citroides Gummy stem blight resistance
GS17 Sy-904304 C. lanatus var. lanatus Allsweet type, resistant to Fusarium wilt
race 1 & anthracnose
the number of them is 41. The coverage of the watermelon genome by this
assembly was confirmed using the available EST and BAC sequences. The
assembly contains 99.6% of the 587,291 watermelon ESTs and 92.9% of the
three finished BAC sequences.
SSR, InDel and SV markers generated based on the WWGS and
BAC end sequences, EST-SSR generated based on the SSH cDNA library
sequences, and EST sequences of watermelon fruit development were used
to construct a high-density genetic map using 103 RILs derived from the
cross of PI296341-FR and 97103. Presently, the genetic map consists of 609
SSR markers with 15 linkage groups. The total coverage is 589.1 cM with
an average distance of 0.97 cM. Using this map, Xu et al. (2010) were able
to anchor 189 of the 201 N90 scaffold, which represent 89.04% assembled
sequence, onto 15 chromosomes.
Three gene-prediction methods (cDNA-EST, homology based, and ab
initio) were used to identify protein-coding genes and then built a consensus
gene set by merging all of the results. Xu et al. (2010) predicted 23,738 genes,
with a mean coding sequence size of 1,102 bp and an average of 4.61 exons
per gene Forty-five NBS R-genes were observed. Under an 80% sequence
overlap threshold, they found that 76.14% of the genes were supported by

Table 10-2 SNP density (num/kb) among the 17 watermelon genotypes used for genome sequencing.
GS1 GS10 GS11 GS12 GS13 GS14 GS15 GS16 GS2 GS3 GS4 GS5 GS6 GS7 GS8 GS17 GS9
GS1
GS10 0.46
GS11 7.14 7.21
GS12 0.95 1.02 7.04
GS13 5.36 5.41 4.77 5.38
GS14 0.84 0.92 7.05 1.05 5.38
GS15 7.18 7.25 1.45 7.08 4.9 7.07
GS16 7.21 7.28 1.14 7.12 4.88 7.12 1.3
GS2 0.38 0.45 7.22 1.01 5.42 0.9 7.26 7.29
GS3 0.32 0.51 7.19 1.01 5.41 0.9 7.23 7.26 0.42
GS4 0.38 0.44 7.18 1.03 5.41 0.93 7.23 7.26 0.38 0.43
GS5 0.25 0.53 7.16 1.02 5.39 0.91 7.2 7.24 0.49 0.35 0.49
GS6 0.34 0.54 7.17 1.03 5.4 0.93 7.22 7.25 0.51 0.38 0.5 0.41
GS7 0.34 0.55 7.17 1.01 5.4 0.9 7.22 7.25 0.51 0.39 0.5 0.4 0.39
GS8 0.36 0.51 7.21 1.01 5.41 0.9 7.25 7.29 0.45 0.23 0.48 0.4 0.42 0.42
GS17 0.4 0.47 7.2 1.03 5.41 0.92 7.24 7.28 0.34 0.47 0.38 0.53 0.55 0.55 0.51
GS9 0.43 0.45 7.19 1.04 5.41 0.93 7.23 7.26 0.45 0.51 0.42 0.55 0.57 0.57 0.53 0.43
Watermelon 327
multiple gene finders. About 83.21% of the genes have homologs in the
TrEMBL protein database, and 68.37% can be classified by InterPro. In total,
83.56% of the genes have either known homologs or can be functionally
classified. On the basis of pair-wise protein sequence similarities, they
carried out a gene family clustering analysis on all genes in sequenced
plants. The watermelon genes consist of 15,460 families. Of these, 3,099 are
watermelon unique families, among which 2,543 are single-gene families.
Comparing the gene families of watermelon with that of cucumber and
Arabidopsis, they found 12,216 gene families that were shared by at least
two species, and 9,916 clusters that were shared by all three species. There
were 868 watermelon-specific gene families (Xu et al. 2010).
Additional 16 watermelon lines were resequenced to discover the
diversity and the key agricultural traits and useful genes. Total base
pairs are 65 Gb in length. Resequencing of 16 watermelon germplasm
materials was completed with a sequencing depth of 6.30–18.79x. After
the preliminary analysis, the SNP density of the 16 watermelon inbred
lines was 0.023%–0.729%. The genetic diversity of these 16 genotypes and
watermelon line 97103 were analyzed based on these SNPs. The genetic
diversity of these genotypes is in agreement with the results derived from
the 30 SSR markers.
10.9 Development of BAC Libraries

Genome-wide integrated physical and genetic mapping using bacterial
artificial chromosomes (BAC) (Zhang and Wing 1997) proved to be an efficient
and powerful approach for modern genomics and genetics research. BAC-
based mapping has provided targeted marker development, fine-mapping,
and map-based cloning of genes and QTLs, and large-scale mapping of genes
or ESTs in human (International Human Genome Mapping Consortium 2001)
and several model plant and animal species (Arabidopsis, Marra et al. 1999 and
Chang et al. 2001; Drosophila, Hoskins et al. 2000; indica rice, Tao et al. 2001;
japonica rice, Chen et al. 2002; chicken, Ren et al. 2003). A BAC library was
constructed for watermelon by Joobeur et al. (2006). The BAC clones have
an average insert-size of 106 kb, providing 21 haploid genome equivalents.
The library was used to identify BAC clones that are anchored to probes
evenly distributed on the genomes of melon or Arabidopsis. Twenty-eight
probes (representing 66% of the tested probes) from melon and 30 probes
(65%) from Arabidopsis identified positive BAC clones. This BAC library was
donated by Syngenta Seeds and is available for the public through Clemson
University Genomics Institute (http://www.genome.clemson.edu). Genome-
wide BAC-based map for watermelon is being developed in China by Xu et
al. (2010). This BAC-map will significantly impact the genomic, genetic, and
breeding research of this important agricultural crop. The advanced genomics

Watermelon 329
information developed here will not only provide extensive genetic and
physical mapping of the watermelon genome, but will also be incorporating
useful genes into watermelon cultivars. The striking differences between
wild and cultivated watermelon in terms of resistance to disease and pests
are evident by studies on Fusarium wilt (Netzer and Martyn 1989), root-knot
nematodes (Thies and Levi 2003, 2007), potyviruses (Ling et al. 2009; Harris
et al. 2009a), and whiteflies (Simmons and Levi 2002). Extensive genome
sequencing, as well as fine-mapping of this important crop, with particular
focus on the resistance genes, will greatly contribute to the breeder’s ability
to generate resistant cultivars. Hongbin Zhang and his team (Texas A&M
University) have constructed a binary large-insert, plant-transformation-
competent, BIBAC-library from the DNA of watermelon cultivar Charleston
Gray. The library contains a total of 26,112 clones arrayed as individual clones
in 68 microplates (384-well in each). Analysis of a sample of random clones
showed that the library has an average insert size of 170 kb, representing a
10x coverage of the watermelon haploid genome, thus providing greater than
99% probability of obtaining at least one positive clone from the library with a
single-copy probe. The library was constructed in a binary vector that could be
directly transformed into plants via either Agrobacterium-mediated (Hamilton
et al. 1996; Liu et al. 1999; Tao et al. 2001) or particle bombardment (Zhang
and Chang 2009) method. It could be used for genome-wide or large-scale
functional analysis, molecular breeding, and molecular pharming. The BIBAC
library developed for “Charleston Gray” by Hongbin Zhang (pers. comm.) is
well suited for the development of a physical map (Ren et al. 2005). A total of
~30,000 (~11x) clones (Ren et al. 2005), ~15,000 clones from each library, can
be selected, sequenced, and fingerprinted (Zhang and Chang 2009).
10.10 Stack Holders

There is great interest in Asia (mainly in China, which is the biggest world
producer and consumer of watermelon) in the genomics of this important
vegetable crop. In the USA, watermelon is being produced in the southern
states and there is great interest by growers and by seed companies in
enhancing watermelon cultivars using genomics and genetic tools for
marker development and employment of markers in breeding programs
to enhance disease and pest resistance. The Boyce Thompson Institute
maintains the International Cucurbit Genomics Initiative (ICuGI) website
(http://www.icugi.org), including the genomic data for watermelon.
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11
Cucumber Genomics
Zhonghua Zhang,1 Jun He1 and Sanwen Huang1,*
ABSTRACT
Cucumber is the first cucurbit completely sequenced. This chapter
covers genetic and genomic resources, genome features, comparative
analysis among cucurbits, resistance genes in cucumber genome. As of
the end of 2009, a total of 8,113 expressed sequence tag (EST) sequences
were deposited in GenBank dbEST and an additional 359,108 EST
sequences were obtained. These EST sequences had been assembled
into 81,401 unigenes, which can be accessed at www.icugi.org. A fosmid
library with the insert size of 35–40 Kb was constructed using the
sequenced genotype 9930 and a BAC library with an average insert size
of 101 Kb was also constructed using “Chinese long” inbred line 228.
The sequenced genome of “Chinese long” inbred line 9930 was 243.5
Mb covering more than 96% of the genic regions and contained 26,682
protein-coding and 292 rRNA fragments, 699 tRNA, 238 snoRNA, 192
snRNA, and 171 miRNA genes. Total repeats account for 24% of the
genome, including 10.4% retrotransposon sequences. Chromosome
karyotyping and generation of markers based on the completed
sequence are also discussed.
Keywords: cucumber, BAC library, EST, genome sequencing, SSR
marker
This chapter includes: 1) Genetic and genomic resources for the cucumber
genome project; 2) Whole genome features of cucumber; 3) Comparative
analysis among cucurbits; 4) Pathogen resistance and expanded genes in
cucumber genome; 5) Cucumber as a mode genome for vascular biology.
1
Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing,
100081, China.
*Corresponding author: huangsanwen@caas.net.cn

11.1 Cucumber Genome Initiative

In 2007, the cucumber genome initiative (CuGI) was initiated by the Institute
of Vegetables and Flowers, Chinese Academy of Agricultural Sciences
(IVF-CAAS), and an international consortium was established including
Beijing Genomics Institute, Beijing Normal University, China Agricultural
University, University of California-Davis, University of Wisconsin, Cornell
University, DArT P/L and Wageningen University. The aims of CuGI are: 1)
to obtain a complete sequence of the cucumber genome; 2) to get an indepth
understanding of the genetic and molecular bases of major agronomical
traits; 3) to generate a comprehensive toolbox for molecular breeding; and
4) to create a model for some specific biology and genetics researches.
For the cucumber genome, a novel combinational de novo sequencing
strategy was adopted, taking advantage of the long read/clone length
of Sanger technology and, for the first time, the high sequencing depth,
low unit cost of Illumina GA technology. The sequenced genotype was a
domestic cucumber, C. sativus var. sativus L. “Chinese long” inbred line
9930, which is commonly used in modern cucumber breeding (Staub et
al. 1999).
11.2 A Karyotype for Cucumber Genome Using Repeat

Sequences
Four types of satellite sequences (Type I, II, III, IV) in the genome of
cucumber have been characterized ( Ganal et al. 1986; Ganal and Hemleben
1988), which together accounted for over 90% of the satellite DNA in the
genome. By aligning whole-genome shotgun reads against the satellite
sequences, it was estimated that Type I/II, Type III and Type IV accounted
for 10.39, 4.04 and 5.53% of the cucumber genome, respectively (Han et al.
2008). In addition, two cucumber-specific repeats CsRP1 and CsRP2, which
are localized in centromeric/pericentromeric regions, were isolated by PCR
(Koo et al. 2005). Based on whole-genome shotgun reads, about 0.72 and
0.48% of the cucumber genome are CsRP1 and CsRP2 repeats (Han et al.
2008). The proportion of 45S rDNA and 5S rDNA sequences was estimated to
be 3.30 and 0.13% respectively in the cucumber genome (Han et al. 2008).
Cytological locations of the above repeat sequences were revealed by
performing fluorescence in situ hybridization (FISH) (Fig. 11-1). Type I/II and
Type IV were located in telomeric heterochromatin regions. Type III and 45s
rDNA were located in cytologically defined cucumber centromeres. CsRP1
was a dispersed repetitive sequence. Its flurorescence in situ hybridization
(FISH) signals were detected on the pericentromeric regions of all but one
chromosome, where they were detected at an intercalary position. The 439
bp of CsRP2 had about 90% sequence homology to the central section of the
ribosomal DNA (rDNA) spacer from cucumber (Koo et al. 2005).

Cucumber Genomics 337
Figure 11-1 Ideogram showing the position and intensity of Type I/II, Type III, Type IV,
45S rDNA/CsRP2 and 5S rDNA on cucumber metaphase chromosomes (Han et al. 2008).
Chromosome nomenclature follows Koo et al. (2005).
Following chromosome nomenclature in Koo et al. (2005), an integrated

karyotype for cucumber was constructed based on the distribution of
these dominant repeat sequences (Fig. 11-1) (Han et al. 2008). The relative
chromosome lengths varied from 11 to 16.97%. All the chromosomes are
metacentric with an arm ratio of 1.02–1.36.
11.3 Develop Genetic Map on the Basis of Genome Sequences

A total of 23,800 putative simple sequence repeat (SSR) sequences were
identified from whole-genome 3x shot-gun sequencing. To reduce the
nonspecific amplification, SSR primers with multiple homologs in the
assembly were excluded. If a sequence contains more than one SSR, only
the one with the longest motif was chosen to reduce marker clustering. A
total of 1,940 with the longest repeat motifs were selected for polymorphism
screening between the parental lines of a recombinant inbred line (RIL)
mapping population (Gy14, a North American processing market type
cucumber cultivar, and PI 183967, an accession of C.s. var. hardwickii
originating from India). Of the 1,940 SSRs, 1,322 (68.1%) were polymorphic
between Gy14 and PI 183967. In addition, 200 previously reported SSRs
(Danin-Poleg et al. 2000; Fazio et al. 2003; Kong et al. 2006) were also tested,
and 40 (24%) showed polymorphism.

Using 77 RI lines from the inter-subspecific cross between Gy14 and

PI183967, 995 SSR loci were mapped in seven linkage groups spanning
over 572.9 cM (Ren et al. 2009). In total, 678 recombination events (bins)
were identified, where 311 (46%) bins were filled by one or more markers
(Table 11-1; Fig. 11-2). Since the cucumber genome size is approximately
367 Mbp, the map defined herein represents average genetic and physical
intervals of ~0.6 cM and ~370 Kb per marker, respectively, making it the
most saturated linkage map in the Cucurbitaceae to date. In addition, 895
diversity array technology (DArT) markers were developed for cucumber
by IVF-CAAS and DArT Pty Ltd ( unpubl.data). They have been integrated
with the above SSR map, thus a total of 1,885 markers are available.
Table 11-1 Summary of the cucumber genetic map with RIL mapping population from the
inter-subspecific cross between Gy14 and PI 183967 (Ren et al. 2009).
Chr. No. Markers cM Density Recombination Filled bins

(cM/marker) events
1 118 96.2 0.82 118 47
2 126 100.2 0.80 113 54
3 187 112.7 0.60 143 70
4 114 37.3 0.33 41 16
5 160 59.9 0.37 70 29
6 203 106.5 0.52 125 68
7 87 60.1 0.69 68 27
Total 995 572.9 0.58 678 311
The above SSR markers were used to examine the genetic affinity of
diverse cucumber inbred lines and evaluate their potential in marker-
assisted selection (MAS). Approximately 65% of the 995 SSRs examined
were polymorphic in these 11 lines. The polymorphism information
content (PIC) values ranged from 0.17 to 0.84, with an average value of
0.44 indicating that the SSR markers employed provided for a robust
discrimination among this germplasm array. Moreover, the SSRs with PIC
values from 0.4 to 0.6 were most common, and ~250 SSRs with PIC value
> 0.5 were highly polymorphic. Therefore, these highly informative SSR
markers would most likely be useful in tracing economically important
traits in breeding populations.
This genentic map might have a broader scope of deployment and use
in cucurbit breeding. An appreciable number of the SSR markers were able
to amplify products in melon [487 (48.9%)], watermelon [258 (25.9%)] and
pumpkin [221 (22.2%)]. Moreover, these SSR markers detected relatively
high levels of polymorphism in these species (melon, 39.6%, watermelon,
46.5%, and pumpkin, 54.8%). Thus, these markers are also potentially useful
in these related crop species.

Chr.1 Chr.2 Chr.3 Chr.4 Chr.5 Chr.6 Chr.7
1 1 0.0 S 1 1 0.0 S 1 1 0.0 S 1 1 0.0 1 2 0.0 1 2 0.0 S 1 1 0.0

2 2 1.4
3 3 2.7 S 3 6 1.8
4 17 2.2 4 1 2.5
5 2 3.9 5 2 3.2 5 1 5.2
6 1 4.9 6 25 5.6
7 9 6.1 7 2 5.9 7 36 4.4 7 12 6.3
8 1 5.3 8 16 5.0 S 8 63 6.0
9 38 7.9 9 13 6.2
S 10 2 7.8 10 1 8.1
11 5 7.1 11 5 7.5 11 1 9.8
12 6 10.9 12 1 8.4
13 1 12.0 13 5 8.5 13 1 9.3
14 1 12.7 14 1 11.1 14 2 9.5
15 4 13.1 15 6 10.8 15 1 12.5
16 3 14.2 16 1 11.1 16 2 11.3 SDR 16 12 11.9 16 1 13.8
17 1 14.9 17 2 13.8 17 1 14.7 17 2 13.2
18 3 12.3 18 1 15.5 18 2 14.5
19 6 16.4 19 5 13.2 19 1 16.2
20 1 17.0 20 1 18.9
21 3 17.7
22 1 18.5 22 2 20.4 22 1 15.5 22 1 18.5
23 7 19.0 23 1 21.6 23 2 20.4 23 1 21.1
24 4 20.4 24 3 22.5 24 3 17.3 24 1 22.3 24 1 20.1
26 1 21.7 26 1 21.0
27 8 19.6 27 4 24.6
28 3 23.4 28 2 26.9 28 2 25.3
29 1 27.6 29 1 23.8 29 1 26.0
30 2 28.6 30 3 23.0 30 1 24.9
31 1 27.8
32 1 26.3 32 2 26.5 32 2 26.6 32 3 29.1
33 2 25.8 33 1 27.3 33 2 28.0
34 1 28.1
35 1 28.1 35 3 33.6 35 1 31.5
36 2 32.3 36 1 30.4 36 1 32.2
37 3 29.3 37 2 36.0 37 2 29.1 37 6 34.4 37 3 31.3
38 2 30.0 L 38 34 35.1
39 1 37.5 39 1 31.0 39 3 33.3 39 2 35.6
40 1 32.3 40 2 31.7 40 2 33.7
41 1 37.3 41 3 35.6
42 4 33.5 42 1 39.8 42 1 33.0 42 5 36.3 42 4 35.6
43 1 34.5 43 1 41.1 43 1 38.3 43 1 39.0
44 1 41.8 44 2 39.0 44 2 37.2 44 1 39.6
45 1 35.7 45 3 42.9
46 7 39.6
47 1 37.1 47 3 44.9
48 1 37.0
49 6 38.5 49 3 46.2 49 4 43.2
50 4 39.4
51 1 41.3 51 2 48.4 51 2 39.0 51 3 46.0
52 1 43.6
53 5 40.7 53 1 48.4
54 1 48.9 54 1 45.3
55 3 43.3 55 1 49.6 55 4 48.6
56 3 44.4 56 1 52.8 56 1 42.1 56 2 50.3 56 3 46.7 56 1 50.9
57 7 47.4 57 1 51.6
58 1 46.1 58 1 43.9 58 4 47.9
59 5 48.7 59 4 52.9
60 7 57.5 60 1 45.4 60 3 53.1 60 4 49.7
61 1 48.3 61 6 58.2 61 3 53.8 61 6 50.5 SDR
62 1 59.3 L 62 4 54.5 62 2 55.0
63 13 60.8 63 2 47.0 63 1 51.6
64 1 50.3 64 1 52.7 64 3 56.4
65 2 48.7 65 3 53.4
66 1 61.5 66 5 49.4 66 3 54.3 66 1 58.5
67 2 59.3
68 1 51.1 68 2 55.7 L 68 9 60.1
69 2 52.4 69 1 62.9 69 1 52.7 69 1 56.4
70 2 64.1 70 1 59.9 70 6 57.9
71 1 53.3 71 4 66.3 71 2 54.0 71 1 58.6
73 3 54.8 73 8 55.9 73 1 59.8

74 1 56.6 74 9 62.5
75 3 55.7
76 3 58.1
77 1 70.0
78 1 58.0 78 1 59.0 78 1 66.7
79 1 72.2 79 4 68.0
80 2 73.0
81 5 61.1 81 3 74.0
82 2 61.6
83 1 75.5 83 7 62.4 83 1 70.2
84 4 63.5 84 4 76.4 84 1 63.8 84 1 72.3
L 85 5 64.5 85 2 78.0
86 4 78.6 86 1 73.8
87 1 78.9
88 3 66.6 88 1 80.6 88 1 66.2 88 1 75.1
89 2 68.1 89 1 81.0 89 1 67.0
90 1 82.2 90 1 67.8
91 3 68.2 91 3 77.3
92 7 83.8 92 4 69.7 92 3 78.8
93 1 85.2 93 2 80.2
94 1 81.1
95 4 73.0 SDR 95 1 72.0 95 2 82.3
96 1 87.6 96 1 72.8
97 2 73.8
98 4 84.5
99 1 90.9 99 1 85.1
100 1 78.2 100 1 86.3
101 1 92.2 101 2 77.1 101 2 87.6
102 4 78.2
104 2 83.5 L 104 1 94.3 104 5 89.8

105 2 84.1 105 5 95.1 105 1 80.2
106 3 86.0 106 2 81.1
107 3 83.3 107 2 92.5
108 5 84.7 108 7 93.4
109 1 85.5 109 3 94.2
111 2 90.0 111 1 97.7 111 5 86.9 111 1 95.8

112 1 88.6 112 1 96.6
113 1 100.2
114 5 92.7 114 1 90.0
115 4 98.8
116 1 99.9
118 1 96.2 118 3 92.4 118 7 101.5

119 2 102.1
120 1 93.5 120 1 102.8
121 1 103.5
123 4 96.6 123 5 105.0
L 125 4 106.5
127 1 98.6
128 3 99.8
130 1 101.1
131 1 101.8
132 1 102.7
133 2 103.7
134 1 104.0
L 135 12 105.1
136 3 105.2
137 2 107.7
139 2 109.4
143 1 112.7
cluster S short arm L long arm
Figure 11-2 Cucumber SSR linkage map (Ren et al. 2009). The bin names and genetic distances
in cM are respectively listed on the left and right of the chromosomes. The number of SSR
markers in each filled bin is indicated in the boxes. White boxes indicated a recombination
event with no markers. The short and long arms are indicated with S and L, respectively. SDR
= segregation distortion region.

11.4 Integration of Genetic and Cytogenetic Map

FISH analysis was used to establish the relationships between linkage
groups and chromosomes in cucumber. Three to five SSR markers located
in the distal ends of each linkage group were selected to screen a fosmid
library developed from “Chinese long” inbred line 9930. To identify
individual chromosome, these fosmid clones were FISH-mapped on
mitotic chromosomes (Fig. 11-3A), which were then reprobed with two
tandem repeat sequences (Type III and 45S) (Fig. 11-3B) whose distribution
patterns on each haploid chromosome are known (Han et al. 2008). Using
this strategy, each fosmid clone defining a single locus was assigned
to a chromosome allowing integration of all seven linkage groups into
chromosomes (Fig. 11-3A). The short arm/long arm orientation of each
linkage group could be established based on the positions of fosmid FISH
analysis. i.e., physical locations of the chromosome-specific fosmid clones
represent chromosome locations of corresponding SSR markers used during
fosmid clone screening. Furthermore, the 14 chromosome arm-specific
fosmid clones can also serve as convenient and reliable cytological markers
in the future cytogenetical studies of cucumber.
Figure 11-3 Integration of the seven linkage groups of cucumber with individual chromosomes
(Ren et al. 2009). (A1) Distribution of Type I/II (green) and Type III (red) repeats on cucumber
chromosomes. (A2) DAPI staining was converted to black and white images. (A3) Localization
of chromosome-specific fosmid clones on both arms of individual chromosomes, genetic
location of arm-specific fosmid clones are indicated in Fig. 11-2. (B) Localization of fosmids 4S
(red) and 4L (green) together with Type III (red) and 45S rDNA (green) repeats on the mitotic
chromosomes. Bar = 2.5 µm.
11.5 EST Sequences and BAC/Fosmid Libraries for Cucumber Genome

As of the end of 2009, a total of 8,113 expressed sequence tag (EST) sequences
were deposited in GenBank dbEST. To facilitate the annotation of the
cucumber genome, 359,108 EST sequences were obtained by sequencing

cDNAs from near-isogenic unisexual and bisexual flower buds, respectively,

using the 454-pyrosequencing technology. These EST sequences had been
assembled into 81,401 unigenes, which can be accessed at www.icugi.org.
A fosmid library with the insert size of 35–40 Kb was constructed using
the sequenced genotype 9930 as template by IVF-CAAS. A bacterial artificial
chromosome (BAC) library, which has an average insert size of 101 Kb, was
also constructed using C. sativus var. sativus L. “Chinese long” inbred line
228 as template by IVF-CAAS. The fosmid and BAC libraries represent an
approximate 20 and 10-fold coverage of the cucumber genome, respectively.
Both of them were end-sequenced with standard Sanger methodologies on
ABI-3730 and MegaBACE 1000 sequence analyzers.
11.6 Whole Genome Features

11.6.1 Genome Sequencing and Assembly
A total of 26.5G high-quality base pairs, or 72.2-fold genome coverage, of
which the Sanger reads provided 3.9-fold coverage, and the GA reads 68.3-
fold coverage were generated for the cucumber genome. The Illumina GA
reads ranged in length from 42 to 53 bp. All the reads that were generated
from both ends of clones were mate-paired. The assembled N50 contig
and scaffold sizes were 19.8 Kb and 1.14 Mb, respectively (Table 11-2).
The total assembled genome length was 243.5 Mb; about 30% smaller
than the size estimated by flow cytometry of isolated nuclei stained with
propidium iodide (367 Mb) (Arumuganathan and Earle 1991) and by K-mer
Table 11-2 Cucumber genome statistics regarding genome assembly, transposon annotation,
and gene annotation (Huang et al. 2009).
Assembly Contig Contig total Scaffold N50 Scaffold % sequence

N50a (Kb) (Mb) (Kb) total (Mb) anchored on
chromosome
Sanger + Illumina GA 19.8 226.5 1,140 243.5 72.8%
TE annotation Copies Total length % of assembled % classified b
genome
266,232 54.4 Mb 24.0% 51.5%

Gene annotation Number Average # Exons per With homologs c
CDS length gene
26,682 1,046 bp 4.39 82%

a
N50 represents the length such that 50% of the sequence is contained in contigs/scaffolds
of this length or greater.
b
Classified TEs are those with similarities to known TEs or TE-related proteins.
c
Genes with homologs that were alignable to Swiss-Prot and TrEMBL protein databases (BlastP;
1e-5) or classifiable by InterPro.

depth distribution of sequenced reads (350 Mb) (Huang et al. 2009). The
majority of the remaining 30% unassembled regions of the genome are
likely heterochromatic satellite or rRNA sequences. By aligning the EST
sequences against the assembly, it was estimated that more than 96% of
the genic regions were included in the assembled genome.
Using the above mentioned map, 72.8% of the assembled sequences
can be anchored onto the seven chromosomes. Among the 1,885 markers,
1,763 (93.5%) were uniquely aligned and used for constructing the
pseudochromosomes. The majority (98.7%) of the markers were collinear
with the sequence assembly (Fig. 11-4a). Comparison of the genetic and
physical distances between markers showed recombination suppression
of two 10 Mb regions at either end of chromosome 4, a 20 Mb region
on chromosome 5, and an 8 Mb region on chromosome 7. Using FISH,
segmental inversion within the suppression region on chromosome 5
between Gy14 and PI183967 was detected (Fig. 11-4b) that provides an
explanation for recombination suppression in these regions. These regions
of recombination suppression are additionally useful for studying cucumber
evolution during domestication.
11.6.2 Repetitive Sequences and Transposons

In addition to satellite and rRNA sequences, the cucumber genome contains
a large number of transposable elements (TEs) (Table 11-3). The LTR
retrotransposons (gypsy and copia) made up the majority of the TE classes
and comprised 10.4% of the genome. The repeat content in the cucumber
genome, including unclassified sequences in the repeat library, was
approximately 24% (Table 11-2). The repeats’ divergence-rate (percentage
of substitutions in the matching region compared to the consensus repeats
in constructed libraries) distribution showed a peak at 20%. A fraction of
long terminal repeat (LTR) retrotransposons, long interspersed elements
(LINEs), and DNA transposons (composing 2.3, 0.4, and 0.2% of the genome,
respectively) are of relatively recent origin, having a sequence divergence
rate of less than 5%.
11.6.3 Gene Annotation

In cucumber genome, a small number of genes (26,682) were predicted
(Table 11-2). About 81% of the genes have homologs in the Swiss-Prot/
TrEMBL protein database, and 66% can be classified by InterPro. Altogether,
82% of the genes have either known homologs or can be functionally
classified. In addition to protein-coding genes, there are also 292 rRNA
fragments, 699 tRNA, 238 snoRNA, 192 snRNA, and 171 miRNA genes in
the cucumber genome.

Figure 11-4 Integrated genetic/physical map of cucumber (Huang et al. 2009). (a) Genetic
versus physical distance map of the seven cucumber chromosomes. The genetic map was
constructed using an RIL mapping population from the inter-subspecific cross between Gy14
(domestic cucumber) and PI183967 (wild cucumber). (b) The segmental inversion between the
domestic cultivar Gy14 and the wild accession PI183967 on cucumber chromosome 5 detected
by FISH. 12-2 and 12-7 denote individual fosmid clones. (Scale bars, 1 µm)

Table 11-3 Repeat content in the assembled cucumber genome (Huang et al. 2009).
Type # Copies Length (bp) Fraction (%)

DNA 16,972 2,808,075 1.24
Retrotransposons 119,339 27,538,485 12.16
LTR 91,109 23,622,636 10.43
LINE 16,899 3,937,077 1.74
SINE 195 14,911 0.01
Other 25 2,581 0.00
Unclassified 135,464 26,367,990 11.64
Total 266,232 54,361,644 24.01
Compared to other sequenced plant genomes, the cucumber contains

the smallest number of tandem gene duplications (479), whereas grapevine
has the largest number (5,382). In part, this may contribute to the small
gene number in cucumber.
11.6.4 Genome Duplications

Whole-genome duplication (WGD) is common in angiosperm plants and
produces a tremendous source of raw material for gene genesis. Previous
research has revealed a paleo-hexaploidy (γ) event in the common ancestor
of Arabidopsis and grapevine after the divergence of monocotyledons
and dicotyledons (Jaillon et al. 2007). Subsequently, two WGDs (α and β)
occurred in Arabidopsis (Bowers et al. 2003) and one (p) in poplar (Tuskan
et al. 2006), whereas no recent WGD occurred in grapevine and papaya. By
carrying out a colinear gene-order analysis on the cucumber genome, no
recent WGD and only a few segmental duplication events were identified.
Using the 4DTv (distance-transversion rate at 4-fold degenerate sites)
method, recent WGD was also not detected, but showed ancient duplication
events. Therefore, the small cucumber genome with no recent WGD will
provide an important complement to grape and papaya for the study of
ancestral forms and arrangements of plant genes.
11.7 Comparative Genomics

11.7.1 Chromosomal Evolution in Cucurbits
Melon and cucumber belong to the same genus. Interestingly, cucumber
contains seven chromosomes, whereas melon has 12. Watermelon, their
common distant relative, has 11 chromosomes. By comparing the melon
(Fernandez-Silva et al. 2008; Deleu et al. 2009) and watermelon genetic maps
to the cucumber genome, a high level colinearity among them was observed
(Fig. 11-5a). Interestingly, it was found that cucumber chromosomes 1, 2, 3, 5,

and 6 were colinear to melon chromosomes 2+12, 3+5, 4+6, 9+10, and 8+11,
respectively, indicating that, after speciation, these cucumber chromosomes
each resulted from a fusion of two ancient chromosomes. In addition to
chromosome fusion, the comparison also showed the occurrence of several
inter-chromosome and intra-chromosome rearrangements (Fig. 11-5a).
Figure 11-5 Comparative genomic analysis of cucurbits (Huang et al. 2009). (a) Comparative
analysis of the melon and watermelon genetic maps with the cucumber sequence map. (b)
Syntenic blocks between the cucumber genome (scaffold000089) and a melon BAC sequence
(accession: EF188258.1). Genes are drawn as black arrows with the orientation indicated on
the sequence. Transposable elements (TEs) are illustrated as rectangles; retrotransposable
elements are in red, DNA transposons are in blue and unclassified TEs are in green. Orthologous
sequence regions between the two genomes are displayed.
11.7.2 Cucumber-Melon Microsynteny

To estimate the sequence divergence rate, the four sequenced melon BACs
were compared to the cucumber genome (One example in Fig. 11-5b; Huang
et al. 2009). There are 56 genes inside the melon BACs, 52 of which are
colinear with the cucumber genome. The mean sequence similarity over
coding regions is 95%. Although the gene region similarity is very high, the
repeat content between the two genomes is quite different. New TEs were
frequently inserted in the intergenic regions of both genomes. Thus, only
54% of the BAC sequences could be aligned onto the cucumber genome
with an average of 88% sequence identity. Nonetheless, the highly conserved
gene content and order between the two species make the cucumber genome
useful for genetic analysis of melon.

Using the annotated genes in the four melon BACs, it was estimated
that cucumber and melon diverged about 4~7 million years ago (Mya) based
on the divergence age of Arabidopsis and papaya (54~90 Mya) (Huang et
al. 2009).
11.7.3 Centromere Repositioning in Cucurbit Species

Comparative analysis of the melon genetic map with the cucumber sequence
map revealed that there was a high-level colinearity between cucumber
and melon. However, comparative FISH mapping using common sets of
fosmid clones revealed changes in centromere positions between cucumber
chromosome (chr.) 6 vs. melon chr. 1 and cucumber chr. 7 vs. melon Chr.
2 during evolution (Fig. 11-6; Han et al. 2009). The current centromeres
of all four cucumber and melon chromosomes are found to be associated
with distinct pericentromeric heterochromatin by pachytene chromosome
analysis. Moreover, inactivation of a centromere in the original centromeric
region was associated with a loss or erosion of its affixed pericentromeric
heterochromatin. It can be concluded that centromere activation and
inactivation in cucurbit species were associated with a gain/loss of a large
amount of pericentromeric heterochromatin.
6-1
6-2 7-1
Cen
6-3 7-2
6-4
Cen
6-5/6-6
Cen 7-3
6-6
6-7 7-4 Cen

7-5
6-8
6-9 7-6
6-10
7-7
6-11
7-8
6-12
Cucumber Melon Cucumber Melon
chromosome 6 chromosome I chromosome 7 chromosome II
Figure 11-6 Diagrammatic illustration of the marker orders and centromere positions of two
pairs of cucumber and melon chromosomes (Han et al. 2009).

11.8 Pathogen Resistance Genes in Cucumber

Only 61 nucleotide-binding site (NBS)-containing resistance (R) genes
were identified in cucumber, which could be useful for plant improvement
purposes. This number is close to papaya (55) (Ming et al. 2008) but only a
fraction of that in Arabidopsis (200), poplar (398), and rice (600) (Tuskan et al.
2006). Distribution of NBS genes on chromosomes is not random, with only
five genes located on chr. 1, 6 and 7, whereas 20 on chr. 2 (Fig. 11-7). Three-
quarter of the NBS genes are within 11 clusters, indicating they evolved
through tandem duplications, similar to other known plant genomes.
Figure 11-7 Genomic locations of R genes on the cucumber chromosomes (Huang et al.
2009). Three R genes could not be anchored on specific chromosome.
Eukaryotic translation initiation factors, and particularly the eIF4E

and eIF4G families, were found to confer recessive resistance to plant
RNA virus infections. An eIF4E gene in melon was found to mediate a
recessive resistance against melon necrotic spot virus (Nieto et al. 2006). In
the cucumber genome, three eIF4E and three eIF4G genes were identified,
providing candidates for known recessive resistance genes against RNA
viruses such as zucchini yellow mosaic virus and watermelon mosaic
virus (Wai and Grumet 1995). In some wild melon genotypes, enhanced
expression of two glyoxylate aminotransferase genes (At1 and At2) controls
the resistance to downy mildew, a devastating foliar disease of cucurbits
(Taler et al. 2004). Two Arabidopsis thaliana (At) homologs were also identified
in cucumber, and are, thus, potential candidate genes for downy mildew
resistance.

11.9 Expanded Gene Families in Cucumber

The lipoxygenase (LOX) pathway plays an important role in developmentally
and environmentally regulated processes in plants (Liavonchanka and
Feussner 2006). The LOX gene family has been notably expanded in the
cucumber genome (cucumber 23, Arabidopsis 6, papaya 15, poplar 21,
grape 18, and rice 15). The majority of cucumber LOX genes (19 of the 23)
are distributed in three clusters; the largest contains 11 members that are
arranged in tandem (Fig. 11-8). The other sequenced plant genomes show
no obvious LOX clustering, with the exception of grapevine, which has one
Figure 11-8 Lineage-specific expansion of the lipoxygenase (LOX) family in the cucumber
genome (Huang et al. 2009). The LOX family was divided into two groups, “Type I” and “Type
II”. The two tandem duplicated gene clusters were ordered and displayed on chromosomes
2 and 4, plus one unmapped scaffold of the cucumber genome.

harboring six copies. Fourteen of the LOX genes are specific to the cucumber
lineage. The volatile (E, Z)-2, 6-nonadienal (NDE) gives cucumber its “fresh
green” flavor (Buescher and Buescher 2001) and confers resistance to some
bacteria and fungi (Cho et al. 2004). LOX and one type of hydroperoxide
lyase (9-HPL) synthesize NDE from linolenic acid precursors. Genes
with 9-HPL activity are rarely found in other plants (Matsui et al. 2000).
However, cucumber contains two tandem HPL genes, one of which has
been experimentally confirmed as having 9-HPL activity (Matsui et al. 2000).
The expansion of the LOX gene family and the duplicated HPL genes may
relate to the high level of NDE synthesis in cucumber.
Expansins are cell-wall loosening proteins in plants (Cosgrove 2000).
In cucumber, the expansin subfamily, EXLA, has undergone significant
expansion via tandem duplication (8 genes in cucumber as compared to 1
to 3 in other sequenced genomes); this event may well have contributed to
the development of tendril coiling in cucumber.
Among the sugar transporter proteins, polyol transorter (PLT) gene
family was expanded significantly in cucumber compared with other plants.
A specific gene cluster resulted from tandem duplication contributed mostly
to the expansion.
11.10 Cucumber: A Model Genome for Plant Vascular Biology

Evolution of the plant vascular system, comprised of xylem and phloem
tissues, plays a pivotal role in the emergence of land plants. The sieve tube
system of phloem, the equivalent of the animal arterial system, delivers
nutrients and signaling molecules to developing organs (Lough and Lucas
2006). Cucurbits are the model system for studying the identity and function
of the phloem proteome because it is one of only a few plant species from
which analytical quantities of the translocation stream can be collected.
A BLASTP analysis of 1,209 protein fragments from pumpkin phloem
(Lin et al. 2009) identified 800 genes in the cucumber genome (Huang et
al. 2009). Using these cucumber genes, homologous genes were identified
in other vascular plants as well as the non-vascular moss, Physcomitrella
patens. There are 1,072, 2,458, 2,780, 2,351, 1,944, 3,454, 1,986 and 2,535
homologs in moss, rice, sorghum, Arabidopsis, papaya, cucumber and
grapevine, respectively. These will be an important resource for vascular
biology studies in plants.
11.11 Perspective of Cucumber Genomics

The cucumber is the seventh sequenced plant, following Arabidopsis, the
poplar tree, grapevine, papaya, and the crops rice and sorghum. The
cucumber genome adopted the next generation sequencing technology

(Illumina GA). This makes it possible to carry out rapid and low-cost
sequencing for other important plant species. Equipped with this reference
genome, many diverse cucumber lines can be rapidly sequenced at low cost.
Using these resequenced sequences, many genes or signatures related to
the domestication processes and adaptation to diverse environments can be
detected. These genes or signatures will enable marker-assisted breeding of
high-yielding, disease-resistant, and fresh green-scented cucumber.
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12
Sex Expression in Cucurbits
Rebecca Grumet 1,* and Jessica Taft 1
ABSTRACT
The Cucurbitaceae is one of only a handful of plant families
characterized by predominantly unisexual flower production. Sex types
are highly variable, and a given genus can include both monoecious
and dioecious species. Dioecy is codified by divergent chromosomes
or gene complexes, while monoecy is conferred by a small number of
independently segregating genes. Microscopic analysis of cucumber
(Cucumis sativus) and melon (C. melo) indicates that unisexuality
results from specific suppression of either stamen or carpel primordia
subsequent to initial whorl formation. Sexual identity of a given bud
is caused by hormone balance, primarily ethylene level, which is,
in turn, influenced by genetic, developmental, and environmental
factors. Recently great progress has been made to elucidate underlying
molecular bases for sex determination. Primary sex determination
loci, A and M from melon and cucumber respectively, which cause
suppression of stamens, have been cloned and found to encode a
key enzyme for ethylene biosynthesis, ACS (1-amino-1-cyclopropane
carboxylate synthase). Similarly, the dominant F locus from cucumber,
which prevents suppression of carpels, also encodes an ACS gene. The
dominant G locus from melon, which causes carpel suppression, also
has been cloned and found to encode a WIP family transcription factor.
Further insight has been gained from studies with transgenic melon
expressing ethylene biosynthesis or perception genes, demonstrating
the essential role of ethylene perception and indicating that cross-
talk between the floral whorls is important for the sex determination
process.
Keywords: sex determination, floral development, ethylene, monoecy,
cucumber, melon, Cucumis
1
Department of Horticulture and Graduate Program in Genetics, Michigan State University,
East Lansing, MI 48824, USA; e-mail: taftjess@msu.edu
*Corresponding author: grumet@msu.edu

12.1 Introduction: Sex Forms and Their Evolution

Angiosperm species typically produce bisexual flowers including both
male (pollen-bearing) and female (ovule-bearing) organs, the stamens
and carpels, respectively. Throughout evolution, numerous biochemical,
physiological, and morphological mechanisms have evolved to promote
outcrossing, such as self-incompatibility systems where “own” pollen
is recognized and prevented from successful fertilization; asynchronous
development of eggs and pollen; or physical placement of the stamens,
such that self-pollen is unlikely to come in contact with the pistils (e.g.,
heterostyly) (Ainsworth 2000; Barret 1998). Perhaps the most extreme
morphological alteration is the development of unisexual flowers that bear
only male (staminate) or female (pistillate) organs (Fig. 12-1). Unisexuality
has been estimated to arise more than 100 times within the plant kingdom,
and can take two general forms: monoecy and dioecy (Renner and Ricklefs
1995). In monoecy, separate male and female flowers are formed on the
same plant. Dioecy carries the process further, leading to separate male
and female plants, each bearing flowers of only one sex type.
Petal
Petal
Nectary
Anther
Ovary
Nectary
Ovule
Figure 12-1 Sexual differentiation of cucumber flower buds. Longitudinal sections of cucumber
buds under dissection microscope. A. Male bud at stage 11. B. Female bud at stage 12 (anthesis).
Bud development stages are assigned as per Bai et al. (2004).

Sex Expression in Cucurbits 355
Dioecy occurs only in about 6% of the plant genera, including 5%

of monocot species and 8% of dicot species (Renner and Ricklefs 1995;
Ainsworth 2000). Despite their relative rarity, the species exhibiting
dioecy are widely distributed throughout the plant kingdom, representing
approximately 40% of the plant families. Their frequency, however, is not
evenly distributed; occurrence of dioecy is highly correlated with monoecy,
consistent with the possibility that ability to form unisexual flowers is
an evolutionary prerequisite to dioecy (Renner and Ricklefs 1995). The
Cucurbitaceae is one of a handful of families where unisexual flower
production is widespread, including an estimated 32% dioecious genera
(Renner and Rickhofs 1995).
The Cucurbitaceae species are particularly diverse with respect to
sex types. Most are monoecious, many are dioecious, and only a few
are hermaphrodite (Roy and Saran 1990). In addition to the standard
classification of monecy and dioecy, cucurbit species can exhibit a more
nuanced range of diversity including various combinations of sex types
such as gynomonoecy or gynoedioecy (separate female and bisexual flowers
on the same plant, or separate female and bisexual plants, respectively) or
andromonoecy or androdioecy (separate male and bisexual flowers on the
same plant, or separate male and bisexual plants, respectively) (Table 12-1).
Although androdioecy is considered to be the most rare plant sex system,
with only three confirmed species, there is at least one documented species
in the Cucurbitaceae with this flowering habit, Schizopepon bryoniaefolius
Maxim (Akimoto et al. 1999).
It is generally presumed that the evolution of modified sex systems is
either a driver, or a result of enhanced outcrossing. This assumption has
been tested directly for several populations of cucurbit species. Analysis of
populations of Schizopepon bryoniaefolius in Japan showed that the presence
Table 12-1 Diversity of sex types exhibited by the Cucurbitaceae.

Plant sex type Flower type Alternate flower type terms
A. Single plant
Hermaphrodite Bisexual Hermaphrodite or Perfect
Andromonoecious Male and Bisexual Staminate and Hermaphrodite
Gynomoneious Female and Bisexual Pistillate and Hermaphrodite
Trimonoecious Male, Female, and Bisexual Staminate, Pistillate, Hermphrodite
Monoecious Male and Female Staminate and Pistillate
Gynoecious Female Pistillate
Androecious Male Staminate
B. Separate plants
Dioecious Male and Female Staminate or Pistillate
Gynodioecious Female and Bisexual Pistillate or Hermaphrodite
Androdioecious Male and Bisexual Staminate or Hermaphrodite
Trioecious Male, Female, and Bisexual Staminate, Pistillate, Hermaphrodite

of male plants in androdioecious populations increased heterozygosity

relative to hermaphrodite populations (Akimoto et al. 1999). Similarly,
assessment of the Mediterranean cucurbit species Ecballium elaterium,
which is characterized by monoecious (elatrium) and dioecious (dioecium)
subspecies, showed that the monoecous species were highly inbred while
the dioecious species had greater allelic diversity (Costich and Meagher
1992).
It is also possible that sexual differentiation is driven by other factors,
such as resource allocation. It has been hypothesized that resource limiting
environments would favor female plants that do not have to spend energy
on production of male flowers (Costich 1995). Common garden experiments
and geographic analyses of Ecballium were consistent with this possibility.
The dioecious subspecies generally out-performed the monoecious
subspecies, and dioecy was more frequent in drier locations, while monoecy
was more frequent in wetter locations (Costich 1995). Interestingly, analysis
of occurrence of dioecy across the full range of angiosperms also showed
correlations with tropical distribution, abiotic pollination, shrub growth
form, and climbing growth habit, although the reasons for these correlations
are not known (Renner and Ricklefs 1995).
Dioecious species are typically characterized by an XY type system,
where one sex is homogametic or homomorphic (i.e., XX) and the other
heterogametic or heteromorphic (i.e., XY) (Ainsworth 2000; Voltz and
Renner 2008). The XY system may represent specific genes or distinct
sex chromosomes. The first demonstration of an XY sex determination
system in any organism is credited to the studies by Correns in the
early 1900s as a result of crosses performed between monoeious and
dioecious cucurbit species within the genus Bryonia, B. alba and B. dioica
(Roy and Saran 1990; Voltz and Renner 2008). However, the evolution of
morphologically distinct sex chromosomes, as occurs in animals, is much
rarer in plants (Charlesworth 2002). Despite the existence of hundreds of
dioecious plant species, only a dozen in four plant families are known to
have morphologically distinguishable X and Y chromosomes (Ming et al.
2007). Among the cucurbits, sex chromosomes have been verified only in
the species Coccinia grandis (syn. C. indica) (Roy and Saran 1990; Guha et
al. 2004).
Although the Bryonia example is not associated with morphologically
distinguishable sex chromosomes, it has been suggested that Bryonia species
are in the early stages of evolution of sex chromosomes (Oyama et al. 2009).
Distinct sex chromosomes are thought to evolve as a result of suppression
of recombination at the sex determination locus and neighboring regions
(Charlesworth 2002; Ming et al. 2007). The variability of sex types within
the Bryonia genus, which includes seven dioecious and three monoecious
species, further supports the possibility of an early stage of evolution of sex

differentiation, including sex chromosome differentiation (Voltz and Renner

2008). Studies by Heilbronn and coworkers in the 1940s demonstrated that
members of the Bryonia genus were able to produce offspring with varying
sex types, including crosses between two dioecious species that could yield
monoecious progeny and crosses between monoecious species that could
yield dioecious progeny (Cited in Roy and Saran 1990).
Variation of sex types also occurs within other Cucurbitaceae genera,
for example, the Schizopepon genus includes one monoecious, six dioecious
and one androdioecious species (Akimoto et al. 1999) and the Ecballium
elatum species includes both monoecious and dioecious forms (Roy and
Saran 1990). Recent studies of the Momordica genus, which include 34
dioecious species and 22 monoecious species, suggest that monoecy
arose independently seven times from a dioecious ancestor (Schaefer and
Renner 2009). These species can include “leaky” dieocious males capable of
producing a rare fruit, suggesting flexibility in the sex determination system
or a possible first step towards monoecy. Leaky dioecy is not uncommon
among dioecious cucurbits and reports of such a phenomenon date at least
as far back as studies by Darwin (Schaefer and Renner 2009).
Despite extensive examples of dioecy in the Cucurbitaceae, the species
that have achieved primary economic importance, e.g., cucumber, melon,
watermelon, squashes and pumpkins (Cucumis sativus, Cucumis melo,
Citrullus lanatus, Cucurbita pepo, Cucurbita maxima, Cucurbita moschata) are all
monoecious or variants thereof (Rudich 1990; Robinson and Decker-Walters
1997). Furthermore, sex types within these species are highly plastic and
are typically controlled by a small number of key genes that have allowed
breeders to develop the full range of plant sex types listed in Table 12-1A.
Therefore, the rest of this chapter will focus on monoecy and the associated
underlying mechanisms responsible for sexual differentiation.
12.2 Floral Development and Sex Differentiation

A typical angiosperm flower is comprised of four sequentially developing
whorls, the sepals, petals, stamens and carpels (Coen and Meyerowitz 1991;
Bowman 1997). Elegant studies of homeotic floral mutants in Arabidopsis
and Antirrhinum (snapdragon), which cause conversions among different
organs (i.e., stamens to petals), led to the elucidation of the ABC model of
floral development in which master MADS box transcription factors act
in different combinations to specify whorl identity (Fig. 12-2) (Coen and
Meyerowitz 1991; Bowman 1997). The A factors (APETALA 1 and 2) specify
sepals, A factors combined with B factors (APETALA 3 and PISTILLATA)
specify petals, B factors and C factors (AGAMOUS) specify stamens, and
C factors specify carpels. Thus loss of B factors cause conversion of petals
to sepals and stamens to carpels. A and C act antagonistically, where a loss
of one allows the other to influence all four whorls.

Figure 12-2 The ABC model of floral development. Three sets of MADS box transcription
factors interact in different combinations to specify sequential development of the four floral
organ whorls.
Homologs of some of these factors have been cloned from cucumber.

Three cucumber homologs of the Arabidopsis AGAMOUS (AG), C-function
gene (CAG1, CAG2, and CAG3) were isolated from flower buds at the early
stages of whorl initiation (Perl-Treves et al. 1998). Transcripts of CAG1 and
3 continued to be present at later stages in both male and female buds, as
is typical of C-function genes, but CAG2 was expressed only in carpels of
female buds. Another MADS box gene, ERAF17, was identified and its
expression correlated with induction of female flowers (Ando et al. 2001).
Like CAG2, but unlike standard C-function MADS box factors that promote
stamens and pistils, ERAF17 appears to be associated with development
of pistils but not stamens. These observations suggest involvement of
additional MADS box genes specific to female flower development in
cucumber.
The Cucumis species, cucumber and melon, are two of the best studied
model systems for the physiological and molecular analysis of sexual
differentiation leading to unisexuality in flowering plants. Unisexuality in
cucurbits does not appear to result from failure to initiate any specific whorl.
The development of a typical cucumber flower, from floral primordium
initiation to anthesis takes approximately 20 days (Atsmon and Galun 1960;
Goffinet 1990; Hao et al. 2003). During the first five days (1–2 mm in size),
organ primordia of all four whorls are established (stages 1–5) (Goffinet 1990;
Bai et al. 2004) (Table 12-2). At this stage the flower is bisexual or presexual.
At approximately six days, either the stamens or carpels begin to expand
rapidly. In male flowers, the carpel size remains virtually constant while the
stamens expand rapidly; the reverse is true for female flowers. This analysis
indicates that the period in which sex determination occurs is during the
first five days post initiation of the floral primordium. In male cucumber
flowers, arrest of carpel primordia occurs prior to differentiation of the

Table 12-2 Cucumber floral bud development stages based on Bai et al. (2004).
Non sex-specific Sex specific
development development
Stage Male Female
1 Inflorescence initiation
2 Sepal whorl initiation
3 Petal whorl initiation
4 Stamen whorl initiation
5 Carpel whorl initiation
6 Stamen enlargement Carpel elongation

Carpel arrest
7
Anther differentiation Stigma/ovary
differentiation
DNA damage in anther
8 Stamen enlargement Stamen arrest
9 Microsporophyte initiation Macrosporophyte initiation
10–12 Sex organ maturation
12 Anthesis
ovary, at stage 6 (Bai et al. 2004). In female flowers, initial differentiation of

the anther from the filament at stage 7 is accompanied by DNA degradation
in the primordial anther, followed by stamen arrest at stage 8 (Hao et al.
2003). Similar stages have been documented in melon floral buds (Boualem
et al. 2008).
Analysis of cucumber floral homeotic mutants demonstrated that
inhibition of stamens or pistils depends on whorl position, not specific
organ identity (Kater et al. 2001). A B-function mutation would normally
cause a sepal-sepal-carpel-carpel whorl pattern. The spontaneous B-function
cucumber mutant, green petals (gp), results in expected B-function conversion
of petals to sepals in the second whorl. Effects in the third and fourth whorls,
however, depended on whether the flower was destined to become male
or female. In the third whorl of buds that were destined to be female, the
predicted carpels did not develop. Thus the third whorl failed to develop,
regardless of whether it would form stamens or carpels. This phenotype
indicates that development arrest of the inappropriate sex organ depends on
its position, rather than sexual identity. Similarly, in the node positions that
would normally produce male buds, only the predicted first three whorls
developed, as would be the case for a male flower. However, the third whorl
produced carpels instead of stamens or became indeterminate.
In addition to sex differentiation of individual flowers, many cucurbits
undergo a progressive sequence of sex expression relative to the age of
the plant (Karchi 1970; Roy and Saran 1990; Robinson and Decker-Walters
1997). For example, a cucumber vine typically begins with a vegetative
phase, followed by three flowering phases: male, female and male, and

female. In melons, the male phase is followed by a mixture of bisexual and

male flowers, with bisexual flowers more commonly produced on lateral
branches than the main stem. Thus sex expression within the plant follows
a developmentally controlled program, presumably mediated, at least in
part, by endogenous hormone levels. Grafting experiments of monoecious
and gynoecious lines have indicated that mobile signals can influence sex
expression (Mockaitis and Kivilaan 1964;Takahashi and Suge 1982). Fruit set
also can influence sex expression of subsequent nodes causing a reduction
in carpel-bearing flower production in cucumbers, squashes and gourds
(Schapendon and Brouwer 1984; Stephenson et al. 1988; Krupnick et al.
1999; Avila-Sakar et al. 2001).
12.3 Influence of Hormones on Sex Determination

Sexual differentiation of individual cucurbit flower buds is not strictly
pre-ordained and can be influenced by environmental or hormonal cues.
Exogenous hormones can cause cucumber or melon flowers to undergo sex
conversion such that a given bud typically destined to become one sex type
develops as an alternate type (see for review, Rudich 1990; Perl-Treves 1999).
Increased maleness can be manifested by increased male flower production,
or conversion of female to bisexual flowers; similarly, increased femaleness
can occur via increased female flower production, or conversion of male
flowers to bisexual.
Several different hormones have been shown to influence sex expression.
Effects of gibberellins (GAs) and auxins were the first studied. Application
of GAs promote male flower formation and prevent female flowers from
fully developing (Atsmon et al. 1968; Pike and Peterson 1969), while
inhibitors of GA biosynthesis promote femaleness (Yin and Quinn 1995;
Tolla and Peterson 1979). Higher levels of endogenous GA were present
in isogenic monoecious than gynoecious cucumber lines (Atsmon et al.
1968; Friedlander et al. 1977). Auxin can increase femaleness in cucumbers,
melons and squashes (Galun 1959; Galun et al. 1963; Rudich et al. 1969) and
higher levels of auxin were associated with more female sex types (Galun
et al. 1965; Rudich et al. 1972).
Later studies showed that ethylene also affects sex expression.
Exogenously applied ethylene increased femaleness in cucumber, melon
and squash (McMurray and Miller 1968; Robinson et al. 1969; Rudich et al.
1969; Karchi 1970; Owens et al. 1980; Augustine et al. 1973). Treatment of
gynoecious cucumbers with the ethylene perception inhibitor, silver nitrate
or silver thiosulfate, or inhibitors of ethylene synthesis (aminoethoxyvinyl
glycine; AVG) increased maleness through production of both bisexual
and male flowers in gynoecious cucumber and melon plants, indicating
a possible role of ethylene in both inhibition of stamen development, and

promotion of carpel development (Byers et al. 1972b; Owens et al. 1980).

The feminizing effect of ethylene is not universal, however, as exogenous
ethylene enhances maleness in watermelon (Rudich 1990).
Further studies showed that the effect of ethylene was stronger than
that of GA or auxin. Inhibiting ethylene production or perception was more
effective in increasing maleness in cucumber and that application of GA, and
GAs do not influence sex in melon (Kubicki 1969a; Byers et al. 1972a; Tolla
and Peterson 1979; Yin and Quinn 1995). Similarly, inhibitors of ethylene,
but not auxin were able to modify the sex pattern of gynoecious cucumbers
(Trebitsh et al. 1987). Since auxin can induce ethylene production, it was
suggested that increased femaleness was due to auxin-induced production
of ethylene (Trebitsh et al. 1987). Subsequent studies showing increased
femaleness in cucumber in response to brassinosteroid (BR) application also
implicated that the effect was mediated by BR-induced ethylene production
(Papadopoulou and Grumet 2005). In addition, application of BR also did
not influence sex in melon, further supporting the role of ethylene as the
primary hormonal factor influencing sex determination in cucurbits.
Consistent with the effect of exogenous ethylene, endogenous ethylene
levels were correlated with different sex phenotypes in cucumber; ethylene
evolution from apical tips of gynoecious seedlings was 2–3 fold higher than
from monoecious seedlings (Rudich et al. 1972, 1976; Trebitsh et al. 1987;
Makus et al. 1975; Yamasaki et al. 2001, 2003a). In melon, levels of ethylene
production were similar in gynoecious, monoecious, and andromonoecious
genotypes, however the importance of endogenous ethylene, was
demonstrated by application of hypobaric conditions (Byers et al. 1972b).
Reduction of internal gas concentrations resulted in increased maleness that
could be reversed by the application of ethylene. Reduced ethylene levels
also have been associated with fruit set and subsequent reduction in female
flower production in Cucurbita texana (Krupnick et al. 1999).
Timing and dosage of hormone treatment influences which nodes are
affected. No effect on sex was observed following treatment of cucumber
cotyledons with the ethylene-releasing compound, ethrel (Iwahori et al.
1970). Treatment at first true leaf stage, however, caused production of
female buds, rather than male buds, which occured nine nodes earlier than
for non-treated plants. There is typically a delay of approximately 10 days
from the time of application to first appearance of sex-converted flowers
(Robinson et al. 1969; Karchi 1970; Byers et al. 1972a). Hormones applied
at later stages of plant development result in sex conversion on higher
nodes, while higher doses lead to conversion at lower nodes (Robinson
et al. 1969; Karchi 1970). Similarly, cucumber buds destined to be males
could be converted to females in vitro only when they were removed prior
to expansion of the stamen primordia (Galun et al. 1963). Microscopic
analysis of developing buds indicates stamen primordium differentiation

as the critical stage for chemically inducing sex conversions (Yamasaki et

al. 2003b). These observations are consistent with a model in which only
certain stages in development of the floral primordium are receptive to sex
determination factors, resulting in either continued or suspended growth
of the specific organ, and that sex organ primordia are the critical sites for
hormone perception.
12.4 Inheritance of Sex Expression in Cucumber and Melon

Several genes influencing sex expression have been characterized in melon
and cucumber. Cucumber sex expression is controlled primarily by the F
(Female) and M (Monoecious) loci, which in different combinations produce
hermaphrodite (FFmm), andromonoecious (ffmm), monoecious (ffMM) or
gynoecious (FFMM) plants (Table 12-3) (see for review Perl-Treves 1999).
The dominant F allele is necessary to constitutively produce carpel-bearing
(bisexual or female) flowers, while the absence of F allows for the production
of separate male flowers. The dominant M allele is necessary for suppression
of stamens in carpel bearing flowers to produce female rather than bisexual
flowers. The presence of other genes can further increase the range of
phenotypes; for example, the a allele of Androecious, when combined with
M or m and f can create androecious (all male) plants (Kubicki 1969b). Tr
(Trimonoecious), is a co-dominant gene, which removes the inhibition of
carpel development in male buds to create bisexual flowers, and the In-F
(Intensifier-Female) gene increases femaleness in monoecious genotypes
when crossed with a gynoecious genotype (Robinson and Decker-Walters
1997).
Inheritance of sexual phenotypes in melon is similar to cucumber,
in that there are also two major genes controlling sex expression, the A
(Andromonoecious) and G (Gynoecious) loci (Kenigsbuch and Cohen 1990;

Roy and Saran 1990). Suppression of stamens in carpel-bearing flowers

is associated with the dominant A allele, suggesting that A in melon is
analogous to M in cucumber. However, the suppression vs. constitutive
production of carpels appears to differ. For melon, dominant G allele (rather
than recessive f) is needed to produce separate male flowers (i.e., suppress
carpel development). A third locus, M (Maleness), also contributes to sex
determination and is required in recessive form to stabilize the gynoecious
phenotype (Kenigsbuch and Cohen 1990). Other models have suggested
that at the interplay of at least four genes are required to create stable
hermaphrodites (Kubicki 1969b), and are also consistent with involvement
of four loci for production of hermaphrodites (Tatt and Grumet unpubl.
data). Androecy or near androecy has been observed in rare individual
plants, but, to our knowledge, stable androecious lines have not been
reported (Magdum et al. 1982).
These genetic analyses indicate that for both cucumber and melon, two
primary changes are required to convert from hermaphrodite to monoecious
flowering habit, and are consistent with the need to separately suppress
either male or female functions. Suppression of only the stamens or carpels
results in either gynoecious or andromonoecious plants, respectively. The
complete transition to androecy appears to involve at least one additional
gene in cucumber (Table 12-3).
12.5 Molecular Basis of Sex Expression

As discussed above, much of the initial research studying the effects of
hormones on sex expression in the cucurbits was carried out in the 1960s
and 1970s. In the 1990s and 2000s a resurgence of work investigating sex
expression in Cucumis using molecular genetic and genomic approaches
led to the identification of key genes that function in sex expression, and
development of new models for understanding the sex determination
process. These efforts, which have further established the key role of
ethylene in sex determination, have been accelerated in recent years by new
genomic and ultra high-throughput sequencing technologies.
12.5.1 Ethylene Production

Ethylene is produced from S-adenyosyl methionine (SAM) via a two-
step process mediated by the enzymes, ACC (1-aminocyclopropane-1-
carboxylate) synthase (ACS), and ACC oxidase (ACO) (Johnson and Ecker
1998):
S-AdoMet (SAM) ---------------------> 1-amino-1-cyclopropane carboxylate (ACC) ---------------------> ethylene
ACC synthase (ACS) ACC oxidase (ACO)

ACS and ACO are generally encoded by multigene families whose members
are differentially regulated and can be induced by a variety of internal
signals, including other hormones and ethylene itself (Rottman et al. 1991;
Bleecker and Kende 2000; Salmon-Minkov et al. 2008).
A key breakthrough in the understanding of the mechanism of sex
determination in cucumber came from the discovery that the dominant F
allele, which allows for constitutive production of carpels (i.e., gynoecious
or hermaphrodite plants), completely co-segregated with an additional
copy of an ACS gene, CsACS1G (Trebitsch et al. 1997). CsACS1G is absent in
near-isogenic, monoecious lines and expression of CsACS1G transcript was
observed in apices of gynoecious, but not isogenic monoecious cucumbers
(Trebitsch et al. 1997; Kamachi et al. 2000). This led to the clear implication
that the F locus increases femaleness by regulating endogenous ethylene
production, which in turn promotes carpel development. CsACS1G is located
in tandem with CsACS1, and appears to be the result of recombination
between CsACS1 and a branched chain amino acid transferase gene
(Mibus and Tatlioglu 2004; Knopf and Trebitsh 2006). CsACS1 and 1G show
complete homology in the coding region, 400 bp of the proximal promoter,
and the 3’ untranslated regions, suggesting a recent gene duplication event
(Mibus and Tatlioglu 2004; Knopf and Trebitsh 2006). Thus, the feminizing
effect of F may be a gene dosage effect leading to the increased ethylene
production that has been observed in gynoecious lines and/or differential
regulation resulting from differences in the distal promoter region (Knopf
and Trebitsh 2006).
Two other ACS genes, CsACS2 and CsACS4 also exhibit increased
expression with the transition to the female phase (Kamachi et al. 1997;
Yamasaki et al. 2001, 2003a, b). CsACS2 transcript was not detected prior
to the formation of pistil primordia, after which it was localized to the
ovary, just below the pistil primordia (Yamasaki et al. 2003a; Saito et al.
2007). Timing and level of expression of the CsACS2 gene in the apices of
monoecious and gynoecious cucumbers also coincided with the action of
ethylene in the induction of the first female bud (Kamachi et al. 1997, 2000).
It was suggested that CsACS2 is critical for female flower development,
and that CsACS1 (the F locus) may act to accelerate the timing and increase
levels of CsACS2 via higher ethylene production (Kamachi et al. 2000).
Sex- and ethylene-related differential expression also has been observed
for ACO genes (Kahana et al. 1999). Application of the ethylene releasing
compound, ethrel, caused different effects on distinct ACO family members
and in different sex types, indicating likely functional differentiation among
the ACO genes. ACO3 was expressed more strongly in developing stamens
and pistils of female buds than male. On the other hand, ACO2 was not
expressed in gynoecious apices and levels decreased in apices of monoecious
plants when they reached the stage of female flower production. ACO2

and ACO3 also responded differently to exogenous ethylene, depending

on whether the plant was gynoecious or androeicous.
Further demonstration of the importance of endogenous ethylene in sex
determination was obtained through the production of transgenic melons
constitutively expressing ACS (Papadopoulou et al. 2005). The 35S::ACS
melon plants showed increased ethylene production by leaves and flower
buds, and increased femaleness as measured by earlier and increased
number of bisexual buds. There was also a higher frequency of adjacent
bisexual flowers and adjacent nodes setting fruit on the main stem of field
grown plants (Papadopoulou et al. 2005). These observations suggest that
increased ethylene production in the transgenic melons may interact with
the normal hormone balance associated with fruit set and suppression
of subsequent carpel-bearing flower production as has been observed in
various cucurbits (Krupnick et al. 1999).
New breakthroughs in defining the critical role of ethylene in sex
determination in Cucumis came with cloning of the A locus from melon
(Boualem et al. 2008), and subsequent cloning by two laboratoriess, of the
analogous M locus in cucumber (Boualem et al. 2009; Li et al. 2009). An
extensive set of high resolution mapping, map-based cloning, and targeted
induced local lesions in genomes (TILLING) experiments showed that A,
which causes loss of stamens in carpel-bearing flowers (i.e., monoecy or
gynoecy), encodes a previously unknown ACS gene in melon, CmACS7
(Boualem et al. 2008). Mutational analysis of the segregating F2 population
from a cross of monoecious (AAGG) and andromonoecious (aaGG) lines
identified a missense, alanine to valine mutation (A57V) of a conserved
residue within the ACS active site of the recessive a allele. Enzymatic assays
of the mutant and wild type ACS7 showed that the mutation reduced the
activity to below 50% of wild type, leading to the loss of stamen inhibition
in bisexual flowers (Boualem et al. 2008). In situ hybridization studies of
the ACS7 transcript in male, bisexual and female flowers at stages 4 and 7
found that ACS7 was only expressed in the carpel primordia of bisexual and
female buds at both stages. It was concluded that the formation of female
flowers requires full activity of CmACS7 to inhibit stamens, however, if
activity is < 50%, the stamen primordia continue to develop, resulting in
bisexual flowers (Boualem et al. 2008).
Based on the identification of CmACS7, the M locus in cucumber was
elucidated using an ortholog-based approach, as both A and M act to inhibit
stamens in carpel-bearing flowers in their respective species (Boualem et
al. 2009; Li et al. 2009). BLAST analysis of the cloned product identified the
previously studied CsACS2 gene, whose expression is localized to the ovary
of carpel-bearing buds in monoecious and gynoecious cucumbers (Kamachi
et al. 1997; Yamasaki et al. 2003b; Saito et al. 2007 ), as the predicted homolog
to CmACS7 (Boualem et al. 2009). Sequence and segregation analyses of

CsACS2 in monoecious (MMffAA), gynoecious (MMFFAA), androecious

(MMffaa), and hermaphrodite (mmFFAA) plants showed a single nucleotide
polymorphism (SNP) causing an amino acid change (P209S) linked with
the M locus (Boualem et al. 2009). In a parallel study, Li et al. (2009) used
a map-based cloning approach to identify the M locus in cucumber. Two
candidate genes were found, of those, only CsACS2 exhibited a mutation
that was associated with bisexuality. A point mutation causing a change from
glycine to cysteine in amino acid 33, resulting in reduced ACS enzymatic
activity (Li et al. 2009). Further sequencing of CsACS2 from 28 cucumber
accessions led to identification of a third SNP within hermaphrodite and
andromonoecious lines, S399L (Boualem et al. 2009).
Analysis of predicted structure indicates that all three mutations in
CsACS2 occur in/near key residues that position SAM in the enzyme active
site and/or help with binding of the pyridoxal 5’-phosphate (PLP) cofactor,
and are therefore predicted to cause reduction or loss of enzymatic activity
(Rottman et al. 1991; Huai et al. 2001). This loss or reduction of activity of an
ACS, as was observed in melon, causes a loss of stamen inhibition that allows
for the development of bisexual (mm) vs. female (M) flowers (Boualem et al.
2009; Li et al. 2009). The multiple SNPs observed in cucumber, all of which
are different from the A57V change in melon, indicate that unisexuality
in Cucumis preceded the split between cucumber and melon (Boualem et
al. 2008; Li et al. 2009). Reversion to bisexuality appears to have occurred
independently, and several times within cucumber.
12.5.2 Ethylene Perception

Collectively these studies of the F, M and A loci clearly demonstrate the
importance of ethylene production in regulating the development of both
stamens and carpels, in cucumber and melon, albeit in opposite ways. The
ability of the same hormone to have positive and negative effects on sex
primordia, implicates differential perception and/or downstream responses
between the primordia. Yin and Quinn (1995) proposed the presence of
separate male and female receptors associated with different response
pathways, and that interaction between levels of endogenous hormones
and sensitivity of receptors regulate sex expression. Presumably lower
levels of ethylene are required to stimulate carpel development than to
inhibit stamens, although differences in timing of ethylene production also
may account for differential responses. The path of ethylene perception
and signaling has been well characterized in Arabidopsis thaliana (Wang et
al. 2002; Guo and Ecker 2004) and provided a starting point for analysis
in Cucumis.
Homologs of several Arabidopsis ethylene perception genes, ETR1,
ETR2 and ERS, have been cloned from cucumber and studied for possible

involvement in sex expression (Yamasaki et al. 2000). Higher expression

levels of CsETR1, CsETR2 and CsERS were observed in shoot apices of
gynoecious rather than moneocious genotype, and transcripts increased in
gynoecious plants at 4–5 leaf stage when the first female buds are initiated
(Yamasaki et al. 2000, 2001). Ethylene application increased expression of
these genes in apices of both monoecious (Mf) and gynoecious (MF) apices,
while inhibiton of ethylene with AVG, inhibited expression. In contrast,
ethylene did not promote expression in andromonoecious (mf) plants that
do not exhibit a loss of stamens in carpel-bearing flowers (Yamasaki et al.
2000, 2001).
Ethylene perception has been demonstrated to be critical for carpel-
bearing flower production in melon. Andromonoecious or gynoecious
melons genetically transformed to constitutively express the dominant
negative ethylene perception mutant, the Arabidopsis thaliana etr1-1 (ethylene
triple response) gene exhibited almost complete loss of carpel-bearing nodes
(Little et al. 2007). To examine the necessary location of perception in the
developing floral bud, the etr1-1 gene was introduced under control of
tissue specific promoters (Little et al. 2007). APETELA3 (AP3) is a class B
homeotic floral gene that is expressed in developing stamens and petals
in Arabidopsis (Irish and Yamamoto 1995), while CRABSCLAW (CRC) is a
YABBY transcription factor that drives expression in carpel and nectary
primordia (Bowman and Smyth 1999). If ethylene perception by carpel
primordia is needed to promote carpel development, it would be expected
that inhibition of perception in the CRC::etr1-1 melons would show a loss
of carpel bearing nodes. However, despite expression of the CRC::etr1-1
transgene, melons did not show a reduction in carpel-bearing buds. In
contrast, AP3::etr1-1 plants exhibited a near complete elimination of carpel-
bearing buds on both the main stem and lateral branches. AP3::etr1-1
plants that did form carpel-bearing buds showed defects in pistil and
ovary development, while stamens appeared normal (Little et al. 2007).
These results indicate that ethylene perception by the stamen primordia
is necessary to promote development of carpel primordia, and implicates
involvement of a mechanism that mediates cross talk between the two
whorls.
12.5.3 Other Factors

The G locus in melon, which is responsible for suppression of carpel
development leading to male flower formation, was the final of the four
major Cucumis sex determining genes to be cloned. Positional cloning
using a high resolution genetic map of a segregating F2 population derived
from a gynoecious (AAgg) x monoecious (AAGG) cross, localized the
locus to a 1.4 kb non-coding region including an insertion of a hAT family

transposon in the gynoecious parent (termed Gyno-hAT) (Martin et al. 2009).

Screening of C. melo germplasm accessions showed that all monoecious
and andromonoecious accessions lacked Gyno-hAT, while the recessive
hermaphrodite or gynoecious lines possessed the insertion. Analysis of
the surrounding region for methylation and reduced gene expression as is
typically associated with transposons, led to the identification of a candidate
gene, CmWIP1, with homology to WIP transcription factor genes (Martin
et al. 2009). TILLING analysis of a monoecious line further identified three
missense mutations in CmWIP1 associated with conversion of male flower
nodes to female (Martin et al. 2009). These results are consistent with
recessive g allele associated with hermaphrodite and gynoecious plants.
The identification of CmWIP1, rather than an ethylene biosynthetic gene,
as the melon G locus is consistent with earlier studies that failed to show a
difference in endogenous ethylene production between apices or gynoecious
and monoecious melon genotypes (Byers et al. 1972b).
Expression of CmWIP1 is localized to the carpel primordia of future
staminate flowers at stage 6, while carpel-bearing flowers show low
expression (Martin et al. 2009). Comparison of expression levels of CmWIP1
and CmACS7 showed an antagonistic relationship. No physical interaction
was observed between the promoter of CmACS7 and CmWIP1 protein,
however, indicating that the antagonism is indirect. Thus, expression of
CmWIP1 leads to inhibition of carpel primordia, while methylation-induced
silencing of CmWIP1 allows for carpels to develop.
12.6 Utilization of Sex Forms in Crop Improvement

Manipulation of sex expression can influence fruit quality, yield, cropping
methods, and breeding strategies. Varieties with a long vegetative or male
phase prior to production of carpel-bearing flowers have a long growing
season which can be a disadvantage depending on location (e.g., ability
to avoid frost), as well as timing of harvest to receive premium prices.
Cucumber is one species for which gynoecy, conferred by the F allele,
has been used extensively to produce an earlier yielding crop with more
uniform, concentrated fruit set (Lower and Nienhuis 1990). These traits
are particularly valuable for machine harvesting of pickling cucumbers.
It has also been possible to allow for double cropping in an otherwise
comparatively short season, as is done in some northern US production
areas (Ngouajio and Mennan 2005). Similarly, gynoecy in bitter gourd
(Momordica charantia) has been associated with earlier fruit production and
higher yield (Behera et al. 2009).
To allow for fruit set in the field, seed of a gynoecious variety is typically
sold in combination with a small percent (5–10%) of a monoecious pollinizer
line to provide male flowers for pollination (Robinson and Decker-Walters

1997). Earliness of staminate flower production is desirable to ensure

adequate pollen availability for early fruit set (Walters and Wehner 1994).
In the absence gynoecious breeding lines for squash, cultivars have been
selected to maximize female flower production to increase yield (Robinson
and Decker-Walters 1997). When planted too early in the season, however,
the effect of low temperatures leading to increased femaleness can cause
reduced fruit set due to unavailability of pollen. Gynoecy also can be
combined with parthenocarpy, eliminating the need for pollination, and
increasing yield potential (Rudich et al. 1977). Parthenocarpy in cucumber
is often accompanied by increased fruit quality. It also can confer increased
yield, due to lack of the inhibition of subsequent fruit set that is typically
associated with seeded fruits (Sun et al. 2006).
One of the primary advantages of gynoecy is to facilitate hybrid seed
production by eliminating undesirable self-pollination. The use of an all
female line for hybrid production ensures that the seed harvested is a
product of outcrossing. The inbred gynoecious lines used for maternal
hybrid parents in cucumber can be maintained by application of ethylene
inhibitors to produce male flowers, which are then used to self-pollinate
the gynoecious line (Tolla and Peterson 1979). Hybrid cucumber cultivars
resulting from a cross between gynoecious and monoecious parents results
in hybrid Ff cultivars, which can exhibit a less stable gynoecious phenotype
than homozygous FF plants in field production settings. To minimize
this problem, additional modifying genes, such as In-F, may be included
(Robinson and Decker-Watlers 1997). Alternatively a hermaphrodite FFmm
line may be used as the male parent to provide homozygous FF progeny
(Robinson and Decker-Walters 1997). Development of gynoecious lines
of bitter gourd is currently underway to facilitate hybrid seed production
(Behera et al. 2009).
The lack of an F locus equivalent in melon has made it more difficult
to maintain stable breeding lines of gynoecious melons (Kenigsbuch and
Cohen 1990). In squash, although gynoecious lines are not available,
cultivars with maximized female flower production provide an advantage
in hybrid production by reducing the number of male flowers that must
be removed to prevent inbreeding (Robinson and Decker-Walters 1997).
Conversion to all female flowers by ethylene treatment also is more readily
achieved for those lines that already have a tendency towards a high ratio
female/male flowers, thereby facilitating hybrid seed production on the
maternal parent plants.
Recent efforts have included development of molecular markers to
facilitate breeding for desired sex types. Perhaps in the long term, markers
will be especially useful for the inclusion of modifier genes. Current
examples include sequence related amplified polymorphism (SRAP) and

sequence characterized amplified region (SCAR) markers for the M locus

in cucumber (Li et al. 2008) and the A locus in melon (Feng et al. 2009).
12.7 Concluding Remarks

The extensive sexual diversity of the Cucurbitaceae species provides unique
insights into evolutionary and developmental biology and provides novel
opportunities for crop improvement. Cucurbit species show a range of
evolutionary states from hermaphrodite to monoecious to dioecious, and
back again, often within a given genus or among subspecies of a single
species. In recent years tremendous progress has been made in understanding
the genetic, physiological and molecular underpinnings of the capacity for
unisexual flower development, especially through the cloning of the major
sex expression genes from melon and cucumber. The identification of these
genes definitively clarifies the long-known role of ethylene in regulation of
sex primordia development in these species, and raises new questions about
mechanisms of action and inter-whorl communication. It is anticipated
that with the recent completion of sequencing of the cucumber genome
(Huang et al. 2009), rapidly evolving ultra high-throughput sequencing
technologies, and worldwide genomic efforts, that further evolutionary
and developmental insights are yet to come.
Acknowledgments
We thank Drs. David Dilley and Holly Little for helpful reviews of this
chapter. This work was in part supported by research grant US-3735-
05C from the United States-Israel Binational Agricultural Research and
Development (BARD) Fund.
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13
Future Prospects
Hiroshi Ezura
ABSTRACT
Cucurbit plants are important not only as crops for world food
production but also as model plants for elucidating significant traits
in plant development and responses, such as sex determination and
signaling through the phloem sap. To achieve sustainable production
and to extend our understanding of development and environmental
responses in cucurbit plants, continuous research and development
(R&D) is needed. As described in previous chapters of this book,
significant amounts of knowledge in terms of conventional and
molecular genetics and breeding are currently available for this plant
species. In addition, emerging technologies, such as high-throughput
sequencing technology and high-resolution metabolomics, have
generated a very large amount of data on cucurbit plants. New resources
such as induced mutant lines have also been generated for this plant
species. Effective and efficient integration of these technologies and
resources into conventional genetic and molecular tools is crucial for
achieving successful R&D in cucurbit plants. In this chapter, future
aspects of R&D useful for extending our knowledge and promoting
the use and breeding of cucurbit plants will be discussed.
Keywords: parthenocarpy, omics, metabolome, phenome, high-
throughput sequencing technology
13.1 Parthenocarpy, a Trait for Expansion of Productivity

Enhanced tolerance to biotic stresses, such as pathogens, insects and
weeds, and to abiotic stresses, such as drought, flooding, chilling and high
temperatures in cucurbit plants, is essential for their sustainable production.
Many researchers have targeted these traits, and we have summarized
Gene Research Center, Graduate School of Life and Environmental Sciences, University of
Tsukuba, Ten-nodai 1-1-1, Tsukuba 305-8572, JAPAN; e-mail: ezura@gene.tsukuba.ac.jp

Future Prospects 377
some of their achievements in previous chapters (Chapter 6, 7, 8 and 9) of

this book. Here, we emphasize the importance of parthenocarpic traits for
expanding the production of cucurbit crops in the world.
Parthenocarpy is the natural or artificially induced production of
seedless fruits without fertilization of the ovules. Parthenocarpy occasionally
occurs as a mutation in nature, but it can be induced by genetic engineering,
as reported in tomato (e.g., Rotino et al. 2005; Martinelli et al. 2009) and
eggplant (e.g., Rotino et al. 1997; Acciarri et al. 2002). Parthenocarpy of
some fruits might be of value. Production of seedless fruit when pollination
is unsuccessful may be advantageous to a plant because it provides food.
Seedlessness is a desirable trait in edible fruit with hard seeds, such as
pineapple, banana, orange and grapefruit. Parthenocarpy is also useful
in fruit crops that may be difficult to pollinate or fertilize, such as tomato
and summer squash.
In cucurbit crops, watermelon requires pollination or other stimulation
to induce parthenocarpic fruit development, while cucumber and summer
squash do not. Triploid watermelon has been traditionally used to produce
parthenocarpic seedless fruits. Crossing diploid and tetraploid parental lines
produces the triploid seed. Parthenocarpic fruits are obtained by crossing
diploid pollen to triploid flowers or by chemical treatments (Huitron et
al. 2007). Alternatively crossing soft-X-irradiated pollen to diploid plants
can produce parthenocarpic seedless fruits (Sugiyama et al. 2002). Both
techniques require pollination by insects or by hand to set fruits. However,
insect pollination is sometimes affected by climate, and hand pollination
requires a significant amount of labor. By contrast, some cultivars of
cucumber (Sun et al. 2006) and summer squash (Robinson and Reiners 1999)
do not require pollination or other stimulation to induce parthenocarpic
fruit development. If this type of parthenocarpic trait can be introduced
to watermelon, melon and other cucurbit crops, the production would be
stabilized. In addition, labor would be saved and production of the cucurbit
crops would expand.
Although several studies have been conducted on the genetics of
parthenocarpy in cucumber (Sun et al. 2006) and summer squash (Menezes
et al. 2005), the molecular mechanism of parthenocarpic fruit development
in cucurbit plants is still unclear. Significant functional genomics tools have
recently been developed for cucumber, which is an important cucurbit crop
for studying parthenocarpic fruit development (Ren et al. 2009; Huang
et al. 2009). These tools will facilitate the cloning of genes responsible for
parthenocarpy in cucumber and allow us to fully understand the molecular
mechanism underlying parthenocarpic fruit development in cucumber.
This knowledge will enable us to introduce parthenocarpy to other cucurbit
crops, which will contribute to stable cucurbit fruit production.

13.2 Bioresources for The Enhancement of Genetics and Breeding

Bioresources are fundamental materials for promoting genetics and
breeding in plants. As described in Chapter 1 of this book, major national
and international gene banks are currently collecting and maintaining
the cucurbit germplasms. In addition to the natural cucurbit germplasm,
artificially induced mutant populations have been developed through EMS
mutagenesis in melon using model cultivars of various regions (Tadmor et
al. 2007; Puigdomenech et al. 2007; Ezura and Fukino 2009). These mutant
populations should help to elucidate significant traits in melon. However,
such induced mutant populations should also be generated in other cucurbit
species.
The Convention on Biological Diversity (http://www.cbd.int/
convention/) was established on December 29, 1993, and has been signed
by 193 countries and regions. The Convention on Biological Diversity was
inspired by the world community’s growing commitment to sustainable
development. It represents a dramatic step forward in the conservation
of biological diversity, the sustainable use of its components, and the fair
and equitable sharing of benefits arising from the use of genetic resources.
Under the guidelines at the convention, the international exchange of
the germplasm will be more guarded in the future. However, to achieve
sustainable cucurbit production, geneticists and breeders must continuously
generate new cultivars in response to global environmental changes.
Therefore, we have to establish an open network of the cucurbit germplasm
and a reasonable and fair sharing protocol for research use.
13.3 Profiling Functional and Pharmaceutical Ingredients

Carotenoids are organic pigments naturally occurring in the chloroplast and
chromoplast of plants. They possess health-promoting or disease-preventing
properties for humans. Over 700 carotenoids have been identified,
including the well-known carotenoids, beta-carotene, alpha-carotene,
gamma-carotene, lycopene, lutein, beta-cryptoxanthin, zeaxanthin, and
astaxanthin (Britton et al. 2004). Cucurbitaceae crops, including squash,
pumpkin, watermelon, and melon with orange flesh, are important sources
of carotenoids. The carotenoid content varies in these species; for example,
the principal carotenoids in Cucurbita moschata are beta-carotene and alpha-
carotene, whereas lutein and beta-carotene dominate in C. maxima and C.
pepo (Azevedo-Meleiro and Rodriguez-Amaya 2007).
In addition to the well-known functional ingredients, cucurbit crops
contain other ingredients conducive to human health. Citrulline is a unique
amino acid that was first identified in watermelon (Wada 1930). Citrulline
from watermelon is effectively converted into arginine in humans. Arginine

is the nitrogenous substrate used in the synthesis of nitric oxide and plays an
essential role in cardiovascular and immune functions (Collins et al. 2007).
Melon contains a higher amount of gamma-aminobutyric acid (GABA), a
compound with antihypertensive effects (Yoshimura et al. 2010), in its fruits
(Ezura et al. unpubl. result). Cucumisin is a thermostable alkaline serine
protease found in the juice of melon fruits (Cucumis melo L.) (Kaneko and
Tominaga 1975). Its complete nucleotide sequence and deduced amino acid
sequence have been determined (Yamagata et al. 1994). Cucumisin supports
daily food life by promoting the digestion of meats, but it is reported to
be a major allergen in melon fruits (Cuesta-Herranz et al. 2003). Recently,
the preventive effects of melon extracts, which are rich in superoxide
scavenging activity, on abdominal and liver fat and adipokine imbalance
in high-fat-fed hamsters have been reported (Decorde et al. 2009). Melon
extracts prevented aortic lipids and liver steatosis in a diet-induced model
of atherosclerosis (Decorde et al. 2010). Both reports indicate the benefits
of melon for human health.
Minor cucurbitaceae plants include pharmaceutical ingredients, which
may contribute to human health. Momordica charantia Linn, belonging to
the family of Cucurbitaceae, is a useful medicinal and vegetable plant for
human health and one of the most promising plants for diabetes treatment
(Lee et al. 2009). Cucurbitane-type triterpenoids are the main active
constituents of M. charantia and have a number of potential biological and
pharmacological applications because of their antidiabetic, anti-obesity,
anticancer, anti-HIV, antifeedant and antioviposition activities. Since
the early 1960s, the constituents of bitter melon have been investigated,
and several classes of secondary metabolites, including cucurbitane-type
triterpenoids, have been isolated. Charantin, an anti-diabetic compound,
is a typical cucurbitane-type triterpenoid in M. charantia and a potential
and promising substance for the treatment of diabetes. Citrullus colocynthis
Schrad., endemic in southern Tunisia, is used in folk medicine to treat many
inflammatory diseases (Marzouk et al. 2010). After identification and acute
toxicity assays, C. colocynthis aqueous extracts were screened for analgesic
and anti-inflammatory activities using the acetic acid writhing test in mice
and the carrageenan-induced paw edema assay in rats, respectively. All
extracts displayed analgesic and anti-inflammatory activities at different
doses without inducing acute toxicity. Topical results were obtained with
immature fruits followed by seeds. The stem and root extracts were shown
to possess less significant inhibitory activity than analgesic and anti-
inflammatory models. Based on this study, C. colocynthis is a potentially
useful drug suitable for further evaluation for rheumatoid arthritis, and
its folk medicinal use as an analgesic and anti-inflammatory agent is
validated.

High-resolution metabolomic analyses such as LC-MS and GC-MS

have been applied from model plants to crop species (Arbona et al. 2009).
The novelty of the technique relies on the use of mass signals as markers
for phenotype demarcation independent of putative metabolite identities
and the relatively simple analytical strategy that is applicable to a wide
range of plant materials with no previous optimization. More recently,
these techniques have been used to distinguish more closely related plant
materials, such as transgenic and non-transgenic lettuce (Sobolev et al. 2010)
and 10 varieties of cooked rice (Heuberger et al. 2010).
In Cucurbitaceae crops, a metabolomic approach combining 1H NMR
and gas chromatography-electrospray ionization time-of-flight mass
spectrometry (GC-EI-TOFMS) profiling was first employed to characterize
melon (Cucumis melo L.) fruit (Biais et al. 2009). In a first step, quantitative
1H NMR of polar extracts and principal component analyses (PCA) of
the corresponding data highlighted the major metabolites in fruit flesh,
including sugars, organic acids, and amino acids. In a second step, the
spatial localization of metabolites was investigated using both analytical
techniques. Direct 1H NMR profiling of juice or GC-EI-TOFMS profiling of
tissue extracts collected from different locations in the fruit flesh provided
information on the advantages and disadvantages of each technique for
the analysis of a sugar-rich matrix such as fruit. 1H NMR and GC-EI-
TOFMS data sets were compared independently using PCA and multiblock
hierarchical PCA (HPCA), respectively. In addition, a correlation-based
multiblock HPCA was used for direct comparison of both analytical data
sets. These data analyses revealed several gradients of metabolites in fruit
flesh related to differences in metabolism and indicated the suitability
of multiblock HPCA for the correlation of data from two metabolomics
platforms.
Application of the high-resolution metabolomic analysis will allow for
the discovery of novel functional ingredients. Combined with functional
genomics tools, the biochemical and molecular mechanisms regulating the
contents of the novel functional ingredients will be elucidated. Finally, the
contents of the functional ingredients will be controlled by the effective use
of bioresources and genetic engineering techniques. These new cultivars
will enhance the production of the cucurbit crops and improve their
functionality as foods.
13.4 A New Use of Cucurbit Plants: Phytoremediation

The potential uses of Cucurbitaceae plants for phytoremediation have been
studied. Dieldrin and endrin were extensively used on arable land in Japan
from 1958 to 1971. The uptake of dieldrin and endrin by 32 plant species
of arable crops in 17 families and by 34 cultivars of Cucurbita sp. grown

in contaminated soil was compared. Cucurbits took up more dieldrin and

endrin than the other families, and uptake by zucchini was the highest.
These results suggested that cucurbits, especially zucchini, would make
good candidates for phytoremediation in dieldrin- and endrin-contaminated
fields (Otani et al. 2007). Seven cultivars of Lagenaria siceraria species were
screened for their ability to remediate heptachlor- and heptachlor epoxide-
contaminated soil (Campbell et al. 2009). These seven Lagenaria cultivars
were grown in contaminated and uncontaminated Molokai soil for 13 weeks.
The results showed that all plants tolerated heptachlor and heptachlor
epoxide at levels of 0.169 and 0.376 mu g/g, respectively, in the soil. All
seven Lagenaria cultivars showed some ability to take up heptachlor epoxide
into their vines, with bioaccumulation factors varying from 1.0 to 5.2. The
two contaminants were not detected in the fruits, and heptachlor itself was
not detected in the vines. One local cultivar showed the largest decrease of
heptachlor, from 0.376 down to 0.050 mu g/g dry soil. These two studies
demonstrated that some cucurbit plants are useful for phytoremediation,
which is an alternative use of this plant.
13.5 Comprehensive Use of High-throughput Sequencing

Technology and Bioinformatics
The introduction of high-throughput sequencing technology enables us to
conduct full genome sequencing of model cultivars of each plant species
over a short term with lower costs. Consequently, the cucumber genome
was sequenced first in cucurbit plants by BGI (Huang et al. 2009). The
melon genome is being sequenced by the Spanish Genomics Initiative,
and the International Watermelon Genomics Initiative is sequencing the
watermelon genome (Xu et al. 2010). Sequencing of both genomes will be
completed soon. After their completion, full genome sequencing of specific
cultivars or mutants will be much easier. By comparing the reference
genome sequence and the specific genome sequence, comprehensive DNA
markers for molecular breeding and gene cloning will be obtained, as
demonstrated in rice. A high-throughput sequencer was used for whole-
genome sequencing of an elite Japanese rice cultivar, Koshihikari, which is
closely related to Nipponbare, whose genome sequencing has recently been
completed (Yamamoto et al. 2010). A high-throughput typing array based
on the SNP information by comparison of the two sequences was designed.
Both high-throughput sequencers and typing arrays have detected genome-
wide SNPs, allowing for the evaluation of the genomic composition of
genetically related rice varieties. A combination of traditional map-based
cloning with high-throughput sequencing technology enables us to isolate
the genes responsible for significant traits in cucurbit plants. However, such
high-throughput sequencers generate huge amounts of sequence datasets.

Therefore, we need to establish a bioinformatics pipeline to assemble such

datasets and find necessary information in the assembled datasets.
13.6 Integration of Omics Data into Phenome Data

Excluding proteomic data, omics datasets including genomes, transcriptomes
and metabolomes have been reported for several cucurbit plants, as
described in previous chapters of this book. However, phenome data
are less available in cucurbit plants than in other model plants such as
Arabidopsis and rice (Alonso and Ecker 2006; Kuromori et al. 2009). For
effective use of the omics datasets, a phenome dataset is needed in cucurbit
plants, followed by subsequent integration with the genome, transcriptome
and metabolome datasets. International collaboration was important for
full genome sequencing during the last decade, and it is now needed for
comprehensive integration of omics data in cucurbit plants. International
collaboration will facilitate the promotion of cucurbit science and breeding,
ideally resulting in enhanced cucurbit production.
13.7 High-throughput Genetic Transformation Technology

Expanding the use of the high-throughput sequencing technology and its
integration into omics datasets enabled us to isolate many candidate genes
accounting for significant traits for the cucurbit development and breeding.
Subsequently, we need to validate the functions and roles of the candidate
genes. Agrobacterium-mediated genetic transformation has been used to
validate the functions of isolated genes. Various transformation procedures
have been reported for cucurbit plants including melon, cucumber, squash
and watermelon. However, the current protocols for genetic transformations
are not high-throughput and remain a limiting factor for the validation of
candidate genes in cucurbit plants. Therefore, we need to develop a high-
throughput genetic transformation technique for cucurbit plants.
Engineered Agrobacterium for high-throughput genetic transformation
has been developed and will be a significant tool for the validation of
candidate genes isolated from cucurbit plants. Ezura et al. (2000) observed
that explants produced ethylene during Agrobacterium inoculation. By
adding an ethylene biosynthesis inhibitor, AVG, to the co-cultivation
medium, they reduced ethylene production by the explants, resulting in
increased transformation efficiency. Nonaka et al. (2008a) demonstrated that
the ethylene evolved from a plant inoculated with A. tumefaciens inhibited
vir gene expression in A. tumefaciens via ethylene signal transduction in the
plant, consequently inhibiting genetic transformation. To suppress ethylene
evolution, they introduced 1-aminocyclopropane-1-carboxylate (ACC)
deaminase into A. tumefaciens (Nonaka et al. 2008b). The enzyme cleaves

ACC (the immediate precursor to ethylene) to α-ketobutyrate and ammonia,

and ethylene levels are consequently decreased. Agrobacterium tumefaciens
with ACC deaminase activity, named Super-Agrobacterium, has shown
reduced ethylene evolution and enhanced gene transfer into melon explants.
Super-Agrobacterium showed 3 to 5 times higher genetic transformation
and frequency of Citrullus colocynthis compared to Agrobacterium without
ACC deaminase activity (Ntui et al. 2010). Although we need to test
Super-Agrobacterium for other cucurbit species, it has potential for high-
throughput genetic transformation of the cucurbit plants and for enhancing
the functional validation of isolated genes accounting for significant traits
in cucurbit plants.
13.7 Conclusion
Cucurbit plants are commonly grown in the world. To maintain or further
extend their sustainable production and use, continuous R&D of key
technologies such as an induction of parthenocarpy traits, metabolomic
profiling of functional ingredients for human health, finding new uses such
as phytoremediation, comprehensive use of high-throughput sequencing
technology and informatics, the integration of omics datasets into phenome
data and high-throughput genetic transformation are required. Sharing
bioresources and the information generated by the technologies mentioned
above are also significant means to enhance cucurbit production and
research.
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Index
A biotic stresses 376

bitter melon 68
acorn 94–97, 104, 109, 113, 114, 116, 119–121, bitterness 63, 64, 68, 69, 71
126 BLAST analysis 218
ACS gene 353, 364, 365 botanical varieties 146 ,156, 163
adana 146, 154 bottle gourd 68
agrestis 143–146, 150, 151, 153, 155 Brachypodium 289
Agrobacterium 382, 383 brassinosteroid 361
amino acids 315 bridging species 40
aminoethoxyvinyl glycine 360 Bryonieae 141
amplified fragment length polymorphism bulked segregant analysis 225, 227
(AFLP) 129, 146, 205, 240 bush phenotype 100, 104, 112, 113, 115, 123
androecious 64 β-carotene 22, 23, 38
androgenesis 130
andromonoecious 4, 64, 65, 68, 71, 74, 77, C
226, 228, 288, 292, 302
andromonoecy 4 calabaza 107
angular leaf spot 65, 79, 126, 127 candidate genes 206, 216, 217
antalgic properties 23 cantalupensis 145, 146, 148, 150–154
anthracnose 65, 72, 77–79 carotenoids 121, 315, 316, 321
antibacterial 23 charantin 379
antidiabetic 23 Cheese group 106
antihepatotoxic 17, 24 Chito 145, 146, 151, 155
antihypercholesterolemia 23 chito-dudaim 145
antihypertension 23 chloroplast 4
antimicrobial 17, 24 chromosomal evolution 344
antioxidant 24 citrulline 378
antitumor 23, 25 Citrullus 140–142, 160–162, 164
antiulcerogenic 17 C. andreana 8, 110
antiviral 17, 24 C. anguria 142, 143, 160
aphid 64, 66, 67, 125, 127 C. argyrosperma 22, 28, 32, 33, 39, 40, 43,
Arabidopsis 217, 218 167, 168, 175, 176, 179, 180
auxin 360, 361 C. colocynthis 8, 160–165
AVRDC-World Vegetable Centre 35, 37 C. eccirrhosus 8, 160–163
C. ecuadorensis 108, 110, 128
B C. ficifolia 140, 167, 168, 179
C. foetidissima 28, 32, 33, 43, 167, 168, 179
BAC clones 216, 217, 219 C. hardwickii 21
BAC end sequences 216 C. hystivus 156
bacterial leaf spot rot 126 C. hystrix 7, 9, 12
bacterial wilt 65, 71 C. lanatus 160–162, 164, 165
banana 105, 114, 115 C. lanatus var. citroides 7
Benincasa hispida 140, 165 C. lanatus var. lanatus 7, 12
Benincaseae 140–142 C. lundelliana 110, 126
bin mapping 207 C. maxima 140, 167, 168, 173, 176–179,
bioresources 378, 380, 383 182

C. melo 142–145, 147, 156, 159, 182 diversity array technology (DArT) 338
C. metuliferus 142, 143, 159 DNA marker 238, 240
C. moschata 140, 167, 168, 173–177, 179, double haploid lines (DHL) 244
180 dry matter 93, 106, 107, 109, 111, 115–117,
C. okeechobeensis 110, 126 120–124
C. pepo 140, 167–171, 173, 176, 177, 179
C. rehmii 8 E
C. sativus 142, 143, 156, 157, 159
C. sororia 8 Egusi 310, 316
C. trigonus 21 ethylene 313, 318
classification 93–95, 103, 114, 121 European Central Cucurbits Database 10,
cleaved amplified polymorphic sequence 11
240 evolution 287
climacteric 287, 288, 298, 299 expressed sequence tag (EST) 245, 254
Cm-eIF4E proteins 218 -unigenes 317–319, 322, 323
coat protein (CP) genes 129
F
Coniandreae 141
Conomon 145–147, 151–154 femaleness 4
Consultative Group on International finger printed contigs 292
Agricultural Research 10 flanking markers 216, 218
Corynespora blight 65 flesh bitterness 69
Crookneck 96, 100, 104, 106, 122, 129 flesh color 66, 118
crown rot 67 flesh thickness 118
cucumber 1–7, 9, 12 flexuosus 148
cucumber beetles 127, 128 flowering and fruiting patterns 116
cucumber genome initiative 336 fluorescence in situ hybridization 9, 336
cucumber mosaic virus 65, 67, 79, 107, 108, foliar blights 126
125 fosmid libraries 340
Cucumis 1, 2, 4–7, 9, 11, 12 fruit fly 66, 67
C. anguria 2 fruit quality 63, 64, 66, 67, 79
C. dudaim 145, 146, 149, 151, 154, 155 fruit rot 126
C. melo 2, 5 Fursa 310
C. metuliferus 2 Fusarium wilt 65, 66, 74, 75, 77, 78, 80, 227,
C. picrocarpus 7 232
C. sativus 2, 5
cucumisin 379 G
cucurbit leaf crumple virus 320
Cucurbita 2, 5, 8, 11, 12 gamma-aminobutyric acid 379
C. ficifolia 3, 8 GC-EI-TOFMS 301
C. mixta 2, 3, 8 gene 4, 8, 12, 13
C. maxima 2, 5 genetic diversity 238, 239, 241, 248, 252,
C. maxima X C. moschata 108, 109 253, 262, 271, 276
C. maxima X C. pepo 93, 97, 108 genetic map 9, 199–201, 206, 208, 219
C. moschata 2, 5, 106 genetically-modified organism (GMO) 83
C. moschata X C. martinezii 108 genic male-sterility 69
C. pepo 2, 5 genome 4, 5, 9
Cucurbitaceae 1, 4–6, 9, 140–142, 144, 160, 173 genotyping 240, 246, 270, 271
cucurbitacins 64 geographically-based 241, 253
germplasm 313, 314, 319, 328
D Germplasm Resources Information
Network (GRIN) 10
dioecy 353–357 gibberellins 360
disease resistance 64, 66, 67, 78, 82, 83, 107, Golden Delicious 105
110, 122, 124, 125 gummy stem blight 66, 78, 82, 126, 127

Index 389
gynoecious 4, 64, 66, 68, 70, 79, 80, 288, 292, linked marker 225, 227, 230, 232
302 Lipoxygenase 348
gynogenesis 130 long terminal repeat 342
gynomonoecious 65, 80 Luffa 17–19, 21, 25, 26, 28, 30, 36, 37, 46, 47
L. abyssinica 166
H L. acutangula 17–19, 25, 47
L. aegyptiaca 26, 30, 36, 37, 47
hand-pollination 100, 101
L. astorii 36, 37
haploid 130
L. breviflora 166
hermaphrodite 65, 288
L. cylindica 17
herpetospermae 141
L. forskalii 30
heteromorphic 356
L. graveolans 18
heterosis 118, 123–125
L. graveolens 36, 37
high performance liquid chromatography
L. hermaphrodita 18, 47
249
L. operculata 36, 37
high-information-content fingerprinting
L. quinquefida 36, 37
292
L. sphaerica 166
HindIII 291, 292
L. umbellate 30
homomorphic 356
luffeae 140, 141, 181
Hubbard 105, 108, 114, 119
luffinS1 26
hull-less oil seed pumpkins 111
luffinS2 26
hybrid cultivar 112, 118, 123
luffinS3 26
hypoglycemic 23
lutein 378
lycopene 315, 316
I
lycopene β-cyclase 267, 316
immortalized mapping population 244,
264, 275 M
Immunomodulation 23
MADS box 357, 358
inbred backcross lines 270, 271
male-sterile 65, 66, 69, 71, 75
inbreeding depression 93, 123
map-based cloning 199, 200, 207, 216–218
inheritance of traits 62
marker-assisted selection (MAS) 43, 226
inodorus 145, 146, 149–155
Medicago 274, 275
insect resistance 64, 125, 127
M. truncatula 289
internal transcribed spacer (ITS) 143, 146
meiotic drive 321, 322
International Cucurbit Genomic Initiative
melon 1–7, 9, 12
(ICuGI) 247
melon necrotic spot virus (MNSV) 217
International Watermelon Genomics
Melothria medraspatana 68
Initiative 381
metabolic sink 268
inter simple sequence repeat 146, 240
metabolomics 286, 300, 301
Interspecific hybridization 128
microarrays 286, 299, 300
intestinal antiparasitia 23
microsynteny 266, 272–275, 345
isozyme marker 200
mitochondrial 4, 8
modifying genes 255, 258
J
molecular marker 199–201, 204, 205, 207
Joliffieae 140, 141, 180 molecular techniques 128
Momordica 140–142, 145–147, 151–154, 180,
K 181
M. balsamina 18, 28, 29
Kabocha 95, 106, 114, 119–121 M. charantia var. charantia 28, 35, 45
M. charantia 17, 18, 20, 21, 24, 29
L M. charantia var. minima 29
Lagenaria siceraria 140, 166 M. charantia var. muricata 28, 35, 45
leucine-rich repeat (LRR) 250 M. cochinchinensis 18, 20, 28

M. dioica 18, 20, 28, 29 prolycopene 315

M. sahyadrica 28, 29 proteomics 286, 300
M. subangulata 28, 29 protoplast fusion 42, 47
monoecious 65, 68, 77, 79, 226, 228, 229,
232, 288, 292, 302 Q
monoecy 353–357, 365
morphological marker 199, 200 quantitative trait loci (QTL) 227
mutagenesis 262, 271 detection 238, 240
N R
necrotic spot virus 66, 75 random amplified polymorphic DNA

nematode 318, 319, 326, 329 (RAPD) 128, 146, 205, 240
nested association mapping 276 recombinant 216, 218
non-climacteric 287, 298, 299 recombinant inbred lines (RIL) 244
North American seed catalogs 104 red pumpkin beetle 66, 68
nucleotide binding site (NBS) 250, 266 respiration rate 287
restriction fragment length polymorphism
O (RFLP) 146, 240
reverse transcriptase 294
open-reading frame (ORF) 216 ribosome inactivating proteins 26
Or gene 268 rind 62–64
color 105, 106, 118, 121
P hardness 118
ringspot virus 65–68, 74, 78, 79
papaya ringspot virus 40, 41, 65–68, 74, 78,
root rot 126
79, 108, 125
parthenocarpy 256, 260, 376, 377, 383 S
phenome 293, 301
phenotyping 151 S-adenyosyl methionine 363
phloem 4, 5 saturated map 244, 247, 254, 263, 264, 275
phylogenetic analysis 240, 276 scab 65, 73, 79
physical mapping 329 scallop 104, 112
phytoene desaturase 298 Schizopeponeae 141
pigments 23 SDS-PAGE 46, 47
pickling cucumber 3 Seed Pumpkins 105, 111, 117, 127
pistillate flowers 98 seedlessness 377
plant architecture 239, 240, 248, 255, 269, sequence characterized amplified regions
270 (SCAR) 208, 229, 231, 232, 240
plant hormonal 255 sequence-related amplified polymorphisms
pollen mother cell 36 208, 240
pollen stainabilty 37 sex chromosome 356
pollen sterility 36 sex determination 286–288, 292, 302
pollination techniques 100 sex expression 4, 64–66, 77, 79, 80, 226
pollinators 99, 100, 109 Show Pumpkins 105, 118
pollinizer 368 sicyeae 140, 141, 182
polymorphism 311, 313, 320–322 silver-leaf disorder 42
polymorphism information content 338 silverleaf whitefly 42
Populus 274, 275 simple sequence repeat (SSR) 129, 146, 206,
positional cloning 367 240
potyvirus 261, 266 single nucleotide polymorphism (SNP) 150,
powdery mildew 65–67, 72–75, 77, 79–81, 206, 240
107, 109, 110, 125, 126, 226, 228, 230 single-feature polymorphism 290
Praecitrullus fistulosus 160 size of blossom scar 118
primary gene pool 241 Spaghetti squash 105

Index 391
Spanish Genomics Initiative var. chandalak 146

(MELONOMICS) 289 var. chate 145, 146, 148, 155
squash bug 127 var. chinensis 146
staminate flower 99 var. makuwa 146, 147, 152, 153
starch 117, 119, 120, 121 var. tibish 146, 147
sterility 65, 67, 69, 71 variety classes 104–106
straightneck 96, 100, 104, 106, 109, 114, 122, Vegetable Marrow 104
126, 128 violaxanthin 315
sugar 93, 120–122 viral pathogens 40
summer squash 2, 3, 5
suppression substractive hybridization 319 W
T warty fruit 118

watermelon 1–9, 12, 13
target leaf spot 65, 73, 79 mosaic 65, 67, 68, 70, 76
TILLING 219 mosaic virus 40, 41, 108, 125
transgenic 125, 129, 130 white fly 127
Trichosantheae 141 winter squash 2, 3, 5
trimonoecious 64, 68 whole genome sequencing 324
triploid hybrid 77, 78
Turban group 106 Y
U Yellow Crookneck 96, 104, 122, 129
USDA National Plant Germplasm System Z

(NPGS) 10
Zanonioideae 141
V zeaxanthin 378
zucchini yellow mosaic virus 40, 65–67, 70,
var. acidulous 146, 147 74, 76, 78, 79, 108, 125
var. ameri 146, 148, 154

Color Plate Section

Chapter 1
Figure 1-1 Major and minor cucurbit phylogenetic tree. Chromosome numbers and common
names follow each species name (when available). Molecular clock in million years ago,
if available, was shown on branching points. The tribe to which the species belongs was
shown to the right of vertical bars. Geographical occurrence of species: Green—America;
Black—mainland African; Red—Asia; Blue—Australia. The tree was redrawn after Schaefer
et al. (2009).

Chapter 4
Figure 4-1 Ilustration of the three major squash types grown in North America: acorn (C.
pepo, A) kabocha/buttercup (C. maxima, B) and butternut (C. moschata, C).
Figure 4-2 Pistillate (left) and staminate (right) flowers of monoecious C. maxima.

Figure 4-3 Flower morphology of C. maxima (left), C. pepo (middle) and C. moschata (right),
represented by pistillate flowers.
Figure 4-4 Ilustration of peduncle types in C. maxima (bottom laft), C. moschata (bottom right),
C. pepo subsp. ovifera (top laft), C. pepo subsp. pepo (top right). See text for explanation.

Figure 4-5 Pistillate flower (left) tied off with a Twist-ems tie one day prior to anthesis; pistil-
late flower retied and tagged after pollination.

Chapter 5
Figure 5-1 Distribution map of Cucumis spp. Principal diversity centers based on Kirkbride
(1993).
C. sativus L. and C. sativus L. var. hardwickii (Royle) Alef.
C. hystrix Chakravarty
C. myriocarpus Naudin ▲
C. africanus L.
C. anguria L.
C. dipsaceus Ehrenberg ex Spach
C. zeyheri Sonder
C. ficifolius A. Richard
C. metuliferus E. Meyer ex Naudin
♦ ♦
C. melo L. ssp. Melo and C. melo L. ssp. agrestis (Naudin) Pangalo
C. sagittatus Peyritsch

Figure 5-2 Distribution map of Citrullus spp. Principal diversity centers based on Jeffrey
(2001).
C. colocynthis (L.) Schrad.
C. ecirrhosus Cogn.
C. lanatus ((Thunb.) Matsum & Nakai) var. lanatus ▲ and var. citroides ▲
C. rehmii De Winter ♦

Figure 5-3 Distribution map of Cucurbita spp. Principal diversity centers based on Lira-Saade
(1995) and Sanjur et al. (2002).
C. argyrosperma C. Huber
C. ficifolia Bouché
C. maxima Duchesne ▲
C. moschata Duchesne
C. pepo L.
C. ecuadorensis
C. okeechobeensis (J.K. Small) L.H. Bailey
♦
C. lundelliana L.H. Bailey
C. digitata A. Gray, C. cylindrata L.H. Bailey and C. palmata S. Watson
C. foetidissima Kunth

CONOMON

MAKUWA
CHINENSIS
MOMORDICA
ACIDULUS
TIBISH
Table 5-1 contd....

Table 5-1 contd....
402
CHATE Yes

FLEXUOSUS Yes
sometimes
green
CANTALUPENSIS
usually
sometimes
with warts
RETICULATUS
AMERI
INODORUS
DUDAIM


thick, lignified
non-lignified
lignified

Chapter 6




Figure 6-1 A genetic map of Cucurbita pepo. The new map contains 659 loci: 178 SSR, 244
AFLP, 230 RAPD, five SCAR markers, and two morphological traits (h and B) (From Gong
et al. 2008b).

Chapter 8
Figure 8-3 Comparative analysis of the melon (Cucumis melo L.; n = x = 12) and watermelon
(Citrullus lanatus (Thumb.) Matsum & Nakai; n = x = 11) with the cucumber (Cucumis sativus
L.; n = x = 7) sequence map. The watermelon genetic maps employed in the analysis are
organized into 18 linkage groups. Figure adapted by permission from Macmillan Publishers
Ltd: Nature Genetics; Huang et al. 2009.
Figure 8-4 Overview of microsynteny between melon (Cucumis melo L.) BAC 1-21-10 and
regions in the Arabidopsis thaliana, Medicago truncatula, and Populus trichocarpa (not drawn
to scale). Genes are represented by arrows where gene name, number or ID given above or
below the arrow. Homologous genes are illustrated with the same color and indicated by
narrow connecting lines of the corresponding color. Arrows representing genes that have
one or more ESTs are designate with a spot. Genes without homologs are given in black.
Transposable elements are delineated in gray and indicated by Tn. At1g, At2g, At4g referrers
to A. thaliana chromosomes 1, 2 and 4, respectively. Cm11 referrers to C. melo Linkage Group
11, Pt_XI referrers to Populus trichocarpa Linkage Group XI, and Pt_204 referrers to Populus
unmapped scaffold 204. Mt4 referrers to M. truncatula chromosome 4, consists of BAC
clones AC137837, AC153460 and AC144608, and * = End of scaffold. Figure adapted with
kind permission from Springer Science and Business Media: Deleu et al. (2007), Mol Genet
Genomics 278: 611–622.

Chapter 10
Figure 10-3 Root systems of Citrullus colocynthis, C. lanatus subsp. lanatus “Charleston Gray”
heavily infected with peanut root-knot nematode (RKN), Meloidogyne arenaria race 1, versus
C. lanatus subsp. citroides showing resistance to RKN.
Figure 10-4 Classification of 880 EST-unigenes Illini Red watermelon fruit based on homology
of 880 EST-unigenes to gene sequences in other plant species.

Chapter 11
Figure 11-1 Ideogram showing the position and intensity of Type I/II, Type III, Type IV,
45S rDNA/CsRP2 and 5S rDNA on cucumber metaphase chromosomes (Han et al. 2008).
Chromosome nomenclature follows Koo et al. (2005).
Figure 11-3 Integration of the seven linkage groups of cucumber with individual chromosomes
(Ren et al. 2009). (A1) Distribution of Type I/II (green) and Type III (red) repeats on cucumber
chromosomes. (A2) DAPI staining was converted to black and white images. (A3) Localization
of chromosome-specific fosmid clones on both arms of individual chromosomes, genetic
location of arm-specific fosmid clones are indicated in Figure 11-2. (B) Localization of fosmids
4S (red) and 4L (green) together with Type III (red) and 45S rDNA (green) repeats on the
mitotic chromosomes. Bar = 2.5 µm.

Figure 11-4 Integrated genetic/physical map of cucumber (Huang et al. 2009). (a) Genetic
versus physical distance map of the seven cucumber chromosomes. The genetic map was
constructed using an RIL mapping population from the inter-subspecific cross between Gy14
(domestic cucumber) and PI183967 (wild cucumber). (b) The segmental inversion between the
domestic cultivar Gy14 and the wild accession PI183967 on cucumber chromosome 5 detected
by FISH. 12-2 and 12-7 denote individual fosmid clones. (Scale bars, 1 µm)

Figure 11-5 Comparative genomic analysis of cucurbits (Huang et al. 2009). (a) Comparative
analysis of the melon and watermelon genetic maps with the cucumber sequence map. (b)
Syntenic blocks between the cucumber genome (scaffold000089) and a melon BAC sequence
(accession: EF188258.1). Genes are drawn as black arrows with the orientation indicated on
the sequence. Transposable elements (TEs) are illustrated as rectangles; retrotransposable
elements are in red, DNA transposons are in blue and unclassified TEs are in green. Orthologous
sequence regions between the two genomes are displayed.
6-1
6-2 7-1
Cen
6-3 7-2
6-4
Cen
6-5/6-6
Cen 7-3
6-6
6-7 7-4 Cen

7-5
6-8
6-9 7-6
6-10
7-7
6-11
7-8
6-12
Cucumber Melon Cucumber Melon
chromosome 6 chromosome I chromosome 7 chromosome II
Figure 11-6 Diagrammatic illustration of the marker orders and centromere positions of two
pairs of cucumber and melon chromosomes (Han et al. 2009).

Figure 11-7 Genomic locations of R genes on the cucumber chromosomes (Huang et al. 2009).
Three R genes could not be anchored on specific chromosome.

Figure 11-8 Lineage-specific expansion of the lipoxygenase (LOX) family in the cucumber
genome (Huang et al. 2009). The LOX family was divided into two groups, “Type I” and “Type
II”. The two tandem duplicated gene clusters were ordered and displayed on chromosomes
2 and 4, plus one unmapped scaffold of the cucumber genome.

Chapter 12
Petal
Petal
Nectary
Anther
Ovary
Nectary
Ovule
Figure 12-1 Sexual differentiation of cucumber flower buds. Longitudinal sections of cucumber
buds under dissection microscope. A. Male bud at stage 11. B. Female bud at stage 12 (anthesis).
Bud development stages are assigned as per Bai et al. (2004).

(Genetics, Genomics and Breeding of Crop Plants) Yi-Hong Wang - Tusar Kanti Behera - Chittaranjan Kole - Genetics, Genomics and Breeding of Cucurbits PDF

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Handbook of

Molecular and Cellular

Boca Raton London New York

CRC Press is an imprint of the

© 2011 by Taylor & Francis Group, LLC

No claim to original U.S. Government works

International Standard Book Number-13: 978-1-4398-8195-8 (eBook - PDF)

and the CRC Press Web site at

Part I DNA-Based Technology

Chapter 1 Isolation and Purification of DNA................................................................................ 3

Chapter 2 DNA Cloning Strategies.............................................................................................. 29

Chapter 3 Polymerase Chain Reaction Methodologies................................................................ 37

Chapter 4 Preparation of Nucleic Acid Probes............................................................................ 57

Chapter 5 Southern Blot Hybridization....................................................................................... 77

Chapter 6 Genomic DNA Libraries.............................................................................................97

Chapter 7 DNA Sequencing and Analysis................................................................................. 141

Part II RNA-Based Technology

Chapter 8 Isolation and Purification of RNA............................................................................ 167

Chapter 9 Real-Time PCR and qRT-PCR Methodologies......................................................... 187

Chapter 10 Preparation of RNA Probes...................................................................................... 197

Chapter 11 Northern Blot Hybridization..................................................................................... 211

Chapter 12 cDNA Libraries......................................................................................................... 217

Chapter 13 Differential Display................................................................................................... 261

Chapter 14 Localization of RNA Expression.............................................................................. 273

Part III Protein-Based Technology

Chapter 15 Isolation and Purification of Proteins........................................................................ 291

Chapter 16 Western Blot Hybridization....................................................................................... 313

Chapter 17 Localization of Protein Expression........................................................................... 331

Chapter 18 DNA Footprinting and Gel Retardation Assay......................................................... 337

Chapter 19 Protein–Protein and Protein–Ligand Interactions.................................................... 351

Part IV Metabolites

Chapter 20 Isolation and Purification of Metabolites.................................................................. 367

Chapter 21 Analysis of Metabolites............................................................................................. 381

Part V Genomics, Proteomics, and Metabolomics

Chapter 22 Bioinformatics and Data Analysis............................................................................. 409

Chapter 23 Microarray Platforms................................................................................................ 437

Chapter 24 Microarray Data Collection...................................................................................... 447

Chapter 25 Microarray Data Analysis......................................................................................... 455

Chapter 26 Proteomics: Data Collection and Analysis................................................................ 459

Chapter 27 Metabolomics: Data Collection and Analysis........................................................... 471

Chapter 28 Common Protocols on Membrane Lipid Analysis and Lipidomics.......................... 485

Chapter 29 Integrated Functional Genomics...............................................................................509

Part VI Manipulation of Biological Systems

Chapter 30 Inhibition of Gene Expression................................................................................... 521

Chapter 31 Inhibition of Protein Synthesis.................................................................................. 549

Chapter 32 Gene Transfer and Expression in Animal Cells........................................................ 557

Chapter 34 Tissue Engineering of Fully Differentiated Tissue: Lessons from Work

Chapter 35 Plant Tissue and Cell Culture.................................................................................... 607

Chapter 36 Gene Transfer and Expression in Plants.................................................................... 629

Part VII Macromolecular Analyses

Chapter 37 Microscopy: Light and Confocal............................................................................... 657

Chapter 38 Microscopy: Scanning Electron, Environmental Scanning Electron,

Chapter 39 Laser Capture Microdissection and Whole Genome Amplification......................... 687

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Ramesh Buyyarapu, PhD, is a research scientist working in the Molecular Technology

Omar De la Cruz Cabrera, PhD, is a postdoctoral scholar in the Department of Statistics at

Venkateswara Rao Sripathi is a graduate research assistant in the Department of Natural

© 2012 by Taylor & Francis Group, LLC

Books in this Series:

© 2012 by Taylor & Francis Group, LLC

© 2012 by Taylor & Francis Group, LLC

No claim to original U.S. Government works