DNA Fingerprinting and cultivar identification in sunflower

(Helianthus annuus L.) using genomic SSR markers.

A dissertation report Submitted

In the partial fulfilment of the requirement for the award of degree of

Master of Science in Biotechnology

Submitted by:

Ankita Sharma

MSc BioTechnology
AmityUniversity
2011-2013

Dr. S. Rajkumar
Division of Genomic Resource
National Bureau of Plant Genetic Resources
New Delhi 110 012
ACKNOWLEDGEMENT.

With my immense and heartfelt gratitude I place on record the efforts, inspiring guidance and
constant supervision of Dr. S. Rajkumar (Senior Scientist) DNA Fingerprinting, NBPGR in
bringing up my project work. whose encouragement, able guidance and total support from the
initial to the final level enabled me to develop an understanding of the project and helped me to
complete it.

I respect and thank and Dr. K. C. Bansal Director, NBPGR and Dr. K.V. Bhat, Head, Division
of Genomic Resources for giving me an opportunity to do the project work in National Bureau of
Plant Genetic Resources. I am extremely grateful to him for a valuable experience and exposure
which would help me in understanding and contributing in all my future endeavors in the field of
biotechnology.

I would like to express my gratitude to Dr. P. K. Paul, Director (AIB) who gave me the
opportunity to gain practical knowledge in the field of biotechnology.

I would also like to thank my faculty guide, Dr. Puniti Mathur for her active support without
which it would have been difficult for me to complete the project and make this work a
successful one.

I would not forget to remember Dr. Rajesh kumar and Dr. R. Parimalan of NBPGR for their
unlisted encouragement and moreover their timely support and guidance for the constant
encouragement and support which helped me in completing my project.

Last but not the least, I would also express my thanks to my lab assistant Neeraj Singh and all the
non-staff members who were there all the times to help and assist me.

ANKITA SHARMA
TABLE OF CONTENTS PAGE NO

1. Introduction
2. Materials and methods
3. Results and discussion
4. Conclusion
5. References
6. Appendix
 1. INTRODUCTION

Sunflower is distinguished from other crops by its single stem and conspicuous large
head.radiant and dazzling, the common sunflower has pleased and fascinated mankind for
centuries.Helianthus,the genus name of sunflower(Helianthus annuus L..)is derived from the
greek word "Helios", meaning sun and "anthus" meaning flower. The spanish name for
sunflower, girasol and the French name tournesol literally means turn with the sun, a trait
exhibited by sunflower until anthesis, after which the capitula face the east (fick 1989).

Sunflower is unique in that it has been bred for distinctly different uses: first as a forage
crop, later as an oilseed crop, ediable confection, birdseed, and to a much lesser extent as an
ornamental for home gardens and the cut -flower industry. Sunflower drives most of its
economic importance from the oil extracted from the achenes, with remaining value from the
meal. Sunflower is grown worldwide and performs well in most temperate climates of the
world, with significant production occurring in each of the six crop-producing continents.

Molecular techniques have been used to search for the origin of cultivated sunflower.
Morphological, geographical and archaeological evidence has led to the hypothesis that the
domestic sunflower derived from a wild/weedy form of H. annuus, possibly in the mid west.
Molecular evidence was concordant with this hypothesis with high degrees of enzymatic and
cp DNA sequence similarity observed between wild and domesticated H. annuus, and
domesticated H.annuus contained a subset of the alleles and cpDNA found in wild H. annuus
(Riesberg and seiler 1990). The extensive polymorphism in the wild plants and the virtual
monomorphism in the cultivated line for both isozyme and cpDNA phenotypes further
suggest a single originof the domesticated sunflower from a very limited gene pool.
1.1 ECONOMIC IMPORTANCE

1.1.1 NUTRITIONAL INFORMATION

1.1.1.1 oil and oil quality

The oil extracted from the achenes accounts for about 80%of the total value of the oilseed
sunflower crop(fick and miller1997).oil content depends on both the percentage of hull and the
oil concentration in the kernel.hull percentage aong genotypes ay range from10-60%,while oil
content in the kernel ay range fro 260-270g/kg.most oil seed have a whole achene oil content of
400-500g/kg.sunflower oil is premium oil due to its high unsaturated linoleic fatty
acidconcentration,low linolenic acid content,the absence of significant toxic copounds,and its
excellent nutritional properties. Linoleic acid is an essential fatty acid not synthesized by
humans,and is the precursor of gamma-linolenic and anhydride acids(Dorrell and vick1997).

1.1.1.2 protein

Protein concentration of achenes is of interest for huan and liverstock consumption,coercial

sunflower meal has aprotein concentration of approximately 440g/kg.

1.1.1.3 Tocopherol coposition

Tocopherols are powerful natural fat soluble antioxidants that inhibit lipid oxidation in foods and
biological systems. Alpha tocopherol (vitainE) is the principal tocopherol in sunflower oil
representing ove r90% of the total tocopherols.

1.2 BOTANICAL DESCRIPTION

1.2.1 Taxonomic position

The compositae (asteraceae) is the largest and most diverse family of flowering plants,
comprising one-tenth of all known angiosperm species (Heywood 1978,Funk et al.2005).species
wihin this family are characterized by a compound inflorescence that has the appearance of a
single “composite” flower. Helianthus belongs to the asteraceae subfamily asteroidae, tribe
Heliantheae, subtribe Helianthineae( Panero and Funk 2002).
1.3 DNA FINGERPRINTING AND MOLECULAR MARKER SYSTEM

The use of DNA marker to obtain a genotype specific profile has advantages over
morphological and biochemical methods. The morphological markers are influenced by
environmental factors, are laborious and time consuming. Biochemical markers such as
isozymes and protein patterns though minimally influenced by the environment but offer
limited polymorphism, and donot allow discrimination between closely related
genotypes.
DNA markers overcome most of these disadvantages of morphological and biochemical
markers.
Alec Jeffery et al. (1985) developed the technique of DNA fingerprinting in human being
for the first time. The usefulness of DNA fingerprinting technique for cultivar
identification was demonstrated first by Dallas (1988) in rice.
DNA fingerprinting has many application in crop genetics such as; study of genetic
diversity within texa and study of evolutionary and genetic relationship; tagging of
economically useful traits; assessment of genetic purity and inbred lines and varieties;
selection of recurrent parental genome in back cross; study breeding behaviour of crop
plants and determine isolation distances in seed production programmes; selection of
parents in breeding programmes.
A number of molecular system either alone or in combinations, is available now for DNA
fingerprinting and cultivar identification. Some of these are following.

Restriction fragment length polymorphism (RFLP)

Random amplified polymorphic DNA (RAPD)
Simple sequence repeats (SSR)
Amplified fragment length polymorphism (AFLP)
DNA sequencing
DNA micro-array analysis

1.3.1 IMPORTANCE OF SSR MARKER

SSR (simple sequence repeat), also known as microsatellite or STR(shoet tandem repeats) , is a
class of DNA sequence stratches characterized by highly repeated- di, tri, tetra,penta nucleotid
motifs(e.g.,CACACACA….,CATCATCAT…….,ACGTACGTACGT…OR
TAAAATAAAATAAAA) and spread throughout the genomes. SSR polymorphisms are assayed
by specifically amplifying a small DNA fragment that containsthe repeats via PCR and then by
sizing the amplified fragments on agarose, polyacralamide gel or capillary electrophoresis. The
number of repeats varies among genotypes under investigation and results in a length variation of
the amplified fragments. Being highly polymorphic, highly abundant, co-dominant inheritance,
analytically simple and readily transferable, makes SSR the marker of choice over many other
markers for genomic studies.
Abundant interspread repetitive DNA elements in different eukaryotes were observed
from the limited amount of DNA sequence information in the early 1980’s (Miesfeld et
al.1981; Hamada and Kakunaga 1982). Weber and May (1989) first realized that this
class of sequence represents a vast new pool of potential genetic markers to be used to fill
the gaps in the existing human genetic map and to improve map resolution. The use of
STR in human genetic mapping turned out to be a great success.
SSR polymorphism was first reported in the crop plants in soyabean (Akkaya et al. 1992).
Since than ssr makers have been developed in a number of crop species such as rice
(Morgante and olivieri 1993; wu and Tanksley 1993), barley (Backer and Heun 1995).
SSR markers rapidly displaced RFLP markers not only in plant genome mapping but also
in other applied areas such as genotype identification and variety protection (smith and
Helentjaris 1996), seed purity evaluation and germplasm conservation (Brown and
kresovich 1996), germplasm diversity studies (xiao et al. 1996), quantitative trait
loci(QTL) analysis (Blair and McCouch 1997), pedigree analysis and marker assisted
breeding (ayres et al. 1997), and as anchor points in screening of large insert libraries for
potential gene cloning.
Since sequence information is needed for designing specific primer pairs for individual
ssr markers, the initial development of SSR started with a laborious and costly approach
of screening genomic libraries by hybridizing with SSR probes and sequencing the
hybridized clones. The SSR enrichement procedure of using biotin-labeled SSR-
containing probes in combination with magnetic beads with streptavidin or nitrate filter
(Edwards et al. 1996) in small insert libraries considerably improved the efficiency of
isolating SSR sequence from the genome and hence reduced the time and cost for ssr
development. The growing ability of expressed sequence tag(EST) sequence from a range
of crop plants allows large scale search for SSR motif and design of SSR primers using
computer programs.
The total number of public sunflower SSR markers did not reach the 100 before 2002 (
whitton et al.1997 gedil1996). Yu et al.(2002) developed 131 unique SSR marker from
970 clones isolated from the (CA)n-1 (CT)n-1, (CAA)n-1, (CATA)n-1 enriched genomic
libraries.
 2. MATERIALS AND METHODS

Plant Material:

Authenticated seeds were procured from gene bank NBPGR and Directorate of oil seeds
Research, Hyderabad for sunflower varieties and parental lines. The details of varities and
parental lines used in this study were provided in Table . Total 15 varities and 5 parental lines
were used in the present study. The seeds were grown in petriplate and the seedlings were used
for genomic DNA isolation.

2.1 Genomic DNA Isolation

For genomic DNA isolation, CTAB method was used. CTAB (Cetyl trimethyl ammonium
bromide) is a cationic detergent, which solubilizes cell membranes and forms a complex with
DNA.

DNA isolation protocol:- CTAB method for isolation of DNA

take sample (~ 100 mg)
crush with motorized pestle
add 0.5 ml of 2x CTAB
incubate at 65 in water bath for an hour
cool at room temp and add 0.5 ml of chloroform
vortex for 30 sec and centrifuge (~ 12000rpm)
take out supernatent in another 1.5 ml tube
add 0.5 ml of isopropanol and mix gently
centrifuge (12000rpm, 10 min ) : one can see the pellet
pour off carefully supernatent and add 0.5 ml of 70% ethanol
centifuge 12000rpm for 2- 5 min
Pour off carefully supernatent centrifuge again and remove the extra ethanol by pipette.
Air dry the pellet and dissolve pellet in ~ 150 µl of TE.

2.2 DNA QUANTIFICATION

The isolated genomic DNA was loaded in the 0.8% Agarose gel prepared in1X TAE buffer containing

0.5 g/ml of Etbr. Along with the samples control DNA ( DNA) having variable concentration

viz.200ng, 400ng, 600ng respectively and the samples concentration were calculated comparing with

control DNA.

2.3 PCR Reaction for SSR Markers

DNA amplification was carried out by making final reaction volume of 25 µl containing 1.0 µl
of dNTP mix, 0.6 µl of Taq DNA polymerase (3U/µl), 2.0 µl DNA template (30-40 ng/µl), 0.5
µl of 5 µM each of forward and reverse primer, 2.5 µl of 10X PCR buffer (Taq buffer A) and
18.3 µl of sterilized distilled water. Total thrity three primer pairs from known genomic
microsatellite markers from sunflower were synthesized (Sigma) and used for amplification of
DNA of 20 samples of varities and parental lines of sunflower. The amplification was carried out
in a Thermal Cycler (?). The PCR programme was as follows: 94oC for 5 minutes (Initial
denaturation), 25 cycles of polymerase reaction was set with 94oC for 30sec, 58oC for 30 sec for
intial annealing with touchdown temperature of 1o per cycle up to 54o C , the remaining 20
cycles with annealing temperature of 54o C aand 72oC for 45 sec, with final extension for 7 min
at 72oC with rapid cooling to 4oC..

2.5 Preparation of Metaphor gel

3% Metaphor gel was prepared by suspending 9g Metaphor in 250ml chilled 1X TBE buffer.
Then boil in microwave oven till completely dissolved. The agrose mixture brought to room
temperature and 4 l ethidium bromide was added and casted in horizontal electrophoresis trey
and kept in 4o C for 20 minutes for proper solidification. The trey then placed in 1x TBE buffer
tank and ready for electrophoresis.

Gel Electrophoresis:

The amplified DNA samples were mixed with DNA loading dye (bromophenol blue) in 7:3
proportion and electrophoresis was carried out on 3% Metaphor Gel in 1X TAE buffer at 120
volt for 3 hours.
Data Analysis:

After electrophoresis , the gel was placed in Gel Documentation System (Alpha Imager)
and the molecular weight of the each fragment was identified using in build image software
using 50 bp ladder in the gel. The scoring was done for each varities and parental lines and the
data were subjected to further analysis. The dendrogram was constructed using Jaccard
coefficient based on the binary data generated using software PAST ver. 2.03 (Hammer et al
2001). The genetic data of the markers were generated using GenAlex 6.4
 RESULTS AND DISCUSSION

Genetic Diversity:

Total 20 accessions includes 15 national varities and five parental lines were used for

diversity analysis using SSR markers and to differentiate varities using SSR based fingerprinting

method. Out of 33 primer pairs were screened for above analysis 8 of them are used for analysis

based on reproducibility. Among the 8 locus two of the locus produced high number of allelic

with 11 alleles each (locus SF2 and SF6). The lowest number of alleles were from SF 14 where

only 5 differnt alleles from 20 accessions. Based on the allele frequency the effective number of

alleles were calculated and given in Table. The diversity indices caluculate based on the allele

frequency provided richness of usuage of locus in the sample analyzed. The number of alleles is

directionaly propotional to the diversity index. The loci SF 14 , SF4 and SF 17 showed higher

shanon diversity index compared to others. All the locus studied in the analysis produced

homozygous alleles, hence the test for hypothesis on the heterozygosity could not be done.

However, the expected heterozygosity based on the number of alleles and population size

provided higher values in all the loci studied.

The frequency of alleles for each locus were studied. This provided the representation

and abundance of the alleles in the population studied. Among the alleles, locus SF18 have

alleles with high frequency(0.40) followed by locus SF19 (0.40) all other loci have alleles with

frequency of less than 0.35. This showed maximum number of loci are polymorphic in nature

which is very useful in diversity analysis and fingerprinting of cultivars. The mean values of

allele pattern in the varieties showed maximum number of alleles are unique to the varieties

studied. This was evident by presence large number of mean private alleles (Fig.) in the

accessions studied
 Based on the allele diversity similarity matrix were constructed based on Jaccard

coefficient similarity matrix. The similarity between the varieties ranged from 0- 75%. Among

the varities studies the parental line 17A have high similarity with another parental line R437.

The least similarity was found in many parental lines with released varieties. This showed that

the varities released based on selection is distanly similar with parental lines and hybrids. The

above analysis displayed broad genetic based in the varities of sunflower studied.

Genetic relationship:

The relationship between the varities and the parental lines were depicted in dendrogram
and scatter plot using principal component analysis. The dendrogram developed based on jaccard
similarity matrix and using pair group method. The parental lines were formed separate cluster
particularly some of the CMS lines. The hybrid nature of Phule Raviraj could be able to
identified based on the dendrogram and scatter plot where it was distinctly grouped with the
parental lines 17A and R437. However, the other hybrid
3.3.2 DANDOGRAM:-

0.2

0.4

0.6

0.8
0

1
17 A

73
R 437

86
Phule Ravira
46

RHA-272
18

CMS-17A
45

CMS-234A
R630
57
100

DRSF-113
DRSF-108
22

18

TNAUSUF-1
11

RSFH 130
14

CMS 104 A
CO2
12

Surya
PKVSF-9
19
30

TAS-82
33

67

CO 5 (SFV)
21

67

TNAUSUF-7
GAUSUF-15
55

Morden

Figure1. Dendrogram showing relationship between the cultivars and parental lines of sunflower
 Allele Frequency
0.500
0.400
Frequency

0.300
0.200
0.100
0.000 173
185
196
332
373
389
393
397
400
311
356
378
223
232
249
257
264
183
188
196
367
373
382
391
229
243
249
260
140
144
150
Locus1 Locus2 Locus3 Locus4 Locus5 Locus6 Locus7 Locus8
Locus

Reference Allele Number Number

allele size of of
length range different effective Shanon Expected
alleles alleles information Heterozygosity
Locus N (Na) (Ne) index (I) (He)
SF1 20 6 4.255 1.583 0.765
SF2
20 11 7.143 2.177 0.860
SF4
20 6 3.636 1.469 0.725
SF6
20 11 7.692 2.207 0.870
SF14
20 5 4.255 1.522 0.765
SF17
20 9 6.897 2.056 0.855
SF18
20 7 4.167 1.653 0.760
SF19
20 6 3.922 1.540 0.745
 Allelic Patterns across varities and parental lines
10.000

8.000

6.000
Mean

4.000

2.000

0.000
Varities

Na Na Freq. >= 5% Ne I No. Private Alleles

Principal Coordinates

DRSF-113
R630
TNAUSUF-10

RSFH 130
DRSF-108
Coord. 2

CO 5 (SFV)
SuryPaKVSF-9
CMS 104 A
CMS-17A CO2 TNAUSUF-7
CMS-234A GAUSUF-15 TAS-82
Phule Raviraj
17 A
R 437 Morden
RHA-272
Coord. 1
 4. DISCUSSION

SSR markers were used for the cultivar identification in sunflower hybrids and varieties. SSR
makers are universally accepted in genotype specific fingerprinting and diversity analysis
provides an opportunity for their deployment in genetic purity assessment of commercial
cultivars. Since hybrids are obtained by crossing diverse homozygous parent, they are
heterozygous in genetic constitution. Genetic purity of hybrids could be determined by using
SSR primers. If polymorphism is present the microsattelite between female parent and male
parent, since SSR are codominant in nature hybrid would express both the alleles of that locus
specific to parent using the same SSR primer pair.

5. FUTURE PROSPECT

Cultivated sunflower has undergone significant improvement in yield and other agronomic traits
during the past half century. However, non-GMO sunflower faces major challenges from insect
and herbicide resistant. In several sunflower producing countries, sunflower production has
shifted in recent years to marginal production regions in response to compition from soyabean
and maize. Despite displacement to less fertile growing areas, yields have steadily increased as a
result of improved hybrids.

FUTURE POTENTIAL TO INCREAE PRODUCTIVITY

Sunflower that has been genetically modified (GMO) to incorporate genes from other species for
useful new agronomic traits would, if introduced into commercial cultivation, likely follow a
similar path.

IMPACT OF ADVANCES IN SUNFLOWER GENOMICS

The past decade has been marked with milestone in sunflower genomics research that included
the construction of linkage maps using various molecular markers, such
as,RFLP,RAPD,AFLP,SSR,TRAP and Indel (insertion,deletion), the establishment of genetic
transformation methods and the sequencing of thousand of EST’S (expressed sequence tags) of
the cultivated sunflower and its related species. These milestones have an enormous impact on
addressing problems in both fundamental biology and applied genetic improvement of this crop.
The massive sequence data will offer access for developing DNA markers for gene controlling
economically important traits. These markers will be useful for rapidly moving genes among
elite lines to enhance the target traits via marker-assisted breeding. Marker –assisted selection
evolved from the idea of indirect-selection, which has been a common practice in both plant and
animal breeding.

NEW USES

1 BIOFULES AND LUBRICANTS

Sunflower has been used as a source for biofuel by two quite different systems. It is possible to
use whole sunflower plants for biomass production and conversion in biogas reactors to methane
gas. Halen et al. (2006) tested different sunflower types, and estimated that 6,000 m3 ha-1 of
metane gas is a realistic breedind goal for biogas production from sunflower. In germany, district
biogas reactors have been built to convert biomass to methane as an energy source. Whether high
biomass sources for commercial methane production will be determined by market economics.

Sunflower oil, along with most other vegetable oils, has been recognized as an excellent
biodiesel fuel for several decades. Early utilization of vegetable oils for biodiesel fuel was with
unmodified oil. Vegetable oils are now transesterified to release the fatty acids as methyl or ethyl
esters for use as biodiesel fules. High-oleic sunflower oil, whether directly or as feedstock for
synthetic esters, has great potential both as a biodiesel fuel and as a lubricant due to its superior
wear-reducing properties. Reduced consumption, high quality, low maintenance, and
biodegradability decrease the long term costs associated with disposal of lubricants to the
environment.

High-oleic sunflower oil is in a particularly good position for use as a biofuel of its higher
stability than traditional, high – linoleic sunflower oil.

2. BIO-PHARMING BY SUNFLOWER

Bio- pharming is the production of pharamaceutical products by introduction into a host plant of
a foreign gene that codes for the drug product. Sunflower is a candidate host for producing
pharmaceuticals, and compares favourably with maize, canola, soyabean, and alfalfa for cost of
production (Kusnadi et al.1997).
3. BIOREMEDIATION

Sunflowers have the potential to assist in bioremediation of contaminated soils. Two approaches
have been reported in which sunflowerd play an important role

1. Utilization of sunflower oil to extract harmful chemicals

2. Planting of sunflower (photoremediation) on soils contaminated with heavy metals.
Gong et al.(2006) have reported that sunflower oil has a great capacity to remove PAHs
(polycyclic aromatic hydrocarbons) from contaminated soils. Contaminated soils are transferred
to a column and sunflower oil allowed to percolate down through the soil. More than 90% of
PAHs were removed.
The ability of sunflower to accumulate heavy metal ions such as
cadmium,chromiuim,zinc,mercury and lead from the soil is well known Li et al. (1997)
described the genetic variation in cadmium accumulation by sunflower genotypes, and Van der
Lelie et al.(2001) have reported the use of sunflower as a suitable crop for phytoremediation of
soils polluted by zinc.The symbiotic relationship between sunflower and microbes can be
exploited to enhance heavy metal ion accumulation by the plant, and is environmentally more
favorable than using metal ion chelators, which run the risk of leaching chelated metals into
groundwater.
 APPENDIX

1. Preparation of CTAB Buffer ( 100ml)

CTAB 2.0g

1M Tris Ph 8.0 10.0 ml

0.5M EDTA Ph 8.0 4.0 ml

5M NaCl 28.0 ml

H2O 40.0 ml

Adjust all to Ph 5.0 with HCL and make up to 100ml with H2O.

2. Preparation of 5X TBE buffer

Tris Base 54g

Boric acid 27.5g

0.5M EDTA( pH 8.0) 20ml

Make up to 1L with distilled water

3. Preparation of 50X TAE buffer

Tris base 242g

Acetic acid 57.1ml

0.5M EDTA(pH 8.0) 100ml

Make upto 1L with distilled water

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2. Anashchenko AV (1974) phylogenetic relations in the genus Helianthus L.Bull appl Bot genet
plant breed 64:146-156.
3. Atlagic J(2004) roles of interspecific hybridisation and cytogenetic studies in sunflower
breeding. Helia 27: 1-24
4 Baack E,Whitney KD,Riesberg LH(2005) hybridisation and genomic size evolution: timing
and magnitude of nuclear DNA content increases in Helianthus homoploid species. new phytol
167:623-630
5Beard BH(1981) The sunflower crop. Sci Am 244:150-161.
6.