may present several problems for future ble CE, eds).
ble CE, eds). Salt Lake City: University of Utah Press
research. Our observations in F2 genera-
tions showed small numbers of nonshat-
tering plants, presenting an additional
problem for plant breeders. If the goal is
to incorporate desired wild characters
into productive domesticated lines, large
F2 generations will have to be screened to
identify desirable nonshattering plants.
The knowledge that single genes govern
seed coat color and stem pigmentation
should serve to simplify applied research,
particularly efforts to select parents for
breeding, given the great difficulty in per-
forming crosses, and the present lack of
described molecular markers for the spe-
cies. Our basic research is continuing with
the development of isozyme and RAPD
markers with the intent of mapping the six
linkage groups of S. hispanica. The quali-
tative characters described here will
prove to be valuable as genetic map mark-
ers, contribute to a greater understanding
of the genes involved in the traits of
closed calyxes, and contribute to our gen-
eral understanding of the domestication
process.
From the Department of Botany and Plant Sciences,
University of California, Riverside, Riverside, CA 92521.
Thanks are extended to Dr. Arturo Gomez-Pompa, Dr.
Giles Waines, J. A. Hernandez Gomez, P. McCrowan, and
R. Gentry for support and providing seeds of chia ac-
cessions. Address correspondence to J. P. Cahill at the
address above or e-mail: jpca@citrus.ucr.edu.
2002 The American Genetic Association
References
Ayerza R, 1995. Oil content and fatty acid composition
of chia (Salvia hispanica L.) from five northwestern lo-
cations in Argentina. J Am Oil Chem Soc 72:10791081.
Coates W and Ayerza R, 1996. Production potential of
chia in northwestern Argentina. Indust Crop Prod 5:
229233.
Estilai A, Hashemi A, and Truman K, 1990. Chromo-
some number and meiotic behavior of cultivated chia,
Salvia hispanica ( Lamiaceae). Hort Sci 25:16461647.
Haque MS and Ghoshal KK, 1980. Karyotypes and chro-
mosome morphology in the genus Salvia L. Cytologia
45:627640.
Haque MS and Ghoshal KK, 1981. Floral biology and
breeding system in the genus Salvia L. Proc Indian Natl
Sci Acad 47:716724.
Harvey HR, 1991. Land politics in the valley of Mexico:
a two thousand year perspective. Albuquerque: Uni-
versity of New Mexico Press.
Jones PG and Sutton MJ, 1997. Plant molecular biology:
essential techniques. New York: John Wiley.
Lin KY, Daniel JR, and Whistler RL, 1994. Structure of
chia seed polysaccharide exudate. Carbohyd Polymer
23:1318.
Rojas-Rabiela T, 1988. Las siembras de ayer, la agricul-
tura indigena del siglo XVI. Mexico DF: Secretaria de
Educacion Publica.
Sahagun B, 19501982. Florentine codex: general his-
tory of the things of New Spain. In: Monographs of the
School of American Research (Anderson AJO and Dib-
tain anticancer (vincristine and vinblas-
(Originally written 15751577 or 15781580).
tine) and antihypertension (ajmalicine) al-
Ting IP, Ahmed M, Scora RW, Brown JH, and Arquette
JD, 1996. Terpene composition of chia and chan leaf kaloids, respectively. The most commonly
tissue. Paper presented at the Third International Con- observed flower colors in periwinkle are
ference on New Industrial Crops and Products. Tucson:
University of Arizona.
pink (pink corolla and red eye), red-eyed
(white corolla and red eye), and white. Flo-
Weber CW, Gentry HS, Kohlhepp EA, and McCrohan PR,
1991. The nutritional and chemical evaluation of chia ry (1944) attributed these three corolla
seeds. Ecol Food Nutr 26:119125. colors to the epistatic interaction of two
Received February 23, 2001 genes, R and W, with the R-W-genotype be-
Accepted September 5, 2001
ing pink, R-ww being red-eyed, and rrW-
Corresponding editor: Susan Gabay-Laughnan and rrww being white flowered. Simmonds
(1960) implicated two additional genes, A
and B, in the determination of flower col-
or, with A being complementary to R, with-
Inheritance of Flower Color out which flowers would be white, and B
being a copigmentation gene which blues
in Periwinkle: Orange-Red
the pink pigment in the A-R-W- and A-R-ww
Corolla and White Eye genotypes. In addition to the above three
flower colors, Milo et al. (1985) identified
Y. Sreevalli, R. N. Kulkarni, and
K. Baskaran another flower color, pale pink center.
They attributed this flower color to anoth-
er gene, I, which like gene W is also epi-
The commonly found flower colors in peri-
static to the gene R. The genotypes and
winkle (Catharanthus roseus)pink, white,
red-eyed, and pale pink centerare re- phenotypes of different flower colors ac-
ported to be governed by the epistatic in- cording to above three models are shown
teraction between four genesA, R, W, in Table 1.
and I. The mode of inheritance of an un- Recently we procured another flower
common flower color, orange-red corolla color type from a local dealer in horticul-
and white eye, was studied by crossing an tural plants. This accession, designated as
accession possessing this corolla color OR, had flowers with an orange-red corolla
with a white flowered variety (Nirmal). The and white eye. The mode of inheritance of
phenotype of the F1 plants and segregation orange-red corolla and white eye has not
data of F2 and backcross generations sug- been reported earlier. Also, this flower col-
gested the involvement of two more inter- or could not be explained by earlier mod-
acting and independently inherited genes, els of Flory (1944), Milo et al. (1985), or
one (proposed symbol E) determining the Simmonds (1960). We therefore studied
presence or absence of red eye and an- the mode of inheritance of these two flow-
other (proposed symbol O) determining or- er color traits, namely, orange-red corolla
ange-red corolla. color and white eye, using accession OR,
Periwinkle [Catharanthus roseus ( L.) G. as a part of our inheritance studies in per-
Don], a perennial semishrub native to iwinkle ( Kulkarni et al. 1999a,b, 2001).
Madagascar, is now found in many tropical Extensive chemogenetic, biosynthetic,
and subtropical regions. It is grown as an and, in recent years, molecular genetic
ornamental plant in gardens and parks for studies on the biosynthesis of pigments in
its colored flowers and ever-blooming na- plants, particularly flavonoids, have great-
ture. It is also cultivated as a medicinal ly facilitated our understanding of the
plant for its leaves and roots, which con- pathway of flavonoid biosynthesis, evolu-
Table 1. Genotypes and phenotypes of flower colors in periwinkle according to existing models
Genotype Phenotype Reference
R-W- Pink corolla and red eye Flory (1944)
R-ww White corolla and red eye
rrww White
A-R-W-B- Violet corolla and purple violet eye Simmonds (1960)
A-R-W-bb Pink corolla and purple violet eye
A-R-ww-B- White corolla and purple eye
A-R-wwbb White corolla and reddish purple eye
A-rr----, aaR-----, and aarr---- White corolla
R-W-I-, R-W-ii- Pink corolla and red eye Milo et al. (1985)
R-wwI- Pale pink corolla and red eye
R-wwii White corolla and red eye
rrW-I-, rrwwI-, rrW-ii, and rrwwii White corolla
Brief Communications 55
 Table 2. Phenotypes, genotypes, expected proportion, and observed and expected frequencies of detailed knowledge of the function and in-
plants with different corolla colors in the parental, F1, and F 2 generations of the cross orange-red
corolla and white eye (OR) white corolla (Nirmal) in periwinkle, according to the proposed model
teraction of genes involved in the biosyn-
thesis of pigments in the plant species of
Expected Observed Expected interest is one of the essential prerequi-
Generation Phenotype Genotype proportion frequency frequencya
sites for controlled engineering of flower
Parents color ( Forkmann 1991).
OR Orange-red corolla and white eye eeRRwwOO 1 All All
Nirmal White corolla EErrWWoo 1 All All
F1 Pink corolla and red eye EeRrWwOo 1 All All Materials and Methods
F2 Pink corolla and red eye E-R-W-O- 81/256
E-R-W-oo 27/256 The accession OR was found to be true
Total 108/256 158 175.5
Pink corolla and white eye eeR-W-O- 27//256 breeding for orange-red corolla and white
eeR-W-oo 9/256 eye. It was crossed with a white flowered
Total 36/256 66 58.5 variety, Nirmal. In an earlier study the
Orange-red corolla and red eye E-R-wwO- 27/256
Total 27/256 45 43.9 flower color genotype of Nirmal was found
Orange-red corolla and white eye eeR-wwO- 9/256 to be rrWW ( Kulkarni et al. 1999b). Parent
E-rrwwO- 9/256
eerrwwO- 3/256
plants were raised in a glasshouse from
Total 21/256 34 34.1 seeds obtained by artificial self-pollina-
White corolla and red eye E-R-wwoo 9/256 tion. Reciprocal crosses were made as de-
Total 9/256 12 14.6
White corolla eeR-wwoo 3/256 scribed earlier ( Kulkarni et al. 2001). The
E-rrW-O- 27/256 F1 plants (four plants) were selfed and also
E-rrW-oo 9/256 backcrossed to both parents. Altogether
E-rrwwoo 3/256
eerrW-O- 9/256 416, 106, and 85 plants of F2, and two back-
eerrW-oo 3/256 crosses, F1 OR and F1 Nirmal, respec-
eerrwwoo 1/256
Total 55/256 101 89.4
tively, were scored for corolla and eye col-
Total 1 416 416 or, along with 10 plants each of the
2(df:5) for the expected ratio of 108:36:27:21:9:55 4.703 (P .50.30). parental and F1 generations. Chi-square
G(df:5) 4.694 (P .50.30). and G tests were used for testing the good-
ness-of-fit of observed and expected fre-
a
In the F2 generation, calculated according to the expected ratio of 108:36:27:21:9:55 for the six phenotypic classes
of flower color.
quencies of different phenotypic classes in
the F2 and backcross generations (Sokal
and Rohlf 1981).
tion of species, and evolution of genes in- lecular breeding, which can overcome
volved in flavonoid biosynthesis, as well crossing barriers between species, has Results and Discussion
as their cloning from a number of plant opened up the possibility of molecular en-
species, particularly maize, petunia, and gineering for flower color. The generation Flowers of F1 plants had pink corolla and
snapdragon (Chandler et al. 1989; Coe of transgenic petunia plants with brick red eye similar to the commonly found
1985; Forkmann 1991; Gottlieb and Ford red flowers using the A1 gene from maize pink flowered plants in periwinkle, which
1988; Quattrocchio et al. 1993). In orna- is the first example of the creation of a have pink corolla and red eye. There were
mental plants, where novelty in flower col- novel flower color in a highly directed no differences in the flower colors of F1
or is an important breeding objective, mo- manner (Meyer et al. 1987). However, a plants of reciprocal crosses.
Plants of the F2 generation could be
broadly classified into six flower color cat-
Table 3. Phenotypes, genotypes, expected proportion, and observed and expected frequencies of egories: pink corolla and red eye, pink co-
plants with different flower colors in backcross generations of the cross orange-red corolla and white
eye (OR) white corolla (Nirmal) in periwinkle, according to the proposed model rolla and white eye, orange-red corolla and
red eye, orange-red corolla and white eye,
Expected Observed Expected white corolla and red eye, and white co-
Generation Phenotype Genotype proportion frequency frequencya
rolla. The pink and orange-red flowered
F1 OR Pink corolla and red eye EeR-WwO- 1/4 22 26.5 plants, however, showed some differences
Pink corolla and white eye eeR-WwO- 1/4 23 26.5
Orange-red corolla and red eye EeR-wwO- 1/4 27 26.5
among themselves in the intensities of
Orange-red corolla and white eye eeR-wwO- 1/4 34 26.5 their corolla color. The observed frequen-
Total 1 106 106 cies of these six categories of flower col-
2(df:3) for the expected ratio of 1:1:1:1 3.358 (P .50.30). ors fit a ratio of 108:36:27:21:9:55 ( Table 2).
G(df:3) 3.252 (P .50.30). The progeny of the backcross, F1 OR,
F1 Nirmal Pink corolla and red eye E-RrW-Oo 1/4 produced four types of plants: pink corolla
E-RrW-oo 1/4 and red eye, pink corolla and white eye,
Total 1/2 42 42.5
White corolla E-rrW-Oo 1/4 orange-red corolla and red eye, and or-
E-rrW-oo 1/4 ange-red corolla and white eye. The ob-
Total 1/2 43 42.5 served frequencies of plants with these
Total 1 85 85
four kinds of flowers fit a ratio of 1:1:1:1
2(df:1) for the expected ratio of 1:1 0.012 (P .95.90).
( Table 3). Only two types of plantspink
G(df:1) 0.012 (P .95.90).
corolla and red eye, and white corolla
a
Based on the expected ratio of 1:1:1:1 and 1:1 in the backcrosses F1 OR and F1 Nirmal, respectively. were observed in the ratio of 1:1 in the

56 The Journal of Heredity 2002:93(1)

progeny of the backcross, F1 Nirmal ( Ta-
ble 3). The model used for developing
these ratios in the F2 and backcross gen-
erations is discussed below.
The commonly observed flower colors
in periwinkle were earlier explained by a
three gene (R, W, and I) model by Milo et
al. (1985). According to their model, the R
allele produces three anthocyanidinsA1,
B1, and C1 ( located in the center of the co-
rolla)and is epistatic to the W and I al-
leles, which function only in its presence.
The W allele produces pigments A2, B2,
and C2. The I allele also produces the same
pigments as the W allele, but in smaller
quantities and mainly in the center of the
corolla. The r, w, and i alleles do not pro-
duce any pigments. Earlier Simmonds
(1960) hypothesized that two complemen-
tary genes, A and R, are necessary for the
production of colored flowers, and with-
out either of these genes the flowers
would be white. The phenotype of the par-
ent OR (orange-red corolla and white eye)
and the phenotypes pink corolla and
white eye, and orange-red corolla and red
eye observed in plants of the F2 and one
of the backcross generations ( F1 OR) of
the present study did not fit into the mod-
els proposed by Milo et al. (1985) and Sim-
monds (1960).
The phenotypes of plants of the paren-
tal, F1, F2, and backcross generations of the
present study could be accounted for by
incorporating two more genes, proposed
symbols E and O, in the models of Milo et
al. (1985) and Simmonds (1960). The ge-
notypes of plants observed in the present
study were deduced from the following
observations: (1) From the flower pheno-
type of the parent OR (i.e., colored corol-
la) and earlier genetic studies involving
the variety Nirmal ( Kulkarni et al. 1999b),
it was evident that both parents were ho-
mozygous for the A allele. (2) The occur-
rence of plants with white corolla and red
eye, that is, red-eyed flowers (iiwwR-), in
the F2 generation suggested that the par-
ent OR was homozygous recessive at the
W locus (from earlier studies it is known
that the genotype of the variety Nirmal
with white flowers is AArrWW). (3) The ab-
sence of plants possessing flowers with
pale pink centers (I-wwR-) in the F2 gener-
ation suggested that both parents had the
genotype ii at the I locus. (4) The flower
phenotype (pink corolla and red eye) of F1
plants suggested the existence of the R al-
lele in a homozygous condition in the par-
ent OR. According to both Milo et al.
(1985) and Simmonds (1960), the R allele
produces anthocyanidins, which are locat-
ed in the eye region. Therefore it may be
assumed that the R allele produces pig-
ments in the eye region only in the pres-
ence of another gene, E; in the absence of
the E gene, flowers would have white eye.
With this assumption, the genotypes of pa-
rental accessions OR and Nirmal would be
AAiieeRRww and AAiiEErrWW, respective-
ly. (5) Another gene (proposed symbol O)
responsible for the production of orange-
red colored corollas could be assumed to
be present in the parent OR. Further, the
absence of plants with orange-red corollas
in the progeny of the backcross, F1 Nir-
mal ( Table 3) suggested that the O allele
did not express in the presence of the W
allele, as all the progeny of this backcross
would have at least one W allele. The ex-
pected genotypes of plants of the paren-
tal, F1, F2, and backcross generations
based on the analysis given above are pre-
sented in Tables 2 and 3.
As mentioned earlier, the pink and or-
ange-red flowered plants of segregating
generations showed minor differences in
intensities of their flower color which
could not be further classified. Such minor
unclassifiable differences in flower color in
segregating generations have also been
found in other plants, and have been at-
tributed to modifying genes and differenc-
es in the genetic background ( Bassett et
al. 1990; Cornu and Maizonnier 1983; Got-
tlieb and Ford 1988; Riley 1945).
The phenotypes observed in the pre-
sent study could also, alternatively, be ex-
plained by assuming that the E allele in
the homozygous condition inhibits the ex-
pression of the R allele in the eye region,
leading to the production of flowers with
white eye (EER-), and genotypes EeR- and
eeR- would produce flowers with red eye
(the genotypes of parents OR and Nirmal
would then be AAiiEERRwwOO and
AAiieerrWWoo, respectively). However,
the former explanation appears to be
more likely since plants with pink corolla
and red eye, as well as plants with white
corolla and red eye, are quite common in
nature, while those with colored corolla
and white eye are rare and may have aris-
en from a recessive mutation at the E lo-
cus. In Pelargonium hortorum, where many
varieties have a floret center area different
in color from the remainder of the floret,
white center has been found to be con-
trolled by a single recessive gene ( Nugent
and Snyder 1967), as found in the present
study. Flower color in many ornamental
plants as well as field crops has been
found to be governed by the interaction
between two or more genes ( Ennos and
Clegg 1983; Forkmann 1991; Kumar et al.
2000; Negi and Raghava 1990). The results
of the present study also suggested the in-
volvement of at least six major interacting
and independently inherited genesA, R,
W, I, E, and Oin the determination of
flower color in periwinkle.
From the Central Institute of Medicinal and Aromatic
Plants, Field Station, Allalasandra, Bangalore 560 065,
India. This study and Y. Sreevalli were supported by
financial assistance from the Karnataka State Council
for Science and Technology, Bangalore, India. The au-
thors thank Prof. Sushil Kumar, Director, CIMAP, Luck-
now, India, for his keen interest and encouragement.
Address correspondence to R. N. Kulkarni at the ad-
dress above.
2002 The American Genetic Association
References
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colors in common bean produced by interactions of
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pattern of floral pigmentation in Clarkia gracilis. Hered-
ity 60:237246.
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mar S, 1999a. Inheritance of morphological traits of
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Brief Communications 57
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Koes RE, 1993. Regulatory genes controlling anthocya-
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Received February 20, 2001
Accepted November 26, 2001
Corresponding Editor: Brandon Gaut
Thirty Polymorphic Nuclear
Microsatellite Loci From
Black Walnut
K. Woeste, R. Burns, O. Rhodes,
and C. Michler
Black walnut (Juglans nigra L) is a large
tree, native to the eastern United States,
that is prized for its high-quality timber and
edible nut. Thirty (GA/CT)n nuclear micro-
satellite markers were identified from black
walnut for use in population genetic stud-
ies, genome mapping, DNA genotyping of
important clones, studies of gene flow,
and tree breeding. The markers were poly-
morphic based on a diversity panel of 10
black walnut individuals from eight Mid-
western U.S. states.
Black walnut (Juglans nigra L.) is a large
tree that is native throughout the eastern
United States from New England to Texas
( Fowells 1965). Black walnut is prized as
a multipurpose species: it provides valu-
able timber, produces a high-quality edible
nut, and is attractive to wildlife ( USDA
Forest Service Fire Effects Information Sys-
tem website). More than 15 million acres
of timberland in 30 states contain black
walnut (Schmidt and Kingsley 1997). The
vast majority of this species exists in nat-
ural stands, with a small percentage grown
in plantations. An estimated 15 million cu-
bic feet of black walnut are harvested an-
nually in the United States ( USDA Forest
Service Forest Inventory and Analysis
website). In 1997, 29 million pounds of in-
shell black walnut nuts were purchased
for processing and about 2 million pounds
of nutmeat were sold ( Hammonds 1998).
Nearly all processed nuts came from un-
cultivated trees growing in wild popula-
tions (Reid 1990).
There have been successful genetic im-
58 The Journal of Heredity 2002:93(1)
provement efforts in black walnut for both
timber and nut traits ( Beineke 1989; Funk
1979; Tourjee 1998). There is now a need
for DNA-based genetic markers to investi-
gate critical problems in black walnut
breeding and conservation. For example,
breeders need a method for genotyping
important cultivars to verify their identity
( Bish C, personal communication). Efforts
to understand the wild black walnut germ-
plasm have been largely limited to prove-
nance tests ( Bresnan et al. 1992; Rink
1997). Provenance tests, and the associ-
ated morphological and phenological
data, have provided important tools for
breeders and foresters where they are
available, well designed, and well main-
tained (Guries et al. 1981), but provenance
tests of black walnut are expensive and
time consuming because the species has
a long juvenility and mature trees are
large. Genetic markers such as restriction
fragment length polymorphisms (RFLPs;
Fjellstrom and Parfitt 1994; Fjellstrom et al.
1994), random amplified polymorphic
DNA (RAPDs; Woeste et al. 1996), internal
transcribed spacer ( ITS) sequence poly-
morphisms (Potter D, personal communi-
cation), and allozymes (Arulsekar et al.
1985) have been identified for several
members of the Juglandaceae, and they
permit a rapid parsing of the genetic dif-
ferentiation within J. nigra. While these
markers are more informative than phe-
notypes in terms of their ability to identify
species substructure and diversity, micro-
satellite DNA markers [simple sequence
length polymorphisms (SSLPs)] can pro-
vide greater levels of resolution in a cost-
effective manner. Microsatellites over-
come some of the limitations of other
marker systems (Goldstein and Pollock
1997), and a large number of methods for
statistical analysis of microsatellite data
are available ( Luikart and England 1999).
We intend to use the microsatellites
published here as part of a larger effort to
understand the genetics of black walnut.
While the commercial value of this species
both for nuts and timber is based almost
entirely on exploitation of the wild re-
source, there are large gaps in our under-
standing of the genetic structure of wild
populations of black walnut, and there is
no published information on the effects of
timber and nut harvests on the long-term
health of the species. In addition, the
markers will be used for genetic mapping,
DNA genotyping (fingerprinting) of impor-
tant clones, and studies of gene flow in
seed orchards as part of a breeding effort.
Materials and Methods
DNA was isolated from the leaves of three
black walnut selections using Nucleon
Phytopure DNA extraction columns
(Amersham, Buckinghamshire, UK). The
trees were part of a walnut breeding and
genetics program in the Department of
Forestry and Natural Resources at Purdue
University. A pooled DNA sample from
these trees was used by Genetic Identifi-
cation Services (San Diego, CA) to create
an enriched (GA/CT )n microsatellite li-
brary. The library was plated on a selec-
tive medium, and 1500 colonies were ro-
botically picked (Genetix, Hampshire, UK)
into 96-well plates, miniprepped (Qiagen
REAL 96 Prep, Valencia, CA), and se-
quenced using an ABI 3700 (Perkin-Elmer,
Foster City, CA). We analyzed the resulting
sequences using Sequencher software
(version 3.1.1; Gene Codes, Ann Arbor, MI)
and discarded candidate sequences if they
contained no discernible microsatellite re-
peat, or if there was insufficient flanking
sequence to construct suitable polymer-
ase chain reaction (PCR) primers. The se-
quences that remained were assigned to
contigs whenever possible. When se-
quence contigs were available, we derived
a consensus sequence for the regions
flanking the microsatellites and used it for
primer design. Primers (1820 bp) for am-
plification of microsatellite-containing se-
quences (100400 bp) were designed using
Primer 0.5 (Whitehead Institute for Bio-
medical Research, Cambridge, MA).
To develop a preliminary screening pan-
el, DNA from 10 J. nigra individuals repre-
senting populations in eight Midwestern
U.S. states was isolated from mature
leaves using an automated nucleic acid ex-
tractor (Autogen, Framingham, MA) and a
CTAB extraction buffer modified with 2
PVP and 2 CTAB. PCR amplification of
primer pairs was performed with an MJ
Research thermal cycler (Waltham, MA)
using 20 l reactions. The PCR reaction
mixture contained 20 ng of DNA template,
1.5 mM MgCl2, 0.4 U AmpliTaq Gold (Per-
kin-Elmer), and 0.8 M (each) primer. All
other components of the PCR mixture
were as recommended by the manufactur-
er (Perkin-Elmer). PCR amplification was
for 50 cycles of 92C for 30 s, 45C for 1
min, and 72C for 1 min. All primers were
annealed at 45C. The reaction products
were then held at 0C until aliquots could
be loaded into 1.5% Trevigels ( Trevigen,
Gaithersburg, MD) containing ethidium
bromide. Electrophoresis was in 1 TAE
buffer, and gels were photographed using