integration of novel ssr and gene-based snp marker loci in ...species gene prediction and isolation...
TRANSCRIPT
Theor Appl Genet (2010) 120:1415–1441
DOI 10.1007/s00122-010-1265-1ORIGINAL PAPER
Integration of novel SSR and gene-based SNP marker loci in the chickpea genetic map and establishment of new anchor points with Medicago truncatula genome
Spurthi N. Nayak · Hongyan Zhu · Nicy Varghese · Subhojit Datta · Hong-Kyu Choi · Ralf Horres · Ruth Jüngling · Jagbir Singh · P. B. Kavi Kishor · S. Sivaramakrishnan · Dave A. Hoisington · Günter Kahl · Peter Winter · Douglas R. Cook · Rajeev K. Varshney
Received: 3 September 2009 / Accepted: 27 December 2009 / Published online: 23 January 2010© The Author(s) 2010. This article is published with open access at Springerlink.com
Abstract This study presents the development and map-ping of simple sequence repeat (SSR) and single nucleotidepolymorphism (SNP) markers in chickpea. The mappingpopulation is based on an inter-speciWc cross betweendomesticated and non-domesticated genotypes of chickpea(Cicer arietinum ICC 4958 £ C. reticulatum PI 489777).This same population has been the focus of previous stud-ies, permitting integration of new and legacy genetic mark-ers into a single genetic map. We report a set of 311 novelSSR markers (designated ICCM—ICRISAT chickpea
microsatellite), obtained from an SSR-enriched genomiclibrary of ICC 4958. Screening of these SSR markers on adiverse panel of 48 chickpea accessions provided 147 poly-morphic markers with 2–21 alleles and polymorphic infor-mation content value 0.04–0.92. Fifty-two of these markerswere polymorphic between parental genotypes of the inter-speciWc population. We also analyzed 233 previously pub-lished (H-series) SSR markers that provided another set of52 polymorphic markers. An additional 71 gene-based SNPmarkers were developed from transcript sequences that are
Communicated by H. T. Nguyen.
Electronic supplementary material The online version of this article (doi:10.1007/s00122-010-1265-1) contains supplementary material, which is available to authorized users.
S. N. Nayak · N. Varghese · J. Singh · D. A. Hoisington · R. K. Varshney (&)Centre of Excellence in Genomics (CEG), International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru 502324, Andhra Pradesh, Indiae-mail: [email protected]
S. N. Nayak · P. B. Kavi KishorDepartment of Genetics, Osmania University, Hyderabad 500007, Andhra Pradesh, India
H. Zhu · S. Datta · H.-K. Choi · D. R. CookDepartment of Plant Pathology, University of California, Davis, CA 95616, USA
H. ZhuDepartment of Plant and Soil Sciences, University of Kentucky, Lexington, KY 40546, USA
S. DattaIndian Institute of Pulses Research, Kanpur 208024, Uttar Pradesh, India
H.-K. ChoiDepartment of Genetic Engineering, Dong-A University, Busan 604-714, South Korea
R. Horres · R. Jüngling · G. KahlUniversity of Frankfurt, Max von Laue Str. 9, 60439 Frankfurt am Main, Germany
J. Singh · S. SivaramakrishnanDepartment of Agricultural Biotechnology, Acharya N.G. Ranga Agricultural University (ANGRAU), Hyderabad 500030, Andhra Pradesh, India
G. Kahl · P. WinterGenXPro GmbH, Frankfurter Innovationszentrum Biotechnologie (FIZ), Altenhöferallee 3, 60438 Frankfurt am Main, Germany
R. K. VarshneyGenomics Towards Gene Discovery Subprogramme, Generation Challenge Programme (GCP), CIMMYT, Int APDO Postal 6-641, 06600 Mexico DF, Mexico
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1416 Theor Appl Genet (2010) 120:1415–1441
highly conserved between chickpea and its near relativeMedicago truncatula. By using these three approaches, 175new marker loci along with 407 previously reported markerloci were integrated to yield an improved genetic map ofchickpea. The integrated map contains 521 loci organizedinto eight linkage groups that span 2,602 cM, with an aver-age inter-marker distance of 4.99 cM. Gene-based markersprovide anchor points for comparing the genomes of Medi-cago and chickpea, and reveal extended synteny betweenthese two species. The combined set of genetic markers andtheir integration into an improved genetic map should facil-itate chickpea genetics and breeding, as well as transla-tional studies between chickpea and Medicago.
Introduction
Chickpea (Cicer arietinum L.) is an annual, self-pollinateddiploid (2n = 2x = 16) species with a relatively smallgenome of 740 Mbp (Arumuganathan and Earle 1991).Chickpea is also the World’s third most widely grown foodlegume. Over 95% of chickpea production area and con-sumption occur in developing countries, with India contrib-uting the largest share (65%), followed by Pakistan (9%),Iran (7%), and Turkey (4%) (FAOSTAT database http://faostat.fao.org/site/567/default.aspx#ancor, 2007). Cytoge-netic and seed protein analyses are consistent with C. retic-ulatum as the wild progenitor of domesticated C. arietinum,with southeastern Turkey as the presumed center of origin(Ladizinsky and Adler 1976). Cultivated chickpea is com-posed of two genetically distinct sub-types that are readilydistinguished based on seed size and color: Desi, composedof small, brown seeded varieties, and Kabuli, composed oflarge, cream seeded varieties. Due to relatively low rates ofpolymorphism between cultivated chickpea accessions,inter-speciWc crosses between C. arietinum and C. reticula-tum have been the primary focus for genetic studies ofagronomic traits (see Singh et al. 2008).
A diverse array of technologies is available to identifyand monitor DNA polymorphism and as a consequencemolecular markers are now routinely used in the breedingprograms of several crop species (Varshney et al. 2006,2007). In the case of chickpea, molecular markers reportedin the literature are almost entirely simple sequence repeat(SSR) loci (Choudhary et al. 2006; Hüttel et al. 1999;Lichtenzveig et al. 2005; Sethy et al. 2003, 2006a, b; Winteret al. 1999). Despite considerable eVort, low rates of bothintra- and inter-speciWc polymorphism have limited thenumber of these SSR markers that have been integratedinto chickpea genetic maps. A primary goal of the currentstudy was to screen additional molecular markers andthereby enhance the marker density of chickpea geneticmaps.
Chickpea is a close relative of the model legume systemMedicago truncatula (Fig. 1, reproduced according to Choiet al. 2004b), and thus should beneWt from the increasinglydetailed description of the structure and function of theM. truncatula genome (Cannon et al. 2006). A pressingtask for chickpea researchers is to use knowledge gainedfrom the study of reference legumes, such as M. truncatula,Lotus japonicus, and soybean, to advance genetic improve-ment of chickpea. Comparative genomics based on ortholo-gous genetic markers oVers means to bridge model andcrop legumes. Alignment of linkage maps and sequencedorthologous regions between several legume species hasrevealed an extensive network of macro- and micro-syn-teny between legume species; importantly, genomic andgenetic comparisons of orthologous nodulation genes inseveral legumes suggests that comparison of genome struc-ture and function may have practical applications to cross-species gene prediction and isolation (see Zhu et al. 2005).The lack of infrastructure (knowledge and physical capac-ity) in chickpea, however, has limited the potential forcross-genome comparisons and has hampered progress inthe area of genomics-assisted breeding (Varshney et al.2009a).
In view of the above, we sought to enhance markerrepertoire and density of genetic maps in chickpea usinga combination of several molecular marker sets. We
Fig. 1 Phylogenetic relationships: Papilionoideae family. This Wgure(taken from Choi et al. 2004b) illustrates the phylogenetic relation ofchickpea with other legumes. Chickpea and Medicago belong toinverted repeat lacking clade (IRLC) of Papilionoideae and consti-tuted the cool season legumes
Tribe Genus Species
Vicia
Lens L. culinaris (lentil)
V. faba (faba bean)Viceae
Cool season legumes
(Galegoid)
Melilotus
Trifolium
Pisum P. sativum (garden pea)
M. officinalis (sweet clover)
T. pratense (red clover)(IRLC) Trifolieae
Lotus
Cicer
Medicago M. sativa (alfalfa)M.truncatula
C. arietinum (chickpea)
L. japonicus
Cicereae
Loteae
(Hologalegina)
Tropicalseason legumes(Phaseoloid)
Phaseolus
Vigna
Glycine
P. vulgaris (common bean)
V. radiata (mung bean)
G. max (soybean)Phaseoleae
Papilionoideae
Cajanus C. cajan (pigeonpea)(Phaseoleae)
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Theor Appl Genet (2010) 120:1415–1441 1417
developed two novel sets of molecular markers based on anSSR-enriched genomic DNA library and gene-based singlenucleotide polymorphism (SNP) markers derived fromcomparison of Medicago and chickpea ESTs. These novelgenetic markers were analyzed together with publishedgenetic markers to develop a dense genetic map of chick-pea. We anticipate that these resources will serve as toolsfor genomics-assisted breeding in chickpea, and enhanceprospects for transfer of knowledge about the structure andfunction of the Medicago genome to chickpea with a Wnalobjective of chickpea improvement.
Materials and methods
Plant material and DNA extraction
The cultivated chickpea germplasm line ICC 4958, belong-ing to Desi type and a parent line of inter-speciWc referencemapping population (C. arietinum ICC 4958 £ C. reticula-tum PI 489777) was used to construct microsatellite-enriched library. While two genotypes of chickpea (ICC4958 and ICC 1882) were used to optimize the polymerasechain reaction (PCR) conditions for newly developed SSRmarkers, an array of 48 genotypes which includes 33 geno-types from cultivated chickpea (C. arietinum) and 15 fromwild species of chickpea (7 genotypes from C. reticulatum,2 genotypes from C. echinospermum and one each fromC. bijugum, C. cuneatum, C. judaicum, C. microphyllum,C. pinnatiWdum, and C. yamashitae) was used to assess thepolymorphism potential of new set of SSR markers(Table 1).
For integrating the markers into the genetic map, theinter-speciWc reference mapping population ICC 4958 £ PI489777 comprising of a total of 131 RILs was used. Whileall 131 RILs were used to score genotyping data for theSSR markers isolated in this study as well as reported byLichtenzveig et al. (2005), a subset of 94 RILs was usedwith gene-based markers.
Total genomic DNA was extracted by employing thestandardized high throughput mini DNA extraction proto-col (as mentioned in Cuc et al. 2008). The quality andquantiWcation of extracted DNA was checked on 1.2% aga-rose. The DNA was normalized to 5 ng/�l for further use.
Construction of SSR-enriched library
To construct a size-fractioned chickpea genomic DNAlibrary, puriWed genomic DNA (100 �g) of ICC 4958 wascompletely digested with MboI or Sau3AI in combinationwith TaqI enzyme. The restricted fragments were separatedon low-melting agarose gels, and the gel zone containing
Table 1 List of 48 chickpea genotypes used for calculating polymor-phic information content (PIC) of newly developed SSR markers
S No. Genotype Botanical variety
Country of origin
Market type
1 ICCV 2 C. arietinum India Kabuli
2 ICC 10673 C. arietinum Turkey Desi
3 ICC 11944 C. arietinum Nepal Desi
4 ICC 12299 C. arietinum Nepal Desi
5 ICC 12379 C. arietinum Iran Desi
6 ICC 1431 C. arietinum India Desi
7 ICC 17116 C. yamashitae Afghanistan Wild
8 ICC 17122 C. bijugum Turkey Wild
9 ICC 17123 C. reticulatum Turkey Wild
10 ICC 17148 C. judaicum Lebanon Wild
11 ICC 17152 C. pinnatiWdum Turkey Wild
12 ICC 17160 C. reticulatum Turkey Wild
13 ICC 17162 C. cuneatum Ethiopia Wild
14 ICC 17248 C. microphyllum Pakistan Wild
15 ICC 1882 C. arietinum India Desi
16 ICC 2679 C. arietinum Iran Desi
17 ICC 283 C. arietinum India Desi
18 ICC 3137 C. arietinum Iran Desi
19 ICC 3239 C. arietinum Iran Desi
20 ICC 3696 C. arietinum Iran Desi
21 ICC 3986 C. arietinum Iran Desi
22 ICC 4853 C. arietinum Unknown Kabuli
23 ICC 4958 C. arietinum India Desi
24 ICC 5002 C. arietinum India Desi
25 ICC 506 EB C. arietinum India Desi
26 ICC 5337 C. arietinum India Kabuli
27 ICC 6263 C. arietinum Russia and CISs Kabuli
28 ICC 7052 C. arietinum Iran Desi
29 ICC 7413 C. arietinum India Pea
30 ICC 8200 C. arietinum Iran Desi
31 ICC 8261 C. arietinum Turkey Kabuli
32 ICC 9644 C. arietinum Afghanistan Desi
33 ICC V95311 C. arietinum India Kabuli
34 JG11 C. arietinum India Desi
35 JG62 C. arietinum India Desi
36 VIJAY C. arietinum India Desi
37 IG 72933 C. reticulatum Turkey Wild
38 IG 72953 C. reticulatum Turkey Wild
39 PI 489777 C. reticulatum Turkey Wild
40 IG 10419 C. arietinum Syria Kabuli
41 IG 6044 C. arietinum Sudan Kabuli
42 IG 6899 C. arietinum Iran Kabuli
43 IG 7148 C. arietinum Algeria Kabuli
44 IG 72971 C. reticulatum Turkey Wild
45 IG 73064 C. echinospermum Turkey Wild
46 IG 73074 C. echinospermum Turkey Wild
47 IG 73082 C. reticulatum Turkey Wild
48 IG 7767 C. arietinum Syria Kabuli
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1418 Theor Appl Genet (2010) 120:1415–1441
the fragments of DNA of size 800–1200 bp were excisedand ligated into Promega pGEM 3Z(f) vector (Promega,Madison, WI, USA). The vector was transformed intoE. coli Sure strain—DH10B (Stratagene, Heidelberg,Germany) by electroporation. Approximately 400,000 cloneswere plated at a density of 20,000 colonies per plate. Themasterplates generated were replica-plated on positivelycharged PVDF macroarrays. Macroarrays were printedusing contact printing technology at RZPD GmbH, Berlin,Germany.
For enriching the genomic DNA library, synthetic oligos(GA)10 and (TAA)10 were enzymatically 3� end-labeledwith digoxigenated oligonucleotides (DIG Oligonucleotide3�-End Labeling Kit; Roche, Mannheim, Germany). Subse-quently, macroarrays/Wlters were hybridized with above-mentioned oligo-probes in Roti-Hybri-Quick buVer (CarlRoth GmbH, Karlsruhe, Germany) including 10 �g/mlsheared, denatured E. coli DNA to minimize non-speciWcbinding. Filters were hybridized at 55°C overnight andwashed three times each for 10 min in 1:2, 1:5, and 1:10dilutions of the hybridization buVer at 60°C. The digoxigenwas detected in a “direct detection assay” performed withthe DIG Wash and Block buVer set, and DIG Luminescentdetection Kit (Roche, Mannheim, Germany) for chemilu-minescent detection with a monospeciWc antibody coupledto alkaline phosphatase in the presence of CSPD. Filterswere exposed to X-ray Wlms (Amersham, Buckingham-shire, UK) with intensifying screens for 4 h or overnight,and the colonies giving strong signals were scraped fromthe master plates; re-grown; spotted on Hybond N mem-branes (Amersham, Buckinghamshire, England) to Wx theDNA by lysis. Hybridization and chemiluminescent detec-tion was done repeatedly to pick the clones with positivesignals. These clones were grown on LB agar plates withampicillin (100 �g/ml) overnight at 37°C. Aliquots of thesecolonies were used for colony PCR.
Development of genomic SSR markers
The colonies with high level of signal were used to isolateplasmid DNA using standard alkaline lysis method(Sambrook and Russell 2001). After checking the qualityof the plasmid DNA on 0.8% agarose gel, the clones weresequenced using the BigDye Terminator cycle sequencingkit on an ABI3700/ABI3730XL (Applied Biosystems Inc.,Foster City, CA, USA). 288 clones were sequenced in bothdirections using standard T7 promoter and SP6 primers and19 clones in one direction by using M13-forward sequenc-ing primer at Macrogen (www.macrogen.com) and ICRI-SAT.
The sequences generated were subjected to CAP3, acontig assembly program (http://pbil.univ-lyon1.fr/cap3.php) in order to deWne unigenes. These unigenes were
subjected to MIcroSAtellite (MISA, Varshney et al. 2002)tool to search microsatellites considering minimum tenrepeat units of mono- (N), and four repeats of di- (NN), tri-(NNN), tetra- (NNNN), penta- (NNNNN) and hexa-(NNNNNN) nucleotides and compound microsatellitespresent within a distance of 100 bp. Primer pairs for SSRswere designed using Primer3 program (http://frodo.wi.mit.edu/) in batch Wle, and the SSR markers developedwere designated as ICCM (ICRISAT Chickpea Microsatel-lite) markers (Table 2).
Development of gene-based SNP markers
PCR primers derived from Medicago ESTs (expressedsequence tags), Medicago BAC (bacterial artiWcial chromo-some)-end sequences, and M. sativa cDNA sequences havebeen described in previous studies (Choi et al. 2004a, b,2006). To design PCR primers based on chickpea ESTs(Buhariwalla et al. 2005), candidate chickpea transcriptswere compared to sequenced Medicago BAC clones (http://www.medicago.org/genome), and transcripts with highnucleotide identity and low copy representation to theMedicago genome were selected for primer designing.Primers were designed from highly conserved codingsequences, to amplify across intron regions (Choi et al.2004a), using the Lasergene PrimerSelect software package(DNAStar Inc., Madison, WI, USA). Details of theseprimer pairs are given in Table 3.
The polymorphic gene-based markers between the par-ents of mapping population were identiWed essentially asmentioned in Choi et al. (2004a). Each pair of correspond-ing sequences from genotypes ICC 4958 and PI 489777was aligned using Sequencher software (Gene Codes, AnnArbor, MI, USA) to detect SNPs. The sequences withSNPs were transferred to DNA Strider 1.2 (Douglas 2008)to identify restriction site that is coincident with SNPs andcleavable ampliWed polymorphic sequence (CAPS) assayfor genotyping the corresponding SNPs were developed.In cases where a suitable restriction enzyme site was notidentiWed, oligonucleotide primers were designed immedi-ately adjacent to the SNP position, which allows for a sin-gle base extension of the SNP site using ABI SNaPshotMultiplexing Kits (Applied Biosystems Inc., Foster City,CA, USA).
Genotyping assay
For both ICCM as well as H-series SSR markers, theforward primers were anchored with M13 tail (CACGACGTTGTAAAACGAC). PCR amplicons generated bySSR markers were analyzed on capillary electrophoresis,while for gene-based SNP markers the CAPS or SNaPshotassays were used for genotyping. For SSR genotyping,
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Theor Appl Genet (2010) 120:1415–1441 1419
Tab
le2
Sim
ple
sequ
ence
rep
eats
(SS
R)
isol
ated
fro
m m
icro
sate
llite
-enr
iche
d li
brar
y of
chi
ckpe
a
Mar
ker
nam
eG
enB
ank
IDS
SR m
otif
aFo
rwar
d pr
imer
(5�
–3�)
Rev
erse
pri
mer
(5�
–3�)
Pro
duct
si
ze (
bp)
Num
ber
of a
llele
sbP
IC
valu
e
ICC
M00
01a
FI85
6656
(AT
)5G
AA
CT
TG
CT
TG
GG
GA
CA
GG
GA
GG
TG
AG
CT
GA
AG
TG
AG
GC
246
20.
05
ICC
M00
01b
FI85
6656
(CT
)4tc
(CT
)4T
GC
AC
AA
CG
GC
TA
TG
TC
TT
CG
AG
GT
GA
GC
TG
AA
GT
GA
GG
C11
89
0.71
ICC
M00
02FI
8566
54(T
C)6
TC
GT
TC
TC
CG
TT
AT
GT
GT
GC
AT
GC
CT
CC
AA
TA
GC
AT
AC
GG
262
20.
04
ICC
M00
03FI
8565
38(C
T)6
AA
TG
GA
AG
AA
CG
TC
AG
GG
TG
TT
CC
AC
TG
GG
GC
AA
AA
TA
AG
252
70.
59
ICC
M00
04FI
8565
39(A
T)4
CA
CT
CA
CA
CA
CG
CA
CT
TT
CA
AA
AA
GA
GA
AG
CC
CA
CC
AA
AA
213
40.
39
ICC
M00
05a
FI85
6657
(TC
)4N
(CT
)4N
(TC
)4T
GC
TG
AG
CG
AT
CT
GA
GG
AT
AG
CA
GC
AA
AA
AT
CA
GC
AC
AA
A27
7N
A
ICC
M00
05b
FI8
5665
7(A
T)4
TT
GG
TG
CA
GA
TT
TT
TG
TT
GC
AG
AT
TT
GG
GG
GA
TA
AA
AG
GG
241
10.
00
ICC
M00
07a
FI85
6661
(CT
C)4
N(T
C)4
N(T
C)4
TC
TA
CA
TT
CT
CT
CC
GT
GC
CC
GA
GG
AG
TG
TA
GG
GG
AG
AG
GG
242
NA
ICC
M00
07b
FI85
6661
(TC
CT
C)4
CT
CT
CC
CC
AC
TC
TC
CC
TT
TC
TG
AG
TA
GG
AT
CG
TA
GT
AG
GG
GG
202
NA
ICC
M00
08a
FI8
5654
0(A
)10N
(A)1
0C
TC
AT
GG
TG
GC
AT
TG
AG
AA
AT
CC
TT
GA
AT
TT
TT
GA
GA
CA
CG
A23
02
0.05
ICC
M00
08b
FI85
6540
(GA
)4N
(AG
)5t(
GA
)4T
CG
TG
TC
TC
AA
AA
AT
TC
AA
GG
AC
AC
TC
GA
CC
AC
CA
CT
GC
TA
A23
52
0.32
ICC
M00
08c
FI85
6540
(CT
)4G
GT
GG
TT
TG
TG
GA
GG
TT
GA
TA
GG
CA
AC
CA
TT
CA
TC
CT
TG
T24
52
0.05
ICC
M00
09a
FI85
6541
(GA
)4C
AC
TT
CA
AA
AG
AG
TG
TT
TG
AT
TT
GA
GG
TT
TG
AA
AG
AT
GA
GT
GG
TT
TT
G18
93
0.10
ICC
M00
09b
FI8
5654
1(A
)11
AA
AA
TA
TG
GA
AA
GT
CG
GG
CA
TG
CA
TT
TT
CT
TA
GC
GG
TT
TT
T25
7N
A
ICC
M00
10a
FI85
6663
(CT
)4G
AC
GG
AA
AT
AC
GG
CT
GG
TT
AG
CT
GC
AA
TA
TA
CT
CC
GC
CT
C11
31
0.00
ICC
M00
10b
FI85
6662
(AT
)4A
CG
CC
AA
TT
CT
TT
TG
AG
CA
CT
CA
GC
AC
TG
GT
GG
AA
CC
AT
A14
212
0.80
ICC
M00
14a
FI85
6542
(GA
)5C
GT
GG
TT
GT
GT
TG
TT
TG
AG
GT
GT
GT
TC
AT
CT
CC
CT
CT
CC
C13
41
0.00
ICC
M00
14b
FI8
5654
2(T
A)5
TT
TG
GG
AA
CC
TT
TC
TC
AT
CA
AC
CA
AA
TC
AT
TT
TT
GT
GC
AC
TG
254
30.
16
ICC
M00
19a
FI85
6544
(AG
)18
TT
CC
GG
TT
GT
TG
AG
TA
GA
GA
AA
AC
TT
CC
AC
TT
GT
TC
CA
TC
CG
189
NA
ICC
M00
19b
FI85
6544
(AG
)4C
GG
AT
GG
AA
CA
AG
TG
GA
AG
TT
CT
CG
TA
CC
GG
AC
CA
GA
GT
C15
36
0.62
ICC
M00
21a
FI85
6697
(CA
)4N
(AT
)4N
(AT
T)5
AA
AC
CG
CA
TT
GA
GG
AA
TG
AC
TT
TG
CC
AC
GA
TA
TG
TT
CA
GG
232
NA
ICC
M00
21b
FI8
5669
6(A
T)4
TC
TT
TC
TA
AG
CA
GT
CT
AG
GA
TA
CG
AA
GG
AA
GG
GT
GG
GA
TA
AT
TG
G22
9N
A
ICC
M00
22FI
8566
99(A
T)4
TA
AA
CC
GC
AT
TG
AC
GA
AT
GA
TG
AA
TT
CG
CA
AG
AA
TC
AA
AT
G12
318
0.89
ICC
M00
24FI
8565
45(A
T)4
CA
CT
CA
CA
CA
CG
CA
CT
TT
CA
AA
AA
GA
GA
AG
CC
CA
CC
AA
AA
A21
43
0.08
ICC
M00
26FI
8567
14(A
G)4
CC
GG
AC
AT
TG
TT
CT
GA
AG
GT
TC
TG
TG
CA
GT
GG
AG
CT
AT
GG
216
20.
05
ICC
M00
29FI
8567
21(A
G)4
N(G
A)4
N(G
A)4
a(A
G)5
CT
TC
CC
CT
TT
CC
AA
AA
TT
GA
TC
GT
TC
AC
AG
GT
TT
TC
CC
TC
244
10.
00
ICC
M00
30a
FI85
6722
(TC
)4N
(CT
)4N
(TC
)4G
GC
AA
CG
TA
CG
GG
AT
AA
AT
GG
CA
GC
AA
AA
AT
CA
GC
AC
AA
A27
11
0.00
ICC
M00
30b
FI85
6722
(T)1
0T
TG
CT
GC
TG
AT
TT
TT
GT
TG
CT
CA
TC
CA
TC
AT
TC
TA
AA
TG
TG
TC
A25
73
0.09
ICC
M00
32FI
8565
46(G
A)4
TC
TC
TT
GA
CA
CA
AG
TC
TG
CA
CA
TC
CC
AC
TC
AC
AT
GT
AG
CG
AA
A23
21
0.00
ICC
M00
34FI
8566
51(G
A)1
1T
TT
GT
TT
GC
GG
AG
GA
AT
AG
GT
CA
CC
TC
AC
CA
CA
CT
TC
TT
TT
C25
96
0.33
ICC
M00
35FI
8565
47(G
T)4
TG
AG
GG
TA
AA
TA
AA
TT
GG
TG
GC
AT
GA
TT
TC
CG
AG
GA
CA
GT
GG
101
10.
00
ICC
M00
37FI
8565
48(A
G)4
N(G
A)4
N(A
G)4
N(G
A)5
CA
AG
CA
AT
GG
GA
GA
CA
CC
TT
CC
AC
CA
CT
TC
AA
CG
TC
TC
CT
212
NA
ICC
M00
42FI
8567
40(C
A)4
TT
CG
TG
AT
AT
TC
AT
CA
AG
GT
GG
TT
TG
AC
GT
AC
TC
TG
TG
TT
TT
GT
TT
259
30.
08
ICC
M00
43FI
8565
52(G
A)6
AA
GA
TT
GG
AT
TC
CA
CA
AC
GC
AC
AA
CC
CA
CC
CA
CA
CA
AA
C27
47
0.44
123
1420 Theor Appl Genet (2010) 120:1415–1441
Tab
le2
cont
inue
d
Mar
ker
nam
eG
enB
ank
IDS
SR m
otif
aFo
rwar
d pr
imer
(5�
–3�)
Rev
erse
pri
mer
(5�
–3�)
Pro
duct
si
ze (
bp)
Num
ber
of a
llele
sbP
IC
valu
e
ICC
M00
45F
I856
553
(TA
)4N
(TC
)12
TG
AA
TC
CC
TC
AA
TC
CT
GT
GG
CT
TA
TC
TC
CC
CA
GT
TG
CC
AG
272
50.
31
ICC
M00
52FI
8567
68(T
AT
)6C
GT
CG
TG
TC
CA
TC
TG
TC
TT
GC
CA
GG
TG
CA
AT
AG
GG
AA
AT
C27
52
0.04
ICC
M00
53FI
8567
71(G
A)3
2t(A
G)4
TG
CG
GT
CG
AC
TC
TA
GA
GG
AT
CT
GA
GA
AG
GA
AG
CC
AA
CA
GG
173
10.
00
ICC
M00
59a
FI85
6779
(GA
A)4
CG
GT
CG
AT
CT
GA
GG
AT
CA
CT
AG
GG
TT
CA
AC
TG
TT
CA
GG
TT
TG
226
NA
ICC
M00
59b
FI85
6779
(AG
)4N
(GA
)9N
(GA
)4C
AA
AC
CT
GA
AC
AG
TT
GA
AC
CC
AG
GT
TC
TC
CC
TC
GC
TC
TC
TC
172
30.
23
ICC
M00
60FI
8567
80(A
T)4
N(C
T)4
TG
AT
GA
AC
AT
GC
TA
AC
AA
AC
TA
AC
AA
CC
AA
GA
CA
AC
TG
GT
GG
AG
GT
260
20.
04
ICC
M00
61FI
8567
98(A
T)6
N(T
TA
)4(T
AA
)4N
(TT
A)1
3A
TG
CG
GT
AA
TC
CT
TG
AC
TG
GT
GA
AA
TT
GG
GT
TG
GA
AG
TT
TG
275
50.
23
ICC
M00
62F
I856
801
(AA
T)4
N(A
AT
)6A
TG
CA
AC
GC
CT
AA
GT
CT
CG
TT
AG
GT
AC
GT
TT
GG
GG
TC
CT
G26
63
0.09
ICC
M00
63F
I856
802
(TT
A)6
TT
GA
TT
TA
TC
CT
GT
GA
TG
CT
TT
TT
GG
CA
GT
CT
GG
TC
AT
GT
GA
AA
194
20.
04
ICC
M00
65a
FI85
6807
(TC
)6C
AT
TC
CC
GC
AA
AG
CT
TG
TA
TG
GT
GC
CT
TG
GA
GA
AA
AT
CA
A13
7N
A
ICC
M00
65b
FI85
6807
(TC
)4C
AC
GG
TG
TC
TT
CC
AC
AT
GA
CT
AA
GC
CA
AT
AC
CA
AG
AG
GC
G19
52
0.08
ICC
M00
66FI
8565
55(T
C)4
AC
CA
TG
CT
CA
CC
AA
CT
CA
CA
TC
TC
TT
GA
CA
CA
AG
TC
TG
CA
CA
T24
21
0.00
ICC
M00
67FI
8565
56(T
C)4
AA
CT
GC
AA
CC
TC
TC
TC
GC
TC
TC
TC
TT
GA
CA
CA
AG
TC
TG
CA
CA
T20
41
0.00
ICC
M00
68FI
8565
57(A
TT
)22
TC
TT
CT
TT
GC
TA
TC
TG
TC
TC
GC
TG
CA
TG
TC
AA
AC
AT
TA
GA
CA
AC
TT
T22
714
0.87
ICC
M00
69FI
8565
58(A
TT
)22
TC
TT
CT
TT
GC
TA
TC
TG
TC
TC
GC
TG
CA
TG
TC
AA
AC
AT
TA
GA
CA
AC
TT
T22
713
0.82
ICC
M00
72F
I856
813
(GA
A)4
CA
AG
AC
CT
TG
AA
CA
GG
AG
GC
TC
TT
TC
AA
TT
TT
CT
AA
CA
AT
TT
CA
TC
200
20.
14
ICC
M00
73a
FI85
6828
(TA
)4N
(A)1
1N(T
A)4
TC
GA
TC
TG
AG
GA
TC
TT
TG
GT
GT
GG
AT
AC
TA
TT
AA
CG
AA
AA
AC
TA
GC
G21
96
0.23
ICC
M00
73b
FI85
6828
(TC
)4C
TT
GT
CC
AC
CC
CA
CA
TT
CT
TT
AG
AG
AA
AT
GG
GG
GA
AG
GG
T21
71
0.00
ICC
M00
74a
FI85
6830
(A)1
1N(T
C)6
AA
AT
CC
CA
AA
TT
TA
GA
GC
GG
AA
CC
TT
TG
AA
AA
AG
GC
GG
TT
183
90.
40
ICC
M00
74b
FI85
6830
(T)1
5A
AC
CG
CC
TT
TT
TC
AA
AG
GT
TC
CT
TC
CA
GG
GG
AA
AA
AG
AA
A26
4N
A
ICC
M00
75FI
8565
59(A
G)1
6C
TT
TG
TA
AC
AA
TA
AA
AT
GC
AA
AG
TA
AA
GG
AA
GC
AC
AG
TC
TG
CA
CA
AA
163
40.
26
ICC
M00
76FI
8565
60(A
TA
)17N
(TA
A)5
CT
CA
TC
GA
AT
AG
AA
CC
TA
CC
GA
CC
GC
TA
CA
CC
TA
CA
AC
GG
TA
A27
03
0.08
ICC
M00
77a
FI85
6561
(AG
)4N
(T)1
0C
CA
CA
AG
AA
GA
CA
AA
GG
GG
AA
AA
AA
GA
TG
CT
AA
AA
CT
AA
AC
CA
AA
GA
217
10.
00
ICC
M00
77b
FI85
6561
(A)1
0G
CC
GA
GA
AA
AT
AA
AT
TT
CA
CC
AG
CC
GC
GA
CC
AT
TA
AT
TC
TA
A12
42
0.04
ICC
M00
78a
FI85
6832
(TA
T)4
g(A
TT
)6ag
(TA
T)5
N(A
T)4
CT
GA
GG
AC
GT
TG
GG
AA
TA
CG
AA
AA
AC
TA
AT
CT
CG
TG
TT
CA
AA
TC
C28
03
0.22
ICC
M00
78b
FI85
6832
(TC
)4A
AT
CC
CA
AC
GG
TG
AG
AG
AT
GG
GA
CA
AG
GA
GT
GG
AA
GG
GA
279
150.
62
ICC
M00
79FI
8565
62(T
TA
)6G
AG
CT
AA
CG
CC
TT
CG
CT
AG
AG
AG
AG
GG
AT
TA
AA
AC
AA
AT
AG
AG
GA
A17
03
0.08
ICC
M00
80FI
8565
63(T
)10
CT
GC
GG
TG
AC
TC
TG
AG
GA
TG
AG
AA
TC
AC
GG
GT
GT
TT
CA
AG
173
30.
15
ICC
M00
81FI
8565
64(T
AA
)6G
AG
AG
GG
AT
TA
AA
AC
AA
AT
AG
AG
GA
AG
AG
CT
AA
CG
CC
TT
CG
CT
AG
A17
03
0.08
ICC
M00
82FI
8568
33(C
T)1
9N(T
C)4
TC
AC
GA
TC
TC
AC
AG
AG
CC
AC
TC
CG
TG
AT
TC
TG
AG
CA
AC
AG
260
30.
08
ICC
M00
83a
FI85
6835
(AT
)4N
(CT
)7C
GC
TC
AC
AC
CA
TC
TC
AC
TT
CG
AA
TG
GA
GG
AA
AT
AC
AG
AG
TG
C27
3N
A
ICC
M00
83b
FI85
6837
(A)1
3A
TT
GA
AA
AA
CC
AC
GC
AC
AC
AA
GC
GA
CG
AC
AG
TG
AC
CT
TC
T14
01
0.00
123
Theor Appl Genet (2010) 120:1415–1441 1421
Tab
le2
cont
inue
d
Mar
ker
nam
eG
enB
ank
IDSS
R m
otif
aF
orw
ard
prim
er (
5�–3
�)R
ever
se p
rim
er (
5�–3
�)Pr
oduc
t si
ze (
bp)
Num
ber
of a
llel
esb
PIC
va
lue
ICC
M00
83c
FI85
6837
(T)1
1T
GT
TT
GT
TG
CC
TA
GC
CA
CT
GT
TG
GA
CA
GA
TT
TT
TG
TT
GT
TT
GT
T19
51
0.00
ICC
M00
84F
I856
993
(GA
)5T
TT
TG
AT
TG
AG
CA
TG
CA
AT
GT
GA
AC
CT
TT
TG
AG
GT
CT
GT
TG
C24
62
0.04
ICC
M00
85FI
8568
55(T
)10N
(TA
T)1
4G
TG
GT
CC
AT
CT
GT
CT
CG
GT
TG
GA
AA
AG
GA
GA
AA
GT
TG
TG
GG
210
NA
ICC
M00
86a
FI85
6856
(TA
)4N
(T)1
1T
GT
GC
AT
TG
AG
CC
TA
TT
GG
TG
AC
AA
CA
GC
GG
CA
TA
AT
CA
A18
2N
A
ICC
M00
86b
FI85
6856
(AC
)4C
TT
CC
CC
TT
TA
CC
CC
GT
TT
AA
AA
GA
AA
TC
GA
CA
AT
AA
AA
GA
GT
GA
175
NA
ICC
M00
88FI
8568
61(T
)10N
(AT
)5A
AA
GG
AA
GG
GA
AG
GA
AA
TG
CG
AG
TT
TG
GG
CA
GG
CA
AT
AA
A24
02
0.19
ICC
M00
89a
FI85
6863
(A)1
2A
AC
AC
CG
AC
TT
TC
CA
AA
AC
GT
TT
GG
GA
AA
TA
CA
AC
CT
TT
GA
A20
49
0.45
ICC
M00
89b
FI85
6862
(TA
T)2
0G
GG
AT
AT
CG
CC
AA
TA
TA
TT
TT
AT
AC
CT
TT
GG
CA
AC
AA
AT
CC
TT
TG
A12
6N
A
ICC
M00
90a
FI85
6864
(TT
A)6
AC
GG
GA
CT
TG
GA
TG
AC
TT
TC
AG
AC
GC
GT
GC
TT
TC
TT
CC
TA
257
NA
ICC
M00
90b
FI85
6864
(TC
)5C
TG
CC
TA
GG
AA
GA
AA
GC
AC
GA
AA
AT
AA
AT
GC
GC
CG
TA
TG
C16
03
0.39
ICC
M00
93a
FI85
6871
(TA
T)2
0C
TT
CT
GT
TA
TT
AT
CG
CC
GC
CA
GC
AT
CA
TG
GA
GC
AG
AG
AG
G22
3N
A
ICC
M00
93b
FI85
6871
(AT
)4T
AC
CC
TT
TC
TC
TC
CC
CA
CC
TT
CA
GT
AG
TC
GG
GC
AA
TA
GA
TG
A12
9N
A
ICC
M00
93c
FI85
6870
(AA
AT
)4C
CA
CT
TT
TA
GG
CG
CA
CT
TC
TC
GA
CT
CA
TT
TT
TC
AC
GG
AC
A19
73
0.24
ICC
M00
94FI
8568
72(A
T)4
AG
AG
GC
AA
AC
AA
GA
AC
CG
AA
AA
GG
GT
TA
GT
GG
AG
GA
AT
TA
TG
AA
279
30.
08
ICC
M00
95a
FI85
6875
(TC
)4C
TC
TC
CA
TC
CC
AT
CC
GA
CT
AG
GA
AG
CC
AT
AT
CC
AG
AG
GG
T18
1N
A
ICC
M00
95b
FI85
6874
(AT
TC
)4C
GG
GA
CA
TT
TC
CG
TT
AA
AA
AC
GA
GT
CG
TT
TT
CT
TG
GC
TT
C17
11
0.00
ICC
M00
96FI
8568
77(A
T)4
N(C
T)4
N(T
C)4
AC
AC
CC
CA
CC
CT
AA
TT
AC
AC
TG
AG
AG
GT
AC
GA
AG
CA
CG
AG
G17
09
0.47
ICC
M00
97a
FI85
6669
(AT
T)1
2N(T
A)4
TG
AG
GA
CT
GC
CA
TA
CT
CC
AG
TC
CC
CT
TT
AT
GA
GG
GC
TT
TT
276
30.
09
ICC
M00
97b
FI85
6668
(CT
T)4
TC
CA
AT
TC
CA
AA
AC
AC
AC
CA
CC
TG
AG
GA
GT
AA
AA
GA
CG
GG
130
10.
00
ICC
M01
01a
FI85
6675
(A)1
3T
AA
CT
GA
GT
TT
CG
GG
TT
CC
GC
TT
AA
CG
GA
CG
TT
GT
AG
GG
C24
7N
A
ICC
M01
01b
FI85
6674
(AG
)4G
AA
GA
CA
AA
AA
GG
GG
CA
CA
AC
CG
AT
TG
TT
TC
AA
GA
CC
AG
A10
52
0.04
ICC
M01
02FI
8566
76(T
)12
CA
CC
AA
AA
AG
GG
AA
CT
TT
CG
AA
AA
AT
AG
GG
TG
GG
GA
GG
G16
71
0.00
ICC
M01
03FI
8566
78(A
)11
AT
GG
GG
GA
AT
CG
GA
GA
CT
AA
GG
AT
AG
GG
AG
GA
GG
GA
AC
AG
110
30.
18
ICC
M01
04F
I856
566
(TT
A)1
1C
CA
AA
CC
TC
CA
AA
AA
TC
TG
CT
CA
TT
TT
TG
AT
TC
AT
TC
TT
GG
G27
88
0.48
ICC
M01
05FI
8565
67(A
T)4
TG
CT
TC
CT
TT
TC
AA
TC
AC
CA
TG
AC
AA
AG
GA
CA
AA
TA
AG
TG
TT
TT
A28
01
0.00
ICC
M01
06FI
8566
81(A
TA
)7T
GG
AA
TT
GC
TA
CC
GA
AT
AT
GG
AC
GA
TC
GG
AG
AG
AA
CG
AG
AA
267
NA
ICC
M01
07a
FI85
6682
(TC
G)4
GC
AA
AA
AG
TG
TT
GC
TT
CC
GT
AA
GC
AC
AT
TG
CC
AC
TA
GC
AT
234
10.
00
ICC
M01
07b
FI85
6682
(TA
)4G
CA
AA
AA
GT
GT
TG
CT
TC
CG
TA
AG
CA
CA
TT
GC
CA
CT
AG
CA
T23
43
0.16
ICC
M01
10FI
8565
68(A
T)4
N(T
TA
)7A
GA
GG
CA
AA
CA
AG
AA
CC
GA
AG
TA
AA
GA
GG
GG
CA
GC
TG
TT
G18
3N
A
ICC
M01
15FI
8567
06(T
C)4
AC
CC
TA
AG
GG
CT
CG
TT
TG
AT
TA
GG
GA
TG
GA
GA
GG
AG
AG
CA
245
10.
00
ICC
M01
16F
I856
709
(T)1
1T
TT
TG
GT
GC
AA
GA
GA
AT
GG
GG
TC
TT
TC
AA
GA
GG
TC
GC
AG
C17
71
0.00
ICC
M01
17FI
8565
72(T
AA
)4A
GT
GA
CC
AG
GA
AA
CA
CG
GT
CG
CA
GA
GA
TT
GA
AT
TT
TG
CC
A25
41
0.00
ICC
M01
18FI
8565
73(T
)11
AA
TT
GG
GA
AG
GA
AA
GC
GA
GT
TC
GC
CA
TT
GC
AA
TA
AT
CA
AA
279
NA
123
1422 Theor Appl Genet (2010) 120:1415–1441
Tab
le2
cont
inue
d
Mar
ker
nam
eG
enB
ank
IDSS
R m
otif
aFo
rwar
d pr
imer
(5�
–3�)
Rev
erse
pri
mer
(5�
–3�)
Pro
duct
si
ze (
bp)
Num
ber
of a
llele
sbP
IC
valu
e
ICC
M01
19FI
8567
10(G
A)4
TT
AT
TG
GA
TG
AG
TG
GA
TG
CG
AT
TA
CG
TA
AC
CC
AA
CG
TC
GC
192
NA
ICC
M01
20a
FI85
6574
(TT
A)1
2T
GT
CT
CG
AT
AA
GA
GT
TT
GT
TA
TT
TT
TC
CG
TT
TT
GT
TT
CA
TA
TT
CA
AA
CT
CG
220
120.
75
ICC
M01
20b
FI85
6574
(AT
T)1
3C
GA
GT
TT
GA
AT
AT
GA
AA
CA
AA
AC
GG
CT
TG
TA
GC
TA
GG
CT
CG
AC
TC
166
50.
29
ICC
M01
21a
FI85
6575
(TA
TT
)4C
AA
AA
AT
TT
GG
AT
TC
GG
GA
GC
TA
TT
GC
AC
CT
GG
GG
AT
AC
G23
82
0.09
ICC
M01
21b
FI85
6575
(A)1
0N(A
TA
)17
AC
GT
AT
CC
CC
AG
GT
GC
AA
TA
AA
CA
GA
TG
TA
GA
AG
GT
AT
AA
TC
CA
TG
A22
41
0.00
ICC
M01
22FI
8565
76(A
AT
)4G
CA
AC
CT
GC
CA
TC
CA
TA
CT
TA
GT
GA
AT
CA
AA
TG
AT
GA
AA
GC
A27
51
0.00
ICC
M01
23a
FI85
6577
(TT
A)1
4G
GA
TG
GT
CT
GC
TG
GA
AT
CA
TA
AA
GA
CA
AC
AA
AA
AG
AC
AA
TC
AT
GT
250
90.
76
ICC
M01
23b
FI85
6577
(TA
A)2
6T
TT
TT
AC
AT
GA
TT
GT
CT
TT
TT
GT
TG
TG
AG
GA
CT
AA
GA
TA
AT
AG
CA
AT
CC
AA
201
NA
ICC
M01
24FI
8565
78(A
AT
)4aa
a(A
AT
)4C
CT
CG
GG
AA
TT
CA
AC
TA
CC
AT
CA
AA
AA
TC
CA
CT
TT
CC
AC
CA
279
50.
16
ICC
M01
25a
FI85
6579
(AA
AT
)4C
GA
TC
TG
AG
GA
TC
AA
CT
TG
TG
AA
CT
AA
CC
AC
CG
TC
GA
CC
AT
C25
3N
A
ICC
M01
25b
FI85
6579
(TT
A)5
TA
TT
TA
TG
GT
CT
GG
TC
CG
GC
CG
CT
AC
CA
AA
TA
TG
GA
AC
GA
CT
251
50.
19
ICC
M01
27FI
8565
81(T
AA
)27
TG
TT
GA
AC
GA
AT
TT
AC
TC
AT
CG
GG
TG
GG
CT
CC
TA
TT
GT
TT
GA
269
60.
40
ICC
M01
28a
FI85
6731
(TA
)4N
(TA
T)4
AA
CC
CT
AA
TT
TA
TT
TG
CA
CT
AT
TA
TC
AT
CA
AA
AT
AC
GG
TA
GT
AG
GA
TA
AG
AT
GA
161
NA
ICC
M01
28b
FI85
6730
(A)1
0A
TT
TG
GA
CG
AT
GT
GT
CG
CT
TT
TA
TA
GC
CC
CT
GC
TT
TG
CT
G26
61
0.00
ICC
M01
30a
FI85
6734
(AA
T)2
2G
GA
TT
TC
GA
CT
TT
TA
TC
CC
TT
TT
TC
GG
AC
TG
GA
AT
CA
AA
AG
CT
C26
83
0.10
ICC
M01
30b
FI85
6734
(AT
T)5
GA
GC
TT
TT
GA
TT
CC
AG
TC
CG
TG
TA
GG
GG
TG
CA
TG
GT
GT
AA
122
10.
00
ICC
M01
31FI
8565
82(A
T)4
N(T
TA
)8A
GA
GC
CA
AA
CA
AG
AA
CC
GA
AG
TA
AA
GA
GG
GG
CA
GC
TG
TT
G19
2N
A
ICC
M01
34FI
8567
48(A
)11
TT
TT
GG
AG
GC
AG
CT
TT
GA
GT
TG
AA
GA
CA
GA
GA
CG
GT
GC
AT
109
30.
08
ICC
M01
38a
FI85
6753
(AG
)4A
TA
AA
TA
GC
CG
GC
CA
CA
AG
AC
CG
AT
TG
TT
TC
AA
GA
CC
AG
A11
91
0.00
ICC
M01
38b
FI85
6752
(AT
A)1
1C
TG
TT
GC
CG
AT
TT
TA
TA
TT
AT
TT
TT
AA
AT
GT
GT
TG
TT
CT
GG
CC
GT
168
NA
ICC
M01
39FI
8567
55(A
TT
C)4
TC
AC
GA
TT
TG
AA
TG
GT
CG
TG
CG
TT
TT
CC
CA
GC
TT
CA
AC
AT
151
NA
ICC
M01
41F
I856
757
(AA
T)2
0A
AG
GT
AT
GA
TG
CA
GT
TC
CC
GG
GG
CG
GA
GG
GT
AA
TT
AT
TG
T24
7N
A
ICC
M01
42a
FI85
6759
(AC
)4G
CA
TT
GC
CA
AT
AT
CG
AA
GG
TA
GT
AG
GT
GC
CA
AA
TG
CA
TC
C20
61
0.00
ICC
M01
42b
FI85
6759
(AT
T)6
GG
AT
GC
AT
TT
GG
CA
CC
TA
CT
GA
GG
TT
GG
TG
TA
GA
AT
AG
AA
TG
GA
137
NA
ICC
M01
42c
FI85
6758
(AC
)7G
GA
GG
TC
CC
GA
GT
CT
AA
AC
CT
CT
TC
AA
GT
CT
GC
TT
GA
CG
TG
T10
51
0.00
ICC
M01
43FI
8567
61(A
C)5
CT
GC
GG
TC
GA
TC
TA
GA
GG
AT
AA
GG
TT
GA
AG
GA
TG
AT
TG
CG
257
NA
ICC
M01
50a
FI85
6583
(AT
TC
)4T
GA
TT
GA
AA
TG
GT
CG
TG
TC
CA
GT
CG
TT
TT
CT
CG
GC
TT
CA
A27
91
0.00
ICC
M01
50b
FI85
6583
(TA
A)8
N(A
T)4
GT
AA
AG
AG
GG
GC
AG
CT
GT
TG
AG
AG
GC
AA
AC
AA
GA
AC
CG
AA
192
50.
16
ICC
M01
52FI
8567
92(T
)13c
(CT
T)4
N(A
G)5
CG
TC
TC
AC
GA
AG
GA
GA
AG
TG
AA
AA
AT
TC
AC
TT
GC
TA
AT
AT
TT
CA
CA
197
10.
00
ICC
M01
54FI
8567
94(A
)11
AG
CT
TG
TT
CG
AC
AG
CA
GG
AT
TC
GA
TG
TG
AA
TA
TG
CC
CT
CT
T24
71
0.00
ICC
M01
55FI
8567
96(A
)11
GA
CG
GC
AG
GA
TT
AA
CT
GC
AT
TT
CG
AT
GT
GA
AT
AT
GC
CC
TC
T24
02
0.04
ICC
M01
56a
FI85
6797
(AT
)7N
(CA
)4T
GC
AT
TC
CC
CT
TT
AA
TT
TG
GC
AG
TG
GT
GG
GG
AG
AC
AA
AA
C28
02
0.04
ICC
M01
56b
FI85
6797
(AT
A)5
2N(A
AT
)6G
TT
TC
TC
CG
TC
CG
CA
CT
TA
TA
AA
CA
GT
GT
AA
AT
TG
TT
GT
TG
GA
AA
276
20.
08
ICC
M01
57F
I856
584
(GA
)8T
TT
GT
TT
TG
AG
GC
AA
CC
AC
AA
AA
CA
AA
CC
CA
GT
GG
GA
GG
T25
81
0.00
123
Theor Appl Genet (2010) 120:1415–1441 1423
Tab
le2
cont
inue
d
Mar
ker
nam
eG
enB
ank
IDS
SR m
otif
aFo
rwar
d pr
imer
(5�
–3�)
Rev
erse
pri
mer
(5�
–3�)
Pro
duct
si
ze (
bp)
Num
ber
of a
llele
sbP
IC
valu
e
ICC
M01
58FI
8565
85(A
TA
)18N
(TC
T)1
6C
CA
AA
AC
TG
AC
AA
GT
CC
CG
TG
GG
AA
AA
TA
TG
AG
GA
AG
TT
TG
C27
51
0.00
ICC
M01
59F
I856
814
(T)1
7N(T
)10
TG
AA
AA
AT
CG
GA
AA
CC
CT
AC
CC
CT
TG
TT
TT
CA
GG
CG
AT
TG
T24
76
0.46
ICC
M01
60FI
8568
16(A
AC
)4N
(TA
A)2
5T
TG
CT
TG
AA
AC
AA
CC
TT
TC
GC
GG
GT
AC
AA
CC
GT
AG
CA
AA
T26
321
0.93
ICC
M01
61a
FI85
6818
(AT
)4G
AT
GG
TC
AT
CG
GT
TC
GA
TT
CA
AC
GA
GG
CC
CT
CT
GT
AA
CG
267
40.
12
ICC
M01
61b
FI85
6817
(TA
A)4
N(A
AT
)4A
CT
GT
CA
GG
AA
GG
AA
CG
GT
GT
CC
GT
AA
CA
AA
AA
TT
GT
GA
AG
AA
A27
93
0.14
ICC
M01
62a
FI85
6586
(AT
T)1
2T
AG
CG
CA
GT
CG
AT
CT
GA
GG
AA
CG
TA
TC
CC
CA
GG
TG
CA
AT
A27
2N
A
ICC
M01
62b
FI85
6586
(AA
AT
)4G
CA
AG
GT
CT
TT
CC
CT
TG
TC
AC
GC
CG
CC
AA
TT
TT
AT
TT
TT
A27
81
0.00
ICC
M01
62c
FI85
6586
(T)1
1N(T
A)4
TA
AA
AA
TA
AA
AT
TG
GC
GG
CG
TC
GT
GT
TA
GG
GT
GT
TT
AG
GG
A26
73
0.21
ICC
M01
62d
FI85
6586
(T)1
0G
AC
TC
TG
CT
GG
GG
AC
AA
TT
TC
GG
GT
TT
AG
AG
AC
CC
AC
TC
A22
51
0.00
ICC
M01
63FI
8568
20(T
C)4
CA
AC
GA
AT
TT
CA
TG
CT
GT
GG
TA
GG
GA
TG
GA
GA
GG
AG
AG
CA
274
10.
00
ICC
M01
65FI
8568
21(T
)11
CG
GA
CG
TA
CA
CC
TT
TC
GT
TC
TG
CT
TC
CG
AA
TA
AC
AT
AA
AG
CA
128
NA
ICC
M01
66a
FI85
6824
(T)1
1G
CC
TA
CT
CG
CG
GA
TT
TT
TA
TC
CC
AG
GT
GC
AA
TA
GG
GA
AA
TC
276
30.
09
ICC
M01
66b
FI85
6824
(AA
AT
)7T
GG
GG
AT
AC
GT
AG
GA
GC
AA
GT
TG
GA
TT
CG
GG
AG
TC
GA
TT
A23
3N
A
ICC
M01
66c
FI85
6824
(AT
)5G
CC
TA
CT
CG
CG
GA
TT
TT
TA
TC
CC
AG
GT
GC
AA
TA
GG
GA
AA
TC
276
20.
04
ICC
M01
66d
FI85
6823
(AT
)4G
AC
TC
CC
CT
AC
CA
CC
TC
AC
AC
GG
AC
GC
GA
CA
AA
AA
CT
AC
275
10.
00
ICC
M01
66e
FI85
6823
(TA
T)4
8A
AT
AA
AA
AT
GC
GG
AA
GT
GG
GT
GT
GA
GG
TG
GT
AG
GG
GA
GT
C27
61
0.00
ICC
M01
67FI
8565
88(A
TA
)48
GA
GT
GT
AC
GG
GG
AT
TA
TA
TG
AT
GA
TC
AA
GA
AA
AG
GA
AC
CA
AG
GC
235
NA
ICC
M01
69FI
8565
89(T
TA
)30
AG
AG
GC
AA
AC
AA
GA
AC
CG
AA
GT
AA
AG
AG
GG
GC
AG
CT
GT
TG
251
NA
ICC
M01
70a
FI85
6839
(TA
)4G
CT
TT
GT
TG
CT
TT
CG
TT
CT
TT
TA
AA
GT
GT
TT
GG
GG
TG
AG
TG
G22
6N
A
ICC
M01
70b
FI85
6839
(C)1
0C
TC
GC
AT
TC
CT
TT
TC
CA
CT
CG
GG
GG
AA
AG
TA
TG
GG
AT
GA
G15
4N
A
ICC
M01
71FI
8565
90(A
TT
C)4
GA
CC
GG
GA
TC
GT
GT
CA
TA
AA
CG
TT
TT
CC
CA
GC
TT
CA
AC
AT
166
10.
00
ICC
M01
72F
I856
841
(AT
)4N
(AT
)4G
CA
GT
CG
AT
CT
GA
GG
AT
CA
AG
TT
CA
CA
AG
AT
GT
TT
TC
AG
AA
CA
AA
G27
8N
A
ICC
M01
74FI
8565
91(A
)11
CA
GC
GA
CC
TC
CT
AC
TG
GG
TA
CA
AA
AA
TG
GA
GG
AT
TT
TT
CC
TT
175
NA
ICC
M01
76FI
8568
47(T
A)4
AT
AG
GC
TA
GA
CC
GT
CC
GA
CA
TC
TG
AA
AT
AT
GA
TG
CA
GC
CG
273
50.
16
ICC
M01
77a
FI85
6849
(AA
T)2
8c(A
TA
)27c
(TA
A)4
CT
TG
AG
TT
CA
AG
CC
AG
AG
AG
GG
CG
TT
AT
TA
CT
GT
TA
CA
AA
TG
GC
A27
91
0.00
ICC
M01
77b
FI85
6848
(TA
T)6
N(T
AT
)11N
(TA
T)4
N(T
TA
)6N
(TA
T)8
CC
CT
TC
TT
CC
AT
TT
CC
GA
AT
GG
GG
AG
GA
GA
AC
GA
AA
AA
GA
273
NA
ICC
M01
78F
I856
592
(AA
T)1
3A
GT
TT
GG
GT
TT
CA
CC
GC
CT
GA
AC
GC
GC
TC
TG
TT
CA
TA
AT
280
110.
83
ICC
M01
79F
I856
850
(TA
T)4
AA
AG
GC
CA
GT
TT
AC
CC
GA
CT
AT
TT
GA
TG
CA
GC
AA
GC
AG
TG
214
NA
ICC
M01
80F
I856
852
(TA
T)4
AG
TC
CC
TG
AT
CT
CC
CG
AA
GT
AT
TT
GA
TG
CA
GC
AA
GC
AG
TG
179
10.
00
ICC
M01
81FI
8565
93(A
TA
)5C
GG
GT
GT
GG
AT
AG
CA
AG
TT
TT
CT
CT
CC
TT
CC
TA
AT
AA
AA
CA
AA
CA
103
10.
00
ICC
M01
83F
I856
595
(TA
T)1
5T
GA
GG
AC
TA
AG
AT
AA
TA
GC
AA
TC
CA
AT
TT
TT
AC
AT
GA
TT
GT
CT
TT
TT
GT
TG
168
10.
00
ICC
M01
85F
I856
597
(T)1
1A
AA
GT
TT
GG
CC
TG
GT
CT
GG
CA
TT
CC
AT
AT
TC
AG
TA
GC
AT
TC
CA
250
60.
46
ICC
M01
87F
I856
599
(TA
T)6
TG
AC
CA
TC
AA
TC
CA
TT
TC
TT
TT
CT
GT
TG
AC
GT
CT
AA
TT
TT
GT
CC
G28
0N
A
123
1424 Theor Appl Genet (2010) 120:1415–1441
Tab
le2
cont
inue
d
Mar
ker
nam
eG
enB
ank
IDSS
R m
otif
aFo
rwar
d pr
imer
(5�
–3�)
Rev
erse
pri
mer
(5�
–3�)
Prod
uct
size
(bp
)N
umbe
r of
alle
lesb
PIC
va
lue
ICC
M01
89FI
8566
01(T
AT
)62
TC
CA
GT
TC
CA
AA
TG
GC
AT
AA
CC
CT
TG
AG
TT
CA
AG
CC
AG
AG
273
30.
21
ICC
M01
90a
FI85
6602
(AT
A)6
N(A
TA
)9G
GG
GG
AT
TG
TC
TG
AG
TT
TC
AA
AA
AG
GC
TG
GA
GA
CA
CC
TC
A19
57
0.53
ICC
M01
90b
FI85
6602
(TA
T)5
TT
TA
TT
GC
AG
GA
AG
CG
GT
TT
CA
CC
AC
TA
TC
AA
AT
GC
CC
CT
273
10.
00
ICC
M01
91FI
8566
03(T
)10
CC
TT
AC
GC
AT
AA
TC
GA
CT
CC
AA
AT
TC
AA
TT
GA
GT
CG
CC
AC
C13
05
0.37
ICC
M01
92a
FI85
6879
(TA
T)1
5c(A
TT
)15
GC
TG
CC
CA
AA
TT
TT
GA
CA
TT
AC
CG
GG
GA
TC
AA
AT
TC
TT
CT
T27
911
0.88
ICC
M01
92b
FI85
6878
(TA
A)1
5tg(
AT
A)1
5C
GG
AC
GG
GG
AT
AA
TT
CT
TC
TG
CT
GC
CC
AA
AT
TT
TG
AC
AT
TA
279
150.
76
ICC
M01
93a
FI85
6881
(TA
A)5
N(T
)10
TG
AA
CT
TT
CA
AA
AC
CA
AA
CC
AA
TT
GT
GA
CA
AT
TT
GA
GG
GT
TC
T26
8N
A
ICC
M01
93b
FI85
6881
(AA
T)7
TC
GA
TT
AT
AG
CT
TT
AT
CT
TT
AC
CC
TT
TA
AA
AG
TG
TT
GG
GA
GG
GG
TT
C24
21
0.00
ICC
M01
94FI
8568
83(A
)10N
(T)1
3C
GA
TT
GT
CC
TA
GT
TT
TA
AA
AA
GA
AA
CG
AC
TT
CC
TG
AA
GG
AA
CG
AA
200
40.
22
ICC
M01
96FI
8566
05(A
TA
)5N
(AG
)6G
TC
GG
GT
GT
GG
AT
AG
CA
AG
TA
AC
AC
AA
TT
CC
TC
AA
AT
AA
CA
AA
CT
154
60.
47
ICC
M01
97a
FI85
6885
(T)1
3C
GC
GT
CT
AG
CA
AA
AC
AA
GA
AT
TC
TC
GG
CC
TA
TA
AA
CA
TC
AA
280
30.
08
ICC
M01
97b
FI85
6884
(TA
TT
)7G
AT
TC
CG
GA
GT
CC
AT
TA
CC
AC
TA
TT
GC
AC
CT
GG
GG
AT
AC
G24
1N
A
ICC
M01
98FI
8568
86(A
)15
CC
AT
CC
GA
GA
AA
AC
TC
GA
AA
CA
AC
GG
TA
TC
CA
TC
GG
AA
TC
163
10.
00
ICC
M01
99a
FI85
6889
(A)1
0g(
AG
AA
)4A
CC
AA
GC
AG
AC
CA
CA
AC
AA
TG
TT
TT
CC
CG
GC
TT
CA
AC
AT
265
10.
00
ICC
M01
99b
FI85
6889
(CA
TT
)5A
CC
AA
GC
AG
AC
CA
CA
AC
AA
TG
TT
TT
CC
CG
GC
TT
CA
AC
AT
265
10.
00
ICC
M01
99c
FI85
6888
(TT
A)1
5T
TA
GA
GG
CA
AA
CC
AG
AA
CC
GA
TC
TT
GA
AG
TG
GG
CA
AA
AC
G24
115
0.86
ICC
M02
00FI
8568
90(T
AA
)4A
CG
GA
GT
GA
CC
AG
GA
AA
CA
CG
CA
GA
CC
TA
CA
GA
AA
CA
GA
GG
AA
231
20.
04
ICC
M02
01FI
8568
92(T
AT
)7(T
TA
TT
G)6
a
(GT
TA
TT
)4A
TA
GA
GA
GA
CC
CA
AA
CC
GC
CG
CC
AA
AG
GC
AA
AA
GA
GA
TT
G13
5N
A
ICC
M02
02a
FI85
6894
(T)1
0N(T
)10
CG
CC
GA
TC
CA
TT
AT
AC
TG
AC
TT
GC
CT
CT
GA
TT
CT
GG
TT
CA
192
20.
04
ICC
M02
02b
FI85
6894
(TT
A)1
3T
GA
AC
CA
GA
AT
CA
GA
GG
CA
AC
CA
AT
TT
GG
TC
CG
GT
TT
TT
A20
712
0.77
ICC
M02
03FI
8566
06(C
T)6
TG
GA
CG
TA
GG
TT
GT
TG
TG
GA
TT
GG
TA
TC
AG
TG
AC
CT
CG
CA
194
10.
00
ICC
M02
04F
I856
607
(TC
)7C
AC
AT
AC
AC
TC
CC
CA
AT
CC
CT
GC
AG
AC
TG
TT
TG
GT
TC
GA
G25
81
0.00
ICC
M02
05FI
8566
08(T
A)9
CG
AC
CA
TG
AT
TC
TC
TG
AT
GT
GC
AC
CT
CT
GC
AT
TT
CT
TC
AA
AC
A26
38
0.26
ICC
M02
07F
I856
610
(TA
)4T
CA
AC
CA
TA
AA
GC
AC
TC
CC
CG
GC
CA
TT
TG
TG
TT
TT
GT
AT
GG
182
20.
04
ICC
M02
10FI
8568
98(T
A)4
N(T
G)4
N(C
CA
)4N
(AG
)16
GA
CC
AT
TG
CC
CA
CT
TC
AA
CT
AG
TT
CT
GC
GA
GA
GG
AA
TG
GA
253
NA
ICC
M02
12a
FI85
6902
(T)1
0N(A
T)4
N(T
A)5
N(T
A)4
TC
CT
AT
AC
CG
AA
AA
CC
CC
AT
TC
AA
AA
TG
GA
TG
GA
TT
GT
GG
G27
02
0.04
ICC
M02
12b
FI85
6901
(GA
)4A
TT
GC
CG
TT
GA
GA
GA
AG
TC
GT
CG
GT
CA
CC
AC
AC
TA
CC
AA
A18
31
0.00
ICC
M02
12c
FI85
6901
(AG
)8T
TT
GG
TA
GT
GT
GG
TG
AC
CG
AA
AC
CC
AA
AA
AC
GT
GG
AC
TC
A16
16
0.32
ICC
M02
14a
FI85
6612
(CT
)6N
(AC
)4C
TC
TT
CA
AT
AG
CC
CC
AT
CC
AC
GT
TG
GA
GA
GG
CT
GA
AA
CA
T24
91
0.00
ICC
M02
14b
FI85
6612
(TT
TA
)5A
TG
TT
TC
AG
CC
TC
TC
CA
AC
GT
CG
CA
CC
TG
AA
CT
CT
CT
GT
G27
6N
A
ICC
M02
15a
FI85
6613
(TC
)5C
CT
TC
AG
TG
TT
TG
GC
TC
AC
AC
TC
CA
GG
AA
TC
CA
CA
GC
AT
T25
33
0.15
ICC
M02
15b
FI85
6613
(T)1
1A
GA
AT
GC
TG
TG
GA
TT
CC
TG
GG
CA
AG
CC
CA
AA
AC
TT
CA
AG
A27
61
0.00
123
Theor Appl Genet (2010) 120:1415–1441 1425
Tab
le2
cont
inue
d
Mar
ker
nam
eG
enB
ank
IDS
SR m
otif
aFo
rwar
d pr
imer
(5�
–3�)
Rev
erse
pri
mer
(5�
–3�)
Prod
uct
size
(bp
)N
umbe
r of
alle
lesb
PIC
va
lue
ICC
M02
16a
FI85
6614
(TG
)4C
GG
GA
CT
TT
CA
TC
TG
CT
GT
TG
TG
GG
AC
AT
CC
TC
CA
AG
AA
A20
02
0.04
ICC
M02
16b
FI85
6614
(AG
)5A
AA
GC
TG
GT
GG
TC
GA
GC
TA
AG
AC
CA
CC
GA
AC
CA
GG
AT
AA
A27
73
0.09
ICC
M02
19a
FI85
6906
(AC
C)4
CC
TT
TT
AA
GG
GC
TG
AA
GG
CT
TG
AA
AG
AA
TG
TG
GG
GG
AG
AG
203
NA
ICC
M02
19b
FI85
6906
(CT
)5T
CA
TC
CT
AC
CC
AA
TT
GC
TC
CG
TA
GT
GG
GG
TA
GG
GG
AT
GG
T24
03
0.10
ICC
M02
19c
FI85
6905
(CT
)4C
TC
AC
CC
AC
CA
CA
CC
TA
TC
CG
CG
AA
GG
GA
GA
GA
AG
GA
AG
T27
8N
A
ICC
M02
19d
FI85
6905
(TC
)5C
TC
AC
CC
AC
CA
CA
CC
TA
TC
CG
CG
AA
GG
GA
GA
GA
AG
GA
AG
T27
8N
A
ICC
M02
20F
I856
908
(TA
)4N
(CT
)4T
CA
AC
CA
TA
AA
GC
AC
TC
CC
CG
GC
CA
TT
TG
TG
TT
TT
GT
AT
GG
183
10.
00
ICC
M02
22FI
8566
17(C
T)5
TC
CG
AT
TG
GA
TT
TT
CA
GG
AC
GG
TA
TC
AG
TG
AC
CT
CG
CC
AT
210
10.
00
ICC
M02
23a
FI85
6912
(TC
T)4
TA
CA
AC
TT
TT
GA
CA
CC
GC
GA
AG
TG
GC
AG
TA
TG
CG
TT
GA
GA
224
NA
ICC
M02
23b
FI85
6911
(TC
)4G
CT
CT
GT
CG
GT
CT
TC
CT
GT
CA
CA
AA
GC
GC
TC
GA
AT
AA
GG
A20
8N
A
ICC
M02
24FI
8569
14(C
T)4
AC
CA
CC
TT
GC
TC
AT
CC
TC
AC
GA
GT
AG
GA
GG
TG
CG
AA
AA
CG
274
160.
74
ICC
M02
25FI
8569
15(C
T)4
AC
GT
CC
GG
AT
TT
GT
TC
TC
AC
AT
TA
TA
GG
AA
GA
TG
GC
GG
GG
252
60.
27
ICC
M02
26a
FI85
6537
(A)1
2C
CA
AC
GA
CG
CG
GA
TA
AA
TA
AA
TG
CC
CA
CC
CA
TA
AT
TT
CA
273
10.
00
ICC
M02
26b
FI85
6537
(TA
)4G
AA
AA
AG
CG
CT
GT
AA
AT
GG
CC
CT
CG
CA
TT
TT
GT
TC
TC
AA
AG
145
10.
00
ICC
M02
28F
I856
619
(CT
)6T
GG
AC
GT
AG
GT
TG
TT
GT
GG
AG
GA
CC
GG
GA
GT
CC
CT
TA
TT
A27
41
0.00
ICC
M02
29F
I856
916
(C)1
0T
GT
CT
TA
TT
CC
TC
CT
CC
CC
CA
GG
GG
TT
TT
TG
GG
TT
AC
CA
G15
89
0.69
ICC
M02
31a
FI85
6919
(TT
A)2
1N(T
C)4
CT
GG
GG
AT
AC
GT
AG
GA
GC
AA
GG
AG
TG
AG
AT
AA
GA
AA
GA
GA
GG
AG
G27
8N
A
ICC
M02
31b
FI85
6919
(TC
)4A
TC
CC
AC
CT
TA
CC
AC
CC
TT
CG
GA
AT
GA
GG
AG
GG
AT
TG
AG
A27
92
0.05
ICC
M02
32FI
8569
20(T
)10
AC
GG
GA
AG
TT
TC
TG
GG
TC
TT
TA
GC
GG
AG
AA
AC
AG
GA
CT
GG
253
30.
09
ICC
M02
33a
FI85
6922
(TA
)4N
(TT
A)4
t(T
TA
)4C
TC
AC
CA
CT
AG
GG
AT
GG
GA
AA
GA
CT
CC
CG
AG
GG
AT
TG
AC
T24
2N
A
ICC
M02
33b
FI85
6922
(A)1
0A
GT
CA
AT
CC
CT
CG
GG
AG
TC
TT
GT
TG
AG
GG
CC
TT
AG
AT
TG
G25
6N
A
ICC
M02
34a
FI85
6925
(TT
A)4
GG
GA
CT
AC
TT
TC
GC
GA
TT
CA
GT
GG
GT
AA
TC
CG
TG
CG
TA
AT
229
10.
00
ICC
M02
34b
FI85
6924
(TA
)4C
AG
GC
TA
TG
TC
AT
CT
CG
TC
GC
CT
GA
CT
GC
CA
CA
AG
TT
TC
A27
01
0.00
ICC
M02
34c
FI85
6924
(TC
G)4
TG
CT
TC
CG
TA
CA
GG
CT
AT
GT
CC
CT
GA
CT
GC
CA
CA
AG
TT
TC
A28
01
0.00
ICC
M02
35a
FI85
6927
(TC
)4T
CG
CT
CA
TG
AC
AG
AC
TC
GA
CG
TA
GC
AG
GT
GA
GA
TG
CA
CG
A17
35
0.32
ICC
M02
35b
FI85
6926
(GT
)4c(
GT
)4G
CC
GA
CC
CG
AT
TA
CC
TT
AC
TG
AG
AG
CT
AA
GG
GG
AA
GG
TG
G17
6N
A
ICC
M02
36a
FI85
6621
(CG
C)7
GC
TT
GT
TG
CC
CT
GT
AT
TG
GT
AA
AA
CG
CA
AG
CA
AA
GC
AA
GT
151
NA
ICC
M02
36b
FI85
6621
(AT
T)4
N(A
)10
AC
TT
GC
TT
TG
CT
TG
CG
TT
TT
TT
TG
TG
GG
TT
GG
TT
GA
TT
TT
219
20.
04
ICC
M02
36c
FI85
6621
(A)1
0N(T
A)4
AA
AA
TC
AA
CC
AA
CC
CA
CA
AA
AT
CA
CT
CT
AC
CG
TC
AA
AA
CG
A24
42
0.04
ICC
M02
37a
FI85
6622
(AT
)4N
(AT
T)6
TC
AA
CC
AT
CC
CT
AA
AA
AC
AT
TT
GT
TC
CT
TT
GC
CA
TT
TA
TG
TT
TT
G18
46
0.34
ICC
M02
37b
FI85
6622
(A)1
3C
AA
AA
CA
TA
AA
TG
GC
AA
AG
GA
AT
CG
TG
TT
GT
AT
TG
TG
CC
GA
T15
52
0.04
ICC
M02
37c
FI85
6622
(AG
)4N
(GA
)4T
GT
GT
TT
CT
CG
AT
GG
CA
GA
GC
TT
TT
TC
TC
CC
TT
TC
CA
CC
A12
01
0.00
ICC
M02
38FI
8566
23(T
C)4
N(T
C)4
AC
CA
TA
GA
CG
AA
CC
AC
CA
CC
CC
AA
GG
GG
GT
AC
AA
CT
TG
TG
T21
51
0.00
ICC
M02
40a
FI85
6650
(TA
)12
AC
CC
GA
AC
CC
GC
AA
AT
AA
TA
GC
AA
TG
AG
AC
TG
GG
GT
TT
TC
248
NA
123
1426 Theor Appl Genet (2010) 120:1415–1441
Tab
le2
cont
inue
d
Mar
ker n
ame
Gen
Ban
k ID
SSR
mot
ifa
For
war
d pr
imer
(5�
–3�)
Rev
erse
pri
mer
(5�
–3�)
Prod
uct
size
(bp
)N
umbe
r of
all
eles
bP
IC
valu
e
ICC
M02
40b
FI8
5665
0(T
A)4
AC
CC
GA
AC
CC
GC
AA
AT
AA
TA
GC
AA
TG
AG
AC
TG
GG
GT
TT
TC
248
190.
77
ICC
M02
42a
FI8
5692
9(A
AT
)18
TG
CA
TT
CA
TC
TG
TT
TC
GC
TC
GA
AA
AT
AT
TT
GT
GG
TT
AT
CC
GA
TT
TT
263
80.
74
ICC
M02
42b
FI8
5692
8(A
)11
AT
CC
GC
AA
CA
CA
AC
AA
AA
CA
CC
CT
AC
TC
GT
AA
TC
GA
CT
CT
CG
252
40.
21
ICC
M02
42c
FI8
5692
8(T
AT
)5C
AA
GT
GC
AA
TA
GG
GA
AA
TC
CA
AT
AG
GG
CT
TT
CC
AC
CG
AT
TT
210
NA
ICC
M02
43a
FI8
5693
1(A
T)5
N(A
T)4
N(A
T)8
TC
AG
GA
AC
AG
AC
GG
AA
CT
TT
TT
GG
GT
TC
AA
AT
CC
TA
TT
GG
GC
277
30.
08
ICC
M02
43b
FI8
5693
1(A
T)4
AT
TT
GC
GC
CC
AA
TA
GG
AT
TT
TT
TT
TC
TA
TC
GG
AA
TA
TC
TC
AT
TT
TC
T28
01
0.00
ICC
M02
43c
FI8
5693
0(G
A)4
1N(A
G)1
0A
CG
AC
GA
TT
CT
GG
AT
TT
TG
GA
GT
TT
TG
GT
AG
GG
GG
TC
GA
G23
716
0.88
ICC
M02
44a
FI8
5693
3(A
T)8
N(T
TA
)4(T
AA
)4N
(TT
A)6
N(A
TT
)4A
TG
CG
GT
AA
TC
CT
TG
AC
TG
GT
GC
AG
GG
AG
TG
AA
TG
TG
TG
T25
25
0.25
ICC
M02
44b
FI8
5693
3(T
A)5
CA
CA
CA
TT
CA
CT
CC
CT
GC
AA
GA
TG
GA
AG
GG
AG
GG
GT
AA
AA
268
NA
ICC
M02
45F
I856
935
(AG
)5G
CG
GC
TG
GT
TT
AA
GA
GT
GA
GC
CA
AC
AC
GA
CC
CA
AA
TC
AA
T18
26
0.53
ICC
M02
46a
FI8
5693
7(A
T)4
TC
TG
AC
AG
CT
CT
TG
CC
TT
GA
AA
CA
CC
CA
GA
CC
CC
TT
TC
AT
280
40.
12
ICC
M02
46b
FI8
5693
7(T
A)4
TG
AA
GA
GG
AA
GA
GA
CG
GG
AG
AA
TC
CA
TT
TA
CG
GG
GG
TA
GC
268
NA
ICC
M02
46c
FI8
5693
7(T
AT
TT
)4T
GA
AG
AG
GA
AG
AG
AC
GG
GA
GA
AT
CC
AT
TT
AC
GG
GG
GT
AG
C26
81
0.00
ICC
M02
46d
FI8
5693
6(T
C)4
GA
TC
AC
GG
TT
AC
GA
AT
GC
AA
TA
AG
GT
TC
CC
AT
TG
GC
TC
TG
209
10.
00
ICC
M02
47F
I856
626
(TT
A)8
CC
TC
AA
TT
CA
TT
TT
TC
TT
CG
GT
TT
CC
CG
AT
AA
AC
CA
TC
TG
TT
136
20.
06
ICC
M02
49F
I856
627
(T)1
2N(T
AA
)29
TT
TC
TT
CG
CA
TG
GG
CT
TA
AC
GG
AG
AT
TT
GT
TG
GG
TA
GG
CT
C19
35
0.16
ICC
M02
50F
I856
940
(TA
T)4
0T
TT
CA
AA
CA
CA
AT
CT
GA
AC
GA
GA
CC
AC
CT
TC
GG
GT
AG
GA
TA
CA
231
20.
04
ICC
M02
51a
FI8
5694
3(A
C)4
TC
CC
TG
CT
AT
AC
AC
CC
AT
CC
TG
GG
CA
TA
TA
TG
GA
TC
AC
GA
252
10.
00
ICC
M02
51b
FI8
5694
3(C
G)4
CT
AC
AC
CC
GC
CA
AC
CT
CT
AC
AA
GT
GT
AT
GT
GA
CC
GA
GC
CC
261
10.
00
ICC
M02
51c
FI8
5694
3(C
A)4
AA
CC
CA
TA
AT
AC
GC
GC
TC
AC
GG
GG
TG
GT
AA
GG
TA
GG
AG
GA
206
20.
08
ICC
M02
52a
FI8
5694
5(C
CT
)4T
CT
AC
CT
CT
CC
GC
TC
TT
CC
AT
GG
TG
AT
AG
GT
GG
TG
GT
TG
A20
31
0.00
ICC
M02
52b
FI8
5694
4(T
)11
TT
GA
CG
GT
GG
GG
GT
AT
AC
AT
TC
CA
CA
CA
CT
CC
CA
CT
AC
CA
267
NA
ICC
M02
53F
I856
946
(AT
)5T
CC
CT
TA
CA
AG
CA
TT
CC
CT
GT
GG
GG
AC
CG
TT
TT
TC
AC
TT
A11
0N
A
ICC
M02
54F
I856
628
(TA
A)4
N(T
AA
)29
GC
CA
AG
CC
CA
TT
AA
AA
CA
CT
CG
TT
GT
TA
AA
AA
CC
GC
GT
TG
273
10.
00
ICC
M02
55F
I856
629
(AT
A)8
N(A
AT
)4N
(AT
)4G
GC
TA
CC
GA
AT
AT
GG
AA
TG
CT
GG
CC
TG
AC
CT
AC
TT
AT
GG
C27
22
0.04
ICC
M02
56a
FI8
5694
9(A
T)4
AC
CG
CT
CA
TT
TC
CA
TA
CG
TC
TG
GA
TC
AA
GA
GG
GA
GG
AT
TG
195
NA
ICC
M02
56b
FI8
5694
8(C
T)4
TT
TC
TC
TT
TT
GG
TG
GT
TG
CC
TT
AA
GG
TT
TG
CC
AC
TC
CT
GG
247
40.
17
ICC
M02
56C
FI8
5694
8(T
TA
)19
TT
AA
GC
CA
GA
CG
TG
GG
AA
AC
AG
AA
AG
AA
GG
GA
AA
TG
GG
GA
236
20.
08
ICC
M02
57F
I856
630
(AT
A)4
4N(A
AT
)11
TC
GC
TT
CC
AA
CA
TT
CA
AA
AA
CA
AT
TG
CA
CT
TA
TA
GC
AC
AA
AC
A25
52
0.04
ICC
M02
58F
I856
950
(AT
A)1
1T
GC
AT
AG
GG
AA
AT
CA
AA
AC
AC
AT
TA
TT
TC
AC
CG
TC
GT
GT
CC
A27
11
0.00
ICC
M02
59F
I856
631
(TT
A)1
5A
GA
GG
CA
AA
CA
AG
AA
CC
GA
AC
GA
AG
CC
GA
GA
AA
AT
GA
CT
C26
12
0.04
ICC
M02
61F
I856
633
(TA
A)2
3G
TC
CG
GG
GA
TT
CA
GT
AG
GA
TC
AA
GC
CA
CG
GA
AC
TT
GT
TT
T23
2N
A
ICC
M02
63a
FI8
5663
5(T
AT
T)7
CG
GG
GA
TA
AA
TC
AA
CA
CA
CC
GG
GC
AA
GG
TC
TT
AC
CC
TT
GT
265
20.
08
123
Theor Appl Genet (2010) 120:1415–1441 1427
Tab
le2
cont
inue
d
Mar
ker
nam
eG
enB
ank
IDS
SR m
otif
aFo
rwar
d pr
imer
(5�
–3�)
Rev
erse
pri
mer
(5�
–3�)
Prod
uct
size
(bp
)N
umbe
r of
all
eles
bP
IC
valu
e
ICC
M02
63b
FI85
6635
(AT
A)1
9G
GT
AG
AA
AA
TA
TT
TA
TG
TG
TT
GA
CC
GC
TC
GT
TC
AC
AT
AC
CG
CC
AT
A26
01
0.00
ICC
M02
65a
FI85
6636
(AT
)5G
GA
AC
TC
GG
GA
TT
GA
AA
TA
GT
CT
TG
CA
AA
GA
AA
AC
AA
TT
TT
AG
GA
214
NA
ICC
M02
65b
FI85
6636
(A)1
3C
GT
TT
AA
TC
CT
AA
AA
TT
GT
TT
TC
TT
TG
AC
GG
CG
AC
AA
CC
AT
TA
AT
TC
190
10.
00
ICC
M02
65c
FI85
6636
(TA
T)9
a(A
TT
)10N
(AT
T)1
1N(A
AT
)4T
AC
CG
CC
AC
GT
TA
CG
TT
TT
TG
AA
AA
TA
TT
TG
TG
TG
TT
GA
CC
GA
248
40.
14
ICC
M02
66FI
8569
55(T
AT
)31
GA
AT
CG
TG
AG
GG
GG
AG
AT
TT
GG
GG
GA
AT
CA
AA
AG
GC
AT
AG
269
NA
ICC
M02
67a
FI85
6637
(TA
TT
)4C
AA
CG
TC
CG
TT
AA
AA
CG
GT
TA
TG
GG
GA
TA
CG
TA
GG
AG
CA
AG
179
10.
00
ICC
M02
67b
FI85
6637
(TT
A)4
N(T
AA
)8N
(AT
A)1
2C
TT
GC
TC
CT
AC
GT
AT
CC
CC
AA
GT
CT
CG
TT
CA
CA
TA
CC
GC
C27
21
0.00
ICC
M02
68FI
8569
57(C
T)7
N(T
C)4
TT
CA
TC
TC
TG
CC
CA
AA
CT
CC
TG
GG
TA
GA
TG
GA
AG
GA
GT
GG
220
10.
00
ICC
M02
69a
FI85
6959
(AC
)4C
CC
TC
TT
TA
CA
CC
CC
AC
CT
TG
TA
GT
GG
AG
TG
GG
GC
AG
GT
A12
92
0.04
ICC
M02
69b
FI85
6959
(TC
)5A
AC
AT
CA
CT
AA
CC
CT
CC
CC
CA
GG
TG
TG
GG
TG
GT
AG
GA
GT
G20
9N
A
ICC
M02
70F
I856
638
(TA
T)1
6T
CA
CA
TA
CC
GC
CA
CA
AT
AC
GA
CG
TA
TC
CC
CA
GG
TG
CA
AT
A27
61
0.00
ICC
M02
71F
I856
639
(GT
)4A
CC
CG
GG
TA
TA
AG
GT
TC
CA
CT
GC
TT
GT
TT
TT
CA
TT
TT
CA
TT
TT
C24
03
0.11
ICC
M02
72a
FI85
6961
(A)1
2T
TT
CC
AC
TT
GG
AA
CA
GG
CT
CA
AT
GG
AC
GA
TG
GT
TG
GG
TT
A28
03
0.12
ICC
M02
72b
FI85
6960
(GA
)10N
(AG
)20
CG
CG
GT
TG
AG
TT
AG
AG
TG
GT
CA
AA
TC
GG
GG
AT
TT
TG
TT
TG
175
120.
74
ICC
M02
72c
FI85
6960
(GA
)4C
GC
GA
TT
AT
TA
CC
CA
CG
TT
TG
GA
AA
GG
AG
GT
AC
CG
GA
GT
C24
93
0.09
ICC
M02
73FI
8569
63(T
GA
)4T
GT
AA
CT
CA
TC
AT
CG
CC
AG
CA
GA
CG
TG
TA
GA
CA
GA
TG
CC
C10
82
0.04
ICC
M02
74F
I856
640
(TC
)9(T
A)1
5G
AC
CC
TA
CC
CC
GC
AA
GT
AA
TT
TT
TT
GT
CC
AC
AC
TC
AC
AC
CT
265
NA
ICC
M02
76F
I856
965
(C)1
0C
TC
CT
AC
AC
TG
CC
TC
CC
CT
CT
CA
TG
CT
TA
CT
CC
GT
TG
CA
G22
2N
A
ICC
M02
77FI
8566
42(T
TA
)11
GG
CA
AA
CA
AT
AA
CC
GA
AA
AC
AG
TA
AA
GA
GG
GC
CA
GC
TG
TT
G19
66
0.30
ICC
M02
78a
FI85
6643
(TT
A)4
AT
AG
GG
GA
CC
AA
AA
CT
GC
AA
GT
GG
GT
AA
TC
CG
TG
CG
TA
AT
203
10.
00
ICC
M02
78b
FI85
6643
(AT
)4A
AA
AT
AC
AC
AT
CC
TG
AC
TG
CC
AT
TT
TG
CT
TA
GA
CT
TG
TA
GG
CA
TT
159
20.
17
ICC
M02
80FI
8569
69(T
)11
AC
TA
GA
TG
GT
CG
CA
TC
CT
GG
GG
TG
AA
GG
TG
TG
GA
TG
AG
GT
280
10.
00
ICC
M02
81a
FI85
6644
(AC
)9T
TC
AA
CC
CT
CC
CT
AC
AC
GT
TG
TT
CT
CT
TC
TT
GC
TG
GT
GC
C23
51
0.00
ICC
M02
81b
FI85
6644
(AA
T)5
N(A
)11
TG
GA
AC
AA
CC
AA
GA
CC
TT
CA
GC
TG
CC
AC
AA
AC
AC
TG
AG
AA
264
20.
04
ICC
M02
82a
FI85
6645
(CG
C)7
CC
TC
GT
TG
TT
GC
CC
TG
TA
TT
AA
AA
CG
CA
AG
CA
AA
GC
AA
GT
154
30.
23
ICC
M02
82b
FI85
6645
(AT
T)4
N(A
)10
AC
TT
GC
TT
TG
CT
TG
CG
TT
TT
TT
TG
TG
GG
TT
GG
TT
GA
AT
TT
T21
9N
A
ICC
M02
82c
FI85
6645
(A)1
0N(T
A)4
AA
AA
TT
CA
AC
CA
AC
CC
AC
AA
AA
TC
AC
TC
TA
CC
GT
CA
AA
AC
GA
250
40.
25
ICC
M02
84a
FI85
6647
(AT
)4C
GT
AT
CT
AC
AC
CC
GC
AC
TC
AT
GG
AA
AA
TC
CA
CT
TT
GA
TT
GG
257
30.
08
ICC
M02
84b
FI85
6647
(TA
)4C
GT
AT
CT
AC
AC
CC
GC
AC
TC
AT
GG
AA
AA
TC
CA
CT
TT
GA
TT
GG
257
90.
30
ICC
M02
85FI
8569
71(A
TT
C)5
TG
AG
GA
CA
AG
AT
TC
CG
TT
CA
AA
CA
TG
CG
GG
TT
GT
TT
TC
TC
267
10.
00
ICC
M02
86a
FI85
6648
(GA
)5A
GC
AT
CA
CG
CA
TA
CA
GC
TT
GA
CA
TT
GG
CT
CC
AT
TG
TT
TG
G25
42
0.04
ICC
M02
86b
FI85
6648
(AG
)4A
CC
CC
CA
AA
AT
GC
TG
TA
GT
GA
CG
CC
CC
TT
TA
CT
GT
TA
CG
A27
61
0.00
123
1428 Theor Appl Genet (2010) 120:1415–1441
Tab
le2
cont
inue
d
Mar
ker
nam
es s
tart
with
preW
x IC
CM
, whi
ch r
epre
sent
IC
RIS
AT
Chi
ckpe
a M
icro
sate
llite
s
NA
not
am
pliW
eda
SSR
mot
ifs
havi
ng “
N”
nucl
eoti
de r
epre
sent
the
inte
rrup
tion
of
few
bas
e pa
irs
betw
een
two
sam
e/diV
eren
t SS
R m
otif
sb
Num
ber
of a
llele
s is
cal
cula
ted
base
d on
scr
eeni
ng 4
8 ch
ickp
ea g
enot
ypes
usi
ng to
uch-
dow
n P
CR
proW
le o
f 61
–51°
C
Mar
ker
nam
eG
enB
ank
IDSS
R m
otif
aFo
rwar
d pr
imer
(5�
–3�)
Rev
erse
pri
mer
(5�
–3�)
Prod
uct
size
(bp
)N
umbe
r of
all
eles
bP
IC
valu
e
ICC
M02
88FI
8569
72(T
AA
)4N
(TT
A)
4N(T
TA
)4T
TA
TT
TT
TC
GG
AT
CC
AA
CG
CG
TG
AT
TT
TT
GT
TC
GG
CC
AT
T27
820
0.88
ICC
M02
89FI
8569
76(T
)13N
(AC
A)4
CA
GC
CT
CC
AT
GG
CA
TA
GA
TA
AT
GC
TT
GA
AT
GA
GT
GC
AA
CA
A21
915
0.83
ICC
M02
90FI
8569
76(A
)15
TT
GT
TG
CA
CT
CA
TT
CA
AG
CA
TT
TT
TA
TT
GG
GG
CA
TT
GA
GC
244
60.
38
ICC
M02
91FI
8569
78(A
T)4
AA
GT
AT
TC
AA
TT
AT
AC
GT
GC
AC
AA
AA
TC
AT
CC
TT
GT
TA
AG
TC
AA
CC
AC
TT
249
10.
00
ICC
M02
92FI
8569
78(T
AA
)6T
GG
TT
GA
CT
TA
AC
AA
GG
AT
GA
GT
GT
CC
TC
AA
GC
AG
AG
GT
GG
TT
C26
71
0.00
ICC
M02
93FI
8569
82(T
AA
)15t
g(A
TA
)15
AG
TG
AT
GC
CA
CG
AG
AA
TT
GC
CT
GG
TT
CG
GA
AT
TG
TC
AT
CC
250
50.
24
ICC
M02
94FI
8569
86(T
TA
)15
AG
AG
GC
AA
AC
AA
GA
AC
CG
AA
CA
CC
CA
AT
TT
TG
TC
CG
AT
TT
185
NA
ICC
M02
95F
I856
987
(T)1
0G
AG
GC
AC
CA
AA
TT
CG
TA
TC
CC
AA
AA
TT
TT
CT
AA
TT
CA
CC
AA
GA
CT
TC
256
30.
09
ICC
M02
96FI
8569
87(T
GA
TT
)4C
GC
CA
AG
TT
TT
AC
TA
TG
TG
CT
GT
GT
CT
GG
AT
GT
TA
CA
TA
AA
CA
CT
CT
T22
71
0.00
ICC
M02
97F
I856
987
(TA
A)1
8C
AT
GA
TT
TG
AT
TT
GA
TT
TG
AT
TT
CG
GA
GT
GG
GA
AA
CC
TT
AA
GC
C27
13
0.08
ICC
M02
98F
I856
989
(TC
)4N
(AA
T)4
GT
GC
AC
TT
GT
TC
AG
CG
TT
GT
CG
CA
AA
CA
CA
CA
TT
CC
TC
TG
221
50.
33
ICC
M02
99F
I856
989
(CT
T)7
N(T
CT
)4T
TA
TG
AA
GC
CG
AA
GC
TC
GT
TG
AG
CA
GT
AA
AC
GT
AC
CC
CC
A27
2N
A
ICC
M03
00FI
8569
90(A
)10
AT
GG
CC
AA
AA
TG
AA
CT
CC
AG
AA
AA
GA
GA
AG
GT
TC
CA
TC
GG
173
20.
10
ICC
M03
01FI
8569
92(A
)10
AT
GG
CC
AA
AA
TG
AA
CT
CC
AG
AA
AA
GA
GA
AG
GT
TC
CA
TC
GG
173
10.
00
123
Theor Appl Genet (2010) 120:1415–1441 1429
Tab
le3
Lis
t of
gene
-bas
ed S
NP
anc
hor
mar
kers
use
d in
com
para
tive
map
ping
of
chic
kpea
and
Med
icag
o
Mar
ker
nam
eT
empl
ate
sequ
ence
ac
cess
ion
no.
Typ
eS
eque
nced
M
t BA
C
acce
ssio
n no
.
Lin
kage
gr
oup
in
M. t
runc
atul
a
Lin
kage
gr
oup
in
chic
kpea
Gen
otyp
ing
met
hod
Res
tric
tion
en
zym
eF
orw
ard
prim
er (
5�–3
�)R
ever
se p
rim
er (
5�–3
�)
CA
LT
LA
W12
6242
Mt/E
STA
C14
9080
14
CA
PS
Alu
IG
TG
GA
AG
GC
AC
CA
TT
GA
TT
GA
CA
AC
TC
TT
CT
TC
TC
AG
CC
TC
TT
CA
AA
TG
C
CD
C2
AW
1717
50M
t/EST
AC
1444
811
4SN
aPsh
otN
/AC
AA
CT
TT
GC
AA
GG
GT
GT
TG
CT
TT
CT
AC
TA
AC
AC
CT
GG
CC
AC
AC
AT
CT
TC
A
Cys
Pr2
AI9
7463
5M
t/EST
AC
1480
981
4C
AP
ST
aq I
CC
AA
AA
AC
TT
GC
TT
CT
AT
AC
TC
TT
CA
TT
CG
AC
AA
AA
CC
CA
CC
CA
GA
CA
AA
TC
AA
CT
AG
HR
IPA
W12
6332
Mt/E
STN
A1
4SN
aPsh
otN
/AG
GA
AA
AA
TT
TA
TC
CT
CC
AA
AT
TG
GG
GG
TA
AA
AA
AT
AG
CA
GT
GC
AC
CA
AA
AA
GT
GC
TG
ppPF
AI9
7468
5M
t/EST
NA
14
SNaP
shot
N/A
TC
TC
GC
CA
CC
AA
CA
AC
AA
CT
AC
AA
AA
AT
TG
TT
CA
TG
AA
CA
CT
CA
CT
TG
AA
GC
CA
RE
PA
A66
0953
Mt/E
STA
C13
3709
14
CA
PS
Taq
IC
TC
CA
TT
TC
CC
GT
TC
GT
TC
GC
AC
CG
GT
TG
CC
CT
CC
AG
AC
RL
3A
I974
458
Mt/E
STA
C12
5473
14
CA
PS
Hin
d II
IT
TC
AA
TC
GA
TA
AG
AA
TA
CT
GC
TT
TG
TG
TG
TG
TC
AT
AC
CA
GC
CT
TG
TC
7670
0T
C76
700
Mt/E
STA
C12
2171
14
CA
PS
Hin
c II
CC
AA
AG
AC
CC
AG
TT
CG
TG
TT
GT
TG
CA
TG
GT
TG
AC
AT
CA
GG
TC
8660
6T
C86
606
Mt/E
STA
C13
9748
14
SNaP
shot
N/A
TG
AA
AA
TG
GC
AA
TG
TG
GA
GA
TT
GG
GA
TT
CC
TC
AT
CC
TC
AG
TC
8727
0T
C87
270
Mt/E
STA
C12
4216
14
CA
PS
Dde
IT
GG
CT
TC
TT
TC
GG
TC
TC
TG
TT
CA
AA
AC
CA
CG
CA
TT
TG
GT
A
TC
8872
7T
C88
727
Mt/E
STA
C13
7895
16
SNaP
shot
N/A
AT
CA
GG
CA
GA
AC
TT
GC
TC
GT
AG
AG
TG
CC
CG
GT
AT
AT
TC
CT
DS
IA
A66
0976
Mt/E
STA
C12
2168
14
SNaP
shot
N/A
GA
AG
CC
AA
AA
AG
TA
TG
AA
GG
GC
CA
CG
CA
CC
AT
GG
TG
CA
TA
AA
AC
TC
AA
CC
AA
GA
CA
TC
Chi
tina
seII
NA
NA
»A
C13
7554
21
PfaV
and
Kah
l (20
03)
AG
CA
CA
TG
AA
CC
AA
TC
CA
CA
CA
TG
GT
TG
CA
AT
TT
CA
CG
TC
AC
CO
AI9
7423
0M
t/EST
AC
1212
362
1C
AP
SS
tu I
GA
AG
AT
GG
CG
CA
AA
AA
GA
AA
GT
CG
AT
GT
GT
CG
TG
TC
AT
CT
CG
TT
AA
GT
TC
CC
T
AJ0
0504
3A
J005
043
Ca/
ES
T»
AC
1271
702
1SN
aPsh
otN
/AT
TT
TC
GG
GA
AC
TG
CT
TA
TG
GC
AT
GG
AA
GT
GG
AC
CG
TT
TT
T
CP
CB
2A
W19
1283
Mt/E
STN
A2
1C
AP
SD
ra I
AG
AA
AG
AG
TG
AA
GT
CT
GT
GG
AT
CT
AC
AT
CG
GA
TG
AA
CA
GC
CA
CA
CA
CC
TA
AT
GT
AA
TC
DM
I-1
AY
4977
71G
ene
AC
1405
502
1C
AP
SH
inc
IIA
CC
CT
TC
TT
TC
CT
TG
GC
AT
TA
CC
GC
AT
AC
AA
CG
CT
AA
AC
C
TC
7748
8T
C77
488
Mt/E
STA
C12
6013
21
CA
PS
AX
III
AT
GC
TT
GC
AG
AT
CC
TG
CT
TT
GA
TC
TG
CC
TC
AA
CT
CC
AA
GC
1433
PA
I974
411
Mt/E
STA
C14
4342
35
SNaP
shot
N/A
AA
GG
TT
TT
CT
AC
CT
TA
AG
AT
GA
AG
GG
AG
GT
TT
AG
CA
AG
AT
TG
CA
GG
CA
CG
A
AF
4575
90A
F45
7590
Ca/
ES
T»
AC
1227
283
5SN
aPsh
otN
/AC
TC
CC
TC
TC
TA
TC
GC
CC
TC
TT
GT
GA
AA
GG
GT
TG
GT
GT
GA
A
AIG
PA
W12
5928
Mt/E
STN
A3
5C
AP
SB
SPW
IC
TG
AT
AG
GG
CC
AG
GA
GG
CA
GG
GA
AG
AG
TT
TT
TT
AG
CA
TT
TG
GA
CG
AA
TG
GT
TG
GT
Cys
Pr1
AI9
7459
5M
t/EST
AC
1310
263
5C
AP
SE
ar I
GA
GA
AT
TC
AA
AG
AA
GA
AA
TT
AA
GA
CA
AA
GA
GA
AG
AA
TT
CA
TG
GG
GA
GC
AA
AG
T
Ms/
U51
5A
J410
128
Ms/
EST
NA
35
SNaP
shot
N/A
GT
TA
AG
GG
AA
CC
AT
GA
CA
AC
CA
CA
CC
AG
GC
TC
GG
AT
TG
AG
CA
GG
GT
TT
GT
Ms/
U83
AJ4
1015
9M
s/E
STN
A3
5SN
aPsh
otN
/AT
GG
AA
AA
CT
TG
AT
GG
AT
CC
TG
CT
CA
TG
AT
AG
TC
CT
GC
AA
CA
AG
TC
CA
GC
A
P40
AJ0
0675
9C
a/cD
NA
»A
C14
3340
35
PfaV
and
Kah
l (20
03)
TT
TT
TA
AA
CG
CC
GC
AA
TG
AC
AG
AA
AG
CA
AT
GG
TG
GG
AA
T
TC
8036
2T
C80
362
Mt/E
STA
C13
7828
35
CA
PS
Ase
IG
CT
TG
AG
CC
TC
CA
GA
GA
TT
GT
GA
TA
AG
CC
TT
GC
AG
TG
TG
C
TR
PT
AA
6603
62M
t/EST
AC
1221
703
5SN
apsh
otN
/AC
AA
CA
AC
CT
AG
TA
TA
GC
GA
TC
AG
TG
CA
TT
CA
AA
GC
CC
AC
CA
AG
TT
DN
AB
PA
I737
524
Mt/E
STA
C12
1244
46
CA
PS
AX
III
CC
CT
AT
GA
GC
TT
GG
GT
TT
GT
CT
CT
CA
TG
GC
AT
AC
GT
GT
TC
AG
C
ES
T94
8A
A66
1051
Mt/E
STA
C14
5021
46
CA
PS
Dde
IG
CA
GG
GG
TT
TC
GC
TC
CA
GT
GA
AC
TT
AA
TG
AA
TG
AT
TG
GA
AG
GT
TT
AG
CG
Ms/
U40
AJ4
1012
0M
s/E
STN
A4
6SN
aPsh
otN
/AA
AG
AA
TG
AG
GA
AG
AG
GA
AT
TC
AA
CA
GG
TT
CC
TA
AG
GA
AC
AA
TG
AT
GA
CA
MT
U07
AI7
3761
0M
t/EST
NA
46
CA
PS
Bsm
IC
AG
AC
AC
CC
AA
AG
AA
TT
AC
CA
GA
AG
AT
GA
CC
AG
AG
CC
TA
AT
AC
TA
TT
TA
TG
AC
T
TC
7875
6T
C78
756
Mt/E
STA
C14
4502
46
CA
PS
Alu
IG
GA
GG
AG
GA
GG
AA
GA
TC
AG
GA
TG
CT
GG
AG
AG
AT
GG
TG
AG
C
TC
7972
6T
C79
726
Mt/E
STA
C13
0805
46
CA
PS
Cla
IT
GC
TG
AA
GG
GA
GG
AT
TC
AA
GA
CA
GC
AT
TG
TG
AG
CA
AC
CA
C
TC
8859
8T
C88
598
Mt/E
STA
C13
5316
46
CA
PS
Bcl
IA
GA
TG
GG
GA
GA
TT
GA
TG
CT
GA
AG
CA
AC
AT
GC
AG
TG
AG
CT
G
TG
DH
AA
6607
42M
t/EST
NA
46
SNaP
shot
N/A
CG
GT
GG
CT
TC
AT
CG
GT
TC
TG
AC
GT
GT
AT
TG
TA
AT
CA
GC
AG
GA
GT
A
tRA
LS
AW
1262
82M
t/EST
AC
1254
764
6C
AP
SM
sp I
GG
TC
TG
CG
AG
CT
GT
TT
TT
GG
AG
AA
GG
CA
AT
TC
CC
TC
CT
CA
GC
TA
AA
AG
TG
123
1430 Theor Appl Genet (2010) 120:1415–1441
Tab
le3
cont
inue
d
Mar
ker
nam
eT
empl
ate
sequ
ence
ac
cess
ion
no.
Typ
eS
eque
nced
M
t BA
C
acce
ssio
n no
.
Lin
kage
gr
oup
in
M. t
runc
atul
a
Lin
kage
gr
oup
in
chic
kpea
Gen
otyp
ing
met
hod
Res
tric
tion
enzy
me
For
war
d pr
imer
(5�
–3�)
Rev
erse
pri
mer
(5�
–3�)
U71
AJ4
1015
0M
s/E
ST
NA
58
SN
aPsh
otN
/AG
CT
GA
AG
CT
GA
AG
GT
TT
TC
GC
GC
AT
TT
TT
AT
GG
AT
GA
AG
AG
A
X60
755
X60
755
Ca/
ES
T»
AC
1376
695
8S
NaP
shot
N/A
AG
GT
GC
CA
TA
GG
AA
GA
CA
CG
CC
CA
AT
CT
TT
TT
CT
CC
CA
CA
AB
0250
02A
B02
5002
Ca/
ES
T»
AC
1227
275
2C
AP
SE
coR
VT
TC
CT
CG
AT
CA
TG
TC
GA
AC
TT
CG
TG
CA
CC
AG
CT
TC
GT
AG
TA
AJ0
0504
1A
J005
041
Ca/
ES
T»
AC
1227
275
2C
AP
SD
ra I
AG
CG
TC
TA
GC
CA
GC
AT
CA
AT
CC
AT
GG
CT
TC
TT
AC
CC
TT
CA
AJ4
0464
0A
J404
640
Ca/
ES
T»
AC
1314
555
2C
AP
SPs
t IT
GG
AG
AG
AA
TG
AG
GG
GA
AT
GT
CA
AA
GG
AT
GC
CA
AA
TC
AC
A
CY
SK
AW
2079
85M
t/E
ST
AC
1353
205
8S
NaP
shot
N/A
GG
AA
TT
GC
TA
AA
GA
TG
TT
AC
AG
AA
TT
GA
AA
TG
AG
GA
CA
CT
CT
GT
CC
AG
GT
GT
GA
CY
SS
AW
1271
54M
t/E
ST
AC
1353
205
8C
AP
SB
gl I
IC
TG
AT
GC
AG
AA
GA
GA
AG
GG
GC
TT
AT
CA
CC
AA
TT
CA
GC
TC
CA
AA
AG
CT
AA
TA
GA
AT
GA
DK
242R
AQ
9172
11M
t/B
ES
NA
52
CA
PS
Bsm
A I
CG
TA
TG
TT
TA
AT
CC
GT
TA
GT
CC
GT
CT
TG
CT
TG
CT
TA
GA
TA
TT
TG
GC
AC
TT
CA
FE
NR
AW
1275
93M
t/E
ST
AC
1380
105
8C
AP
SH
ph I
AT
GC
TT
AT
GC
CA
AA
AG
AT
CC
AA
AT
GC
CT
CA
CA
GC
AA
AG
TC
GA
GC
CT
GA
AG
T
Ms/
U39
3A
J410
119
Ms/
ES
TN
A5
2S
NaP
shot
N/A
AT
TG
GA
GA
AG
GG
CA
AT
CC
TC
CA
CC
AT
CC
CT
TC
AT
TC
TA
TC
CA
TC
CC
AA
GA
Ms/
U89
AJ4
1016
4M
s/E
ST
NA
58
CA
PS
Eco
R V
CA
TA
TG
AA
CA
GT
TG
AA
AG
TG
GA
TG
AC
AA
TA
TG
GC
CA
AC
AT
TA
AA
AA
AT
GG
CA
TC
MO
AW
1275
21M
t/E
ST
AC
1419
235
5C
AP
SH
ph I
GT
CT
AC
GG
CG
AA
CA
TT
GG
CG
TA
AA
AT
GC
CA
AT
TG
CA
GC
AA
CA
TT
GA
TG
TT
CT
CA
AC
A
TC
8736
9T
C87
369
Mt/
ES
TA
C12
4591
62
CA
PS
Pvu
IA
TA
GT
GG
AC
TT
CG
GC
GA
GA
AT
GA
CG
GG
GA
TC
TT
TT
CT
TT
G
AG
TA
W12
6002
Mt/
ES
TN
A7
3C
AP
SX
ba I
GA
TT
TG
GG
CC
TC
AT
TC
CT
TC
TT
GT
GT
GC
AC
CT
GA
AG
GG
GG
AA
AA
TT
GC
CC
AC
AT
TG
A
AJ0
0491
7A
J004
917
Ca/
ES
T»
AC
1365
057
3C
AP
SD
de I
CC
AA
GG
AA
AC
CA
GT
GG
AT
GT
GC
AG
CA
TC
AA
GA
TC
AC
GA
AA
AJ0
1273
9A
J012
739
Ca/
ES
T»
AC
1308
017
3C
AP
SD
ra I
CT
TC
AG
TC
AG
GA
GG
GA
GA
CG
TG
CA
AA
TT
TC
GC
TA
CA
AG
GA
AJ2
9181
6A
J291
816
Ca/
ES
T»
AC
1400
257
3S
NaP
shot
N/A
TT
GG
AG
GT
GC
TG
GT
GA
TG
TA
TG
CA
AA
TG
CT
TG
CC
AA
AT
AG
DK
225L
AQ
9171
91M
t/E
ST
AC
1379
947
3C
AP
SH
inc
IIT
GT
CC
TT
GC
TT
CT
TA
TC
CT
TC
CT
TC
AA
GC
AG
CA
CA
AC
AA
CT
TA
CA
AC
AA
CT
C
EN
OL
AA
6605
34M
t/E
ST
NA
73
CA
PS
Dde
IT
TC
CA
TC
AA
GG
CC
CG
TC
AG
AT
TG
CA
CC
AA
CC
CC
AT
TC
AT
T
MS/
U38
0A
J410
118
Ms/
ES
TN
A7
3C
AP
ST
aq I
CA
CT
CA
TT
GC
AA
TT
TC
CA
TG
CT
TC
AC
AG
TT
GT
TG
TA
GC
AA
GG
GC
AC
A
RN
AH
AI9
7450
3M
t/E
ST
AC
1238
997
3C
AP
SM
bo I
IG
CT
TC
CA
CC
AG
CT
GA
TA
CA
CG
TT
AG
CC
CT
AG
CA
AG
AA
TG
TC
AC
TG
TC
7688
1T
C76
881
Mt/
ES
TA
C13
6505
73
CA
PS
Hph
IT
TC
TT
GG
GG
AA
CA
GA
AC
AG
GG
CA
GC
AT
CA
AG
AT
CA
CG
AA
A
TC
7863
8T
C78
638
Mt/
ES
TA
C13
5795
73
CA
PS
Acc
IT
GA
TT
GC
AA
AG
CA
GG
TT
GA
GC
GG
CA
TT
CA
GT
AG
GA
AG
CT
C
TC
8443
1T
C84
431
Mt/
ES
TA
C13
9355
73
CA
PS
Nsi
IA
GA
AA
GG
AC
TT
GG
GC
AA
CC
TC
CA
AT
AC
GT
GC
CT
CT
TT
GG
T
TC
8621
2T
C86
212
Mt/
ES
TA
C12
4963
73
CA
PS
Hin
c II
GT
GG
CT
CC
TT
GT
GT
GC
TG
TA
AG
CC
AG
GA
TG
AG
GC
AC
TC
TA
TC
8851
2T
C88
512
Mt/
ES
TA
C13
6974
73
SN
aPsh
otN
/AC
TA
CT
GT
TG
GA
CG
GG
TT
GC
TA
AC
CT
GG
GG
CA
TT
TG
TG
TA
A
TC
8872
6T
C88
726
Mt/
ES
TA
C12
1242
73
SN
aPsh
otN
/AG
AG
GA
GA
AT
AC
GC
CA
TC
CA
AG
GA
TA
GT
GT
GG
GC
TG
CA
TC
T
AJ2
7627
0A
J276
270
Ca/
ES
T»
AC
1227
268
7S
NaP
shot
N/A
GC
TG
CG
AA
TT
TT
GA
CC
TA
GC
TG
CC
TT
TG
CA
GA
TT
CA
GT
TG
AJ2
7627
5A
J276
275
Ca/
ES
T»
AC
1400
328
7S
NaP
shot
N/A
TT
TT
TG
CC
TG
GA
CC
AA
AT
TC
AA
GG
CT
TG
TA
CT
GC
CC
TT
GA
AJ4
8961
4A
J489
614
Ca/
ES
T»
AC
1380
878
7S
NaP
shot
N/A
CG
AG
GA
AG
AA
TG
GC
AA
AG
AG
AA
CA
AG
CC
AA
TG
AG
GG
AG
TG
CoA
-OA
I974
546
Mt/
ES
TA
C11
9408
87
CA
PS
Nla
III
TT
TG
GG
GG
AA
AT
AA
TG
GA
AG
TC
TC
TC
GG
GC
AA
TG
TT
GA
AA
AA
TC
CP
OX
2A
W12
7442
Mt/
ES
TN
A8
7S
NaP
shot
N/A
AA
AG
AA
TC
AT
GT
CC
TC
AA
GC
TG
CT
TC
TT
CT
TG
TG
GA
AG
TC
AG
CA
ES
T67
1A
A66
0779
Mt/
ES
TN
A8
7C
AP
SH
pa I
IG
GT
GT
TA
TC
TA
TG
AA
AG
AG
GC
CT
CA
TT
GG
TG
TC
CT
TG
GG
TA
TG
TA
GA
AA
AG
CC
TT
CA
C
FIS
-1A
I974
522
Mt/
ES
TA
C12
2726
87
SN
aPsh
otN
/AT
CA
GT
GA
TT
GA
GG
GT
TT
TT
CT
AC
GC
TG
TT
TC
AT
CA
AC
TT
CA
GC
AA
CT
TT
Ms/
U82
AJ4
1015
8M
s/E
ST
NA
87
SN
aPsh
otN
/AT
TC
GT
CA
AC
GA
GA
TG
TT
TG
TG
TT
GG
CC
AA
AT
TG
AA
GA
AA
AG
AG
GA
AT
GA
AG
GT
123
Theor Appl Genet (2010) 120:1415–1441 1431
PCR was carried out in 5 �l reaction volume in GeneAmp®
PCR System 9700 thermal cycler (Applied Biosystems,Foster City, CA, USA). The reaction mixture containedWnal concentration of 5 ng/�l of template DNA, 0.5 mMdNTPs, 0.5 �M of M13 tailed forward, 1 �M of reverseprimer, 1 �M of M13 labeled primer, 0.75 mM of MgCl2,0.1 U of Taq DNA polymerase (AmpliTaq Gold), and 1£PCR buVer (AmpliTaq Gold). An initial denaturation wasgiven for 15 min at 94°C. Subsequently, ten touch-downPCR cycles comprising of 94°C for 20 s, 61/60/55°C(depending on the marker as given in Table 2, ESMTable 1) for 20 s, and 72°C for 30 s were performed. Thesecycles were followed by 35 cycles of 94°C for 10 s withconstant annealing temperature of 54/56/48°C (dependingon marker and touch-down proWles as given in Table 2,ESM Table 1) for 20 s, and 72°C for 30 s, and a Wnal exten-sion was carried out at 72°C for 20 min. The ampliWedproducts were separated by capillary electrophoresis usingABI PRISM® 3700 DNA analyzer, and allele calling wascarried out as given in Varshney et al. (2009b).
For SNP genotyping, in CAPS assay, 1.5 �l PCR prod-uct of 94 RILs was digested with the corresponding restric-tion enzymes. Each digestion reaction contained 2–5 U ofthe corresponding restriction enzyme and 1£ compatiblebuVer in a total volume of 10 �l. Enzyme digestions wereincubated at the appropriate temperature for at least 4 h.Digestion products were separated and scored as mentionedin Choi et al. (2004a). In case of SNaPshot assay, the ABISNaPshot Multiplexing Kits was used following the sameprotocol as suggested by the manufacturer, except that0.5 �l SNaPshot mix for a single marker was used (seeChoi et al. 2004a).
Polymorphism assessment of SSR markers
While ICCM-series markers were screened on the panel of48 diverse genotypes including the parents of the inter-spe-ciWc mapping population (Table 1), the H-series markerswere screened on only two parental genotypes (ICC 4958and PI 489777). Allelic data obtained for the SSR markerswere subjected to AlleloBin program (http://www.icrisat.org/gt-bt/download_allelobin.htm) for allele calling basedon the repeat units of SSR motif for corresponding markers.In case of ICCM markers, the binned allelic data were usedto calculate polymorphic information content (PIC) valueof the markers by using the PowerMarker V3.25 program(http://statgen.ncsu.edu/powermarker/).
Linkage analysis and map construction
Genotyping data for both ICCM- and H-series polymorphicmarkers were generated on 131 recombinant inbred lines(RILs) of the mapping population and for 94 RILs in caseT
able
3co
ntin
ued
“»”
deno
tes
that
the
puta
tive
ort
holo
gs o
f th
e ch
ickp
ea g
enes
are
loca
ted
in th
e co
rres
pond
ing
M. t
runc
atul
a se
quen
ces
(Gen
bank
acc
essi
on n
umbe
rs).
TC
#’s
are
from
TIG
R g
ene
inde
x da
taba
se (
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runc
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Gen
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ES
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AC
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nkno
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stN
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GT
GT
GA
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AA
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GG
CA
GC
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TC
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CG
CC
AA
AG
GA
CT
CA
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A
123
1432 Theor Appl Genet (2010) 120:1415–1441
of gene-based SNP markers. In addition, marker genotyp-ing data for 407 marker loci were compiled (Huettel et al.2002; PfaV and Kahl 2003; Tekeoglu et al. 2000; Winteret al. 1999, 2000).
Marker genotyping data were analyzed using the �2 testto assess the goodness-of-Wt to the expected 1:1 segregationratio for each marker. Subsequently, genotyping data for allthe markers, including those with distorted segregation,were used for linkage analysis using MAPMAKER/EXP3.0 (Lander et al. 1987). Marker loci were Wrst divided intolinkage groups at a LOD score of 16 and a recombinationfraction of 0.37 by two-point analysis using the ‘group’command. Marker order in the linkage groups was deter-mined using the multi-point analysis ‘try’ command of theprogram. Most likely order of the loci within the group wasdetermined using multipoint ‘compare’ command. Theungrouped marker loci were also attempted to integrate intogenetic map at a smaller LOD value (up to 6). The map dis-tances were calculated by applying the ‘Kosambi’ mappingfunction (Kosambi 1944) as per MAPMAKER/EXP 3.0program. Residual heterozygosity was not considered inlinkage mapping.
Results
Isolation and characterization of simple sequence repeats
A genomic DNA library composed of ca. 400,000 cloneswas constructed from the ICC 4958 genotype. Hybridiza-tion of this library with GA and TAA oligo probes yielded359 clones that were sequenced and assembled into a set of115 contig and 342 singleton DNA sequences, which werefer to as genome survey sequences (GSS). Thesesequences were submitted to National Centre for Biotech-nology Information (NCBI) and respective GenBank acces-sions are mentioned in Table 2.
Two hundred and ninety-nine of the 457 GSSs weredetermined to contain a total of 643 SSRs, with 165 GSSscontaining more than one SSR. As depicted in Fig. 2, di-and tri-nucleotide repeats were the most abundant (39 and40%, respectively), with mono-nucleotide and tetra-nucleo-tide repeats representing 16 and 3% of cases, respectively.Other types of SSRs had <1% representation. In terms ofrepeat motifs, the tri-nucleotide repeat motif TAA/ATTwas most common, accounting 36.8% of all repeat, fol-lowed by the di-nucleotide repeat GA/CT at 19.2%.
These SSR loci were categorized into two groups basedon the length of their SSR tracts: Class I SSRs (>20 nucleo-tides in length) and Class II containing SSRs (>12 but <20nucleotides in length) (Fig. 3). Considering only perfectSSRs, which is the set of SSRs that contain a single motif(e.g., TAA), we observed uneven distribution between
Classes I and II. In particular, the longer Class I SSRs weresubstantially enriched for tri-nucleotide repeats, which rep-resented 77% of all Class I repeats. A similar uneven distri-bution was noted for other repeats, but most notably thepenta-nucleotide repeats, which comprised 55% of all ClassII repeats and less than 2% of all Class I repeats.
Similarity analysis was performed for all 457 GSSsusing BLASTN and BLASTX algorithms, and signiWcantsimilarity was determined at an Expect value threshold of·1E–05 (Table 4). Relatively few of the GSS sequenceshad E values that surpassed this score, irrespective of thespecies data set under analysis. This is consistent with theexpectation that randomly selected short genomicsequences only occasionally correspond to gene coding
Fig. 2 Frequency of microsatellites based on type of repeat motifs inmicrosatellite-enriched library of chickpea. Frequency of tri-nucleo-tide repeats were higher among the chickpea microsatellite markersfollowed by di-nucleotide repeats. N, mono-nucleotide repeats; NN,di-nucleotide repeats; NNN, tri-nucleotide repeats; NNNN, tetra-nucleotide repeats; NNNNN, penta-nucleotide repeats, NNNNNN,hexa-nucleotide repeats
200
250
300
0
50
100
150
Nu
mb
er o
f S
SR
s
N NN NNN NNNN NNNNN NNNNNN
Type of microsatellites
Fig. 3 Distribution of Class I and Class II repeats in newly isolatedchickpea microsatellites. Class I microsatellites are with >20 nucleo-tides in length and Class II repeats contain perfect SSRs with >12 but<20 nucleotides in length. Among Class I repeats, tri-nucleotiderepeats were abundant followed by di-nucleotide repeats, while inClass II repeats, penta-nucleotide repeats contributed highest, followedby hexa-repeats. N, mono-nucleotide repeats; NN, di-nucleotiderepeats; NNN, tri-nucleotide repeats; NNNN, tetra-nucleotide repeats;NNNNN, penta-nucleotide repeats, NNNNNN, hexa-nucleotiderepeats
70%
80%
90%
100%
NNNNNN
NNNNN
20%
30%
40%
50%
60% NNNN
NNN
NN
N
0%
10%
Class I Class II
123
Theor Appl Genet (2010) 120:1415–1441 1433
regions that will match EST data sets. Nevertheless, incases where BLAST hits with e-value lower than 1E–05threshold were recorded, the degree of similarity, expressedas either nucleotide identity of deduced protein similarity,was highest for phylogenetically related species, decreasingin rank order of phylogenetic distance (i.e., Medicago >lotus > soybean = cowpea = common bean > poplar >Arabidopsis > rice). Among these sequences, 40 were iden-tiWed as related sequences in all three analyzed cool seasonlegumes, i.e., chickpea, Medicago, and Lotus (Hologaleginaclade; see Fig. 1), while 29 sequences had similarity withall three analyzed warm season legumes, i.e., soybean,common bean, and cowpea (Phaseoleae clade). Only 21sequences were identiWed as similar sequences in bothHologalegina and Phaseoleae species. Two of these GSSs(FI856609 and FI856659) showed signiWcant similaritywith sequences of all the plant species analyzed in thepresent study (see ESM Table 2).
With the objective of annotating these newly isolatedGSSs, all 457 GSSs were analyzed for BLASTX analysisusing UniProt database. 137 of these GSSs (29.9%) showedhomology to the UniProt database at a relatively relaxedcutoV value of · 1E–05. Among these, 84 unique proteinsequences were used for deriving respective gene ontology(GO) (see ESM Table 3). The GO studies permitted assign-ment of 64 sequences to biological process, 64 to cellularcomponent, and 67 to molecular function ontologies.According to the GO schema, single proteins typically havemore than one Ontology assignment.
Development of novel SSR genetic markers
All SSR containing GSSs (299) were analyzed by means ofPrimer3, yielding a list of potential oligonucleotide primersfrom which 311 primer pairs were selected and synthesized.Where feasible primer pairs were designed for more thanone SSR in a single GSS with the goal of increasing theconversion of GSSs into useable genetic markers.
Primer pairs were screened for ampliWcation of DNAfrom two chickpea genotypes, i.e., ICC 4958 and ICC 1882(Table 2). This analysis provided a set of 234 markers(75%) with scorable amplicons. Screening of these 234markers on 48 genotypes of chickpea further deWned a sub-set of 147 polymorphic markers (62.82%), with allele con-tent ranging from 2 to 21 and an average of Wve alleles permarker. Among these 147 polymorphic sites, 56 were poly-morphic exclusively in wild species, 8 were polymorphicexclusively in cultivated and 83 of them were polymorphicacross wild and cultivated species of chickpea.
We refer to these new polymorphic SSR markers asICCM (ICRISAT Chickpea Microsatellite) markers. Allelicdata obtained from 48 genotypes were used to calculate thePIC value of each ICCM marker, and thus infer the dis-criminatory power of these ICCM markers. PIC valuesranged from 0.04 to 0.92 with an average of 0.26. Twenty-six markers displayed the minimum PIC value of 0.04 each,while marker ICCM0160 had both the highest PIC value(0.92) and the highest number of alleles (21), followed bymarker ICCM0022 with 18 alleles and a PIC value of 0.89
Table 4 Functional annotation of ICCM sequences with EST databases
The database were downloaded from NCBI in May–June, 2008
BLASTN, nucleotide BLAST; BLASTX, protein BLAST; EST, expressed sequence tags; Ca, Cicer arietinum; Mt, Medicago truncatula; Lj, Lotusjaponicus; Pv, Phaseolus vulgaris; Vu, Vigna unguiculata; Gm, Glycine max; Ah, Arachis hypogaea; At, Arabidopsis thaliana; Os, Oryza sativa;Pa, Populus alba
BLAST algorithm
Database Number of entries in database searched
Number of sequences showingsimilarity
Number of sequences withsigniWcant similarity (<1E–05)
Percentage of sequences with expected values <1E–05
Median expected values
BLASTN Ca_EST 7,097 450 26 5.69 2E–60
Mt_EST 249,625 449 76 16.63 5E–22
Lj_EST 158,135 449 48 10.50 1E–16
Pv_EST 83,448 449 35 7.66 4E–26
Vu_EST 183,757 440 49 10.72 1E–14
Gm_EST 880,561 440 73 15.97 3E–14
Ah_EST 41,489 227 14 3.06 6E–15
At_EST 1,527,298 444 44 9.63 1E–11
Os_EST 1,220,877 285 20 4.38 2E–19
Pa_EST 418,223 452 48 10.50 1E–12
BLASTX Uniprot 385,721 409 137 29.98 3E–12
123
1434 Theor Appl Genet (2010) 120:1415–1441
(Table 2). As has been observed in previous studies ofSSRs from plant species (Temnykh et al. 2001), Class ISSRs (41 of 57) were on average more polymorphic thatClass II SSRs (106 of 177), with mean PIC values of 0.38and 0.22, respectively. Nevertheless, a higher fraction ofthe polymorphic SSRs identiWed in this study were fromClass II (106) compared to Class I (41), owing to theincreased abundance of Class II SSRs in our data set. Con-sistent with their overall abundance in Class I SSRs(Fig. 3), tri-nucleotide repeats (20) constituted major part ofthe Class I polymorphic sites, with compound repeats (18)comprising the next largest fraction of Class I ICCM mark-ers. In contrast, di-nucleotide repeats were relatively rare inthe total Class II data set, but comprised the largest fractionof polymorphic Class II ICCM markers (47); similar toClass I markers, compound repeats (30) constituted of thesecond most common fraction of Class II polymorphicsites.
In addition to the ICCM markers developed in this study,we also analyzed a set of 233 markers developed primarilyby Lichtenzveig et al. (2005); these are the so-called “H-series” SSR markers. One-hundred Wfty-three H-seriesmarkers yielded scorable amplicons in two PCR proWles(ESM Table 1). Both the ICCM and H-series SSR markerswere tested for polymorphism between chickpea ICC 4958and PI 489777, the parents of the inter-speciWc mappingpopulation. From this analysis we identiWed 104 SSRs (52ICCM and 52 H-series) that were suitable as genetic mark-ers in the inter-speciWc cross, with polymorphism rates of33.9 and 22.2% for the H-series and ICCM SSR markers,respectively.
Development of gene-based SNP markers
A set of 246 gene-speciWc primers, developed earlier byChoi et al. (2004a) based on gene sequences of M. trunca-tula and M. sativa, were used to amplify DNA of the paren-tal genotypes of the inter-speciWc mapping population ofchickpea. One-hundred four (»42%) of these primer pairsshowed strong single fragments on 1% agarose gels; theseamplicons were re-sequenced in both mapping parents ofthe inter-speciWc cross (ICC 4958 and PI 489777), quality-scored, and trimmed to yield 96 pairs of high qualitysequences. Additional 25 primer pairs were designed basedon chickpea EST sequences that possessed high similarityto previously mapped Medicago genes, yielding 18 addi-tional high-quality sequence pairs. Alignment of the 114ICC 4958 and PI 489777 sequence pairs revealed SNPs in80 (»70%) genes. Seventy-one of these genes containedSNPs that could be converted to reliable genotyping assaysusing either CAPS or SNaPshot protocols (Table 3). Twoadditional gene-based markers, P40 and chitinase II, werealso used for genetic analysis; these genes were previously
mapped in chickpea by PfaV and Kahl (2003), while theirputative orthologs have been mapped in M. truncatula byChoi et al. (2004a).
Construction and features of the genetic map
The inter-speciWc cross between ICC 4958 £ PI 489777 ismaintained as an advanced recombinant inbred populationthat has been used in numerous genetic studies (Huettelet al. 2002; PfaV and Kahl 2003; Winter et al. 2000).Although the number of markers previously analyzed inthis population is relatively large (407 loci), a high percent-age of the markers are anonymous sequences (e.g., RFLP)and/or exhibit dominant patterns of inheritance (e.g.,AFLP). Thus, in many cases, these legacy genetic maps arebased on molecular markers that are either diYcult to applyor to reproduce. With the intent of extending this geneticmap, and enhancing the number of easily scorable markers,we genotyped the 123 new molecular markers (52 ICCMSSR loci and 71 gene-based SNP loci) and 52 previouslypublished H-series SSR loci described above, and com-bined the genotype data with that of the 407 previouslypublished loci. Linkage relationships were evaluated usingMAPMAKER/EXP 3.0.
As shown in Fig. 4, 47 (90.3%) of 52 ICCM marker loci,46 (88.4%) of 52 H-series SSR loci, all (100%) of 71 gene-based marker loci, and 357 (87.7%) of 407 legacy markerloci coalesced to yield eight linkage groups, in agreementwith eight chickpea chromosomes. The linkage groupswere numbered according to Winter et al. (2000), usingmarker loci that were common to both studies. This revisedgenetic map contains 521 marker loci, with an averageinter-marker distance of 4.99 cM and spanning 2,602.1 cM.Considering the 740-Mbp physical size of the chickpeagenome (Arumuganathan and Earle 1991), and ignoring thefact that rates can vary widely within the genome, 1 cM dis-tance in the present map equates to roughly 285 kbp. Withthe exception of linkage group (LG) 8, which has relativelyfew genetic markers (25 markers), the average number ofmarkers per linkage group was 71 § 8.9. LG 8 was also theshortest linkage group based on genetic distance, spanning124.7 cM; however, in general LG size was not well corre-lated with the number of markers. As described below,comparative mapping with Medicago truncatula revealedthat the entirety of chickpea LG8 corresponds to one arm ofMedicago Chr5, adding further credibility to its assignmentas a physically short linkage group.
Comparative linkage analysis between Medicago and chickpea genomes
As shown in Fig. 5, the 71 gene-based SNP markers aredistributed among eight major linkage groups of chickpea,
123
Theor Appl Genet (2010) 120:1415–1441 1435
facilitating comparison of genome structure betweenM. truncatula and chickpea. The respective M. truncatulaand chickpea LGs are numbered according to Choi et al.(2004a) and Winter et al. (2000). Alignment of conservedgenes between the two genetic maps reveals a high level ofsynteny between the two genomes. In particular, theM. truncatula linkage groups 1, 2, 3, 4, 7, and 8 correspondto chickpea linkage groups 4, 1, 5, 6, 3, and 7, respectively.Despite the overall high level of synteny between these sixpairs of linkage groups, intra-chromosomal segment rear-rangements reduce co-linearity (but not synteny) betweenM. truncatula LG1 (MtLG1) and chickpea LG4 (CaLG4).In contrast to the conserved synteny noted for Mt–Calinkage group pairs 1–4, 2–1, 3–5, 4–6, 7–3, and 8–7,
one-to-one relationships do not hold true for M. truncatulalinkage groups 5 and 6 and chickpea linkage groups 2 and8. In particular, M. truncatula LG5 can be aligned withboth chickpea LG2 and LG8. We note that CaLG8 appearsto be derived entirely from one arm of MtLG5, consistentwith its short genetic distance and small number of geneticmarkers, described above. In several cases, conservedmarkers mapped to non-syntenic positions between the twogenomes (e.g., CDC2 and TC88727 on MtLG1, DNABP onMtLG4, and TCMO on MtLG5), which may reXect translo-cation or duplication events involving single genes or smallchromosomal segments, or the mapped loci may corre-spond to paralogous genes. Mt-LG6 could not be eVectivelyaligned to any of the chickpea linkage groups (Fig. 5),
Fig. 4 An integrated genetic map of chickpea based on recombinantinbred lines of C. arietinum (ICC 4958) £ C. reticulatum (PI 489777).Map was constructed using MAPMAKER/EXP 3.0 with Kosambimapping function. Distances between the loci (in cM) are shown to theleft of the linkage group and all the loci are at the right side of the map.Newly developed SSR markers developed from microsatellite-
enriched library (ICCM-series) are bold and italicized; SSR markerstaken from Lichtenzveig et al. (2005) are bold, italicized, and under-lined; SNP markers which were used as the anchor markers in compar-ative mapping of chickpea and Medicago were depicted as bold andunderlined. Linkage groups (LGs) are designated according to the mapof Winter et al. (2000)
LG1 LG2 LG3 LG4
123
1436 Theor Appl Genet (2010) 120:1415–1441
consistent previous reports describing Mt LG6 as rich inheterochromatin (Kulikova et al. 2001) and having a rela-tively low content of transcribed genes (Choi et al. 2004a).
Comparison of resistance gene homologs (RGH) between Medicago and chickpea
The majority of functionally characterized disease resis-tance (R) genes encode a nucleotide-binding site (NBS) anda leucine-rich repeat (LRR) region (Hulbert et al. 2001).NBS-LRR genes have been deeply surveyed and character-ized in M. truncatula (Zhu et al. 2002; Ameline-Torregrosaet al. 2008), with >330 NBS-LRR genes having knowngenetic positions. In contrast, chickpea RGHs are not thor-oughly surveyed, and only a limited number of sequencesfrom degenerate PCR are available in the public databases(Meyers et al. 1999; Huettel et al. 2002). Nevertheless,several phylogenetically distinct RGH classes have beenplaced on the genetic map of chickpea (Huettel et al. 2002),thus facilitating the comparative genome analysis presentedhere.
Comparative phylogenetic analysis of RGH sequencesfrom M. truncatula with those from chickpea is illustrated
in Fig. 6. To highlight the comparison, only those M. trun-catula sequences that are relevant to the mapped chickpeasequences are shown. In the TIR-NBS-LRR subfamily,chickpea RGH-G (CAC86496 on CaLG6; Huettel et al.2002) is highly similar to several M. truncatula TIR-NBS-LRR genes located on MtLG4, in a region syntenic to CicerLG6 that also contains chickpea RGH-G (Huettel et al.2002). Similarly, chickpea RGH-B (CAC86491; Huettelet al. 2002) is a CC-NBS-LRR gene that is closely relatedto several CC-NBS-LRR genes located in a cluster at thetop of MtLG3, in a region of the Medicago genome syn-tenic with the terminus of CaLG5 that contains RGH-B(Huettel et al. 2002). A lack of synteny was observed forchickpea RGH-D (TIR-NBS-LRRs represented bysequences CAC86454, CAC86455, CAC86493, AF186626,and AF186629; Huettel et al. 2002), which is located at thetop of CaLG2; the closest homologs of RGH-D in M. trun-catula (i.e., BAC AC144658) are localized to the distalregion of MtLG4. We note that the bottom of CaLG2 har-bors numerous active resistance genes against two of themost important diseases of chickpea (Fusarium wilt andAscochyta blight). At present, no RGHs have been reportedmapped close to these resistance phenotypes (Winter et al.
Fig. 4 continued
LG5 LG6 LG7 LG8
123
Theor Appl Genet (2010) 120:1415–1441 1437
Fig. 5 Comparative map of Medicago and chickpea. Gene-based SNPmarkers (marked in red color) were used as the anchor markers in com-parative analysis of chickpea and Medicago genome. The resistancegene homologs (RGH) are depicted as oval structures and their homo-
logs in Medicago are shown with connecting dotted lines. Solid linesshow the macrosynteny observed across chickpea and Medicago withrespect to 71 gene-based markers
Ca-LG1
DK238RAC127170
DMI1
DK490L
Ca-LG4Mt-LG1*CDC2TC87270
CysPr2STMS24
TS72003D10
CysPr2TC87270
ppPF
TC76700
NCAS
R-15J11LGA11
STMS21
GAA40
ACCO
Mt-LG2
DMI1ACCO
SAMS
AC137554TC77488DK293R
ppPF
STMS26TA2TR20
GA2
TA130GAA47
REP
CALTL
RL3DSI
005E08
TC86606HRIP
CALTL
RL3
*CDC2
DSI
REPTC88727
ACCO
C32
AJ005043
TC77488
PGKI
CPCB2Chitinase II
TR43TA1
TA113
H2A08
Ca-LG3Mt-LG7
Ca-LG6
Mt-LG4
Ca-LG5Mt-LG3
REP
TC76700
HRIPTC86606
STMS13CPCB2
TC80362AC146744
STMS8
TRPT
R-18D24DK501R
R-13B3R
TS35
*TCMOP40AC131249
AC145027AC142396
TA14
TR44
TR1
*DNABP
TGDHTE016
R 43I21L
AC144658TC84431
DK322L
TC88512MS/U380
AC140025
GA31
TS19
TR56
AF457590
GAA42
TS43TA5
1433P
MS/U83STMS22
STMS20
TC80362
DK201R
GSb
DK417L
AC1227281433P
AC143340
TRPT
RG
H-B TA80
TA176
TC79726
TC88598tRALS
TC78756
MTU07EST948
TGDHR-43I21L
DK379L
TGDHTC79726*DNABP
TC78756
TC88598
tRALS
MTU07AC144502
DK322L
TC88726
RNAHAC136505
TC78638
ENOLTC86212
AGT
TA64
AGTTC78638
TC76881AJ004917
TR56
TC84431
TC88726
TC88512
MS/U380AJ291816
MS/L591RNAH
Ca-LG7Mt-LG8
MS/U515
STMS20AIGP
CysPr1
AC122728
MS/U515
MS/U83
AIGPCysPr1
STMS2
GA34
TA106GA9
MS/U40
*TC88727
RG
H-G
EST948MTU07
MS/U40
AC144502AC135160
ENOL
UNK21AC130801
TA76
TR26ENOLTC86212
TA64
AJ012739
AJ276275
Mt-LG6 Ca-LG2 Ca-LG8Mt-LG5
TA18
TAA58
TA180
MS/U82
R-AW257033
DK455L
R-40H12L
NTRBI
SDP1
CDC16
AC140032
MS/U82 TA78
AC137669
Ms/U89
TC87369 TA194
DK242
*AJ004960
TC87369 RG
H-D
TA110
AB025002
AAMCTT02CYSKMS/U89GAA46
TS45
FENR
SHMTDK242
TE019DK012L
LB1
FENR
CYSK
Ms/U71X60755
Ms/U71
TS12
EST671
COA0AJ489614AJ276270FIS-1
STMS6
AC138087AC122726
MRS
EST671COA-0
FIS-1
MS/U82
CPOX2
CPOX2
TA78
H2E13
AC122727
AC131455
R-36N1L
R-18A5RR-3F12R
004G07DK321L
003E11
AJ005041
AJ404640
MS/U393
TA96TA27
GA16
LB1
DK139L
DK355L
*TCMO
TS12
123
1438 Theor Appl Genet (2010) 120:1415–1441
2000; PfaV and Kahl 2003; Sharma et al. 2004). Moreover,the low frequency of comparative molecular markersaround these R gene regions in both M. truncatula andchickpea complicate precise statements regarding the rela-tionship of these genome regions.
Discussion
SSR markers have become common place for plant genet-ics and breeding applications. Despite the fact that hun-dreds of SSR markers have been identiWed and tested inchickpea (Hüttel et al. 1999; Sethy et al. 2006a, 2006b;Winter et al. 1999; Lichtenzveig et al. 2005), the narrowgenetic background of cultivated chickpea germplasm haslimited their application, and thus there exists a need todevelop a larger set of novel genetic markers. With theobjective of enriching the marker repertoire of chickpea, we
have contributed novel SSR markers derived from a geno-mic library enriched for GA and TAA repeat motifs and aset of gene-based SNP markers. The basis of our markerdiscovery work was C. arietinum genotype ICC 4958,which is being used as a reference genotype for genomicand genetic resource by the chickpea community.
In the present study, 65.4% of hybridizing genomicclones in our SSR-enriched library yielded 643 SSRs. Thisrate of SSR recovery is comparable with previous studies,for example in peanut where 68% of hybridizing clonesyielded SSRs (Cuc et al. 2008). Moreover, the relativelyhigh abundance of tri- and di-nucleotide repeats that weobserved is consistent with previous studies in chickpea(Hüttel et al. 1999; Lichtenzveig et al. 2005; Winter et al.1999). Among the SSRs identiWed here, the most commonSSR motifs were TAA/ATT repeats and GA/CT repeats.This result reXects the fact that our enrichment targetedTAA and GA motifs, and it is consistent with previous
Fig. 6 Comparison of RGH sequences in Medicago and chickpea. Tohighlight the comparison between the chickpea and Medicago RGHs,only those Medicago sequences that are relevant to the mapped chick-pea sequences have been shown in this Wgure. In the TIR-NBS-LRRsubfamily, chickpea RGH-G (CAC86496 on Ca-LG6) was found highlysimilar to several Medicago TIR-NBS-LRR genes (a) located on BAC
clones AC144502 and AC135160. AC144502 and AC135160 wereclosely linked on Mt-LG4, in a region syntenic to Ca-LG6 thatalso contained chickpea RGH-G. In the CC-NBS-LRR (b) subfamily(Ca-LG5), chickpea RGH-B (CAC86491) was closely related to severalCC-NBS-LRR genes located on Medicago BAC clones AC145027,AC142396, AC130810, AC146744, and AC131249
C CAC86491Mt-AC146744.1Mt-AC146744.2Mt-AC130810.1Mt-AC142396.1
Mt-AC145027.3Mt-AC145027.4Mt-AC145027.2
Mt-AC145027.1Ca-AF186628
Ca-CAC86494Ca-CAC86490
Ca_AF186623
Ca-CAC86495Ca-CAC86457Ca-CAC86456
Mt-AC131249.1Mt-AC131249.2Ca-CAC86491
Ca-CAC86455AC143338.3
Ca-AF186624Ca-AF186626Ca-CAC86454
Ca-CAC86493Ca-AF186629
AC144658.1.2AC144658.2.2
AC144658.3.1AC144658.5
AC144658.4.2Ca-AF186627
Ca-CAC86452
AC135160.1Ca-CAC86496
AC135160.2AC144502.1AC144502.4
a TIR-NBS-LRR b CC-NBS-LRR
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studies in chickpea (Hüttel et al. 1999; Lichtenzveig et al.2005; Winter et al. 1999), other legume species (Akkayaet al. 1992; Cregan et al. 1994; Mun et al. 2006), and even incereal species (Varshney et al. 2002; Jayashree et al. 2006).
Temnykh et al. (2001) developed a scheme to classifySSRs according to length, in which Class I and Class II SSRsare greater than or less than 20 bp, respectively. This divisionbased on sequence length has practical utility, because ClassI SSRs are generally more polymorphic and thus more desir-able as genetic markers. The majority of SSRs isolated fromour SSR-enriched library belong to Class II, though asexpected the Class I SSRs had higher rates of polymorphism.A useful measure of polymorphic potential for any geneticmarker is its polymorphism information content value, orPIC value. PIC values provide information on the probabilitythat a given marker will be polymorphic between any twoindividuals in a population, and thus are a function both ofallele frequencies and allele number. Screening of the ICCM-series markers on 48 genotypes revealed that average PICvalue of SSR markers having Class I repeats (0.38) washigher than that of Class II repeats (0.22). The majority of theClass I repeats were tri-nucleotide repeats, consistent withthe known utility of tri-nucleotide repeats as genetic markersin plants (Varshney et al. 2005).
Polymorphic information content value was also ana-lyzed in relation to repeat unit type and length. Among di-,tri-, and tetra-nucleotide repeats, tri-nucleotide repeatsshowed higher polymorphism (average PIC = 0.33) withaverage allele number of 5.7 per marker. Markers withmono-nucleotide repeats showed the least polymorphism(average PIC = 0.197). Relatively longer repeats appear tohave contributed to the higher level of polymorphism ascompared to di-nucleotide repeats (Gupta and Varshney2000). It was also observed that among tri-nucleotide SSRs,the SSR markers based on (TAA/TTA) repeat motifs dis-played higher polymorphism (average PIC = 0.35) with anaverage allele number of 6.12 per marker. Similarly, amongdi-nucleotide repeats SSR markers based on TA/AT repeatmotifs had a higher average PIC value (0.27) compared toothers with an average of 6.1 alleles. In fact, the earlierstudies in chickpea also revealed the abundance of TAA/TTA (tri-nucleotide) and TA/GA (di-nucleotide) SSRmotifs and the extensive polymorphism found with markerscontaining these repeat motifs (Hüttel et al. 1999; Lichtenz-veig et al. 2005). PIC values of compound SSRs (averagePIC = 0.29) were comparable with tri-nucleotide repeatswith 5.68 alleles per marker. This can be attributed to thefact that the markers with compound SSRs have more thanone SSR motif, which increases their chances to be poly-morphic markers.
We assessed the potential identity of SSR-relatedsequences by performing BLAST analyses versus plantEST data sets, and based on GeneOntology analysis
through UniProt. Less than one-third of the SSR-associatedGSS sequences had signiWcant hits in these databases,though were hits were recorded the derived annotations adda potentially useful data type to the marker metadata. Notsurprisingly, chickpea GSS sequences (from which theSSRs were derived) had higher similarity to ESTs fromother legume species, and overall higher similarity to dicotoutgroups (i.e., poplar and Arabidopsis) than to monocot(i.e., rice) data sets.
Comprehensive genetic map of chickpea
An inter-speciWc mapping population derived from ICC4958 (C. arietinum) and PI 489777 (C. reticulatum) wasused to incorporate novel microsatellite and gene basedmarkers. This mapping population has been widely used inpast by chickpea community in order to incorporate severalhundred microsatellite markers (Winter et al. 2000) andgene-based markers (PfaV and Kahl 2003). The diversegenetic background of the parents provides for higher ratesof polymorphism not only at the genetic level but also atphenotypic levels such as resistance to Fusarium wilt(Winter et al. 2000) and Ascochyta blight (Rakshit et al.2003), facilitating trait mapping. Therefore, this populationis generally considered as the international referencemapping population.
The present genetic map of chickpea represents 521marker loci, spanning 2,602 cM with an average inter-marker distance of 4.99 cM. The order of common markerloci deWned in present map agrees with earlier reports fromWinter et al. (2000). However, the current map diVers con-siderably from that of Winter et al. (2000) in having eightlinkage groups, in agreement with eight chromosomes,whereas the Winter et al. (2000) map was composed of 16linkage groups. There are probably at least two factors thatcontribute to this condensation of linkage groups: Wrst, thenew markers identiWed in the present study act as bridgepoints between the Winter et al. linkage groups, and sec-ond, essentially all of the markers mapped in the currentstudy behave a co-dominant genetic features, which addsconsiderable power to the genetic evaluation compared to ahigh fraction of dominant markers in earlier studies. Impor-tantly, the comparative analyses to Medicago support asimple assignment of eight chickpea linkage groups to eightchromosomes.
Comparative mapping of chickpea and Medicago
Mappig of the gene-based markers from Medicago in thegenetic map of chickpea showed not only a high level ofmacrosynteny but also revealed features of structural diver-gence between the two genomes. Six of the eight linkagegroups display a one-to-one correspondence between the
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Medicago and chickpea, suggesting that these linkagegroups reXect the genome of the common Galegoid cladelegume ancestor. Medicago LG5 and LG6, and chickpeaLG2 and LG4, appear to have a more complicated ancestry,consisting of a minimum of several chromosomal translo-cation events. Thus, Mt-LG 5 is essentially a composite ofportions of LG2 and LG8 of chickpea. Several researchgroups have compared genome structure between Medi-cago and various crop legumes (see Zhu et al. 2005). Ourcurrent results extend the comparative network to includechickpea, by demonstrating broad conservation of genomemacrostructure between chickpea and Medicago.
One goal of comparative genetic analyses is to transferinformation from well-characterized reference species toless well-characterized crops with an eye toward cropimprovement. Among the agronomic targets in chickpea isresistance to several economically important pathogens;candidate genes for disease resistance are the conservedfamily of NBS-LRR resistance gene homologs (RGH). Sev-eral phylogenetically distinct RGH classes have been placedon the genetic map of chickpea (Huettel et al. 2002), thusfacilitating the comparative genome analysis between chick-pea and Medicago. In particular, we have documented twocases of syntenic NBS-LRR clusters that contain co-phyleticgenes in each species. Interestingly, Ca-LG2 is known toharbor active resistance genes against Fusarium wilt andAscochyta blight. At present, no RGHs have been reportedmapped close to these resistance phenotypes. Nevertheless,the facts that a single conserved gene (TC87369) maps to thetop terminal region of both Mt-LG6 and Ca-LG2, and thatboth linkage groups are rich in NBS-LRR genes and/oractive disease resistance genes (Sharma et al. 2004; Zhuet al. 2002), may suggest shared ancestry of Mt-LG6 andCa-LG2, though such speculation needs to be veriWed bymore detailed study of the respective genome regions.
Similar observations of NBS-LRR synteny have beenmade for resistance gene homologs within the Solanaceae(Grube et al. 2000) and between Medicago and pea (Pisumsativum) (Zhu et al. 2002). However, the limited numbersof comparative molecular markers (gene-based SNPs)around these R gene regions in both Medicago and chick-pea precludes precise statements regarding the relationshipof these genome regions. Although the current analysis isbased on a relatively small number of comparative markers,the potential of more detailed analyses to predict gene con-tent and chromosomal structure in chickpea by reference toMedicago seems clear.
Conclusion
A set of 311 novel microsatellite markers were developedfrom microsatellite-enriched library in order to increase the
genomic resources in chickpea. In total 147 potential SSRmarker loci were found based on diversity pattern of SSRloci on a panel of 48 diverse chickpea genotypes. Thesemarkers should have utility for genetic analysis of a rangeof chickpea mapping populations and as anchor markers incomparative mapping to other legumes.
Acknowledgments Thanks are due to Prathima Juvvadi, GudipatiSrivani and Abdul Gafoor for their technical assistance. Financial sup-port from Tropical Legume I of Generation Challenge Program (GCP,http://www.generationcp.org) of CGIAR and National Fund of IndianCouncil of Agricultural Research of Government of India are grate-fully acknowledged. The Wnancial support by National Science Foun-dation Grant 0110206 to DRC is acknowledged. Council of ScientiWcand Industrial Research (CSIR), Government of India is acknowledgedfor providing fellowship to SNN and the Indian Council of ResearchNATP-HRD program for providing fellowship to SD. We thankDr. Fred Muehlbauer of the USDA-ARS for providing the chickpearecombinant inbred population and genetic marker data.
Open Access This article is distributed under the terms of the Crea-tive Commons Attribution Noncommercial License which permits anynoncommercial use, distribution, and reproduction in any medium,provided the original author(s) and source are credited.
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