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. 2010 Jan 23;120(7):1415–1441. doi: 10.1007/s00122-010-1265-1

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 1,2, Hongyan Zhu 3,4, Nicy Varghese 1, Subhojit Datta 3,5, Hong-Kyu Choi 3,6, Ralf Horres 7, Ruth Jüngling 7, Jagbir Singh 1,8, P B Kavi Kishor 2, S Sivaramakrishnan 8, Dave A Hoisington 1, Günter Kahl 7,9, Peter Winter 9, Douglas R Cook 3, Rajeev K Varshney 1,10,✉
PMCID: PMC2854349  PMID: 20098978

Abstract

This study presents the development and mapping of simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers in chickpea. The mapping population is based on an inter-specific cross between domesticated and non-domesticated genotypes of chickpea (Cicer arietinum ICC 4958 × C. reticulatum PI 489777). This same population has been the focus of previous studies, permitting integration of new and legacy genetic markers into a single genetic map. We report a set of 311 novel SSR markers (designated ICCM—ICRISAT chickpea microsatellite), obtained from an SSR-enriched genomic library of ICC 4958. Screening of these SSR markers on a diverse panel of 48 chickpea accessions provided 147 polymorphic markers with 2–21 alleles and polymorphic information content value 0.04–0.92. Fifty-two of these markers were polymorphic between parental genotypes of the inter-specific population. We also analyzed 233 previously published (H-series) SSR markers that provided another set of 52 polymorphic markers. An additional 71 gene-based SNP markers were developed from transcript sequences that are highly conserved between chickpea and its near relative Medicago truncatula. By using these three approaches, 175 new marker loci along with 407 previously reported marker loci were integrated to yield an improved genetic map of chickpea. The integrated map contains 521 loci organized into eight linkage groups that span 2,602 cM, with an average inter-marker distance of 4.99 cM. Gene-based markers provide anchor points for comparing the genomes of Medicago and chickpea, and reveal extended synteny between these two species. The combined set of genetic markers and their integration into an improved genetic map should facilitate chickpea genetics and breeding, as well as translational studies between chickpea and Medicago.

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.

Introduction

Chickpea (Cicer arietinum L.) is an annual, self-pollinated diploid (2n = 2x = 16) species with a relatively small genome of 740 Mbp (Arumuganathan and Earle 1991). Chickpea is also the World’s third most widely grown food legume. Over 95% of chickpea production area and consumption occur in developing countries, with India contributing 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). Cytogenetic and seed protein analyses are consistent with C. reticulatum as the wild progenitor of domesticated C. arietinum, with southeastern Turkey as the presumed center of origin (Ladizinsky and Adler 1976). Cultivated chickpea is composed of two genetically distinct sub-types that are readily distinguished based on seed size and color: Desi, composed of small, brown seeded varieties, and Kabuli, composed of large, cream seeded varieties. Due to relatively low rates of polymorphism between cultivated chickpea accessions, inter-specific crosses between C. arietinum and C. reticulatum have been the primary focus for genetic studies of agronomic traits (see Singh et al. 2008).

A diverse array of technologies is available to identify and monitor DNA polymorphism and as a consequence molecular markers are now routinely used in the breeding programs of several crop species (Varshney et al. 2006, 2007). In the case of chickpea, molecular markers reported in 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; Winter et al. 1999). Despite considerable effort, low rates of both intra- and inter-specific polymorphism have limited the number of these SSR markers that have been integrated into chickpea genetic maps. A primary goal of the current study was to screen additional molecular markers and thereby enhance the marker density of chickpea genetic maps.

Chickpea is a close relative of the model legume system Medicago truncatula (Fig. 1, reproduced according to Choi et al. 2004b), and thus should benefit from the increasingly detailed description of the structure and function of the M. truncatula genome (Cannon et al. 2006). A pressing task for chickpea researchers is to use knowledge gained from the study of reference legumes, such as M. truncatula, Lotus japonicus, and soybean, to advance genetic improvement of chickpea. Comparative genomics based on orthologous genetic markers offers means to bridge model and crop legumes. Alignment of linkage maps and sequenced orthologous regions between several legume species has revealed an extensive network of macro- and micro-synteny between legume species; importantly, genomic and genetic comparisons of orthologous nodulation genes in several legumes suggests that comparison of genome structure 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 capacity) in chickpea, however, has limited the potential for cross-genome comparisons and has hampered progress in the area of genomics-assisted breeding (Varshney et al. 2009a).

Fig. 1.

Fig. 1

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Phylogenetic relationships: Papilionoideae family. This figure (taken from Choi et al. 2004b) illustrates the phylogenetic relation of chickpea with other legumes. Chickpea and Medicago belong to inverted repeat lacking clade (IRLC) of Papilionoideae and constituted the cool season legumes

In view of the above, we sought to enhance marker repertoire and density of genetic maps in chickpea using a combination of several molecular marker sets. We developed two novel sets of molecular markers based on an SSR-enriched genomic DNA library and gene-based single nucleotide polymorphism (SNP) markers derived from comparison of Medicago and chickpea ESTs. These novel genetic markers were analyzed together with published genetic markers to develop a dense genetic map of chickpea. We anticipate that these resources will serve as tools for genomics-assisted breeding in chickpea, and enhance prospects for transfer of knowledge about the structure and function of the Medicago genome to chickpea with a final objective of chickpea improvement.

Materials and methods

Plant material and DNA extraction

The cultivated chickpea germplasm line ICC 4958, belonging to Desi type and a parent line of inter-specific reference mapping population (C. arietinum ICC 4958 × C. reticulatum PI 489777) was used to construct microsatellite-enriched library. While two genotypes of chickpea (ICC 4958 and ICC 1882) were used to optimize the polymerase chain reaction (PCR) conditions for newly developed SSR markers, an array of 48 genotypes which includes 33 genotypes from cultivated chickpea (C. arietinum) and 15 from wild species of chickpea (7 genotypes from C. reticulatum, 2 genotypes from C. echinospermum and one each from C. bijugum, C. cuneatum, C. judaicum, C. microphyllum, C. pinnatifidum, and C. yamashitae) was used to assess the polymorphism potential of new set of SSR markers (Table 1).

Table 1.

List of 48 chickpea genotypes used for calculating polymorphic 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. pinnatifidum 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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For integrating the markers into the genetic map, the inter-specific reference mapping population ICC 4958 × PI 489777 comprising of a total of 131 RILs was used. While all 131 RILs were used to score genotyping data for the SSR markers isolated in this study as well as reported by Lichtenzveig et al. (2005), a subset of 94 RILs was used with gene-based markers.

Total genomic DNA was extracted by employing the standardized high throughput mini DNA extraction protocol (as mentioned in Cuc et al. 2008). The quality and quantification of extracted DNA was checked on 1.2% agarose. The DNA was normalized to 5 ng/μl for further use.

Construction of SSR-enriched library

To construct a size-fractioned chickpea genomic DNA library, purified genomic DNA (100 μg) of ICC 4958 was completely digested with MboI or Sau3AI in combination with TaqI enzyme. The restricted fragments were separated on low-melting agarose gels, and the gel zone containing the fragments of DNA of size 800–1200 bp were excised and ligated into Promega pGEM 3Z(f) vector (Promega, Madison, WI, USA). The vector was transformed into E. coli Sure strain—DH10B (Stratagene, Heidelberg, Germany) by electroporation. Approximately 400,000 clones were plated at a density of 20,000 colonies per plate. The masterplates generated were replica-plated on positively charged PVDF macroarrays. Macroarrays were printed using 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-labeled with digoxigenated oligonucleotides (DIG Oligonucleotide 3′-End Labeling Kit; Roche, Mannheim, Germany). Subsequently, macroarrays/filters were hybridized with above-mentioned oligo-probes in Roti-Hybri-Quick buffer (Carl Roth GmbH, Karlsruhe, Germany) including 10 μg/ml sheared, denatured E. coli DNA to minimize non-specific binding. Filters were hybridized at 55°C overnight and washed three times each for 10 min in 1:2, 1:5, and 1:10 dilutions of the hybridization buffer at 60°C. The digoxigen was detected in a “direct detection assay” performed with the DIG Wash and Block buffer set, and DIG Luminescent detection Kit (Roche, Mannheim, Germany) for chemiluminescent detection with a monospecific antibody coupled to alkaline phosphatase in the presence of CSPD. Filters were exposed to X-ray films (Amersham, Buckinghamshire, UK) with intensifying screens for 4 h or overnight, and the colonies giving strong signals were scraped from the master plates; re-grown; spotted on Hybond N membranes (Amersham, Buckinghamshire, England) to fix the DNA by lysis. Hybridization and chemiluminescent detection was done repeatedly to pick the clones with positive signals. These clones were grown on LB agar plates with ampicillin (100 μg/ml) overnight at 37°C. Aliquots of these colonies were used for colony PCR.

Development of genomic SSR markers

The colonies with high level of signal were used to isolate plasmid DNA using standard alkaline lysis method (Sambrook and Russell 2001). After checking the quality of the plasmid DNA on 0.8% agarose gel, the clones were sequenced using the BigDye Terminator cycle sequencing kit on an ABI3700/ABI3730XL (Applied Biosystems Inc., Foster City, CA, USA). 288 clones were sequenced in both directions using standard T7 promoter and SP6 primers and 19 clones in one direction by using M13-forward sequencing primer at Macrogen (www.macrogen.com) and ICRISAT.

The sequences generated were subjected to CAP3, a contig assembly program (http://pbil.univ-lyon1.fr/cap3.php) in order to define unigenes. These unigenes were subjected to MIcroSAtellite (MISA, Varshney et al. 2002) tool to search microsatellites considering minimum ten repeat units of mono- (N), and four repeats of di- (NN), tri- (NNN), tetra- (NNNN), penta- (NNNNN) and hexa- (NNNNNN) nucleotides and compound microsatellites present within a distance of 100 bp. Primer pairs for SSRs were designed using Primer3 program (http://frodo.wi.mit.edu/) in batch file, and the SSR markers developed were designated as ICCM (ICRISAT Chickpea Microsatellite) markers (Table 2).

Table 2.

Simple sequence repeats (SSR) isolated from microsatellite-enriched library of chickpea

Marker name GenBank ID SSR motifa Forward primer (5′–3′) Reverse primer (5′–3′) Product size (bp) Number of allelesb PIC value
ICCM0001a FI856656 (AT)5 GAACTTGCTTGGGGACAGG GAGGTGAGCTGAAGTGAGGC 246 2 0.05
ICCM0001b FI856656 (CT)4tc(CT)4 TGCACAACGGCTATGTCTTC GAGGTGAGCTGAAGTGAGGC 118 9 0.71
ICCM0002 FI856654 (TC)6 TCGTTCTCCGTTATGTGTGC ATGCCTCCAATAGCATACGG 262 2 0.04
ICCM0003 FI856538 (CT)6 AATGGAAGAACGTCAGGGTG TTCCACTGGGGCAAAATAAG 252 7 0.59
ICCM0004 FI856539 (AT)4 CACTCACACACGCACTTTCA AAAAGAGAAGCCCACCAAAA 213 4 0.39
ICCM0005a FI856657 (TC)4N(CT)4N(TC)4 TGCTGAGCGATCTGAGGATA GCAGCAAAAATCAGCACAAA 277 NA  
ICCM0005b FI856657 (AT)4 TTGGTGCAGATTTTTGTTGC AGATTTGGGGGATAAAAGGG 241 1 0.00
ICCM0007a FI856661 (CTC)4N(TC)4N(TC)4 TCTACATTCTCTCCGTGCCC GAGGAGTGTAGGGGAGAGGG 242 NA  
ICCM0007b FI856661 (TCCTC)4 CTCTCCCCACTCTCCCTTTC TGAGTAGGATCGTAGTAGGGGG 202 NA  
ICCM0008a FI856540 (A)10N(A)10 CTCATGGTGGCATTGAGAAA TCCTTGAATTTTTGAGACACGA 230 2 0.05
ICCM0008b FI856540 (GA)4N(AG)5t(GA)4 TCGTGTCTCAAAAATTCAAGGA CACTCGACCACCACTGCTAA 235 2 0.32
ICCM0008c FI856540 (CT)4 GGTGGTTTGTGGAGGTTGAT AGGCAACCATTCATCCTTGT 245 2 0.05
ICCM0009a FI856541 (GA)4 CACTTCAAAAGAGTGTTTGATTTGA GGTTTGAAAGATGAGTGGTTTTG 189 3 0.10
ICCM0009b FI856541 (A)11 AAAATATGGAAAGTCGGGCA TGCATTTTCTTAGCGGTTTTT 257 NA  
ICCM0010a FI856663 (CT)4 GACGGAAATACGGCTGGTTA GCTGCAATATACTCCGCCTC 113 1 0.00
ICCM0010b FI856662 (AT)4 ACGCCAATTCTTTTGAGCAC TCAGCACTGGTGGAACCATA 142 12 0.80
ICCM0014a FI856542 (GA)5 CGTGGTTGTGTTGTTTGAGG TGTGTTCATCTCCCTCTCCC 134 1 0.00
ICCM0014b FI856542 (TA)5 TTTGGGAACCTTTCTCATCAA CCAAATCATTTTTGTGCACTG 254 3 0.16
ICCM0019a FI856544 (AG)18 TTCCGGTTGTTGAGTAGAGAAA ACTTCCACTTGTTCCATCCG 189 NA  
ICCM0019b FI856544 (AG)4 CGGATGGAACAAGTGGAAGT TCTCGTACCGGACCAGAGTC 153 6 0.62
ICCM0021a FI856697 (CA)4N(AT)4N(ATT)5 AAACCGCATTGAGGAATGAC TTTGCCACGATATGTTCAGG 232 NA  
ICCM0021b FI856696 (AT)4 TCTTTCTAAGCAGTCTAGGATACGA AGGAAGGGTGGGATAATTGG 229 NA  
ICCM0022 FI856699 (AT)4 TAAACCGCATTGACGAATGA TGAATTCGCAAGAATCAAATG 123 18 0.89
ICCM0024 FI856545 (AT)4 CACTCACACACGCACTTTCA AAAAGAGAAGCCCACCAAAAA 214 3 0.08
ICCM0026 FI856714 (AG)4 CCGGACATTGTTCTGAAGGT TCTGTGCAGTGGAGCTATGG 216 2 0.05
ICCM0029 FI856721 (AG)4N(GA)4N(GA)4a(AG)5 CTTCCCCTTTCCAAAATTGA TCGTTCACAGGTTTTCCCTC 244 1 0.00
ICCM0030a FI856722 (TC)4N(CT)4N(TC)4 GGCAACGTACGGGATAAATG GCAGCAAAAATCAGCACAAA 271 1 0.00
ICCM0030b FI856722 (T)10 TTGCTGCTGATTTTTGTTGC TCATCCATCATTCTAAATGTGTCA 257 3 0.09
ICCM0032 FI856546 (GA)4 TCTCTTGACACAAGTCTGCACAT CCCACTCACATGTAGCGAAA 232 1 0.00
ICCM0034 FI856651 (GA)11 TTTGTTTGCGGAGGAATAGG TCACCTCACCACACTTCTTTTC 259 6 0.33
ICCM0035 FI856547 (GT)4 TGAGGGTAAATAAATTGGTGGC ATGATTTCCGAGGACAGTGG 101 1 0.00
ICCM0037 FI856548 (AG)4N(GA)4N(AG)4N(GA)5 CAAGCAATGGGAGACACCTT CCACCACTTCAACGTCTCCT 212 NA  
ICCM0042 FI856740 (CA)4 TTCGTGATATTCATCAAGGTGG TTTGACGTACTCTGTGTTTTGTTT 259 3 0.08
ICCM0043 FI856552 (GA)6 AAGATTGGATTCCACAACGC ACAACCCACCCACACAAAC 274 7 0.44
ICCM0045 FI856553 (TA)4N(TC)12 TGAATCCCTCAATCCTGTGG CTTATCTCCCCAGTTGCCAG 272 5 0.31
ICCM0052 FI856768 (TAT)6 CGTCGTGTCCATCTGTCTTG CCAGGTGCAATAGGGAAATC 275 2 0.04
ICCM0053 FI856771 (GA)32t(AG)4 TGCGGTCGACTCTAGAGGAT CTGAGAAGGAAGCCAACAGG 173 1 0.00
ICCM0059a FI856779 (GAA)4 CGGTCGATCTGAGGATCACTA GGGTTCAACTGTTCAGGTTTG 226 NA  
ICCM0059b FI856779 (AG)4N(GA)9N(GA)4 CAAACCTGAACAGTTGAACCC AGGTTCTCCCTCGCTCTCTC 172 3 0.23
ICCM0060 FI856780 (AT)4N(CT)4 TGATGAACATGCTAACAAACTAACAA CCAAGACAACTGGTGGAGGT 260 2 0.04
ICCM0061 FI856798 (AT)6N(TTA)4(TAA)4N(TTA)13 ATGCGGTAATCCTTGACTGG TGAAATTGGGTTGGAAGTTTG 275 5 0.23
ICCM0062 FI856801 (AAT)4N(AAT)6 ATGCAACGCCTAAGTCTCGT TAGGTACGTTTGGGGTCCTG 266 3 0.09
ICCM0063 FI856802 (TTA)6 TTGATTTATCCTGTGATGCTTTTT GGCAGTCTGGTCATGTGAAA 194 2 0.04
ICCM0065a FI856807 (TC)6 CATTCCCGCAAAGCTTGTAT GGTGCCTTGGAGAAAATCAA 137 NA  
ICCM0065b FI856807 (TC)4 CACGGTGTCTTCCACATGAC TAAGCCAATACCAAGAGGCG 195 2 0.08
ICCM0066 FI856555 (TC)4 ACCATGCTCACCAACTCACA TCTCTTGACACAAGTCTGCACAT 242 1 0.00
ICCM0067 FI856556 (TC)4 AACTGCAACCTCTCTCGCTC TCTCTTGACACAAGTCTGCACAT 204 1 0.00
ICCM0068 FI856557 (ATT)22 TCTTCTTTGCTATCTGTCTCGC TGCATGTCAAACATTAGACAACTTT 227 14 0.87
ICCM0069 FI856558 (ATT)22 TCTTCTTTGCTATCTGTCTCGC TGCATGTCAAACATTAGACAACTTT 227 13 0.82
ICCM0072 FI856813 (GAA)4 CAAGACCTTGAACAGGAGGC TCTTTCAATTTTCTAACAATTTCATC 200 2 0.14
ICCM0073a FI856828 (TA)4N(A)11N(TA)4 TCGATCTGAGGATCTTTGGTG TGGATACTATTAACGAAAAACTAGCG 219 6 0.23
ICCM0073b FI856828 (TC)4 CTTGTCCACCCCACATTCTT TAGAGAAATGGGGGAAGGGT 217 1 0.00
ICCM0074a FI856830 (A)11N(TC)6 AAATCCCAAATTTAGAGCGG AACCTTTGAAAAAGGCGGTT 183 9 0.40
ICCM0074b FI856830 (T)15 AACCGCCTTTTTCAAAGGTT CCTTCCAGGGGAAAAAGAAA 264 NA  
ICCM0075 FI856559 (AG)16 CTTTGTAACAATAAAATGCAAAGTAAA GGAAGCACAGTCTGCACAAA 163 4 0.26
ICCM0076 FI856560 (ATA)17N(TAA)5 CTCATCGAATAGAACCTACCGA CCGCTACACCTACAACGGTAA 270 3 0.08
ICCM0077a FI856561 (AG)4N(T)10 CCACAAGAAGACAAAGGGGA AAAAAGATGCTAAAACTAAACCAAAGA 217 1 0.00
ICCM0077b FI856561 (A)10 GCCGAGAAAATAAATTTCACCA GCCGCGACCATTAATTCTAA 124 2 0.04
ICCM0078a FI856832 (TAT)4 g(ATT)6ag(TAT)5N(AT)4 CTGAGGACGTTGGGAATACG AAAAACTAATCTCGTGTTCAAATCC 280 3 0.22
ICCM0078b FI856832 (TC)4 AATCCCAACGGTGAGAGATG GGACAAGGAGTGGAAGGGA 279 15 0.62
ICCM0079 FI856562 (TTA)6 GAGCTAACGCCTTCGCTAGA GAGAGGGATTAAAACAAATAGAGGAA 170 3 0.08
ICCM0080 FI856563 (T)10 CTGCGGTGACTCTGAGGAT GAGAATCACGGGTGTTTCAAG 173 3 0.15
ICCM0081 FI856564 (TAA)6 GAGAGGGATTAAAACAAATAGAGGAA GAGCTAACGCCTTCGCTAGA 170 3 0.08
ICCM0082 FI856833 (CT)19N(TC)4 TCACGATCTCACAGAGCCAC TCCGTGATTCTGAGCAACAG 260 3 0.08
ICCM0083a FI856835 (AT)4N(CT)7 CGCTCACACCATCTCACTTC GAATGGAGGAAATACAGAGTGC 273 NA  
ICCM0083b FI856837 (A)13 ATTGAAAAACCACGCACACA AGCGACGACAGTGACCTTCT 140 1 0.00
ICCM0083c FI856837 (T)11 TGTTTGTTGCCTAGCCACTG TTGGACAGATTTTTGTTGTTTGTT 195 1 0.00
ICCM0084 FI856993 (GA)5 TTTTGATTGAGCATGCAATGT GAACCTTTTGAGGTCTGTTGC 246 2 0.04
ICCM0085 FI856855 (T)10N(TAT)14 GTGGTCCATCTGTCTCGGTT GGAAAAGGAGAAAGTTGTGGG 210 NA  
ICCM0086a FI856856 (TA)4N(T)11 TGTGCATTGAGCCTATTGGT GACAACAGCGGCATAATCAA 182 NA  
ICCM0086b FI856856 (AC)4 CTTCCCCTTTACCCCGTTTA AAAGAAATCGACAATAAAAGAGTGA 175 NA  
ICCM0088 FI856861 (T)10N(AT)5 AAAGGAAGGGAAGGAAATGC GAGTTTGGGCAGGCAATAAA 240 2 0.19
ICCM0089a FI856863 (A)12 AACACCGACTTTCCAAAACG TTTGGGAAATACAACCTTTGAA 204 9 0.45
ICCM0089b FI856862 (TAT)20 GGGATATCGCCAATATATTTTATACC TTTGGCAACAAATCCTTTGA 126 NA  
ICCM0090a FI856864 (TTA)6 ACGGGACTTGGATGACTTTC AGACGCGTGCTTTCTTCCTA 257 NA  
ICCM0090b FI856864 (TC)5 CTGCCTAGGAAGAAAGCACG AAAATAAATGCGCCGTATGC 160 3 0.39
ICCM0093a FI856871 (TAT)20 CTTCTGTTATTATCGCCGCC AGCATCATGGAGCAGAGAGG 223 NA  
ICCM0093b FI856871 (AT)4 TACCCTTTCTCTCCCCACCT TCAGTAGTCGGGCAATAGATGA 129 NA  
ICCM0093c FI856870 (AAAT)4 CCACTTTTAGGCGCACTTCT CGACTCATTTTTCACGGACA 197 3 0.24
ICCM0094 FI856872 (AT)4 AGAGGCAAACAAGAACCGAA AAGGGTTAGTGGAGGAATTATGAA 279 3 0.08
ICCM0095a FI856875 (TC)4 CTCTCCATCCCATCCGACTA GGAAGCCATATCCAGAGGGT 181 NA  
ICCM0095b FI856874 (ATTC)4 CGGGACATTTCCGTTAAAAA CGAGTCGTTTTCTTGGCTTC 171 1 0.00
ICCM0096 FI856877 (AT)4N(CT)4N(TC)4 ACACCCCACCCTAATTACACT GAGAGGTACGAAGCACGAGG 170 9 0.47
ICCM0097a FI856669 (ATT)12N(TA)4 TGAGGACTGCCATACTCCAG TCCCCTTTATGAGGGCTTTT 276 3 0.09
ICCM0097b FI856668 (CTT)4 TCCAATTCCAAAACACACCA CCTGAGGAGTAAAAGACGGG 130 1 0.00
ICCM0101a FI856675 (A)13 TAACTGAGTTTCGGGTTCCG CTTAACGGACGTTGTAGGGC 247 NA  
ICCM0101b FI856674 (AG)4 GAAGACAAAAAGGGGCACAA CCGATTGTTTCAAGACCAGA 105 2 0.04
ICCM0102 FI856676 (T)12 CACCAAAAAGGGAACTTTCG AAAAATAGGGTGGGGAGGG 167 1 0.00
ICCM0103 FI856678 (A)11 ATGGGGGAATCGGAGACTAA GGATAGGGAGGAGGGAACAG 110 3 0.18
ICCM0104 FI856566 (TTA)11 CCAAACCTCCAAAAATCTGC TCATTTTTGATTCATTCTTGGG 278 8 0.48
ICCM0105 FI856567 (AT)4 TGCTTCCTTTTCAATCACCA TGACAAAGGACAAATAAGTGTTTTA 280 1 0.00
ICCM0106 FI856681 (ATA)7 TGGAATTGCTACCGAATATGG ACGATCGGAGAGAACGAGAA 267 NA  
ICCM0107a FI856682 (TCG)4 GCAAAAAGTGTTGCTTCCGT AAGCACATTGCCACTAGCAT 234 1 0.00
ICCM0107b FI856682 (TA)4 GCAAAAAGTGTTGCTTCCGT AAGCACATTGCCACTAGCAT 234 3 0.16
ICCM0110 FI856568 (AT)4N(TTA)7 AGAGGCAAACAAGAACCGAA GTAAAGAGGGGCAGCTGTTG 183 NA  
ICCM0115 FI856706 (TC)4 ACCCTAAGGGCTCGTTTGAT TAGGGATGGAGAGGAGAGCA 245 1 0.00
ICCM0116 FI856709 (T)11 TTTTGGTGCAAGAGAATGGG GTCTTTCAAGAGGTCGCAGC 177 1 0.00
ICCM0117 FI856572 (TAA)4 AGTGACCAGGAAACACGGTC GCAGAGATTGAATTTTGCCA 254 1 0.00
ICCM0118 FI856573 (T)11 AATTGGGAAGGAAAGCGAGT TCGCCATTGCAATAATCAAA 279 NA  
ICCM0119 FI856710 (GA)4 TTATTGGATGAGTGGATGCG ATTACGTAACCCAACGTCGC 192 NA  
ICCM0120a FI856574 (TTA)12 TGTCTCGATAAGAGTTTGTTATTTTTC CGTTTTGTTTCATATTCAAACTCG 220 12 0.75
ICCM0120b FI856574 (ATT)13 CGAGTTTGAATATGAAACAAAACG GCTTGTAGCTAGGCTCGACTC 166 5 0.29
ICCM0121a FI856575 (TATT)4 CAAAAATTTGGATTCGGGAG CTATTGCACCTGGGGATACG 238 2 0.09
ICCM0121b FI856575 (A)10N(ATA)17 ACGTATCCCCAGGTGCAATA AACAGATGTAGAAGGTATAATCCATGA 224 1 0.00
ICCM0122 FI856576 (AAT)4 GCAACCTGCCATCCATACTT AGTGAATCAAATGATGAAAGCA 275 1 0.00
ICCM0123a FI856577 (TTA)14 GGATGGTCTGCTGGAATCAT AAAGACAACAAAAAGACAATCATGT 250 9 0.76
ICCM0123b FI856577 (TAA)26 TTTTTACATGATTGTCTTTTTGTTG TGAGGACTAAGATAATAGCAATCCAA 201 NA  
ICCM0124 FI856578 (AAT)4aaa(AAT)4 CCTCGGGAATTCAACTACCA TCAAAAATCCACTTTCCACCA 279 5 0.16
ICCM0125a FI856579 (AAAT)4 CGATCTGAGGATCAACTTGTGA ACTAACCACCGTCGACCATC 253 NA  
ICCM0125b FI856579 (TTA)5 TATTTATGGTCTGGTCCGGC CGCTACCAAATATGGAACGACT 251 5 0.19
ICCM0127 FI856581 (TAA)27 TGTTGAACGAATTTACTCATCG GGTGGGCTCCTATTGTTTGA 269 6 0.40
ICCM0128a FI856731 (TA)4N(TAT)4 AACCCTAATTTATTTGCACTATTATCA TCAAAATACGGTAGTAGGATAAGATGA 161 NA  
ICCM0128b FI856730 (A)10 ATTTGGACGATGTGTCGCTT TTATAGCCCCTGCTTTGCTG 266 1 0.00
ICCM0130a FI856734 (AAT)22 GGATTTCGACTTTTATCCCTTTTT CGGACTGGAATCAAAAGCTC 268 3 0.10
ICCM0130b FI856734 (ATT)5 GAGCTTTTGATTCCAGTCCG TGTAGGGGTGCATGGTGTAA 122 1 0.00
ICCM0131 FI856582 (AT)4N(TTA)8 AGAGCCAAACAAGAACCGAA GTAAAGAGGGGCAGCTGTTG 192 NA  
ICCM0134 FI856748 (A)11 TTTTGGAGGCAGCTTTGAGT TGAAGACAGAGACGGTGCAT 109 3 0.08
ICCM0138a FI856753 (AG)4 ATAAATAGCCGGCCACAAGA CCGATTGTTTCAAGACCAGA 119 1 0.00
ICCM0138b FI856752 (ATA)11 CTGTTGCCGATTTTATATTATTTTT AAATGTGTTGTTCTGGCCGT 168 NA  
ICCM0139 FI856755 (ATTC)4 TCACGATTTGAATGGTCGTG CGTTTTCCCAGCTTCAACAT 151 NA  
ICCM0141 FI856757 (AAT)20 AAGGTATGATGCAGTTCCCG GGGCGGAGGGTAATTATTGT 247 NA  
ICCM0142a FI856759 (AC)4 GCATTGCCAATATCGAAGGT AGTAGGTGCCAAATGCATCC 206 1 0.00
ICCM0142b FI856759 (ATT)6 GGATGCATTTGGCACCTACT GAGGTTGGTGTAGAATAGAATGGA 137 NA  
ICCM0142c FI856758 (AC)7 GGAGGTCCCGAGTCTAAACC TCTTCAAGTCTGCTTGACGTGT 105 1 0.00
ICCM0143 FI856761 (AC)5 CTGCGGTCGATCTAGAGGAT AAGGTTGAAGGATGATTGCG 257 NA  
ICCM0150a FI856583 (ATTC)4 TGATTGAAATGGTCGTGTCC AGTCGTTTTCTCGGCTTCAA 279 1 0.00
ICCM0150b FI856583 (TAA)8N(AT)4 GTAAAGAGGGGCAGCTGTTG AGAGGCAAACAAGAACCGAA 192 5 0.16
ICCM0152 FI856792 (T)13c(CTT)4N(AG)5 CGTCTCACGAAGGAGAAGTG AAAAATTCACTTGCTAATATTTCACA 197 1 0.00
ICCM0154 FI856794 (A)11 AGCTTGTTCGACAGCAGGAT TCGATGTGAATATGCCCTCTT 247 1 0.00
ICCM0155 FI856796 (A)11 GACGGCAGGATTAACTGCAT TTCGATGTGAATATGCCCTCT 240 2 0.04
ICCM0156a FI856797 (AT)7N(CA)4 TGCATTCCCCTTTAATTTGG CAGTGGTGGGGAGACAAAAC 280 2 0.04
ICCM0156b FI856797 (ATA)52N(AAT)6 GTTTCTCCGTCCGCACTTAT AAACAGTGTAAATTGTTGTTGGAAA 276 2 0.08
ICCM0157 FI856584 (GA)8 TTTGTTTTGAGGCAACCACA AAACAAACCCAGTGGGAGGT 258 1 0.00
ICCM0158 FI856585 (ATA)18N(TCT)16 CCAAAACTGACAAGTCCCGT GGGAAAATATGAGGAAGTTTGC 275 1 0.00
ICCM0159 FI856814 (T)17N(T)10 TGAAAAATCGGAAACCCTACC CCTTGTTTTCAGGCGATTGT 247 6 0.46
ICCM0160 FI856816 (AAC)4N(TAA)25 TTGCTTGAAACAACCTTTCG CGGGTACAACCGTAGCAAAT 263 21 0.93
ICCM0161a FI856818 (AT)4 GATGGTCATCGGTTCGATTC AACGAGGCCCTCTGTAACG 267 4 0.12
ICCM0161b FI856817 (TAA)4N(AAT)4 ACTGTCAGGAAGGAACGGTG TCCGTAACAAAAATTGTGAAGAAA 279 3 0.14
ICCM0162a FI856586 (ATT)12 TAGCGCAGTCGATCTGAGGA ACGTATCCCCAGGTGCAATA 272 NA  
ICCM0162b FI856586 (AAAT)4 GCAAGGTCTTTCCCTTGTCA CGCCGCCAATTTTATTTTTA 278 1 0.00
ICCM0162c FI856586 (T)11N(TA)4 TAAAAATAAAATTGGCGGCG TCGTGTTAGGGTGTTTAGGGA 267 3 0.21
ICCM0162d FI856586 (T)10 GACTCTGCTGGGGACAATTT CGGGTTTAGAGACCCACTCA 225 1 0.00
ICCM0163 FI856820 (TC)4 CAACGAATTTCATGCTGTGG TAGGGATGGAGAGGAGAGCA 274 1 0.00
ICCM0165 FI856821 (T)11 CGGACGTACACCTTTCGTTC TGCTTCCGAATAACATAAAGCA 128 NA  
ICCM0166a FI856824 (T)11 GCCTACTCGCGGATTTTTATC CCAGGTGCAATAGGGAAATC 276 3 0.09
ICCM0166b FI856824 (AAAT)7 TGGGGATACGTAGGAGCAAG TTGGATTCGGGAGTCGATTA 233 NA  
ICCM0166c FI856824 (AT)5 GCCTACTCGCGGATTTTTATC CCAGGTGCAATAGGGAAATC 276 2 0.04
ICCM0166d FI856823 (AT)4 GACTCCCCTACCACCTCACA CGGACGCGACAAAAACTAC 275 1 0.00
ICCM0166e FI856823 (TAT)48 AATAAAAATGCGGAAGTGGG TGTGAGGTGGTAGGGGAGTC 276 1 0.00
ICCM0167 FI856588 (ATA)48 GAGTGTACGGGGATTATATGATGA TCAAGAAAAGGAACCAAGGC 235 NA  
ICCM0169 FI856589 (TTA)30 AGAGGCAAACAAGAACCGAA GTAAAGAGGGGCAGCTGTTG 251 NA  
ICCM0170a FI856839 (TA)4 GCTTTGTTGCTTTCGTTCTTTT AAAGTGTTTGGGGTGAGTGG 226 NA  
ICCM0170b FI856839 (C)10 CTCGCATTCCTTTTCCACTC GGGGGAAAGTATGGGATGAG 154 NA  
ICCM0171 FI856590 (ATTC)4 GACCGGGATCGTGTCATAAA CGTTTTCCCAGCTTCAACAT 166 1 0.00
ICCM0172 FI856841 (AT)4N(AT)4 GCAGTCGATCTGAGGATCAAG TTCACAAGATGTTTTCAGAACAAAG 278 NA  
ICCM0174 FI856591 (A)11 CAGCGACCTCCTACTGGGTA CAAAAATGGAGGATTTTTCCTT 175 NA  
ICCM0176 FI856847 (TA)4 ATAGGCTAGACCGTCCGACA TCTGAAATATGATGCAGCCG 273 5 0.16
ICCM0177a FI856849 (AAT)28c(ATA)27c(TAA)4 CTTGAGTTCAAGCCAGAGAGG GCGTTATTACTGTTACAAATGGCA 279 1 0.00
ICCM0177b FI856848 (TAT)6N(TAT)11N(TAT)4N(TTA)6N(TAT)8 CCCTTCTTCCATTTCCGAAT GGGGAGGAGAACGAAAAAGA 273 NA  
ICCM0178 FI856592 (AAT)13 AGTTTGGGTTTCACCGCCT GAACGCGCTCTGTTCATAAT 280 11 0.83
ICCM0179 FI856850 (TAT)4 AAAGGCCAGTTTACCCGACT ATTTGATGCAGCAAGCAGTG 214 NA  
ICCM0180 FI856852 (TAT)4 AGTCCCTGATCTCCCGAAGT ATTTGATGCAGCAAGCAGTG 179 1 0.00
ICCM0181 FI856593 (ATA)5 CGGGTGTGGATAGCAAGTTT TCTCTCCTTCCTAATAAAACAAACA 103 1 0.00
ICCM0183 FI856595 (TAT)15 TGAGGACTAAGATAATAGCAATCCAA TTTTTACATGATTGTCTTTTTGTTG 168 1 0.00
ICCM0185 FI856597 (T)11 AAAGTTTGGCCTGGTCTGG CATTCCATATTCAGTAGCATTCCA 250 6 0.46
ICCM0187 FI856599 (TAT)6 TGACCATCAATCCATTTCTTTTC TGTTGACGTCTAATTTTGTCCG 280 NA  
ICCM0189 FI856601 (TAT)62 TCCAGTTCCAAATGGCATAA CCCTTGAGTTCAAGCCAGAG 273 3 0.21
ICCM0190a FI856602 (ATA)6N(ATA)9 GGGGGATTGTCTGAGTTTCA AAAAGGCTGGAGACACCTCA 195 7 0.53
ICCM0190b FI856602 (TAT)5 TTTATTGCAGGAAGCGGTTT CACCACTATCAAATGCCCCT 273 1 0.00
ICCM0191 FI856603 (T)10 CCTTACGCATAATCGACTCCA AATTCAATTGAGTCGCCACC 130 5 0.37
ICCM0192a FI856879 (TAT)15c(ATT)15 GCTGCCCAAATTTTGACATTA CCGGGGATCAAATTCTTCTT 279 11 0.88
ICCM0192b FI856878 (TAA)15tg(ATA)15 CGGACGGGGATAATTCTTCT GCTGCCCAAATTTTGACATTA 279 15 0.76
ICCM0193a FI856881 (TAA)5N(T)10 TGAACTTTCAAAACCAAACCAA TTGTGACAATTTGAGGGTTCT 268 NA  
ICCM0193b FI856881 (AAT)7 TCGATTATAGCTTTATCTTTACCCTTT AAAAGTGTTGGGAGGGGTTC 242 1 0.00
ICCM0194 FI856883 (A)10N(T)13 CGATTGTCCTAGTTTTAAAAAGAAA CGACTTCCTGAAGGAACGAA 200 4 0.22
ICCM0196 FI856605 (ATA)5N(AG)6 GTCGGGTGTGGATAGCAAGT AACACAATTCCTCAAATAACAAACT 154 6 0.47
ICCM0197a FI856885 (T)13 CGCGTCTAGCAAAACAAGAA TTCTCGGCCTATAAACATCAA 280 3 0.08
ICCM0197b FI856884 (TATT)7 GATTCCGGAGTCCATTACCA CTATTGCACCTGGGGATACG 241 NA  
ICCM0198 FI856886 (A)15 CCATCCGAGAAAACTCGAAA CAACGGTATCCATCGGAATC 163 1 0.00
ICCM0199a FI856889 (A)10 g(AGAA)4 ACCAAGCAGACCACAACAAT GTTTTCCCGGCTTCAACAT 265 1 0.00
ICCM0199b FI856889 (CATT)5 ACCAAGCAGACCACAACAAT GTTTTCCCGGCTTCAACAT 265 1 0.00
ICCM0199c FI856888 (TTA)15 TTAGAGGCAAACCAGAACCG ATCTTGAAGTGGGCAAAACG 241 15 0.86
ICCM0200 FI856890 (TAA)4 ACGGAGTGACCAGGAAACAC GCAGACCTACAGAAACAGAGGAA 231 2 0.04
ICCM0201 FI856892 (TAT)7(TTATTG)6a(GTTATT)4 ATAGAGAGACCCAAACCGCC GCCAAAGGCAAAAGAGATTG 135 NA  
ICCM0202a FI856894 (T)10N(T)10 CGCCGATCCATTATACTGAC TTGCCTCTGATTCTGGTTCA 192 2 0.04
ICCM0202b FI856894 (TTA)13 TGAACCAGAATCAGAGGCAA CCAATTTGGTCCGGTTTTTA 207 12 0.77
ICCM0203 FI856606 (CT)6 TGGACGTAGGTTGTTGTGGA TTGGTATCAGTGACCTCGCA 194 1 0.00
ICCM0204 FI856607 (TC)7 CACATACACTCCCCAATCCC TGCAGACTGTTTGGTTCGAG 258 1 0.00
ICCM0205 FI856608 (TA)9 CGACCATGATTCTCTGATGTG CACCTCTGCATTTCTTCAAACA 263 8 0.26
ICCM0207 FI856610 (TA)4 TCAACCATAAAGCACTCCCC GGCCATTTGTGTTTTGTATGG 182 2 0.04
ICCM0210 FI856898 (TA)4N(TG)4N(CCA)4N(AG)16 GACCATTGCCCACTTCAACT AGTTCTGCGAGAGGAATGGA 253 NA  
ICCM0212a FI856902 (T)10N(AT)4N(TA)5N(TA)4 TCCTATACCGAAAACCCCATT CAAAATGGATGGATTGTGGG 270 2 0.04
ICCM0212b FI856901 (GA)4 ATTGCCGTTGAGAGAAGTCG TCGGTCACCACACTACCAAA 183 1 0.00
ICCM0212c FI856901 (AG)8 TTTGGTAGTGTGGTGACCGA AACCCAAAAACGTGGACTCA 161 6 0.32
ICCM0214a FI856612 (CT)6N(AC)4 CTCTTCAATAGCCCCATCCA CGTTGGAGAGGCTGAAACAT 249 1 0.00
ICCM0214b FI856612 (TTTA)5 ATGTTTCAGCCTCTCCAACG TCGCACCTGAACTCTCTGTG 276 NA  
ICCM0215a FI856613 (TC)5 CCTTCAGTGTTTGGCTCACA CTCCAGGAATCCACAGCATT 253 3 0.15
ICCM0215b FI856613 (T)11 AGAATGCTGTGGATTCCTGG GCAAGCCCAAAACTTCAAGA 276 1 0.00
ICCM0216a FI856614 (TG)4 CGGGACTTTCATCTGCTGTT GTGGGACATCCTCCAAGAAA 200 2 0.04
ICCM0216b FI856614 (AG)5 AAAGCTGGTGGTCGAGCTAA GACCACCGAACCAGGATAAA 277 3 0.09
ICCM0219a FI856906 (ACC)4 CCTTTTAAGGGCTGAAGGCT TGAAAGAATGTGGGGGAGAG 203 NA  
ICCM0219b FI856906 (CT)5 TCATCCTACCCAATTGCTCC GTAGTGGGGTAGGGGATGGT 240 3 0.10
ICCM0219c FI856905 (CT)4 CTCACCCACCACACCTATCC GCGAAGGGAGAGAAGGAAGT 278 NA  
ICCM0219d FI856905 (TC)5 CTCACCCACCACACCTATCC GCGAAGGGAGAGAAGGAAGT 278 NA  
ICCM0220 FI856908 (TA)4N(CT)4 TCAACCATAAAGCACTCCCC GGCCATTTGTGTTTTGTATGG 183 1 0.00
ICCM0222 FI856617 (CT)5 TCCGATTGGATTTTCAGGAC GGTATCAGTGACCTCGCCAT 210 1 0.00
ICCM0223a FI856912 (TCT)4 TACAACTTTTGACACCGCGA AGTGGCAGTATGCGTTGAGA 224 NA  
ICCM0223b FI856911 (TC)4 GCTCTGTCGGTCTTCCTGTC ACAAAGCGCTCGAATAAGGA 208 NA  
ICCM0224 FI856914 (CT)4 ACCACCTTGCTCATCCTCAC GAGTAGGAGGTGCGAAAACG 274 16 0.74
ICCM0225 FI856915 (CT)4 ACGTCCGGATTTGTTCTCAC ATTATAGGAAGATGGCGGGG 252 6 0.27
ICCM0226a FI856537 (A)12 CCAACGACGCGGATAAATA AATGCCCACCCATAATTTCA 273 1 0.00
ICCM0226b FI856537 (TA)4 GAAAAAGCGCTGTAAATGGC CCTCGCATTTTGTTCTCAAAG 145 1 0.00
ICCM0228 FI856619 (CT)6 TGGACGTAGGTTGTTGTGGA GGACCGGGAGTCCCTTATTA 274 1 0.00
ICCM0229 FI856916 (C)10 TGTCTTATTCCTCCTCCCCC AGGGGTTTTTGGGTTACCAG 158 9 0.69
ICCM0231a FI856919 (TTA)21N(TC)4 CTGGGGATACGTAGGAGCAA GGAGTGAGATAAGAAAGAGAGGAGG 278 NA  
ICCM0231b FI856919 (TC)4 ATCCCACCTTACCACCCTTC GGAATGAGGAGGGATTGAGA 279 2 0.05
ICCM0232 FI856920 (T)10 ACGGGAAGTTTCTGGGTCTT TAGCGGAGAAACAGGACTGG 253 3 0.09
ICCM0233a FI856922 (TA)4N(TTA)4t(TTA)4 CTCACCACTAGGGATGGGAA AGACTCCCGAGGGATTGACT 242 NA  
ICCM0233b FI856922 (A)10 AGTCAATCCCTCGGGAGTCT TGTTGAGGGCCTTAGATTGG 256 NA  
ICCM0234a FI856925 (TTA)4 GGGACTACTTTCGCGATTCA GTGGGTAATCCGTGCGTAAT 229 1 0.00
ICCM0234b FI856924 (TA)4 CAGGCTATGTCATCTCGTCG CCTGACTGCCACAAGTTTCA 270 1 0.00
ICCM0234c FI856924 (TCG)4 TGCTTCCGTACAGGCTATGTC CCTGACTGCCACAAGTTTCA 280 1 0.00
ICCM0235a FI856927 (TC)4 TCGCTCATGACAGACTCGAC GTAGCAGGTGAGATGCACGA 173 5 0.32
ICCM0235b FI856926 (GT)4c(GT)4 GCCGACCCGATTACCTTACT GAGAGCTAAGGGGAAGGTGG 176 NA  
ICCM0236a FI856621 (CGC)7 GCTTGTTGCCCTGTATTGGT AAAACGCAAGCAAAGCAAGT 151 NA  
ICCM0236b FI856621 (ATT)4N(A)10 ACTTGCTTTGCTTGCGTTTT TTTGTGGGTTGGTTGATTTT 219 2 0.04
ICCM0236c FI856621 (A)10N(TA)4 AAAATCAACCAACCCACAAA ATCACTCTACCGTCAAAACGA 244 2 0.04
ICCM0237a FI856622 (AT)4N(ATT)6 TCAACCATCCCTAAAAACATTTG TTCCTTTGCCATTTATGTTTTG 184 6 0.34
ICCM0237b FI856622 (A)13 CAAAACATAAATGGCAAAGGAA TCGTGTTGTATTGTGCCGAT 155 2 0.04
ICCM0237c FI856622 (AG)4N(GA)4 TGTGTTTCTCGATGGCAGAG CTTTTTCTCCCTTTCCACCA 120 1 0.00
ICCM0238 FI856623 (TC)4N(TC)4 ACCATAGACGAACCACCACC CCAAGGGGGTACAACTTGTGT 215 1 0.00
ICCM0240a FI856650 (TA)12 ACCCGAACCCGCAAATAATA GCAATGAGACTGGGGTTTTC 248 NA  
ICCM0240b FI856650 (TA)4 ACCCGAACCCGCAAATAATA GCAATGAGACTGGGGTTTTC 248 19 0.77
ICCM0242a FI856929 (AAT)18 TGCATTCATCTGTTTCGCTC GAAAATATTTGTGGTTATCCGATTTT 263 8 0.74
ICCM0242b FI856928 (A)11 ATCCGCAACACAACAAAACA CCCTACTCGTAATCGACTCTCG 252 4 0.21
ICCM0242c FI856928 (TAT)5 CAAGTGCAATAGGGAAATCCA ATAGGGCTTTCCACCGATTT 210 NA  
ICCM0243a FI856931 (AT)5N(AT)4N(AT)8 TCAGGAACAGACGGAACTTTTT GGGTTCAAATCCTATTGGGC 277 3 0.08
ICCM0243b FI856931 (AT)4 ATTTGCGCCCAATAGGATTT TTTTTCTATCGGAATATCTCATTTTCT 280 1 0.00
ICCM0243c FI856930 (GA)41N(AG)10 ACGACGATTCTGGATTTTGG AGTTTTGGTAGGGGGTCGAG 237 16 0.88
ICCM0244a FI856933 (AT)8N(TTA)4(TAA)4N(TTA)6N(ATT)4 ATGCGGTAATCCTTGACTGG TGCAGGGAGTGAATGTGTGT 252 5 0.25
ICCM0244b FI856933 (TA)5 CACACATTCACTCCCTGCAA GATGGAAGGGAGGGGTAAAA 268 NA  
ICCM0245 FI856935 (AG)5 GCGGCTGGTTTAAGAGTGAG CCAACACGACCCAAATCAAT 182 6 0.53
ICCM0246a FI856937 (AT)4 TCTGACAGCTCTTGCCTTGA AACACCCAGACCCCTTTCAT 280 4 0.12
ICCM0246b FI856937 (TA)4 TGAAGAGGAAGAGACGGGAG AATCCATTTACGGGGGTAGC 268 NA  
ICCM0246c FI856937 (TATTT)4 TGAAGAGGAAGAGACGGGAG AATCCATTTACGGGGGTAGC 268 1 0.00
ICCM0246d FI856936 (TC)4 GATCACGGTTACGAATGCAA TAAGGTTCCCATTGGCTCTG 209 1 0.00
ICCM0247 FI856626 (TTA)8 CCTCAATTCATTTTTCTTCGG TTTCCCGATAAACCATCTGTT 136 2 0.06
ICCM0249 FI856627 (T)12N(TAA)29 TTTCTTCGCATGGGCTTAAC GGAGATTTGTTGGGTAGGCTC 193 5 0.16
ICCM0250 FI856940 (TAT)40 TTTCAAACACAATCTGAACGAGA CCACCTTCGGGTAGGATACA 231 2 0.04
ICCM0251a FI856943 (AC)4 TCCCTGCTATACACCCATCC TGGGCATATATGGATCACGA 252 1 0.00
ICCM0251b FI856943 (CG)4 CTACACCCGCCAACCTCTAC AAGTGTATGTGACCGAGCCC 261 1 0.00
ICCM0251c FI856943 (CA)4 AACCCATAATACGCGCTCAC GGGGTGGTAAGGTAGGAGGA 206 2 0.08
ICCM0252a FI856945 (CCT)4 TCTACCTCTCCGCTCTTCCA TGGTGATAGGTGGTGGTTGA 203 1 0.00
ICCM0252b FI856944 (T)11 TTGACGGTGGGGGTATACAT TCCACACACTCCCACTACCA 267 NA  
ICCM0253 FI856946 (AT)5 TCCCTTACAAGCATTCCCTG TGGGGACCGTTTTTCACTTA 110 NA  
ICCM0254 FI856628 (TAA)4N(TAA)29 GCCAAGCCCATTAAAACACT CGTTGTTAAAAACCGCGTTG 273 1 0.00
ICCM0255 FI856629 (ATA)8N(AAT)4N(AT)4 GGCTACCGAATATGGAATGC TGGCCTGACCTACTTATGGC 272 2 0.04
ICCM0256a FI856949 (AT)4 ACCGCTCATTTCCATACGTC TGGATCAAGAGGGAGGATTG 195 NA  
ICCM0256b FI856948 (CT)4 TTTCTCTTTTGGTGGTTGCC TTAAGGTTTGCCACTCCTGG 247 4 0.17
ICCM0256C FI856948 (TTA)19 TTAAGCCAGACGTGGGAAAC AGAAAGAAGGGAAATGGGGA 236 2 0.08
ICCM0257 FI856630 (ATA)44N(AAT)11 TCGCTTCCAACATTCAAAAA CAATTGCACTTATAGCACAAACA 255 2 0.04
ICCM0258 FI856950 (ATA)11 TGCATAGGGAAATCAAAACACA TTATTTCACCGTCGTGTCCA 271 1 0.00
ICCM0259 FI856631 (TTA)15 AGAGGCAAACAAGAACCGAA CGAAGCCGAGAAAATGACTC 261 2 0.04
ICCM0261 FI856633 (TAA)23 GTCCGGGGATTCAGTAGGAT CAAGCCACGGAACTTGTTTT 232 NA  
ICCM0263a FI856635 (TATT)7 CGGGGATAAATCAACACACC GGGCAAGGTCTTACCCTTGT 265 2 0.08
ICCM0263b FI856635 (ATA)19 GGTAGAAAATATTTATGTGTTGACCG CTCGTTCACATACCGCCATA 260 1 0.00
ICCM0265a FI856636 (AT)5 GGAACTCGGGATTGAAATAGTC TTGCAAAGAAAACAATTTTAGGA 214 NA  
ICCM0265b FI856636 (A)13 CGTTTAATCCTAAAATTGTTTTCTTTG ACGGCGACAACCATTAATTC 190 1 0.00
ICCM0265c FI856636 (TAT)9a(ATT)10N(ATT)11N(AAT)4 TACCGCCACGTTACGTTTTT GAAAATATTTGTGTGTTGACCGA 248 4 0.14
ICCM0266 FI856955 (TAT)31 GAATCGTGAGGGGGAGATTT GGGGGAATCAAAAGGCATAG 269 NA  
ICCM0267a FI856637 (TATT)4 CAACGTCCGTTAAAACGGTTA TGGGGATACGTAGGAGCAAG 179 1 0.00
ICCM0267b FI856637 (TTA)4N(TAA)8N(ATA)12 CTTGCTCCTACGTATCCCCA AGTCTCGTTCACATACCGCC 272 1 0.00
ICCM0268 FI856957 (CT)7N(TC)4 TTCATCTCTGCCCAAACTCC TGGGTAGATGGAAGGAGTGG 220 1 0.00
ICCM0269a FI856959 (AC)4 CCCTCTTTACACCCCACCTT GTAGTGGAGTGGGGCAGGTA 129 2 0.04
ICCM0269b FI856959 (TC)5 AACATCACTAACCCTCCCCC AGGTGTGGGTGGTAGGAGTG 209 NA  
ICCM0270 FI856638 (TAT)16 TCACATACCGCCACAATACG ACGTATCCCCAGGTGCAATA 276 1 0.00
ICCM0271 FI856639 (GT)4 ACCCGGGTATAAGGTTCCAC TGCTTGTTTTTCATTTTCATTTTC 240 3 0.11
ICCM0272a FI856961 (A)12 TTTCCACTTGGAACAGGCTC AATGGACGATGGTTGGGTTA 280 3 0.12
ICCM0272b FI856960 (GA)10N(AG)20 CGCGGTTGAGTTAGAGTGGT CAAATCGGGGATTTTGTTTG 175 12 0.74
ICCM0272c FI856960 (GA)4 CGCGATTATTACCCACGTTT GGAAAGGAGGTACCGGAGTC 249 3 0.09
ICCM0273 FI856963 (TGA)4 TGTAACTCATCATCGCCAGC AGACGTGTAGACAGATGCCC 108 2 0.04
ICCM0274 FI856640 (TC)9(TA)15 GACCCTACCCCGCAAGTAAT TTTTTGTCCACACTCACACCT 265 NA  
ICCM0276 FI856965 (C)10 CTCCTACACTGCCTCCCCTC TCATGCTTACTCCGTTGCAG 222 NA  
ICCM0277 FI856642 (TTA)11 GGCAAACAATAACCGAAAACA GTAAAGAGGGCCAGCTGTTG 196 6 0.30
ICCM0278a FI856643 (TTA)4 ATAGGGGACCAAAACTGCAA GTGGGTAATCCGTGCGTAAT 203 1 0.00
ICCM0278b FI856643 (AT)4 AAAATACACATCCTGACTGCCA TTTTGCTTAGACTTGTAGGCATT 159 2 0.17
ICCM0280 FI856969 (T)11 ACTAGATGGTCGCATCCTGG GGTGAAGGTGTGGATGAGGT 280 1 0.00
ICCM0281a FI856644 (AC)9 TTCAACCCTCCCTACACGTT GTTCTCTTCTTGCTGGTGCC 235 1 0.00
ICCM0281b FI856644 (AAT)5N(A)11 TGGAACAACCAAGACCTTCA GCTGCCACAAACACTGAGAA 264 2 0.04
ICCM0282a FI856645 (CGC)7 CCTCGTTGTTGCCCTGTATT AAAACGCAAGCAAAGCAAGT 154 3 0.23
ICCM0282b FI856645 (ATT)4N(A)10 ACTTGCTTTGCTTGCGTTTT TTTGTGGGTTGGTTGAATTTT 219 NA  
ICCM0282c FI856645 (A)10N(TA)4 AAAATTCAACCAACCCACAAA ATCACTCTACCGTCAAAACGA 250 4 0.25
ICCM0284a FI856647 (AT)4 CGTATCTACACCCGCACTCA TGGAAAATCCACTTTGATTGG 257 3 0.08
ICCM0284b FI856647 (TA)4 CGTATCTACACCCGCACTCA TGGAAAATCCACTTTGATTGG 257 9 0.30
ICCM0285 FI856971 (ATTC)5 TGAGGACAAGATTCCGTTCA AACATGCGGGTTGTTTTCTC 267 1 0.00
ICCM0286a FI856648 (GA)5 AGCATCACGCATACAGCTTG ACATTGGCTCCATTGTTTGG 254 2 0.04
ICCM0286b FI856648 (AG)4 ACCCCCAAAATGCTGTAGTG ACGCCCCTTTACTGTTACGA 276 1 0.00
ICCM0288 FI856972 (TAA)4N(TTA)4N(TTA)4 TTATTTTTCGGATCCAACGC GTGATTTTTGTTCGGCCATT 278 20 0.88
ICCM0289 FI856976 (T)13N(ACA)4 CAGCCTCCATGGCATAGATAA TGCTTGAATGAGTGCAACAA 219 15 0.83
ICCM0290 FI856976 (A)15 TTGTTGCACTCATTCAAGCA TTTTTATTGGGGCATTGAGC 244 6 0.38
ICCM0291 FI856978 (AT)4 AAGTATTCAATTATACGTGCACAAAA TCATCCTTGTTAAGTCAACCACTT 249 1 0.00
ICCM0292 FI856978 (TAA)6 TGGTTGACTTAACAAGGATGAGTG TCCTCAAGCAGAGGTGGTTC 267 1 0.00
ICCM0293 FI856982 (TAA)15tg(ATA)15 AGTGATGCCACGAGAATTGC CTGGTTCGGAATTGTCATCC 250 5 0.24
ICCM0294 FI856986 (TTA)15 AGAGGCAAACAAGAACCGAA CACCCAATTTTGTCCGATTT 185 NA  
ICCM0295 FI856987 (T)10 GAGGCACCAAATTCGTATCC CAAAATTTTCTAATTCACCAAGACTTC 256 3 0.09
ICCM0296 FI856987 (TGATT)4 CGCCAAGTTTTACTATGTGCTG TGTCTGGATGTTACATAAACACTCTT 227 1 0.00
ICCM0297 FI856987 (TAA)18 CATGATTTGATTTGATTTGATTTC GGAGTGGGAAACCTTAAGCC 271 3 0.08
ICCM0298 FI856989 (TC)4N(AAT)4 GTGCACTTGTTCAGCGTTGT CGCAAACACACATTCCTCTG 221 5 0.33
ICCM0299 FI856989 (CTT)7N(TCT)4 TTATGAAGCCGAAGCTCGTT GAGCAGTAAACGTACCCCCA 272 NA  
ICCM0300 FI856990 (A)10 ATGGCCAAAATGAACTCCAG AAAAGAGAAGGTTCCATCGG 173 2 0.10
ICCM0301 FI856992 (A)10 ATGGCCAAAATGAACTCCAG AAAAGAGAAGGTTCCATCGG 173 1 0.00
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Marker names start with prefix ICCM, which represent ICRISAT Chickpea Microsatellites

NA not amplified

aSSR motifs having “N” nucleotide represent the interruption of few base pairs between two same/different SSR motifs

bNumber of alleles is calculated based on screening 48 chickpea genotypes using touch-down PCR profile of 61–51°C

Development of gene-based SNP markers

PCR primers derived from Medicago ESTs (expressed sequence tags), Medicago BAC (bacterial artificial chromosome)-end sequences, and M. sativa cDNA sequences have been described in previous studies (Choi et al. 2004a, b, 2006). To design PCR primers based on chickpea ESTs (Buhariwalla et al. 2005), candidate chickpea transcripts were compared to sequenced Medicago BAC clones (http://www.medicago.org/genome), and transcripts with high nucleotide identity and low copy representation to the Medicago genome were selected for primer designing. Primers were designed from highly conserved coding sequences, to amplify across intron regions (Choi et al. 2004a), using the Lasergene PrimerSelect software package (DNAStar Inc., Madison, WI, USA). Details of these primer pairs are given in Table 3.

Table 3.

List of gene-based SNP anchor markers used in comparative mapping of chickpea and Medicago

Marker name Template sequence accession no. Type Sequenced Mt BAC accession no. Linkage group in M. truncatula Linkage group in chickpea Genotyping method Restriction enzyme Forward primer (5′–3′) Reverse primer (5′–3′)
CALTL AW126242 Mt/EST AC149080 1 4 CAPS Alu I GTGGAAGGCACCATTGATTGACAAC TCTTCTTCTCAGCCTCTTCAAATGC
CDC2 AW171750 Mt/EST AC144481 1 4 SNaPshot N/A CAACTTTGCAAGGGTGTTGCTTTCT ACTAACACCTGGCCACACATCTTCA
CysPr2 AI974635 Mt/EST AC148098 1 4 CAPS Taq I CCAAAAACTTGCTTCTATACTCTTCATTC GACAAAACCCACCCAGACAAATCAACTAG
HRIP AW126332 Mt/EST NA 1 4 SNaPshot N/A GGAAAAATTTATCCTCCAAATTGGGGGTA AAAAATAGCAGTGCACCAAAAAGTGCTG
ppPF AI974685 Mt/EST NA 1 4 SNaPshot N/A TCTCGCCACCAACAACAACTAC AAAAATTGTTCATGAACACTCACTTGAAGCCA
REP AA660953 Mt/EST AC133709 1 4 CAPS Taq I CTCCATTTCCCGTTCGTTCG CACCGGTTGCCCTCCAGAC
RL3 AI974458 Mt/EST AC125473 1 4 CAPS Hind III TTCAATCGATAAGAATACTGCTTTG TGTGTGTCATACCAGCCTTG
TC76700 TC76700 Mt/EST AC122171 1 4 CAPS Hinc II CCAAAGACCCAGTTCGTGTT GTTGCATGGTTGACATCAGG
TC86606 TC86606 Mt/EST AC139748 1 4 SNaPshot N/A TGAAAATGGCAATGTGGAGA TTGGGATTCCTCATCCTCAG
TC87270 TC87270 Mt/EST AC124216 1 4 CAPS Dde I TGGCTTCTTTCGGTCTCTGT TCAAAACCACGCATTTGGTA
TC88727 TC88727 Mt/EST AC137895 1 6 SNaPshot N/A ATCAGGCAGAACTTGCTCGT AGAGTGCCCGGTATATTCCT
DSI AA660976 Mt/EST AC122168 1 4 SNaPshot N/A GAAGCCAAAAAGTATGAAGGGCCACGCAC CATGGTGCATAAAACTCAACCAAGACATC
ChitinaseII NA NA ~AC137554 2 1 Pfaff and Kahl (2003) AGCACATGAACCAATCCACA CATGGTTGCAATTTCACGTC
ACCO AI974230 Mt/EST AC121236 2 1 CAPS Stu I GAAGATGGCGCAAAAAGAAAGT CGATGTGTCGTGTCATCTCGTTAAGTTCCCT
AJ005043 AJ005043 Ca/EST ~AC127170 2 1 SNaPshot N/A TTTTCGGGAACTGCTTATGG CATGGAAGTGGACCGTTTTT
CPCB2 AW191283 Mt/EST NA 2 1 CAPS Dra I AGAAAGAGTGAAGTCTGTGGATCTACATC GGATGAACAGCCACACACCTAATGTAATC
DMI-1 AY497771 Gene AC140550 2 1 CAPS Hinc II ACCCTTCTTTCCTTGGCATT ACCGCATACAACGCTAAACC
TC77488 TC77488 Mt/EST AC126013 2 1 CAPS Afl III ATGCTTGCAGATCCTGCTTT GATCTGCCTCAACTCCAAGC
1433P AI974411 Mt/EST AC144342 3 5 SNaPshot N/A AAGGTTTTCTACCTTAAGATGAAGGGAG GTTTAGCAAGATTGCAGGCACGA
AF457590 AF457590 Ca/EST ~AC122728 3 5 SNaPshot N/A CTCCCTCTCTATCGCCCTCT TGTGAAAGGGTTGGTGTGAA
AIGP AW125928 Mt/EST NA 3 5 CAPS BSPW I CTGATAGGGCCAGGAGGCAGGGAAGA GTTTTTTAGCATTTGGACGAATGGTTGGT
CysPr1 AI974595 Mt/EST AC131026 3 5 CAPS Ear I GAGAATTCAAAGAAGAAATTAAGACAAAGA GAAGAATTCATGGGGAGCAAAGT
Ms/U515 AJ410128 Ms/EST NA 3 5 SNaPshot N/A GTTAAGGGAACCATGACAACCACA CCAGGCTCGGATTGAGCAGGGTTTGT
Ms/U83 AJ410159 Ms/EST NA 3 5 SNaPshot N/A TGGAAAACTTGATGGATCCTGCTCA TGATAGTCCTGCAACAAGTCCAGCA
P40 AJ006759 Ca/cDNA ~AC143340 3 5 Pfaff and Kahl (2003) TTTTTAAACGCCGCAATGA CAGAAAGCAATGGTGGGAAT
TC80362 TC80362 Mt/EST AC137828 3 5 CAPS Ase I GCTTGAGCCTCCAGAGATTG TGATAAGCCTTGCAGTGTGC
TRPT AA660362 Mt/EST AC122170 3 5 SNapshot N/A CAACAACCTAGTATAGCGATCAGTG CATTCAAAGCCCACCAAGTT
DNABP AI737524 Mt/EST AC121244 4 6 CAPS Afl III CCCTATGAGCTTGGGTTTGTCT CTCATGGCATACGTGTTCAGC
EST948 AA661051 Mt/EST AC145021 4 6 CAPS Dde I GCAGGGGTTTCGCTCCAGTG AACTTAATGAATGATTGGAAGGTTTAGCG
Ms/U40 AJ410120 Ms/EST NA 4 6 SNaPshot N/A AAGAATGAGGAAGAGGAATTCAACA GGTTCCTAAGGAACAATGATGACA
MTU07 AI737610 Mt/EST NA 4 6 CAPS Bsm I CAGACACCCAAAGAATTACCAGAA GATGACCAGAGCCTAATACTATTTATGACT
TC78756 TC78756 Mt/EST AC144502 4 6 CAPS Alu I GGAGGAGGAGGAAGATCAGG ATGCTGGAGAGATGGTGAGC
TC79726 TC79726 Mt/EST AC130805 4 6 CAPS Cla I TGCTGAAGGGAGGATTCAAG ACAGCATTGTGAGCAACCAC
TC88598 TC88598 Mt/EST AC135316 4 6 CAPS Bcl I AGATGGGGAGATTGATGCTG AAGCAACATGCAGTGAGCTG
TGDH AA660742 Mt/EST NA 4 6 SNaPshot N/A CGGTGGCTTCATCGGTTCT GACGTGTATTGTAATCAGCAGGAGTA
tRALS AW126282 Mt/EST AC125476 4 6 CAPS Msp I GGTCTGCGAGCTGTTTTTGGAGAAG GCAATTCCCTCCTCAGCTAAAAGTG
U71 AJ410150 Ms/EST NA 5 8 SNaPshot N/A GCTGAAGCTGAAGGTTTTCG CGCATTTTTATGGATGAAGAGA
X60755 X60755 Ca/EST ~AC137669 5 8 SNaPshot N/A AGGTGCCATAGGAAGACACG CCCAATCTTTTTCTCCCACA
AB025002 AB025002 Ca/EST ~AC122727 5 2 CAPS EcoR V TTCCTCGATCATGTCGAACTT CGTGCACCAGCTTCGTAGTA
AJ005041 AJ005041 Ca/EST ~AC122727 5 2 CAPS Dra I AGCGTCTAGCCAGCATCAAT CCATGGCTTCTTACCCTTCA
AJ404640 AJ404640 Ca/EST ~AC131455 5 2 CAPS Pst I TGGAGAGAATGAGGGGAATG TCAAAGGATGCCAAATCACA
CYSK AW207985 Mt/EST AC135320 5 8 SNaPshot N/A GGAATTGCTAAAGATGTTACAGAATTGA AATGAGGACACTCTGTCCAGGTGTGA
CYSS AW127154 Mt/EST AC135320 5 8 CAPS Bgl II CTGATGCAGAAGAGAAGGGGCTTATCA CCAATTCAGCTCCAAAAGCTAATAGAATGA
DK242R AQ917211 Mt/BES NA 5 2 CAPS BsmA I CGTATGTTTAATCCGTTAGTCCGTCTT GCTTGCTTAGATATTTGGCACTTCA
FENR AW127593 Mt/EST AC138010 5 8 CAPS Hph I ATGCTTATGCCAAAAGATCCAAATGC CTCACAGCAAAGTCGAGCCTGAAGT
Ms/U393 AJ410119 Ms/EST NA 5 2 SNaPshot N/A ATTGGAGAAGGGCAATCCTCCACCA TCCCTTCATTCTATCCATCCCAAGA
Ms/U89 AJ410164 Ms/EST NA 5 8 CAPS EcoR V CATATGAACAGTTGAAAGTGGATGA CAATATGGCCAACATTAAAAAATGGCA
TCMO AW127521 Mt/EST AC141923 5 5 CAPS Hph I GTCTACGGCGAACATTGGCGTAAAATGC CAATTGCAGCAACATTGATGTTCTCAACA
TC87369 TC87369 Mt/EST AC124591 6 2 CAPS Pvu I ATAGTGGACTTCGGCGAGAA TGACGGGGATCTTTTCTTTG
AGT AW126002 Mt/EST NA 7 3 CAPS Xba I GATTTGGGCCTCATTCCTTCTTGTGTGCA CCTGAAGGGGGAAAATTGCCCACATTGA
AJ004917 AJ004917 Ca/EST ~AC136505 7 3 CAPS Dde I CCAAGGAAACCAGTGGATGT GCAGCATCAAGATCACGAAA
AJ012739 AJ012739 Ca/EST ~AC130801 7 3 CAPS Dra I CTTCAGTCAGGAGGGAGACG TGCAAATTTCGCTACAAGGA
AJ291816 AJ291816 Ca/EST ~AC140025 7 3 SNaPshot N/A TTGGAGGTGCTGGTGATGTA TGCAAATGCTTGCCAAATAG
DK225L AQ917191 Mt/EST AC137994 7 3 CAPS Hinc II TGTCCTTGCTTCTTATCCTTCCTTCA AGCAGCACAACAACTTACAACAACTC
ENOL AA660534 Mt/EST NA 7 3 CAPS Dde I TTCCATCAAGGCCCGTCAGA TTGCACCAACCCCATTCATT
MS/U380 AJ410118 Ms/EST NA 7 3 CAPS Taq I CACTCATTGCAATTTCCATGCTTCA CAGTTGTTGTAGCAAGGGCACA
RNAH AI974503 Mt/EST AC123899 7 3 CAPS Mbo II GCTTCCACCAGCTGATACACG TTAGCCCTAGCAAGAATGTCACTG
TC76881 TC76881 Mt/EST AC136505 7 3 CAPS Hph I TTCTTGGGGAACAGAACAGG GCAGCATCAAGATCACGAAA
TC78638 TC78638 Mt/EST AC135795 7 3 CAPS Acc I TGATTGCAAAGCAGGTTGAG CGGCATTCAGTAGGAAGCTC
TC84431 TC84431 Mt/EST AC139355 7 3 CAPS Nsi I AGAAAGGACTTGGGCAACCT CCAATACGTGCCTCTTTGGT
TC86212 TC86212 Mt/EST AC124963 7 3 CAPS Hinc II GTGGCTCCTTGTGTGCTGTA AGCCAGGATGAGGCACTCTA
TC88512 TC88512 Mt/EST AC136974 7 3 SNaPshot N/A CTACTGTTGGACGGGTTGCT AACCTGGGGCATTTGTGTAA
TC88726 TC88726 Mt/EST AC121242 7 3 SNaPshot N/A GAGGAGAATACGCCATCCAA GGATAGTGTGGGCTGCATCT
AJ276270 AJ276270 Ca/EST ~AC122726 8 7 SNaPshot N/A GCTGCGAATTTTGACCTAGC TGCCTTTGCAGATTCAGTTG
AJ276275 AJ276275 Ca/EST ~AC140032 8 7 SNaPshot N/A TTTTTGCCTGGACCAAATTC AAGGCTTGTACTGCCCTTGA
AJ489614 AJ489614 Ca/EST ~AC138087 8 7 SNaPshot N/A CGAGGAAGAATGGCAAAGAG AACAAGCCAATGAGGGAGTG
CoA-O AI974546 Mt/EST AC119408 8 7 CAPS Nla III TTTGGGGGAAATAATGGAAGTCT CTCGGGCAATGTTGAAAAATC
CPOX2 AW127442 Mt/EST NA 8 7 SNaPshot N/A AAAGAATCATGTCCTCAAGCTG CTTCTTCTTGTGGAAGTCAGCA
EST671 AA660779 Mt/EST NA 8 7 CAPS Hpa II GGTGTTATCTATGAAAGAGGCCTCATTGG TGTCCTTGGGTATGTAGAAAAGCCTTCAC
FIS-1 AI974522 Mt/EST AC122726 8 7 SNaPshot N/A TCAGTGATTGAGGGTTTTTCTACG CTGTTTCATCAACTTCAGCAACTTT
Ms/U82 AJ410158 Ms/EST NA 8 7 SNaPshot N/A TTCGTCAACGAGATGTTTGTGTTGGC CAAATTGAAGAAAAGAGGAATGAAGGT
AJ004960 AJ004960 Ca/EST ~AC136142 Unknown 2 CAPS BstN I GCCTGGTGTGATCGTTACCT ATGAAACCGGCAAAGACTTG
Ms/L591 AJ410091 Ms/EST ~AC148970 Unknown 3 CAPS Hind III GGCAGCTATAAAATCAAGTATCATGC TGCCACTTCGCCAAAGGACTCATTA
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“~” denotes that the putative orthologs of the chickpea genes are located in the corresponding M. truncatula sequences (Genbank accession numbers). TC#’s are from TIGR gene index database (http://www.tigr.org)

Mt, Medicago truncatula; Ms, Medicago sativa; Ca, Cicer arietinum; NA, not available; N/A, not applicable

The polymorphic gene-based markers between the parents of mapping population were identified essentially as mentioned in Choi et al. (2004a). Each pair of corresponding sequences from genotypes ICC 4958 and PI 489777 was aligned using Sequencher software (Gene Codes, Ann Arbor, MI, USA) to detect SNPs. The sequences with SNPs were transferred to DNA Strider 1.2 (Douglas 2008) to identify restriction site that is coincident with SNPs and cleavable amplified polymorphic sequence (CAPS) assay for genotyping the corresponding SNPs were developed. In cases where a suitable restriction enzyme site was not identified, oligonucleotide primers were designed immediately adjacent to the SNP position, which allows for a single base extension of the SNP site using ABI SNaPshot Multiplexing Kits (Applied Biosystems Inc., Foster City, CA, USA).

Genotyping assay

For both ICCM as well as H-series SSR markers, the forward primers were anchored with M13 tail (CACGACGTTGTAAAACGAC). PCR amplicons generated by SSR markers were analyzed on capillary electrophoresis, while for gene-based SNP markers the CAPS or SNaPshot assays were used for genotyping. For SSR genotyping, PCR was carried out in 5 μl reaction volume in GeneAmp® PCR System 9700 thermal cycler (Applied Biosystems, Foster City, CA, USA). The reaction mixture contained final concentration of 5 ng/μl of template DNA, 0.5 mM dNTPs, 0.5 μM of M13 tailed forward, 1 μM of reverse primer, 1 μM of M13 labeled primer, 0.75 mM of MgCl2, 0.1 U of Taq DNA polymerase (AmpliTaq Gold), and 1× PCR buffer (AmpliTaq Gold). An initial denaturation was given for 15 min at 94°C. Subsequently, ten touch-down PCR cycles comprising of 94°C for 20 s, 61/60/55°C (depending on the marker as given in Table 2, ESM Table 1) for 20 s, and 72°C for 30 s were performed. These cycles were followed by 35 cycles of 94°C for 10 s with constant annealing temperature of 54/56/48°C (depending on marker and touch-down profiles as given in Table 2, ESM Table 1) for 20 s, and 72°C for 30 s, and a final extension was carried out at 72°C for 20 min. The amplified products were separated by capillary electrophoresis using ABI PRISM® 3700 DNA analyzer, and allele calling was carried out as given in Varshney et al. (2009b).

For SNP genotyping, in CAPS assay, 1.5 μl PCR product of 94 RILs was digested with the corresponding restriction enzymes. Each digestion reaction contained 2–5 U of the corresponding restriction enzyme and 1× compatible buffer in a total volume of 10 μl. Enzyme digestions were incubated at the appropriate temperature for at least 4 h. Digestion products were separated and scored as mentioned in Choi et al. (2004a). In case of SNaPshot assay, the ABI SNaPshot Multiplexing Kits was used following the same protocol as suggested by the manufacturer, except that 0.5 μl SNaPshot mix for a single marker was used (see Choi et al. 2004a).

Polymorphism assessment of SSR markers

While ICCM-series markers were screened on the panel of 48 diverse genotypes including the parents of the inter-specific mapping population (Table 1), the H-series markers were screened on only two parental genotypes (ICC 4958 and PI 489777). Allelic data obtained for the SSR markers were subjected to AlleloBin program (http://www.icrisat.org/gt-bt/download_allelobin.htm) for allele calling based on the repeat units of SSR motif for corresponding markers. In case of ICCM markers, the binned allelic data were used to calculate polymorphic information content (PIC) value of 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 polymorphic markers were generated on 131 recombinant inbred lines (RILs) of the mapping population and for 94 RILs in case of gene-based SNP markers. In addition, marker genotyping data for 407 marker loci were compiled (Huettel et al. 2002; Pfaff and Kahl 2003; Tekeoglu et al. 2000; Winter et al. 1999, 2000).

Marker genotyping data were analyzed using the χ2 test to assess the goodness-of-fit to the expected 1:1 segregation ratio for each marker. Subsequently, genotyping data for all the markers, including those with distorted segregation, were used for linkage analysis using MAPMAKER/EXP 3.0 (Lander et al. 1987). Marker loci were first divided into linkage groups at a LOD score of 16 and a recombination fraction of 0.37 by two-point analysis using the ‘group’ command. Marker order in the linkage groups was determined using the multi-point analysis ‘try’ command of the program. Most likely order of the loci within the group was determined using multipoint ‘compare’ command. The ungrouped marker loci were also attempted to integrate into genetic map at a smaller LOD value (up to 6). The map distances were calculated by applying the ‘Kosambi’ mapping function (Kosambi 1944) as per MAPMAKER/EXP 3.0 program. Residual heterozygosity was not considered in linkage mapping.

Results

Isolation and characterization of simple sequence repeats

A genomic DNA library composed of ca. 400,000 clones was constructed from the ICC 4958 genotype. Hybridization of this library with GA and TAA oligo probes yielded 359 clones that were sequenced and assembled into a set of 115 contig and 342 singleton DNA sequences, which we refer to as genome survey sequences (GSS). These sequences were submitted to National Centre for Biotechnology Information (NCBI) and respective GenBank accessions are mentioned in Table 2.

Two hundred and ninety-nine of the 457 GSSs were determined to contain a total of 643 SSRs, with 165 GSSs containing more than one SSR. As depicted in Fig. 2, di- and tri-nucleotide repeats were the most abundant (39 and 40%, respectively), with mono-nucleotide and tetra-nucleotide repeats representing 16 and 3% of cases, respectively. Other types of SSRs had <1% representation. In terms of repeat motifs, the tri-nucleotide repeat motif TAA/ATT was most common, accounting 36.8% of all repeat, followed by the di-nucleotide repeat GA/CT at 19.2%.

Fig. 2.

Fig. 2

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Frequency of microsatellites based on type of repeat motifs in microsatellite-enriched library of chickpea. Frequency of tri-nucleotide repeats were higher among the chickpea microsatellite markers followed 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

These SSR loci were categorized into two groups based on the length of their SSR tracts: Class I SSRs (>20 nucleotides in length) and Class II containing SSRs (>12 but <20 nucleotides in length) (Fig. 3). Considering only perfect SSRs, 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 were substantially enriched for tri-nucleotide repeats, which represented 77% of all Class I repeats. A similar uneven distribution was noted for other repeats, but most notably the penta-nucleotide repeats, which comprised 55% of all Class II repeats and less than 2% of all Class I repeats.

Fig. 3.

Fig. 3

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Distribution of Class I and Class II repeats in newly isolated chickpea microsatellites. Class I microsatellites are with >20 nucleotides in length and Class II repeats contain perfect SSRs with >12 but <20 nucleotides in length. Among Class I repeats, tri-nucleotide repeats were abundant followed by di-nucleotide repeats, while in Class II repeats, penta-nucleotide repeats contributed highest, followed by hexa-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

Similarity analysis was performed for all 457 GSSs using BLASTN and BLASTX algorithms, and significant similarity was determined at an Expect value threshold of ≤1E–05 (Table 4). Relatively few of the GSS sequences had E values that surpassed this score, irrespective of the species data set under analysis. This is consistent with the expectation that randomly selected short genomic sequences only occasionally correspond to gene coding regions that will match EST data sets. Nevertheless, in cases where BLAST hits with e-value lower than 1E–05 threshold were recorded, the degree of similarity, expressed as either nucleotide identity of deduced protein similarity, was highest for phylogenetically related species, decreasing in rank order of phylogenetic distance (i.e., Medicago > lotus > soybean = cowpea = common bean > poplar > Arabidopsis > rice). Among these sequences, 40 were identified as related sequences in all three analyzed cool season legumes, i.e., chickpea, Medicago, and Lotus (Hologalegina clade; see Fig. 1), while 29 sequences had similarity with all three analyzed warm season legumes, i.e., soybean, common bean, and cowpea (Phaseoleae clade). Only 21 sequences were identified as similar sequences in both Hologalegina and Phaseoleae species. Two of these GSSs (FI856609 and FI856659) showed significant similarity with sequences of all the plant species analyzed in the present study (see ESM Table 2).

Table 4.

Functional annotation of ICCM sequences with EST databases

BLAST algorithm Database Number of entries in database searched Number of sequences showing similarity Number of sequences with significant 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
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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, Lotus japonicus; Pv, Phaseolus vulgaris; Vu, Vigna unguiculata; Gm, Glycine max; Ah, Arachis hypogaea; At, Arabidopsis thaliana; Os, Oryza sativa; Pa, Populus alba

With the objective of annotating these newly isolated GSSs, all 457 GSSs were analyzed for BLASTX analysis using UniProt database. 137 of these GSSs (29.9%) showed homology to the UniProt database at a relatively relaxed cutoff value of ≤ 1E–05. Among these, 84 unique protein sequences were used for deriving respective gene ontology (GO) (see ESM Table 3). The GO studies permitted assignment of 64 sequences to biological process, 64 to cellular component, and 67 to molecular function ontologies. According to the GO schema, single proteins typically have more than one Ontology assignment.

Development of novel SSR genetic markers

All SSR containing GSSs (299) were analyzed by means of Primer3, yielding a list of potential oligonucleotide primers from which 311 primer pairs were selected and synthesized. Where feasible primer pairs were designed for more than one SSR in a single GSS with the goal of increasing the conversion of GSSs into useable genetic markers.

Primer pairs were screened for amplification of DNA from 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 234 markers on 48 genotypes of chickpea further defined a subset of 147 polymorphic markers (62.82%), with allele content ranging from 2 to 21 and an average of five alleles per marker. Among these 147 polymorphic sites, 56 were polymorphic exclusively in wild species, 8 were polymorphic exclusively in cultivated and 83 of them were polymorphic across wild and cultivated species of chickpea.

We refer to these new polymorphic SSR markers as ICCM (ICRISAT Chickpea Microsatellite) markers. Allelic data obtained from 48 genotypes were used to calculate the PIC value of each ICCM marker, and thus infer the discriminatory power of these ICCM markers. PIC values ranged 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 by marker ICCM0022 with 18 alleles and a PIC value of 0.89 (Table 2). As has been observed in previous studies of SSRs from plant species (Temnykh et al. 2001), Class I SSRs (41 of 57) were on average more polymorphic that Class II SSRs (106 of 177), with mean PIC values of 0.38 and 0.22, respectively. Nevertheless, a higher fraction of the polymorphic SSRs identified in this study were from Class II (106) compared to Class I (41), owing to the increased abundance of Class II SSRs in our data set. Consistent with their overall abundance in Class I SSRs (Fig. 3), tri-nucleotide repeats (20) constituted major part of the Class I polymorphic sites, with compound repeats (18) comprising the next largest fraction of Class I ICCM markers. In contrast, di-nucleotide repeats were relatively rare in the total Class II data set, but comprised the largest fraction of polymorphic Class II ICCM markers (47); similar to Class I markers, compound repeats (30) constituted of the second most common fraction of Class II polymorphic sites.

In addition to the ICCM markers developed in this study, we also analyzed a set of 233 markers developed primarily by Lichtenzveig et al. (2005); these are the so-called “H-series” SSR markers. One-hundred fifty-three H-series markers yielded scorable amplicons in two PCR profiles (ESM Table 1). Both the ICCM and H-series SSR markers were tested for polymorphism between chickpea ICC 4958 and PI 489777, the parents of the inter-specific mapping population. From this analysis we identified 104 SSRs (52 ICCM and 52 H-series) that were suitable as genetic markers in the inter-specific cross, with polymorphism rates of 33.9 and 22.2% for the H-series and ICCM SSR markers, respectively.

Development of gene-based SNP markers

A set of 246 gene-specific primers, developed earlier by Choi et al. (2004a) based on gene sequences of M. truncatula and M. sativa, were used to amplify DNA of the parental genotypes of the inter-specific mapping population of chickpea. One-hundred four (~42%) of these primer pairs showed strong single fragments on 1% agarose gels; these amplicons were re-sequenced in both mapping parents of the inter-specific cross (ICC 4958 and PI 489777), quality-scored, and trimmed to yield 96 pairs of high quality sequences. Additional 25 primer pairs were designed based on chickpea EST sequences that possessed high similarity to previously mapped Medicago genes, yielding 18 additional high-quality sequence pairs. Alignment of the 114 ICC 4958 and PI 489777 sequence pairs revealed SNPs in 80 (~70%) genes. Seventy-one of these genes contained SNPs that could be converted to reliable genotyping assays using either CAPS or SNaPshot protocols (Table 3). Two additional gene-based markers, P40 and chitinase II, were also used for genetic analysis; these genes were previously mapped in chickpea by Pfaff and Kahl (2003), while their putative orthologs have been mapped in M. truncatula by Choi et al. (2004a).

Construction and features of the genetic map

The inter-specific cross between ICC 4958 × PI 489777 is maintained as an advanced recombinant inbred population that has been used in numerous genetic studies (Huettel et al. 2002; Pfaff and Kahl 2003; Winter et al. 2000). Although the number of markers previously analyzed in this population is relatively large (407 loci), a high percentage 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 are based on molecular markers that are either difficult to apply or to reproduce. With the intent of extending this genetic map, and enhancing the number of easily scorable markers, we genotyped the 123 new molecular markers (52 ICCM SSR loci and 71 gene-based SNP loci) and 52 previously published H-series SSR loci described above, and combined the genotype data with that of the 407 previously published loci. Linkage relationships were evaluated using MAPMAKER/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 marker loci coalesced to yield eight linkage groups, in agreement with eight chickpea chromosomes. The linkage groups were numbered according to Winter et al. (2000), using marker loci that were common to both studies. This revised genetic map contains 521 marker loci, with an average inter-marker distance of 4.99 cM and spanning 2,602.1 cM. Considering the 740-Mbp physical size of the chickpea genome (Arumuganathan and Earle 1991), and ignoring the fact that rates can vary widely within the genome, 1 cM distance in the present map equates to roughly 285 kbp. With the exception of linkage group (LG) 8, which has relatively few genetic markers (25 markers), the average number of markers per linkage group was 71 ± 8.9. LG 8 was also the shortest linkage group based on genetic distance, spanning 124.7 cM; however, in general LG size was not well correlated with the number of markers. As described below, comparative mapping with Medicago truncatula revealed that the entirety of chickpea LG8 corresponds to one arm of Medicago Chr5, adding further credibility to its assignment as a physically short linkage group.

Fig. 4.

Fig. 4

Fig. 4

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An integrated genetic map of chickpea based on recombinant inbred lines of C. arietinum (ICC 4958) × C. reticulatum (PI 489777). Map was constructed using MAPMAKER/EXP 3.0 with Kosambi mapping function. Distances between the loci (in cM) are shown to the left 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 markers taken from Lichtenzveig et al. (2005) are bold, italicized, and underlined; SNP markers which were used as the anchor markers in comparative mapping of chickpea and Medicago were depicted as bold and underlined. Linkage groups (LGs) are designated according to the map of Winter et al. (2000)

Comparative linkage analysis between Medicago and chickpea genomes

As shown in Fig. 5, the 71 gene-based SNP markers are distributed among eight major linkage groups of chickpea, facilitating comparison of genome structure between M. truncatula and chickpea. The respective M. truncatula and chickpea LGs are numbered according to Choi et al. (2004a) and Winter et al. (2000). Alignment of conserved genes between the two genetic maps reveals a high level of synteny between the two genomes. In particular, the M. truncatula linkage groups 1, 2, 3, 4, 7, and 8 correspond to chickpea linkage groups 4, 1, 5, 6, 3, and 7, respectively. Despite the overall high level of synteny between these six pairs of linkage groups, intra-chromosomal segment rearrangements reduce co-linearity (but not synteny) between M. truncatula LG1 (MtLG1) and chickpea LG4 (CaLG4). In contrast to the conserved synteny noted for Mt–Ca linkage 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. truncatula linkage groups 5 and 6 and chickpea linkage groups 2 and 8. In particular, M. truncatula LG5 can be aligned with both chickpea LG2 and LG8. We note that CaLG8 appears to be derived entirely from one arm of MtLG5, consistent with its short genetic distance and small number of genetic markers, described above. In several cases, conserved markers mapped to non-syntenic positions between the two genomes (e.g., CDC2 and TC88727 on MtLG1, DNABP on MtLG4, and TCMO on MtLG5), which may reflect translocation or duplication events involving single genes or small chromosomal segments, or the mapped loci may correspond to paralogous genes. Mt-LG6 could not be effectively aligned to any of the chickpea linkage groups (Fig. 5), consistent previous reports describing Mt LG6 as rich in heterochromatin (Kulikova et al. 2001) and having a relatively low content of transcribed genes (Choi et al. 2004a).

Fig. 5.

Fig. 5

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Comparative map of Medicago and chickpea. Gene-based SNP markers (marked in red color) were used as the anchor markers in comparative analysis of chickpea and Medicago genome. The resistance gene homologs (RGH) are depicted as oval structures and their homologs in Medicago are shown with connecting dotted lines. Solid lines show the macrosynteny observed across chickpea and Medicago with respect to 71 gene-based markers

Comparison of resistance gene homologs (RGH) between Medicago and chickpea

The majority of functionally characterized disease resistance (R) genes encode a nucleotide-binding site (NBS) and a leucine-rich repeat (LRR) region (Hulbert et al. 2001). NBS-LRR genes have been deeply surveyed and characterized in M. truncatula (Zhu et al. 2002; Ameline-Torregrosa et al. 2008), with >330 NBS-LRR genes having known genetic positions. In contrast, chickpea RGHs are not thoroughly surveyed, and only a limited number of sequences from degenerate PCR are available in the public databases (Meyers et al. 1999; Huettel et al. 2002). Nevertheless, several phylogenetically distinct RGH classes have been placed on the genetic map of chickpea (Huettel et al. 2002), thus facilitating the comparative genome analysis presented here.

Comparative phylogenetic analysis of RGH sequences from M. truncatula with those from chickpea is illustrated in Fig. 6. To highlight the comparison, only those M. truncatula sequences that are relevant to the mapped chickpea sequences 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 Cicer LG6 that also contains chickpea RGH-G (Huettel et al. 2002). Similarly, chickpea RGH-B (CAC86491; Huettel et al. 2002) is a CC-NBS-LRR gene that is closely related to several CC-NBS-LRR genes located in a cluster at the top of MtLG3, in a region of the Medicago genome syntenic with the terminus of CaLG5 that contains RGH-B (Huettel et al. 2002). A lack of synteny was observed for chickpea RGH-D (TIR-NBS-LRRs represented by sequences CAC86454, CAC86455, CAC86493, AF186626, and AF186629; Huettel et al. 2002), which is located at the top of CaLG2; the closest homologs of RGH-D in M. truncatula (i.e., BAC AC144658) are localized to the distal region of MtLG4. We note that the bottom of CaLG2 harbors numerous active resistance genes against two of the most important diseases of chickpea (Fusarium wilt and Ascochyta blight). At present, no RGHs have been reported mapped close to these resistance phenotypes (Winter et al. 2000; Pfaff and Kahl 2003; Sharma et al. 2004). Moreover, the low frequency of comparative molecular markers around these R gene regions in both M. truncatula and chickpea complicate precise statements regarding the relationship of these genome regions.

Fig. 6.

Fig. 6

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Comparison of RGH sequences in Medicago and chickpea. To highlight the comparison between the chickpea and Medicago RGHs, only those Medicago sequences that are relevant to the mapped chickpea sequences have been shown in this figure. In the TIR-NBS-LRR subfamily, chickpea RGH-G (CAC86496 on Ca-LG6) was found highly similar to several Medicago TIR-NBS-LRR genes (a) located on BAC clones AC144502 and AC135160. AC144502 and AC135160 were closely linked on Mt-LG4, in a region syntenic to Ca-LG6 that also contained chickpea RGH-G. In the CC-NBS-LRR (b) subfamily (Ca-LG5), chickpea RGH-B (CAC86491) was closely related to several CC-NBS-LRR genes located on Medicago BAC clones AC145027, AC142396, AC130810, AC146744, and AC131249

Discussion

SSR markers have become common place for plant genetics and breeding applications. Despite the fact that hundreds of SSR markers have been identified and tested in chickpea (Hüttel et al. 1999; Sethy et al. 2006a, 2006b; Winter et al. 1999; Lichtenzveig et al. 2005), the narrow genetic background of cultivated chickpea germplasm has limited their application, and thus there exists a need to develop a larger set of novel genetic markers. With the objective of enriching the marker repertoire of chickpea, we have contributed novel SSR markers derived from a genomic library enriched for GA and TAA repeat motifs and a set of gene-based SNP markers. The basis of our marker discovery work was C. arietinum genotype ICC 4958, which is being used as a reference genotype for genomic and genetic resource by the chickpea community.

In the present study, 65.4% of hybridizing genomic clones in our SSR-enriched library yielded 643 SSRs. This rate of SSR recovery is comparable with previous studies, for example in peanut where 68% of hybridizing clones yielded SSRs (Cuc et al. 2008). Moreover, the relatively high abundance of tri- and di-nucleotide repeats that we observed is consistent with previous studies in chickpea (Hüttel et al. 1999; Lichtenzveig et al. 2005; Winter et al. 1999). Among the SSRs identified here, the most common SSR motifs were TAA/ATT repeats and GA/CT repeats. This result reflects the fact that our enrichment targeted TAA and GA motifs, and it is consistent with previous studies in chickpea (Hüttel et al. 1999; Lichtenzveig et al. 2005; Winter et al. 1999), other legume species (Akkaya et al. 1992; Cregan et al. 1994; Mun et al. 2006), and even in cereal species (Varshney et al. 2002; Jayashree et al. 2006).

Temnykh et al. (2001) developed a scheme to classify SSRs according to length, in which Class I and Class II SSRs are greater than or less than 20 bp, respectively. This division based on sequence length has practical utility, because Class I SSRs are generally more polymorphic and thus more desirable as genetic markers. The majority of SSRs isolated from our SSR-enriched library belong to Class II, though as expected the Class I SSRs had higher rates of polymorphism. A useful measure of polymorphic potential for any genetic marker is its polymorphism information content value, or PIC value. PIC values provide information on the probability that a given marker will be polymorphic between any two individuals in a population, and thus are a function both of allele frequencies and allele number. Screening of the ICCM-series markers on 48 genotypes revealed that average PIC value of SSR markers having Class I repeats (0.38) was higher than that of Class II repeats (0.22). The majority of the Class I repeats were tri-nucleotide repeats, consistent with the known utility of tri-nucleotide repeats as genetic markers in plants (Varshney et al. 2005).

Polymorphic information content value was also analyzed in relation to repeat unit type and length. Among di-, tri-, and tetra-nucleotide repeats, tri-nucleotide repeats showed higher polymorphism (average PIC = 0.33) with average allele number of 5.7 per marker. Markers with mono-nucleotide repeats showed the least polymorphism (average PIC = 0.197). Relatively longer repeats appear to have contributed to the higher level of polymorphism as compared to di-nucleotide repeats (Gupta and Varshney 2000). It was also observed that among tri-nucleotide SSRs, the SSR markers based on (TAA/TTA) repeat motifs displayed higher polymorphism (average PIC = 0.35) with an average allele number of 6.12 per marker. Similarly, among di-nucleotide repeats SSR markers based on TA/AT repeat motifs had a higher average PIC value (0.27) compared to others with an average of 6.1 alleles. In fact, the earlier studies in chickpea also revealed the abundance of TAA/TTA (tri-nucleotide) and TA/GA (di-nucleotide) SSR motifs and the extensive polymorphism found with markers containing these repeat motifs (Hüttel et al. 1999; Lichtenzveig et al. 2005). PIC values of compound SSRs (average PIC = 0.29) were comparable with tri-nucleotide repeats with 5.68 alleles per marker. This can be attributed to the fact that the markers with compound SSRs have more than one SSR motif, which increases their chances to be polymorphic markers.

We assessed the potential identity of SSR-related sequences by performing BLAST analyses versus plant EST data sets, and based on GeneOntology analysis through UniProt. Less than one-third of the SSR-associated GSS sequences had significant hits in these databases, though were hits were recorded the derived annotations add a potentially useful data type to the marker metadata. Not surprisingly, chickpea GSS sequences (from which the SSRs were derived) had higher similarity to ESTs from other legume species, and overall higher similarity to dicot outgroups (i.e., poplar and Arabidopsis) than to monocot (i.e., rice) data sets.

Comprehensive genetic map of chickpea

An inter-specific mapping population derived from ICC 4958 (C. arietinum) and PI 489777 (C. reticulatum) was used to incorporate novel microsatellite and gene based markers. This mapping population has been widely used in past by chickpea community in order to incorporate several hundred microsatellite markers (Winter et al. 2000) and gene-based markers (Pfaff and Kahl 2003). The diverse genetic background of the parents provides for higher rates of polymorphism not only at the genetic level but also at phenotypic levels such as resistance to Fusarium wilt (Winter et al. 2000) and Ascochyta blight (Rakshit et al. 2003), facilitating trait mapping. Therefore, this population is generally considered as the international reference mapping population.

The present genetic map of chickpea represents 521 marker loci, spanning 2,602 cM with an average inter-marker distance of 4.99 cM. The order of common marker loci defined in present map agrees with earlier reports from Winter et al. (2000). However, the current map differs considerably from that of Winter et al. (2000) in having eight linkage groups, in agreement with eight chromosomes, whereas the Winter et al. (2000) map was composed of 16 linkage groups. There are probably at least two factors that contribute to this condensation of linkage groups: first, the new markers identified in the present study act as bridge points between the Winter et al. linkage groups, and second, essentially all of the markers mapped in the current study behave a co-dominant genetic features, which adds considerable power to the genetic evaluation compared to a high fraction of dominant markers in earlier studies. Importantly, the comparative analyses to Medicago support a simple assignment of eight chickpea linkage groups to eight chromosomes.

Comparative mapping of chickpea and Medicago

Mappig of the gene-based markers from Medicago in the genetic map of chickpea showed not only a high level of macrosynteny but also revealed features of structural divergence between the two genomes. Six of the eight linkage groups display a one-to-one correspondence between the Medicago and chickpea, suggesting that these linkage groups reflect the genome of the common Galegoid clade legume ancestor. Medicago LG5 and LG6, and chickpea LG2 and LG4, appear to have a more complicated ancestry, consisting of a minimum of several chromosomal translocation events. Thus, Mt-LG 5 is essentially a composite of portions of LG2 and LG8 of chickpea. Several research groups have compared genome structure between Medicago and various crop legumes (see Zhu et al. 2005). Our current results extend the comparative network to include chickpea, by demonstrating broad conservation of genome macrostructure between chickpea and Medicago.

One goal of comparative genetic analyses is to transfer information from well-characterized reference species to less well-characterized crops with an eye toward crop improvement. Among the agronomic targets in chickpea is resistance to several economically important pathogens; candidate genes for disease resistance are the conserved family of NBS-LRR resistance gene homologs (RGH). Several phylogenetically distinct RGH classes have been placed on the genetic map of chickpea (Huettel et al. 2002), thus facilitating the comparative genome analysis between chickpea and Medicago. In particular, we have documented two cases of syntenic NBS-LRR clusters that contain co-phyletic genes in each species. Interestingly, Ca-LG2 is known to harbor active resistance genes against Fusarium wilt and Ascochyta blight. At present, no RGHs have been reported mapped close to these resistance phenotypes. Nevertheless, the facts that a single conserved gene (TC87369) maps to the top terminal region of both Mt-LG6 and Ca-LG2, and that both linkage groups are rich in NBS-LRR genes and/or active disease resistance genes (Sharma et al. 2004; Zhu et al. 2002), may suggest shared ancestry of Mt-LG6 and Ca-LG2, though such speculation needs to be verified by more detailed study of the respective genome regions.

Similar observations of NBS-LRR synteny have been made for resistance gene homologs within the Solanaceae (Grube et al. 2000) and between Medicago and pea (Pisum sativum) (Zhu et al. 2002). However, the limited numbers of comparative molecular markers (gene-based SNPs) around these R gene regions in both Medicago and chickpea precludes precise statements regarding the relationship of these genome regions. Although the current analysis is based on a relatively small number of comparative markers, the potential of more detailed analyses to predict gene content and chromosomal structure in chickpea by reference to Medicago seems clear.

Conclusion

A set of 311 novel microsatellite markers were developed from microsatellite-enriched library in order to increase the genomic resources in chickpea. In total 147 potential SSR marker loci were found based on diversity pattern of SSR loci on a panel of 48 diverse chickpea genotypes. These markers should have utility for genetic analysis of a range of chickpea mapping populations and as anchor markers in comparative mapping to other legumes.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (XLS 32 kb) (32.5KB, xls)
Supplementary material 2 (XLS 689 kb) (689.5KB, xls)
Supplementary material 3 (XLS 66 kb) (66KB, xls)

Acknowledgments

Thanks are due to Prathima Juvvadi, Gudipati Srivani and Abdul Gafoor for their technical assistance. Financial support from Tropical Legume I of Generation Challenge Program (GCP, http://www.generationcp.org) of CGIAR and National Fund of Indian Council of Agricultural Research of Government of India are gratefully acknowledged. The financial support by National Science Foundation Grant 0110206 to DRC is acknowledged. Council of Scientific and Industrial Research (CSIR), Government of India is acknowledged for providing fellowship to SNN and the Indian Council of Research NATP-HRD program for providing fellowship to SD. We thank Dr. Fred Muehlbauer of the USDA-ARS for providing the chickpea recombinant inbred population and genetic marker data.

Open Access

This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.

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