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

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 x 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.

Fig. 1

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

Fig. 2

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

Fig. 3

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

Fig. 4

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)

Fig. 4

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)

Fig. 5

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

Fig. 6

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