Genome-wide discovery of di-nucleotide SSR markers based on whole genome re-sequencing data of Cicer arietinum L. and Cicer reticulatum Ladiz

Simple sequence repeats (SSRs) are valuable genetic markers due to their co-dominant inheritance, multi-allelic and reproducible nature. They have been largely used for exploiting genetic architecture of plant germplasms, phylogenetic analysis, and mapping studies. Among the SSRs, di-nucleotide repeats are the most frequent of the simple repeats distributed throughout the plant genomes. In present study, we aimed to discover and develop di-nucleotide SSR markers by using the whole genome re-sequencing (WGRS) data from Cicer arietinum L. and C. reticulatum Ladiz. A total of 35,329 InDels were obtained in C. arietinum, whereas 44,331 InDels in C. reticulatum. 3387 InDels with 2 bp length were detected in C. arietinum, there were 4704 in C. reticulatum. Among 8091 InDels, 58 di-nucleotide regions that were polymorphic between two species were selected and used for validation. We tested primers for evaluation of genetic diversity in 30 chickpea genotypes including C. arietinum, C. reticulatum, C. echinospermum P.H. Davis, C. anatolicum Alef., C. canariense A. Santos & G.P. Lewis, C. microphyllum Benth., C. multijugum Maesen, C. oxyodon Boiss. & Hohen. and C. songaricum Steph ex DC. A total of 244 alleles were obtained for 58 SSR markers giving an average of 2.36 alleles per locus. The observed heterozygosity was 0.08 while the expected heterozygosity was 0.345. Polymorphism information content was found to be 0.73 across all loci. Phylogenetic tree and principal coordinate analysis clearly divided the accessions into four groups. The SSR markers were also evaluated in 30 genotypes of a RIL population obtained from an interspecific cross between C. arietinum and C. reticulatum. Chi-square (χ(2)) test revealed an expected 1:1 segregation ratio in the population. These results demonstrated the success of SSR identification and marker development for chickpea with the use of WGRS data. The newly developed 58 SSR markers are expected to be useful for chickpea breeders.