Identification of microsatellite markers from Cicer reticulatum: molecular variation and phylogenetic analysis

Microsatellite sequences were cloned and sequenced from Cicer reticulatum, the wild annual progenitor of chickpea (C. arietinum L.). Based on the flanking sequences of the microsatellite motifs, 11 sequence-tagged microsatellite site (STMS) markers were developed. These markers were used for phylogenetic analysis of 29 accessions representing all the nine annual Cicer species. The 11 primer pairs amplified distinct fragments in all the annual species demonstrating high levels of sequence conservation at these loci. Efficient marker transferability (97%) of the C. reticulatum STMS markers across other species of the genus was observed as compared to microsatellite markers from the cultivated species. Variability in the size and number of alleles was obtained with an average of 5.8 alleles per locus. Sequence analysis at three homologous microsatellite loci revealed that the microsatellite allele variation was mainly due to differences in the copy number of the tandem repeats. However, other factors such as (1) point mutations, (2) insertion/deletion events in the flanking region, (3) expansion of closely spaced microsatellites and (4) repeat conversion in the amplified microsatellite loci were also responsible for allelic variation. An unweighted pairgroup method with arithmetic averages (UPGMA)-based dendrogram was obtained, which clearly distinguished all the accessions (except two C. judaicum accessions) from one another and revealed intra- as well as inter-species variability in the genus. An annual Cicer phylogeny was depicted which established the higher similarity between C. arietinum and C. reticulatum. The placement of C. pinnatifidum in the second crossability group and its closeness to C. bijugum was supported. Two species, C. yamashitae and C. chorassanicum, were grouped distinctly and seemed to be genetically diverse from members of the first crossability group. Our data support the distinct placement of C. cuneatum as well as a revised classification regarding its placement.