Development of EST-SSR markers in Cenchrus ciliaris and their applicability in studying the genetic diversity and cross-species transferability

Abstract

Most of the grasses of the genus Cenchrus (20-25 species) and Pennisetum (80-140 species) are distributed throughout the tropical and subtropical regions of the world and reproduce both by sexual and apomictic modes. However, the relationships among the Cenchrus-Pennisetum species are not very clear yet. Molecular markers like expressed sequence tag-simple sequence repeats (EST-SSRs) have been reported to be a better choice for resolving the phylogenetic relationships and to estimate the genetic diversity. The present study describes the identification of EST-SSR markers based on the transcriptome data of Cenchrus ciliaris inflorescence and illustrates the genetic diversity and phylogenetic relationships among these species. Of the 378 primer pairs used across 33 accessions of 21 Cenchrus, Pennisetum, and related grass (Bothriochloa, Dichanthium and Panicum) species, 116 EST-SSR markers were found to be polymorphic with an average polymorphism information content (PIC) of 0.49. Fifty-one EST-SSR loci and 520 alleles showed that where the PIC value is >0.5 there the GAG repeat motif was highly polymorphic. Two EST-SSR markers, CcSSR_80 and CcSSR_102, are polymorphic among the Cenchrus species, while they are absent in Pennisetum and the allied species. Five SSR markers (CcSSR_75, CcSSR_85, CcSSR_87, CcSSR_88 and CcSSR_114) showed 100% cross-transferability among the 21 Cenchrus-Pennisetum species. Species-specific alleles could also be detected for seven species of Cenchrus, Pennisetum and Panicum across 10 SSR markers. Assay of polymorphism across these agamic complexes showed that the three SSR markers (CcSSR_26, CcSSR_97 and CcSSR_109) were associated with Cenchrus-Pennisetum complex, and one (CcSSR_47) with Bothriochloa-Dichanthium complex. Markers with high discriminating power, namely CcSSR_4, CcSSR_38, CcSSR_48, CcSSR_66, CcSSR_67 and CcSSR_70, can be used to estimate the allelic sequence divergence across the sexual and apomictic lineages. Genetic diversity analysis using neighbour-joining (NJ) and principal co-ordinate analysis (PCoA) based approaches showed six and five clusters for the 33 accessions, respectively, having congruence in the pattern of clustering. These accessions were grouped according to their mode of reproduction. Cenchrus and Pennisetum species were grouped separately within the same clade, implying monophyletic group within a 'bristle clade'. Thus, this study showed high discrimination power of microsatellite (EST-SSR) markers to resolve the phylogenetic relationships.