Population Genetic and Structure Analyses of Annual Iranian Medicago orbicularis Indicates Advantageous Use of HTS-Generated SSR Markers

Simple sequence repeat (SSR) markers are among more reliable markers widely used in population genetics and traditionally need intensive labor to develop. High-throughput sequencing (HTS) has enabled researches in screening whole DNA in order to develop dozens of reliable SSR markers to pave route for future studies. Annual medics (Medicago spp.) are specifically beneficial in agriculture, genetics, and pastureland management plans. In order to facilitate study of annual medics using SSRs and to test their effectiveness, HTS screening of whole DNA resulted in developing of hundreds of primer pairs published in this paper. Subsequent testing of candidate primers on Iranian M. orbicularis germplasm was narrowed down to six more variable primer pairs that generated 28 polymorphic bands with the allele size of 200–310 bp. Eleven populations were examined using our SSR markers. The average polymorphic information content was very high (> 0.90) for the primer combinations. Genetic diversity parameters were measured for all populations. A high level of polymorphism of 75% within populations was observed. We hypothesized that the self-pollinating reproductive system of M. orbicularis has led to the reduction of gene flow between populations and ultimately the low diversity among populations (24%). Different genotypes observed by structure analysis as well as regression and correlation analyses showed that genetical similarity was congruent with the geographical proximity. Our results suggest that our HTS-generated SSR markers can be used for the further study of other annual medics.