The development of 204 novel EST-SSRs and their use for genetic diversity analyses in cultivated alfalfa

Cultivated alfalfa (Medicago sativa L.) is one of the most important forage legume in the world. Here, we report the development of 204 novel polymorphic expressed sequence tag simple sequence repeat (EST-SSRs) markers from transcript sequences via global Illumina sequencing for cultivated alfalfa. Among the synthesized 750 pairs of primers, 204 EST-SSR primer pairs showed polymorphisms among 10 alfalfa accessions (including five individual plants in each accession) generating a total of 1222 alleles, with the number of alleles per locus, the observed heterozygosity (Ho), the corrected heterozygosity (He) and the Shannon-Wiener diversity index (H'c) averaging at 5.99, 0.73, 0.71 and 1.14, respectively. Of the 204 novel EST-SSRs in alfalfa, 120 can be in silico mapped onto the eight Medicago truncatula chromosomes (Mt3.5.2). Considering the high polymorphism, these EST-SSRs can be applied to assess genetic diversity, population structure, relatedness, evolution, linkage mapping and cultivar protection of cultivated alfalfa to facilitate alfalfa breeding programs.