Development and verification of EST-SSR markers in Medicago archiducis-nicolai by transcriptome sequencing

Medicago archiducis-nicolai, which is endemic to the Qinghai-Tibet Plateau and its adjacent regions, is a species of Platycarpae in the genus Medicago. This species is characterized by its superior adaptability to extremely harsh environments. In this study, we developed EST-SSR markers of M. archiducis-nicolai using transcriptome sequencing. Polymerase chain reactions verified 350 of 477 randomly chosen EST-SSR primer sets in M. archiducis-nicolai. Of the verified primer sets, 346 were successfully amplified in M. ruthenica, indicating high transferability of the EST-SSR primers to M. ruthenica. The genetic diversity and structure of M. archiducis-nicolai and M. ruthenica populations were characterized using 64 non-genetically linked EST-SSR loci that did not deviate from Hardy-Weinberg equilibrium. Results show significant inter- and intra-species genetic differentiation of the two species, implying geographic distance and environmental selection influenced intra-species genetic differentiation. The EST-SSR markers developed in this study provide useful tools for detailed characterization of the genetic diversity of M. archiducis-nicolai and M. ruthenica and reveal the mechanisms underlying their adaptation to harsh environments.