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Melilotus  is one of the most important legume forages, but the lack of molecular markers has limited the development and utilization of  Melilotus  germplasm resources. In the present study, 151 M clean reads were generated from various genotypes of  Melilotus albus  using Illumina sequencing. A total of 19,263 potential EST-SSRs were identified from 104,358 unigene sequences. Moreover, 18,182 primer pairs were successfully designed, and 550 primer pairs were selected using criteria of base repeat type, fragment length and annealing temperature. In addition, 550 primer pairs were screened by using PCR amplification products and used to assess polymorphisms in 15  M. albus  accessions. A total of 114 primer pairs were detected as being highly polymorphic, and the average polymorphism information content ( PIC ) value was 0.79. Furthermore, those 114 polymorphic primer pairs were used to evaluate the transferability to 18 species of the genus  Melilotus , and 70 EST-SSR markers were found to be transferable among the 18  Melilotus  species. According to the UPGMA dendrogram and STRUCTURE analysis, the 18  Melilotus  species were classified into three clusters. This study offers a valuable resource for the genetic diversity and molecular assisted breeding of germplasm resources in the genus  Melilotus .

Cross-species transferability of EST-SSR markers developed from the transcriptome of Melilotus and their application to population genetics research