Genome-wide development and application of miRNA-SSR markers in Melilotus genus

Genetic diversity of plants is the brace of biodiversity and diversity within species, between species, and of ecosystems. SSR markers are the most preferable molecular marker tool that has been successfully used to study the genetic diversity of plant species. Development of miRNA-SSR markers has been deed in animals but is still limited in plants. In this study, 365 precursors miRNA were extracted from Melilotus albus (Ma) genome and used to design Ma miRNA-SSR primers. 137 Ma primer pairs (79 from known and 58 from novel pre-miRNAs) were obtained. 66 pairs of Ma miRNA-SSR primers were selected with polymorphisms and expected fragment size. The polymorphisms of primers were evaluated in 60 individuals of 15 Ma accessions. A total of 66 primer pairs showed high polymorphism, with average polymorphic information content of 0.49 among 15 Ma accessions and 0.63 among 18 Melilotus species, indicating that these primers have high polymorphisms. The number of alleles produced per primer ranged from 2 to 6 with an average of 3.6 alleles per locus in Ma accessions, and 2 to 10 numbers of alleles with a mean of 5.24 alleles per locus in Melilotus spp. For further studies, the genetic relationship was examined and the cluster analysis showed that 15 Ma accessions were grouped in three groups, on the other hand, 18 Melilotus species clustered into two groups. The analysis of molecular variance (AMOVA) revealed that 64.82% of the variation was found within the species and 35.18% between the species. The population structure analysis showed similar results with PCA analysis in that 18 species were grouped in two groups. In addition, 16,450 miRNA target genes were identified and used for GO and KEGG analysis. This is the first study to develop miRNA-SSR molecular markers in Melilotus spp., which has a great potential for marker-assisted, genetic improvement, genotyping applications, QTL analysis, and molecular-assisted selection studies for plant breeders and other researchers.