Analysis Of Genetic Diversity Using Est-SSR And Identification Of MiRNA In Melilotus Spp.

Melilotus spp.is the important legume forage,while there are few studies on the molecular level research,such as evolution,interspecific relationship and genetic diversity among genus.miRNAs play important roles in post-transcriptionally regulating gene expression during plant growth,development,and other various biological processes.In this study,EST-SSR molecular markers,which were obtained by global sequence data,were identified.Furthermore,these polymorphic primers were used to evaluate the transferability to 18 Melilotus species.Additionally,we used Illumina RNA-Seq technology to identify miRNA and analyze their differential expression based on cDNA libraries from M.albus accessions with different coumarin levels.The main results are as follows:1.A total of 18182 M.albus pairs of SSR primers were designed by using EST sequences.Of these,550 primers were successfully screened based on base repeat types,expected size,annealing temperatures.Moreover,a total of 114 primers showed a high level of polymorphism in 15 M.albus accessions.The polymorphism information content values ranged from 0.16 to 0.92 with an average value of 0.79.Forthemore,these 114 EST-SSR markers were used to evaluate the transferability in 18 Melilotus species,and 70 highest-SSR markers were found to be polymorphic with 61.4% transferability among these species.Based on UPGMA cluster analysis,we found that the 18 species were divided into three clusters.Cluster I,II and III consisted of 10,4 and 4 species,respectively.Population structure analysis suggested that the optimum number of groups was three,which is similar to cluster analysis.2.A total of 417 known miRNAs and 76 novel miRNAs were identified form RNA-Seq result of five M.albus genotypes.Furthermore,the target genes of miRNAs were predicted and 4196 target genes were identified.Then these target genes were successfully annotated in GO and KEGG databases.Of these,three miRNAs and their two target genes were identified as being involved in coumarin biosynthesis by shikimate O-hydroxycinnamoyltransferase(HCT).To validate RNA-Seq result,qRT-PCR was performed on 28 miRNAs and 32 potential target genes.We also found that both positive and negative expression changing pattern between miRNAs and their related target genes.