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Melilotus, an annual or biennial herb, belongs to the tribe Trifolieae (Leguminosae) and consists of 19 species. As an important green manure crop, diverse Melilotus species have different values as feed and medicine. To identify different Melilotus species, we examined the efficiency of five candidate regions as barcodes, including the internal transcribed spacer (ITS) and two chloroplast loci, rbcL and matK, and two non-coding loci, trnH-psbA and trnL-F. In total, 198 individuals from 98 accessions representing 18 Melilotus species were sequenced for these five potential barcodes. Based on inter-specific divergence, we analysed sequences and confirmed that each candidate barcode was able to identify some of the 18 species. The resolution of a single barcode and its combinations ranged from 33.33% to 88.89%. Analysis of pairwise distances showed that matK+rbcL+trnL-F+trnH-psbA+ITS (MRTPI) had the greatest value and rbcL the least. Barcode gap values and similarity value analyses confirmed these trends. The results indicated that an ITS region, successfully identifying 13 of 18 species, was the most appropriate single barcode and that the combination of all five potential barcodes identified 16 of the 18 species. We conclude that MRTPI is the most effective tool for Melilotus species identification. Taking full advantage of the barcode system, a clear taxonomic relationship can be applied to identify Melilotus species and enhance their practical production. [ABSTRACT FROM AUTHOR]

Melilotus is one of the most important legume plants, in part due to its production of coumarin, but the genetic diversity among the 18 species remains unclear. In the present study, the analysis of genetic diversity of the Melilotus species was performed with simple sequence repeat (SSR) markers. From the PCR amplification, we selected 18 out of 70 primers that were used in previous studies. Three hundred twenty-six sampled individuals were assayed to study the genetic diversity and polymorphisms based on the SSR markers. All analyzed markers were polymorphic, and 287 alleles were identified, with 15.94 alleles per locus detected. The polymorphism information content ( PIC ) values ranged from 0.71 to 0.93, with an average of 0.87, which indicates that the markers were highly informative. Based on the unweighted pair-group method with arithmetic mean (UPGMA) cluster analysis, we found that the 18 species were divided into two clusters. M. italicus and M.​ speciosus from cluster A and M. indicus and M. segetalis from cluster B were closely related. Population structure analysis suggested that the optimum number of groups was three. From the analysis of molecular variance, 17.79% of the variance was due to species differences, 31.61% of the variance was due to differences among populations within species and the remaining 50.60% was due to differences within populations. The results of the present study showed that these SSR markers will benefit the Melilotus research community for genetics and breeding. Furthermore, this study also established the foundation for future breeding programs, genetic improvement and coumarin production in the Melilotus species. [ABSTRACT FROM AUTHOR]