Molecular marker development and genetic diversity exploration in Medicago polymorpha

Medicago polymorpha L. (bur clover), an invasive plant species of the genus Medicago, has been traditionally used in China as an edible vegetable crop because of its high nutritive value. However, few molecular markers for M. polymorpha have been identified. Using the recently published high-quality reference genome of M. polymorpha, we performed a specific-locus amplified fragment sequencing (SLAF-seq) analysis of 10 M. polymorpha accessions to identify molecular markers and explore genetic diversity. A total of 52,237 high-quality single nucleotide polymorphisms (SNPs) were developed. These SNPs were mostly distributed on pseudochromosome 3, least distributed on pseudochromosome 7, and relatively evenly distributed on five other pseudochromosomes of M. polymorpha. Phenotypic analysis showed that there was a great difference in phenotypic traits among different M. polymorpha accessions. Moreover, clustering all M. polymorpha accessions based on their phenotypic traits revealed three groups. Both phylogenetic analysis and principal component analysis (PCA) of all M. polymorpha accessions based on SNP markers consistently indicated that all M. polymorpha accessions could be divided into three distinct groups (I, II, and III). Subsequent genetic diversity analysis for the 10 M. polymorpha accessions validated the effectiveness of the M. polymorpha germplasm molecular markers in China. Additionally, SSR mining analysis was also performed to identify polymorphic SSR motifs, which could provide valuable candidate markers for the further breeding of M. polymorpha. Since M. polymorpha genetics have not been actively studied, the molecular markers generated from our research will be useful for further research on M. polymorpha resource utilization and marker-assisted breeding.