Molecular markers have been widely utilized in population genetics, diagnostic taxonomy, and genetic mapping, and can be applied to cultivar discrimination during field selection processes for alfalfa. In this study, whole-genome sequencing information was obtained for seven alfalfa lines and cultivars developed in Korea, including ‘Alfaone (MS001)’, using Next-Generation Sequencing (NGS). Single nucleotide polymorphism (SNP) analysis revealed that ‘Alfaone (MS001)’ could be distinguished from other lines and cultivars using six SNP loci. Specifically, only two SNP loci were sufficient to differentiate ‘Alfaone (MS001)’ from major lines and cultivars such as ‘MS002’ and ‘Alfaking (MSCB07)’. This set of SNP barcodes provides a reliable standard for alfalfa cultivar discrimination, contributing to domestic cultivar protection and the advancement of the Korea forage industry. Furthermore, the development of distinguishing markers across alfalfa cultivars will enhance genetic resource identification and support the breeding of high-quality new cultivars.

Molecular markers have been widely applied in population genetics, diagnostic taxonomy, and genetic mapping, and they can also be used for classifying varieties of Italian ryegrass during field selection. In this study, genome-wide sequence information was generated for 10 Italian ryegrass cultivars (40 samples), including ‘Kowinearly’ (KW), using next-generation sequencing (NGS). Single nucleotide polymorphism (SNP) analysis revealed that only three SNP loci were sufficient to distinguish KW from the other cultivars. Furthermore, 21 alternative barcode sets, each consisting of three SNPs, were identified. These SNP barcode sets provide a reliable criterion for cultivar discrimination in Italian ryegrass and can contribute to the protection of domestic varieties and the advancement of the forage industry in Korea. More broadly, the development of distinguishing markers across Italian ryegrass cultivars will enhance genetic resource identification and support the breeding of high-quality new varieties.