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.

This study reports the development of a new alfalfa (Medicago sativa L.) variety, ‘Alfaone’, at the Forage Production Systems Division, National Institute of Animal Science, Rural Development Administration, Korea, from 2015 to 2023. The variety originated from an artificial cross between Xun Lu (maternal parent) and RadarⅡ Brand (paternal parent), followed by pedigree selection and performance testing. The elite line ‘MsCB01’ was subsequently released as ‘Alfaone’. Regional adaptability trials were conducted for two years (2022–2023) across four representative sites in Korea (Cheonan, Pyeongchang, Jeongeup, and Jinju) to evaluate agronomic traits, forage yield, and quality. Evaluated characteristics included plant height, regrowth ability, winter survival, and lodging resistance. The average dry matter yield of ‘Alfaone’ was 20,811 kg/ha, approximately by about 3% higher than that of the standard cultivar ‘Vernal’ (20,236 kg/ha). Yield superiority was particularly evident in Pyeongchang, suggesting excellent cold tolerance and winter hardiness. Assessment of forage nutritive traits indicated that ‘Alfaone’ was comparable to ‘Vernal’, demonstrating that its yield advantage did not come at the expense of quality. Overall, ‘Alfaone’ is a promising cultivar that combines high productivity with strong adaptability to unfavorable environments, particularly cold-prone regions. Its release is expected to promote the expansion of alfalfa cultivation, enhance forage self-sufficiency, and reduce dependence on imported hay in Korea.