Alfalfa (Medicago sativa L.) is an important forage legume with high feed value and productivity. Because cultivated alfalfa is an outcrossing autotetraploid species with high heterozygosity, phenotype-based cultivar identification can be limited by environmental variation and within-cultivar genetic diversity. In this study, single nucleotide polymorphism (SNP) markers were developed for the identification of the Korean alfalfa cultivar ‘Alfaking (MSCB07)’. Newly generated genotyping-by-sequencing (GBS) datasets for ‘Vernal 25’ and ‘Common (AF)’ were analyzed together with a previously generated whole-genome sequencing (WGS) dataset of ‘Alfaking (MSCB07)’. After alignment to the reference genome and SNP filtering, 20,375 SNP loci were retained for downstream analysis. Principal component analysis and neighbor-joining tree analysis separated ‘Alfaking (MSCB07)’ from the other analyzed cultivars. Genotype pattern comparison identified two diagnostic barcode groups, and their combined profile distinguished ‘Alfaking (MSCB07)’ as “bb” among the analyzed cultivars. Finally, 54 SNP loci were selected as candidate markers for ‘Alfaking (MSCB07)’ discrimination. These results suggest that the selected SNP markers may be useful for cultivar identification, seed purity control, and cultivar protection of ‘Alfaking (MSCB07)’. Further validation with additional alfalfa cultivars and genetic resources is needed to confirm the broader applicability of these markers.

ABSTRACT
Ⅰ. INTRODUCTION
Ⅱ. MATERIALS AND METHODS
    1. Plant materials
    2. DNA extraction and sequencing data production
    3. Sequencing data preprocessing and SNP analysis
    4. Phylogenetic tree and principal component analysis(PCA)
Ⅲ. RESULTS AND DISCUSSION
    1. Sequencing data production and preprocessing
    2. Read mapping and SNP extraction
    3. Extraction of marker candidates for cultivardiscrimination
Ⅳ. CONCLUSIONS
Ⅴ. ACKNOWLEDGEMENTS
Ⅵ. REFERENCES

• Chang-Woo Min(Forages Production Systems Division, National Institute of Animal Science, RDA, Cheonan 31000, Republic of Korea)
• Bo Ram Choi(Forages Production Systems Division, National Institute of Animal Science, RDA, Cheonan 31000, Republic of Korea)
• Yowook Song(Forages Production Systems Division, National Institute of Animal Science, RDA, Cheonan 31000, Republic of Korea)
• Hyung Soo Park(Forages Production Systems Division, National Institute of Animal Science, RDA, Cheonan 31000, Republic of Korea)
• Sang-Hoon Lee(Forages Production Systems Division, National Institute of Animal Science, RDA, Cheonan 31000, Republic of Korea)
• Ki-Won Lee(Forages Production Systems Division, National Institute of Animal Science, RDA, Cheonan 31000, Republic of Korea) Corresponding author
• Jun Gyeong Choi(BIOTO, 187, Techno 2-ro, Yuseong-gu, Daejeon 34025, Republic of Korea)
• Yun Ju Kang(BIOTO, 187, Techno 2-ro, Yuseong-gu, Daejeon 34025, Republic of Korea)