Development of KASP molecular markers and fingerprinting based on reduced representation genome sequencing of garlic

In this study, a garlic DNA fingerprinting system based on single nucleotide polymorphisms (SNPs) was developed using representative cultivated garlic accessions. SNP markers were identified based on reduced-representation genome sequencing (GBS) data from 77 garlic accessions. A total of 26,701,817 raw SNPs were detected and subsequently filtered to obtain 7,006 high-quality candidate SNP loci. Among these, KASP primers were successfully designed for 4,297 loci, corresponding to an assay design success rate of 61.3%. Of these candidate loci, 30 generated reliable KASP genotyping results, from which 13 high-quality core SNP markers were finally selected through stepwise screening based on genotyping quality, marker polymorphism, and discriminatory ability. The average minor allele frequency (MAF), expected heterozygosity (He), and polymorphism information content (PIC) of these core markers were 0.235, 0.281, and 0.227, respectively. Based on these 13 core SNP markers, an SNP-based DNA fingerprinting system for garlic was established. Genotyping analysis of the 77 accessions showed that 73 could be effectively distinguished, whereas 4 accessions remained unresolved. Overall, this core SNP marker set showed effective discriminatory power for most garlic accessions and provides a practical basis for germplasm identification, variety protection, and molecular breeding in garlic.