Development of KASP molecular markers and construction of fingerprinting for cowpea (Vigna unguiculata (L.) Walp.) based on ddRAD-Seq.

Cowpea (Vigna unguiculata (L.) Walp.) is a globally important legume crop. However, the scarcity of efficient molecular markers has hindered molecular breeding efforts and the protection of plant breeders' rights. In this study, we employed double-digest restriction-site associated DNA sequencing (ddRAD-seq) to characterize the genetic diversity of 19 cowpea accessions. A total of 791,621 SNPs were identified, of which 13,469 high-quality SNPs were retained after filtering. Population structure and phylogenetic analyses revealed that these accessions clustered into three distinct groups. To facilitate cost-effective and rapid genotyping, we developed a panel of KASP (Kompetitive Allele-Specific PCR) markers. Through rigorous screening for polymorphism and stability, we identified six core KASP markers located in exonic regions. These six markers alone were sufficient to discriminate all 19 accessions. Based on these core markers, we constructed a unique DNA fingerprinting profile and assigned specific QR codes for each accession. This study demonstrates that selecting core KASP markers from ddRAD-seq data is a powerful strategy for germplasm identification. The developed fingerprinting system provides a robust, low-cost tool for seed purity testing, variety authentication, and marker-assisted selection in cowpea breeding programs.