Development of KASP markers for DNA fingerprinting in fiber-type hemp (Cannabis sativa L.) germplasms

Single-nucleotide polymorphisms (SNPs) are the most abundant and informative genomic variations in plants. Kompetitive Allele-Specific PCR (KASP) translates SNP data into a cost-effective, high-throughput genotyping platform, yet validated KASP markers for Cannabis sativa remain scarce. Whole-genome resequencing of 46 fiber-type hemp accessions yielded 11.55 million SNPs. After stringent quality filtering, 8688 loci were deemed KASP-convertible. Twenty-eight SNPs (2–3 per chromosome) were chosen and validated by Sanger sequencing; fifteen polymorphic loci were converted into functional KASP assays (54 % conversion efficiency). The resulting marker panel exhibits high polymorphism information content (PIC = 0.325–0.375) and robust discrimination power. A minimal set of seven markers produced unique genotypic profiles for all 42 germplasms tested, providing a streamlined DNA fingerprint with a cumulative DP = 0.999. We present the first high-resolution KASP marker system for fiber-type hemp. This toolkit fills a critical gap in cultivar identification, supports germplasm management, and lays a molecular foundation for marker-assisted breeding and plant variety protection in hemp. • Stringent SNP filtering: From > 11 million SNPs, 8688 KASP-convertible lociwere retained across 46 fibre-type hemp accessions. • High-quality marker panel: Fifteen KASP markers with superior PIC and DPwere successfully developed (54 % conversion rate). • Minimal yet universal barcode: A seven-marker subset enables rapid DNA fingerprinting and cultivar authentication.