Construction of a DNA fingerprinting system for tea plant (Camellia sinensis) germplasm resources based on KASP-SNP markers: a case study of Damiaoshan tea accessions

• A system based on nine core SNP markers can differentiate among 48 tea varieties. • DNA barcodes and fingerprint profiles are tools to verify cultivar authenticity. • Genome-wide sequencing identified approximately 2.05 million high-quality SNPs. • Integration of KASP technology allowed genotyping standardization in tea plants. • Candidate SNP markers offer a foundation for tea germplasm resource conservation. Tea plant ( Camellia sinensis ) germplasm resources constitute a fundamental basis for tea breeding programs. However, due to extensive and prolonged germplasm exchange and introduction, the issues of homonymy and synonymy are widespread, complicating variety identification and significantly impeding breeding progress as well as the effective protection of plant variety rights. To address this challenge, this study developed a high-resolution DNA fingerprinting system based on the Kompetitive Allele-Specific PCR (KASP) technology platform. Through reduced-representation genome sequencing of 48 tea germplasm accessions, including 46 elite breeding materials derived from the unique Damiaoshan germplasm pool and two widely cultivated control varieties, approximately 60.28 million single nucleotide polymorphism (SNP) loci were identified. Following rigorous bioinformatics filtering, 2.07 million high-quality SNPs were retained for downstream genetic diversity analysis and population structure inference. From this dataset, 208 candidate SNPs were selected through iterative screening based on criteria including flanking sequence conservation, polymorphism information content (PIC > 0.35), and genomic uniqueness, and subsequently used for KASP marker development. Ultimately, 55 functional-associated markers located in exon regions were successfully designed and experimentally validated, enabling stable and reproducible genotyping across all germplasm accessions. Notably, only nine core SNP markers were sufficient to fully discriminate all 48 materials. Using this minimal marker set, a unique DNA fingerprint was established for each variety, and corresponding barcodes and QR codes were generated, thereby realizing a “molecular identity card” system for cultivar authentication. This study not only provides an efficient, accurate, and cost-effective tool for variety identification, supporting breeder’s rights protection and seed quality control, but also establishes a scalable KASP-SNP genotyping workflow, offering robust technical support for the standardized management, intellectual property protection, and sustainable utilization of tea germplasm resources.