Optimization and Application of SSR Molecular Markers in Pepper Variety Identification

Pepper breeding in China faces challenges including narrow genetic backgrounds, variety homogenization, and authenticity issues in the seed market. The original SSR-based variety identification system (NY/T 2475-2013) has limitations such as low polymorphism and uneven marker distribution, failing to meet current efficiency requirements. In this study, 240 pepper varieties were used. Fluorescent capillary electrophoresis was employed to analyze issues in the original system. A total of 150 SSR primer pairs was screened using eight phenotypically distinct varieties, followed by rescreening with 96 varieties from different origins. After marker optimization, a new identification system was established, validated using 144 varieties, and a DNA fingerprint database was constructed. Phenotypic experiments were conducted to evaluate applicability. The new system comprises 26 core primer pairs: 12 newly selected and 14 retained from the original standard. These primers are evenly distributed across chromosomes, with annealing temperatures optimized to 57 °C and grouped into seven multiplex panels. Across 240 pepper varieties, the 26 primer pairs detected 153 allelic variants and 293 genotypes. Average polymorphism information content (PIC) increased from 0.43 to 0.49 compared to the original system, and the discrimination efficiency reached 99.975% among 28,680 pairwise variety combinations. Phylogenetic and population structure analyses divided the 240 pepper varieties into six groups. Varieties with molecular genetic distance approaching zero showed high phenotypic similarity. The newly developed SSR-based system demonstrated excellent performance and strong applicability. It can be used for authenticity and purity identification, parentage testing, and auxiliary selection in DUS testing, supporting new variety rights protection and providing technical support for standardized management of the pepper seed industry.