Development of Genomic SSR Markers to Reveal the Genetic Diversity in Trichosanthes kirilowii from Korea.

Trichosanthes, a genus in the Cucurbitaceae family, includes around 100 species across Asia, Australia, and the Western Pacific. Trichosanthes kirilowii is a perennial herb with distinct male and female plants, both widely used in traditional medicine. Accurate identification of its varieties is essential for ensuring medicinal quality, underscoring the need for reliable genetic markers. In this study, genetic diversity among T. kirilowii accessions was assessed using 53 simple sequence repeat (SSR) markers. Whole-genome sequencing revealed 2,864,449 SSRs, from which 22,614 primer pairs were developed. Based on motif length, tetra-nucleotides were most abundant (33.42%), followed by di-nucleotides (23.53%), tri-nucleotides (14.40%), and hexa-nucleotides (11.99%). Of 100 randomly selected primer sets, 67 produced stable amplification and 53 showed polymorphisms. Polymorphism analysis showed major allele frequency ranged from 0.17 to 0.80 (mean 0.402), number of alleles from 3 to 15 (mean 7.6), observed heterozygosity from 0 to 0.9 (mean 0.30), and polymorphic information content from 0.33 to 0.89 (mean 0.694). Cluster and STRUCTURE analyses consistently categorized accessions into four groups. Minimal marker analysis identified five SSR loci (TK-gSSR-009, TK-gSSR-017, TK-gSSR-022, TK-gSSR-079, and TK-gSSR-097) with complete discriminatory power. These newly developed SSR markers provide valuable tools for species identification, genetic diversity evaluation, population structure analysis, and genetic mapping of T. kirilowii.