Development and Validation of Genome-Wide SSR Markers for Genetic Diversity Analysis of Centella asiatica

(1) Background: Centella asiatica (L.) is a long-lived medicinal plant traditionally recognized for its wound-healing and anti-inflammatory properties. Despite the growing demand for diverse C. asiatica species in Korea, studies on genetic diversity remain limited. (2) Methods: Genome assembly data of C. asiatica from the NCBI database were utilized to develop genomic SSR markers. Genetic diversity and population structure were examined in 30 Korean native C. asiatica accessions using 90 SSR markers. (3) Results: Whole-genome sequencing revealed 376,751 SSR loci, from which 127,528 primer pairs were designed. Among 160 randomly selected primers, 90 showed consistent amplification and displayed high levels of polymorphism. Genetic analyses revealed that the MAF ranged from 0.15 to 1.00 (mean 0.55), the NA ranged from 1 to 15 (mean 5.6), the Ho ranged from 0.00 to 1.00 (mean 0.17), and the PIC values ranged from 0.00 to 0.88 (mean 0.52). Clustering analysis with 90 SSR markers revealed three clusters, whereas population structure analysis indicated two populations among the C. asiatica accessions. Furthermore, two minimum marker sets with five marker combinations were identified and proved useful to differentiate all C. asiatica accessions. (4) Conclusions: The newly developed SSR markers for C. asiatica hold promise for facilitating research endeavors pertaining to variety identification, genetic mapping, and marker-assisted selection.