Evaluation of Centella asiatica Germplasm Resources in Yunnan, China, Based on Phenotypic Traits, SSR Markers, and Chemical Profiling

Centella asiatica (L.) Urb., a perennial herb of the Apiaceae family, is an important medicinal plant with diverse therapeutic applications. In this study, we integrated phenotypic evaluation, SSR markers, and HPLC fingerprinting to assess the diversity of 24 C. asiatica accessions collected from Yunnan Province, China. Considerable phenotypic, genetic, and biochemical diversity was observed. The Shannon–Wiener index ranged from 2.20 to 3.14 for qualitative traits. Principal component analysis identified five components accounting for 82.54% of the total phenotypic variance, with leaf morphology as the primary determinant. Q-type cluster analysis grouped the accessions into five phenotypic clusters. Transcriptome analysis revealed 16,799 SSR loci, with dinucleotide repeats as the most frequent motif (44.31%). UPGMA clustering based on SSR markers delineated five genetic groups that correlated with geographic origin, with accessions from the two northernmost sites forming a distinct cluster. HPLC fingerprints identified 16 common peaks, among which five major bioactive constituents—madecassoside, asiaticoside, kaempferol, madecassic acid, and asiatic acid—were quantified. The total content of these five compounds varied markedly among accessions (4.09–20.18 mg·g−1), with the highest found in Zhaoyang (S5). Most accessions exhibited high chemical similarity (>0.9), except for Yiliang (S12), which displayed a distinct profile. The partial correlation between SSR-based classifications and geographic origin, together with the inconsistency between phenotypic and chemical groupings, indicates that morphological variation is shaped by both environmental factors and complex genetic regulatory mechanisms. These findings provide a scientific basis for germplasm conservation, selective breeding, and the sustainable utilization of C. asiatica.