Integrated chloroplast genomes, RAD-seq, and HPLC-MS/MS fingerprinting for comprehensive evaluation of Hovenia Thunb. woody medicinal resources

Species of the medicinal woody genus Hovenia Thunb. are valued for their seeds with hepatoprotective and anti-alcoholic intoxication properties. However, unclear phylogeny and uncharacterized bioactive component differences between wild species and cultivars hinder standardized development and quality control of this resource. In this study, we integrated morphological characteristics, chloroplast genomes, RAD SNPs, and HPLC-MS/MS fingerprinting to evaluate 14 populations representing three wild species, three cultivars of H. acerba , and one ancient H. dulcis tree. The chloroplast genomes (161,571–161,935 bp) were conserved with low nucleotide diversity ( Pi = 0.00176). Notably, H. trichocarpa lacked a functional rps19 copy at the IRa/LSC junction. RAD analysis assigned 14 populations into three species clusters and detected significant gene flow and introgression ( D = 0.0678–0.3093) between wild populations and cultivars (e.g., P8 vs. P10, f₄ -ratio = 0.0572), as well as among cultivars ( f₄ -ratio = 0.1561–0.5770). Phylogenetic trees derived from chloroplast and nuclear data showed minor discordances, indicating complex evolutionary histories involving hybridization. HPLC-MS/MS quantification of six flavonoids revealed significant interspecific differences. H. dulcis accumulated higher levels of dihydroquercetin and quercetin, whereas H. trichocarpa and H. acerba (including cultivars) were richer in dihydromyricetin and myricetin. Among cultivars, ‘Hongguaizao’ exhibited superior quality, ranking highest or second‑highest in five of the six flavonoids. Our multi‑technology approach clarified the genetic background of major Hovenia taxa and identified ‘Hongguaizao’ as a promising breeding candidate, providing a scientific basis for quality evaluation and resource utilization.