Integrated phenotypic, DNA barcoding, chemotypic, metabolomic, and transcriptomic analyses for quality assessment of Anoectochilus roxburghii and identification of key metabolites and enzyme genes in kinsenoside biosynthesis and leaf non-green colora

Anoectochilus roxburghii is the exclusive medicinal source of kinsenoside and is also valued as an ornamental bonsai material, though quality variability hinders its industrial applications. To address this, we established an integrated quality evaluation system and elucidated the mechanisms underlying superior phytochemical content and pigmentation traits in A. roxburghii through combined phenotypic, DNA barcoding, chemotypic, transcriptomic, and metabolomic analyses. The results showed that nine A. roxburghii varieties exhibited distinct "one variety, one leaf color" patterns, with Hongxia, J6 mu, and Dayuanye exhibiting the most intense pigmentation. Chemotypic analysis revealed that Hongxiadaye leaves contained the highest kinsenoside content among all varieties, while Xiaoyuanye was the only variety containing both kinsenoside and its isomer. Transcriptomic analysis revealed that ACAA , Hbd , and UGT genes were highly expressed in Hongxiadaye, while CoAT , PaaH , and UGT genes, exhibited elevated expression specifically in Xiaoyuanye. Their expression patterns across nine varieties showed significant positive correlations with kinsenoside and its isomer accumulation profiles, respectively. These findings demonstrate the crucial roles of these genes in kinsenoside and its isomer biosynthesis, respectively. Metabolomic profiling identified kaempferol-7-O-glucoside, kaempferol-3-O-galactoside, and cyanidin-O-acetylhexoside as key pigments for A. roxburghii leaf coloration. Transcriptomics revealed that PAL , C4H , 4CL , CHS , F3H , and FLS genes showed peak expression in Hongxiadaye and J6 mu varieties, and DFR , ANS , ANUGT genes were significantly elevated in Hongxiadaye and Dayuanye. Their expression patterns across the nine varieties correlated significantly with the accumulation profiles of these key pigments.These findings showed that these genes play pivotal roles in this pigmentation process. Notably, UGT and BTF genes may have dual regulatory functions in kinsenoside biosynthesis and leaf pigmentation. The findings provide a systematic methodology for quality evaluation of A. roxburghii , and offer fundamental data for precision cultivation based on superior phytochemical and color-quality traits. • Identifying A. roxburghii and its varieties through observation of leaf color. • Differentiating A. roxburghii and its varieties by leaf color and DNA barcodes. • Classification of Chemotypes Based on Kinsenoside and its isomer content. • Screening of indicative coloration metabolites and genes. • Screening of indicative genes involved in kinsenoside and its isomers biosynthesis.