Integrated Multi-Omics Analysis Reveals Key Metabolites and Regulatory Networks Underlying Flower Color Variation in F1 Hybrids of Cymbidium eburneum × Cymbidium insigne.

Background/Objectives: Flower color is a core ornamental trait in orchids (Orchidaceae), arising from the biosynthesis, accumulation, and spatial distribution of pigments under multilayered transcriptional regulation. Methods: We performed parallel RNA-seq transcriptomic and widely targeted metabolomic profiling of 15 samples representing five floral tissue/color categories from an F1 population of Cymbidium eburneum ('Duzhan Chun') × Cymbidium insigne ('Meihua Lan'). Results: Across four prespecified pairwise comparisons, 4891 non-redundant differentially expressed genes (DEGs) and 383 non-redundant differentially accumulated metabolites (DAMs) were identified. WGCNA resolved 12 co-expression modules. Recalculation from archived files identified darkseagreen4 as the module most positively associated with red tissues (r = 0.888), lightgreen as the module most positively associated with yellow tissues (r = 0.916), and plum1 as the module most strongly associated with white tissues in the negative direction (r = -0.983). Genes meeting |MM| ≥ 0.80, |GS| ≥ 0.80, MM p < 0.01, and GS p < 0.01 were retained as color-associated candidates. Anthocyanin derivatives including Cyanidin-3-O-sambubioside-5-O-glucoside, Delphinidin-3-rutinoside, and Peonidin-3,5-O-di-β-glucoside were associated with red floral tissues. Conclusions: Integrated analyses consistently highlighted phenylpropanoid and flavonoid biosynthesis, whereas isoquinoline alkaloid enrichment remains hypothesis-generating. These associations nominate candidates but do not establish causal pigmentation regulators.