Transcriptome and flavonoid metabolome analysis reveals the molecular basis of floral color divergence between the hybrid and their parental species in Paphiopedilum

Background The artificial hybridization of Paphiopedilum began in 1869. With the breakthrough in aseptic sowing techniques, the number of hybrids surged. Over 30,000 hybrids have been registered by the Royal Horticultural Society (RHS). Hybrids derived from Paphiopedilum delenatii typically retain its prominent characteristics: large, symmetrical petals and a magenta-purple labellum. In this study, the hybrid of P. delenatii and P. primulinum exhibited an overall creamy-white coloration. This study focuses on the divergence in floral coloration between the hybrid and their parental lines. Through comparative transcriptome analysis and flavonoid metabolic profiling across three distinct Paphiopedilum , we aim to elucidate the molecular mechanisms underlying how hybrids deviate from the red and yellow-green phenotypes of their parents to develop a cream-colored pigmentation. Results The labellum and dorsal sepal morphology closely resembled P. primulinum , while the petal traits combined the wavy margins of P. primulinum and smooth surface of P. delenatii . Flavonoid metabolome analysis of the floral tissues, a total of 29 flavanones, 93 flavones and flavonols, 13 anthocyanins, 23 isoflavonoids, six flavonoids, and four chalcones and dihydrochalcones were detected. Remarkably, anthocyanin content is significantly higher in P. delenatii compared to P. primulinum and the hybrid. Transcriptome analysis highlighted significant up-regulation of genes in the flavonoid biosynthesis pathway and phenylpropanoid biosynthesis pathway when comparing hybrids to P. primulinum . Conversely, early-stage biosynthetic genes were down-regulated in hybrid relative to P. delenatii , likely contributing to metabolite content differences. Conclusion This study employed multi-omics analysis to compare the floral tissues of P. delenatii and P. primulinum , along with their hybrid progeny. It identified differences in flavonoid metabolite content among them. By analyzing differentially expressed genes (DEGs) within the KEGG flavonoid biosynthesis pathway, the molecular mechanism underlying the divergent floral pigmentation pattern of the hybrid compared to both parental species was explored. Analysis of transcriptomic data, through comparison with the non-redundant (NR) protein database, revealed a closer phylogenetic relationship between Paphiopedilum and the genus Dendrobium , whereas morphologically Paphiopedilum is classified under Cypripedium -related taxa. These findings provide novel insights and molecular evidence for understanding Paphiopedilum floral coloration and phylogenetic relationships.