Integrative Multi-Omics Reveals Genome Evolution and CYP719-Mediated BIA Biosynthesis in Tinospora sagittata

Tinospora sagittata (Oliv.) Gagnep. is an essential medicinal tetraploid plant in the Menispermaceae family. Its tuber, “ Radix Tinosporae ,” is widely used in Traditional Chinese Medicine and is rich in terpenoids and benzylisoquinoline alkaloids (BIAs). To better understand the biosynthesis of these compounds and the evolution of the T. sagittata genome, we performed comparative genomics with 16 other plant species, estimating its evolutionary placement and divergence time within Ranunculales. Genome evolution analyses revealed one round of tandem duplication approximately 1.5 million years ago and one whole-genome duplication (WGD) around 86.9 Mya. WGD contributed to the expansion of the clade-specific cytochrome P450 gene families in Ranunculales. Genome-wide mining identified genes involved in BIA biosynthesis, and transcriptomic profiling was combined with targeted and untargeted metabolomics to analyze gene expression and metabolite accumulation. Finally, one CYP719 gene candidate ( TsA02G014550 ) was functionally characterized to catalyze the formation of (S)-canadine in the jatrorrhizine biosynthetic pathway. Our integrative genomics, transcriptomics, and metabolomics analyses provide new insights into the evolution of the T. sagittata genome and BIA biosynthesis, supporting future sustainable production of these valuable secondary metabolites. • Comparative genomics reveals Tinospora sagittata diverged from Coptis chinensis ~89–109 Mya and experienced an ancient whole-genome duplication ~86.9 Mya, shaping its tetraploid genome. • Integrated transcriptomic and metabolomic analyses identify tissue-specific accumulation of benzylisoquinoline alkaloids (BIAs), with jatrorrhizine most abundant in leaves. • A key cytochrome P450 gene, TsA02G014550 , is functionally characterized as a (S)-canadine synthase, catalyzing a critical step in the jatrorrhizine biosynthetic pathway. • The study provides a genomic resource linking genome evolution, gene family expansion, and BIA biosynthesis, supporting future metabolic engineering in medicinal plants.