Transcription-based dissection of floral identity and trichome biosynthesis pathways in Cannabis sativa L

Cannabis sativa L. has a long history of medicinal and industrial use, with female flowers being the primary source of bioactive compounds such as cannabinoids and terpenoids, which are synthesized in glandular trichomes. Despite its importance, the genetic mechanisms governing flower development, sex determination, and the biosynthesis of these valuable secondary metabolites remain only partially understood. In this study, we conducted an in-depth transcriptomic and comparative genomic analysis to elucidate the molecular networks underlying these key traits. By integrating 117 RNA-Seq datasets and performing phylogenetic analyses across nine distinct C. sativa genomes, we identified 31 orthogroups of MADS-box transcription factors. Expression profiling of these genes revealed distinct sets of candidates associated with male and female flower identity, consistent with the established ABCDE model of floral development. Specifically, genes from the AP3, PI/GLO, and MIKCS clades showed preferential expression in male flowers, while genes from the AGL6, FLC-like, and Bsister clades were highly expressed in female flowers. Furthermore, our investigation into genes related to pollen development highlighted the significant role of sugar metabolism and transport in male fertility. Finally, our analysis of cannabinoid and terpenoid biosynthetic genes confirmed their pronounced expression in trichomes and highlighted a key distinction: while the upstream polyketide, MEV, and MEP pathways and the terpenoid pathway showed conserved expression across chemotypes, the cannabinoid pathway exhibited chemotype-specific expression profiles. Collectively, our findings provide a comprehensive molecular framework for understanding floral development and secondary metabolism in C. sativa , offering valuable targets for future functional studies and advanced breeding programs aimed at optimizing desirable agronomic and medicinal traits.