Terpenoids are key specialized metabolites in Cannabis sativa, shaping cultivar-specific aromas and potentially modulating cannabinoid effects. This study provided a comprehensive analysis of the terpene synthase (TPS) gene family in C. sativa, integrating haplotype-resolved genomic data, volatile terpene profiling, transcriptomics, and functional assays. The comprehensive volatile terpene profiling across 28 spatiotemporal samples spanning weekly developmental intervals and distinct maturity stages from six cultivars identified 227 cannabis volatile terpenes, including 88 monoterpenes and 139 sesquiterpenes, exhibiting distinct tissue, developmental stage, and cultivar-specific patterns. Comparative expression and co-expression network analysis revealed coordinated regulation between MEP/MVA pathways, TPS, and cannabinoid genes, underscoring a shared metabolic foundation. Genome annotation identified 41 full-length CsTPSDK genes, exhibiting extensive expansion and subfamily-specific clusters with structural divergence between haplotypes. Transcript profiling across developmental stages and cultivars distinguished a core set of highly expressed inflorescence-associated CsTPSs from genes exhibiting cultivar-specific regulation. Functional characterization of six previously unreported CsTPSDKs uncovered diverse mono- and sesquiterpene synthase activities, including unexpected substrate promiscuity across subfamilies. These findings deliver the most comprehensive functional annotation of the C. sativa TPS repertoire to date, elucidating the genetic and biochemical bases of terpene diversity and providing a foundation for targeted metabolic engineering and cultivar improvement.

Terpene synthase  /  Cannabis sativa  /  Volatile terpenes  /  Genetic expansion  /  Haplotype-resolved genome