Transcriptomic analysis of fatty acid and cannabinoid synthases in an S1 cannabis progeny segregating for propyl cannabinoid biosynthesis

The biosynthesis of cannabinoids possessing a 5-carbon acyl-tail (cannabigerolic acid (CBGa), tetrahydrocannabinolic acid (THCa), and cannabidiolic acid (CBDa)) in C. sativa is relatively well characterized, but several other families of cannabinoids with various acyl-tail lengths exist. This study aims to deepen our understanding of three carbon (C3, propyl) cannabinoid biosynthesis. Existing theories suggest that two alleles, a truncated BETA-KETOACYL-REDUCTASE (BKR) mutant and an ACYL-LIPID-THIOESTERASE paralog (ALT4) , are responsible for conferring propyl-cannabinoid biosynthesis. This study evaluated several factors across an S1 progeny segregating for varin accumulation. We demonstrate that C3 and C5 dominant chemovars exhbit different patterns in total acid cannabinoid (TAC) accumulation, and that the ratio of C3:C5 cannabinoids in high C3 individuals follows a distinct pattern which correlates with ALT4 expression. In the high-C3 group, we discovered that the butanoic acid ratio was significantly higher than the high-C5 group. We also show that the ratio of C3:C5 cannabinoids is much higher in vegetative leaves than mature flowers. Several genes that are critical to cannabinoid biosynthesis were differentially expressed across 6 weeks of flowering between the C3 and C5 groups, suggesting potential mechanisms for chemotype differentiation. We characterized the expression pattern of ALT4 and demonstrate that neither the overexpression of ALT4 nor the overexpression of a truncated, 7-exon BKR mutant yield an increased propyl cannabinoid ratio. Finally, we demonstrate that nearly all of the C3 individuals had a unique SNP pattern in the BKR locus, while almost none of the C5 individuals had any variation at the BKR locus.