Cannabinoid biosynthesis in Cannabis sativa is governed by a specialized metabolic pathway that culminates in the production of a diverse set of bioactive compounds. The first committed step in this pathway is the synthesis of cannabigerolic acid (CBGA), which serves as a common precursor to other cannabinoids. This step is followed by the enzymatic conversion of CBGA into tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA), and other cannabinoids by a family of cannabinoid oxidocyclase enzymes. Disruption of these enzymes can lead to the accumulation of CBGA, resulting in a CBG-dominant cannabinoid profile. Previous research has identified several independent mutations that, when homozygous, result in this CBGA-dominant cannabinoid profile. In this study we characterized the architecture of the haplotypes surrounding four of these mutations, including a structural variant with a complete deletion of CBDA synthase (CBDAS). Additionally, we compared these haplotypes to homologous regions from more than 200 publicly available genome assemblies to put them in the context of global variation at this important and structurally variable genomic region. We developed high-throughput molecular markers to assay for each of the mutated haplotypes. Further, we generated and characterized more than 30 populations complementing these mutated haplotypes with each other, and with fully functional haplotypes, to characterize haplotype-associated variation in cannabinoid profiles. Together, our findings provide insights into the genetic basis of cannabinoid diversity and offer molecular tools for manipulating cannabinoid profiles in C. sativa.

Learning Objectives:

• Understand how different mutations in cannabinoid synthase genes can result in CBG-dominant C. sativa in the context of the cannabinoid biosynthetic pathway

• Describe the genetic diversity and structural variation among cannabinoid synthase haplotypes across global C. sativa populations