Inheritance and covariation of specialised metabolites among cannabis chemotypes

The major phytocannabinoid bioactives produced by Cannabis sativa L. (cannabis) are Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD), with many minor phytocannabinoids (PCs) also thought to contribute to the pharmacological efficacy. Cannabis typically segregates into three main chemical phenotypes (chemotypes) based on their Δ9-THC/CBD ratio, a highly heritable trait determined by the segregation of two closely related berberine bridge-like enzymes that perform stereoselective oxidative cyclisation on the geranyl moiety of cannabigeroid intermediates. Apart from a small number of metabolome-wide association studies, few attempts have been made to either understand metabolite coupling among Δ9-THC/CBD chemotypes or to examine the inheritance of alternative biomarkers that could be used to discriminate chemotype. Here, we examined the metabolomes of 108 F2 segregants derived from a cross between a Δ9-THC-predominant chemotype I and a CBD-predominant chemotype III plant. Although segregation of the Δ9-THC/CBD ratio followed Mendelian genetics expectations, covariation in the inheritance of minor PCs, including cannabichromene (CBC)-types, indicated changes in cannabinoid synthase product specificity among chemotypes. In addition, several non-PC related metabolites were identified that may serve as potential biomarkers for chemotype prediction. These data have important implications for the pre-breeding and selection of cannabis chemovars and highlight the need to adopt metabolic engineering strategies to optimise PC production.