Origin and Evolution of the Cannabinoid Oxidocyclase Gene Family

Abstract

Cannabis is an ancient crop representing a rapidly increasing legal market, especially for medicinal purposes. Medicinal and psychoactive effects of Cannabis rely on specific terpenophenolic ligands named cannabinoids. Recent whole-genome sequencing efforts have uncovered variation in multiple genes encoding the final steps in cannabinoid biosynthesis. However, the origin, evolution, and phylogenetic relationships of these cannabinoid oxidocyclase genes remain unclear. To elucidate these aspects, we performed comparative genomic analyses of Cannabis, related genera within the Cannabaceae family, and selected outgroup species. Results show that cannabinoid oxidocyclase genes originated in the Cannabis lineage from within a larger gene expansion in the Cannabaceae family. Localization and divergence of oxidocyclase genes in the Cannabis genome revealed two main syntenic blocks, each comprising tandemly repeated cannabinoid oxidocyclase genes. By comparing these blocks with those in genomes from closely related species we propose an evolutionary model for the origin, neofunctionalization, duplication, and diversification of cannabinoid oxidocycloase genes. Based on phylogenetic analyses, we propose a comprehensive classification of three main clades and seven subclades that is intended to aid unequivocal referencing and identification of cannabinoid oxidocyclase genes. Our data suggest that cannabinoid phenotype is primarily determined by presence/absence of single-copy genes. Although wild populations of Cannabis are still unknown, increased sampling of landraces and wild/feral populations across its native geographic range is likely to uncover additional cannabinoid oxidocyclase sequence variants.

The phytochemical diversity of commercial Cannabis in the United States

“…Interesting areas of future study will be to correlate chemotype and genotype directly and determine why other cannabinoids have such low abundance in commercial Cannabis . For example, there are numerous CBC-related genes [ 72 ] but we observe very low levels of CBC (Figs 1 and 2 ), supporting previous claims that CBCA synthase may not be selective for CBC production [ 46 ].…”

“…These likely arise from distinct genotypes. The genes giving rise to the cannabinoid synthases responsible for producing the major cannabinoid acids are highly similar [ 20 , 71 , 72 ]. Copy number variation [ 20 , 73 ] or allelic variation [ 19 ] in the genes encoding these enzymes may explain the observed variation in cannabinoid ratios.…”

Examining population structure across multiple collections of Cannabis

“…However, due to variable coverage of these important gene sequences within the datasets examined, a comprehensive survey across individuals for cannabinoid gene content and the assessment of functional variants was limited. This finding complements previous work where the focus has been on specific gene family phylogenies, such as the cannabinoid phylogeny for CBDAS and THCAS variants [51] [23] and terpene synthase phylogenies [21] [52] [53]. For breeders and growers seeking to maximize the concentration of various cannabinoids, working with breeding material of known genetic constitution is key to creating better products.…”

“…Advances in sequencing technology have resulted in the release of several genomic datasets in the public domain (Table 1). Broad phylogenetic comparisons of the ecotypes and heterotic groups within the Cannabis genus is still lacking with previous approaches limited to gene family phylogenies [19] [20] [21] [22] [23] or limited, regional specific sampling [24] [25]. Drug control laws and prohibition have constrained formal documentation often resulting in unverifiable, anecdotal origins of a given cultivar or plant [7], further constraining genetic diversity analysis.…”

High-throughput methods to identify male Cannabis sativa using various genotyping methods

“…Cannabis sativa is an annual plant in the Cannabaceae family that contains both monoecious and dioecious cultivars; however, in most commercial industrial and medicinal applications, dioecious cultivars are generally preferred. The species can be divided into two types, fiber type (or hemp) used for its fiber or seed oils and drug type (marijuana) that are used for secondary metabolite production (Small and Cronquist 1976 ; McPartland and Guy 2017 ; van Velzen and Schranz 2021 ). For medicinal or drug-type applications, female (sinsemilla) plants are desired for their increased cannabinoid production compared to past monoecious varieties that would produce self-pollinated seed that detracts from medically valued cannabinoid production (Abrams 2017 ).…”