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 meta-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 oxidocyclase gene copy number variation may have less functional relevance than previously thought. Instead, we propose that cannabinoid phenotype is primarily determined by presence/absence of single-copy genes. Increased sampling across Cannabis’ native geographic range is likely to uncover additional cannabinoid oxidocyclase gene sequence variation.Significance statementCannabis genome sequencing efforts have revealed extensive cannabinoid oxidocyclase gene variation. However, phylogenetic relationships and evolution of these genes remains unclear. Our meta analysis of currently available data reveals that these genes comprise three main clades and seven subclades that originated through Cannabis-specific gene duplication and divergence. Our new conceptual and evolutionary framework serves as a reference for future description and functional analyses of cannabinoid oxidocyclases.

Analysis of Sequence Variability and Transcriptional Profile of Cannabinoid synthase Genes in Cannabis sativa L. Chemotypes with a Focus on Cannabichromenic acid synthase

“…Whether (and if) the expression of these sequences correlates with the ability of the different varieties to produce CBCA in some stage of their development and consequently if they should all be considered as actually involved in CBCA biosynthesis in planta, is still to be determined. This is true not only for the sequences identified in this work, but also for all other putative CBCAS sequences released so far [7].…”

“…The genetics of cannabinoid synthesis has been studied for several years and different genes encoding these enzymes are known, each made of a single exon, with THCAS and CBCAS sharing 92% identity at amino acid level and 84% and 83% identity compared to CBDAS, respectively [3][4][5][6][7]. While THCAS and CBDAS have been widely studied at genetic and molecular level, little information is available on CBCAS genes.…”

“…More recently, a comprehensive clade-based classification of all cannabinoid oxidocyclases proposed the naming of all fiber-type THCAS as CBCAS [ 7 ] since they belonged to the same clade of the only functionally CBCAS characterized by Laverty et al [ 6 ]. Based on these results today they are conventionally collectively referred to as CBCAS and, according to this definition, in the present paper we will refer to all fiber-type THCAS sequences with a complete open reading frame as CBCAS .…”

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 minor cannabinoids have such low abundance in commercial Cannabis . For example, there are numerous CBC-related genes (van Velzen and Schranz 2020) but we observe very low levels of CBC (Figures 1-2), supporting previous claims that CBCA synthase may not be selective for CBC production (Vergara et al 2020).…”

“…These likely arise from distinct genotypes. The genes giving rise to the cannabinoid synthases responsible for producing the major cannabinoid acids are highly similar (Vergara et al 2019; van Velzen and Schranz 2020; Vergara et al 2021b). Copy number variation (Vergara et al 2019; Vergara et al 2021b) or allelic variation (Onofri et al 2015) in the genes encoding these enzymes may explain the observed variation in cannabinoid ratios.…”