Complex Variability within the THCA and CBDA Synthase Genes in Cannabis Species

The genes encoding the enzymes THCA and CBDA synthase, enzymes catalyzing the final biosynthetic steps in the production of the cannabinoids Δ-9-tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA), have been subjected to DNA sequencing to better understand the variability of gene structure in different strains of marijuana (Cannabis sp.) and to begin to explore how this variability may have consequences in cannabinoid synthesis. Genomic DNA was extracted from a collection of marijuana samples seized by local law enforcement and made available for molecular analysis. Massively parallel sequencing as well as Sanger sequencing were used to elucidate the sequence of the synthase gene from different seized samples. The sequences were compared with THCA and CBDA synthase gene sequences contained within the GenBank database of NCBI to investigate the variability of these genes and to try and differentiate active from inactive synthase genes known to exist in Cannabis sp.. Results suggest the existence of numerous copies of THCA and CBDA synthase gene sequences in the genome of Cannabis. THCA synthase sequences are less variable than CBDA sequences in terms of SNP polymorphisms visualized and SNPs visualized in THCA synthase are more highly conserved among the seized samples analyzed. Our results suggest the existence of multiple synthase genes that exist combinations of functional and non-functional combinations of genes. It may be difficult to design a simple molecular assay that will distinguish between active and inactive synthase genes in a way that would be useful to growers desiring to propagate plants selected to produce only one or the other of the principal cannabinoids useful for medicinal purposes.