A new Cannabis genome assembly associates elevated cannabidiol (CBD) with hemp introgressed into marijuana

Abstract

Summary Demand for cannabidiol (CBD), the predominant cannabinoid in hemp (Cannabis sativa), has favored cultivars producing unprecedented quantities of CBD. We investigated the ancestry of a new cultivar and cannabinoid synthase genes in relation to cannabinoid inheritance. A nanopore‐based assembly anchored to a high‐resolution linkage map provided a chromosome‐resolved genome for CBDRx, a potent CBD‐type cultivar. We measured cannabinoid synthase expression by cDNA sequencing and conducted a population genetic analysis of diverse Cannabis accessions. Quantitative trait locus mapping of cannabinoids in a hemp × marijuana segregating population was also performed. Cannabinoid synthase paralogs are arranged in tandem arrays embedded in long terminal repeat retrotransposons on chromosome 7. Although CBDRx is predominantly of marijuana ancestry, the genome has cannabidiolic acid synthase (CBDAS) introgressed from hemp and lacks a complete sequence for tetrahydrocannabinolic acid synthase (THCAS). Three additional genomes, including one with complete THCAS, confirmed this genomic structure. Only cannabidiolic acid synthase (CBDAS) was expressed in CBD‐type Cannabis, while both CBDAS and THCAS were expressed in a cultivar with an intermediate tetrahydrocannabinol (THC) : CBD ratio. Although variation among cannabinoid synthase loci might affect the THC : CBD ratio, variability among cultivars in overall cannabinoid content (potency) was also associated with other chromosomes.

Genomic and Chemical Diversity of Commercially Available High-CBD Industrial Hemp Accessions

“…Meanwhile, all the published marijuana-type plants cluster separately (Supplementary Figure 4). These results match prior published data, where CBD-type hemp plants are usually genetically distinct from marijuana (Sawler et al, 2015;Lynch et al, 2016;Grassa et al, 2021).…”

Large-scale whole-genome resequencing unravels the domestication history of Cannabis sativa

“…3B ) that either map both genes and the CBDAS pseudogenes or map THCAS and the CBDAS pseudogenes. The main pattern inferred from our comparative analysis confirms previous structural data based on full genome sequencing of single cultivars ( 18 , 19 ). It is also consistent with published chemotype inheritance models validated among a wide variety of Cannabis accessions ( 16 , 17 , 20 , 43 , 44 ), thus providing complementary evidence for the latter at the genomic sequence level and global validation across a comprehensive panel of Cannabis domestication types distributed worldwide.…”

“…3B) that either map both genes and the CBDAS pseudogenes or map THCAS and the CBDAS pseudogenes. The main pattern inferred from our comparative analysis confirms previous structural data based on full genome sequencing of single cultivars (18,19). It is also consistent with published chemotype inheritance models validated among a wide variety of Cannabis accessions (16,17,20,43,44) As such, the results call into question, from both a biological and functional point of view, the current binary categorization of Cannabis plants as "hemp" or "marijuana" derived from the assignment to a single phenotype [see also (20)].…”

“…When involved in secondary metabolism, the homologs of these genes likely play a major role in chemical plant defense (8). Confirming earlier genetic studies, recent genome assemblies showed that CBDAS and THCAS (and their multiple pseudogenic copies) lie scattered within closely linked loci, in a retrotransposon-rich, highly repetitive region of the genome with suppressed recombination, and with a history of extensive rearrangement and tandem duplication/pseudogenization events (4,(16)(17)(18)(19). Using strict filtering criteria, we mapped the reads of the 104 analyzed genomes to a reference CBDA/THCA hybrid cultivar genome [Jamaican Lion DASH (42)], in which full-length coding sequences for THCAS, CBDAS, and more than 30 pseudogene copies of these genes are assembled.…”

“…1A and table S1). After mapping to the reference CBDRx genome ( 18 ), we identified 12,010,905 putative single-nucleotide polymorphisms (SNPs) that passed filtering criteria across the 104 Cannabis accessions retained for subsequent analyses (fig. S1; see Materials and Methods).…”