A physical and genetic map of Cannabis sativa identifies extensive rearrangements at the THC/CBD acid synthase loci

Abstract

Cannabis sativa is widely cultivated for medicinal, food, industrial, and recreational use, but much remains unknown regarding its genetics, including the molecular determinants of cannabinoid content. Here, we describe a combined physical and genetic map derived from a cross between the drug-type strain Purple Kush and the hemp variety "Finola." The map reveals that cannabinoid biosynthesis genes are generally unlinked but that aromatic prenyltransferase (AP), which produces the substrate for THCA and CBDA synthases (THCAS and CBDAS), is tightly linked to a known marker for total cannabinoid content. We further identify the gene encoding CBCA synthase (CBCAS) and characterize its catalytic activity, providing insight into how cannabinoid diversity arises in cannabis. THCAS and CBDAS (which determine the drug vs. hemp chemotype) are contained within large (>250 kb) retrotransposon-rich regions that are highly nonhomologous between drug-and hemp-type alleles and are furthermore embedded within ∼40 Mb of minimally recombining repetitive DNA. The chromosome structures are similar to those in grains such as wheat, with recombination focused in gene-rich, repeat-depleted regions near chromosome ends. The physical and genetic map should facilitate further dissection of genetic and molecular mechanisms in this commercially and medically important plant.

Genome‐wide analysis of MIKC‐type MADS‐box genes in wheat: pervasive duplications, functional conservation and putative neofunctionalization

“…The genome architecture among different plants varies considerably. Arabidopsis and rice, for example, have relatively small subtelomeric regions (The Arabidopsis Genome Initative, ; Mizuno et al ., ), whereas other plants show increased recombination rates towards the chromosome ends and relatively large distal telomeric regions with increased gene content, very similar to what is seen in wheat (Choulet et al ., ; Glover et al ., ; Li et al ., ; Lambing et al ., ; Grassa et al ., ; Sun et al ., ; Laverty et al ., ). It will be interesting to see whether similar dynamics of MIKC‐type MADS‐box gene duplications can be observed in those species.…”

Development and validation of genetic markers for sex and cannabinoid chemotype in Cannabis sativa L.

“…Other research found a C. sativa plant with a B D /B D genotype and a catalytically inactive CBDAS that accumulates CBGA and essentially no THCA, although mutations in CBDAS and a putative THCAS would also explain this observation (Onofri et al, 2015). A purported active THCAS from CBD-dominant fiber-type hemp was later shown to be a cannabichromenic acid synthase (CBCAS) (Kojoma et al, 2006;Laverty et al, 2019). Lastly, in vitro expression of wild-type CBDAS leads to production of CBDA:THCA in a ratio very close to 20:1 at optimal pH (Zirpel, Kayser, & Stehle, 2018).…”

“…Hemp ( Cannabis sativa L.) is a multiuse crop grown primarily for grain, fiber, and cannabinoids. In recent years, there has been a resurgence in the study of hemp in the United States and across the world in fields including genomics (Grassa et al, ; Laverty et al, ), agronomics (Campbell, Berrada, Hudalla, Amaducci, & McKay, ), and novel end‐uses (Turner, Sloan, & Currell, ; Wang & Xiong, ). The market for hemp (defined as <0.3% tetrahydrocannabinol; THC by dry weight) and marijuana (>0.3% THC), is expected to surpass $26 billion in the US by 2025 (Global Cannabis Report: 2019 Industry Outlook, ).…”

“…The genetic structure of CBDAS and THCAS have been recently elucidated (Grassa et al, 2018;Laverty et al, 2019). While the genes are highly alike, sharing 84% amino acid identity (Onofri, de Meijer, & Mandolino, 2015), they are not allelic or at equivalent loci.…”

The Cannabis Multi-Omics Draft Map Project

“…18 The output spectral library can facilitate both targeted and Data Independent Analysis of plant proteins and is available for download at www.CannabisDraftMap.org Chromosome alignment A recent re-analysis of the CanSat3 genome 19 aligned the sequences into ten separate chromosome files. 20 The Protein Marker node in Proteome Discoverer was used in 4 rounds of reprocessing of the consensus workflow to develop a metric of the number of identified protein entries in this study that are products of each chromosome. Four rounds were necessary due to a limitation in the software that allows a maximum of 3 separate FASTA sequences to be used for output marking.…”