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.

Figure 1.

Comparison of physical and genetic distance in Cannabis sativa and arrangement of sequence features on chromosomes. (A) Median values are indicated for all metacentric linkage groups (Chromosomes 5, 9, and 10 are excluded), scaled to the same physical length. Black points indicate the median increase in genetic distance every 1/100th of the physical distance. Shaded histograms superimposed show density of repeat sequences. Density of genes and GC content are also indicated by blue and purple lines. (B) Values for Chromosome 6, which contains the THCAS/CBDAS loci; here, black points are the representative of individual scaffolds.

Figure 2.

Characterization of CBCAS activity and expression. (A) HPLC analysis of CBCAS activity detected in Pichia pastoris cell cultures. Chromatograms of the CBGA substrate and CBCA standards are shown together with chromatograms of the enzyme reaction in media sampled from Pichia expressing CBCA in the presence of CBGA substrate before and after boiling at 95°C for 10 min. Insets correspond to the UV-absorbance spectrum (top) and the mass spectrum derived from a single quadrapole mass spectrometer (bottom) of the compound that eluted at 10 min. (B) SDS-PAGE analysis of CBCAS expressed in P. pastoris and purified by protein chromatography. (Lane 1) Protein ladder. (2) Concentrated protein fraction exhibiting CBCAS activity. The high-molecular-weight smear is glycosylated CBCAS. (3) Same fraction as lane 2, treated with EndoHf (MW = 70 kDa). (4) EndoHf only. (C) qRT-PCR analysis of CBCAS expression in cannabis tissues. cDNA derived from cannabis tissues was used as a template for PCR reactions using CBCAS-specific primers and EF1α as a reference gene. Differential expression of CBCAS is depicted as fold-change between tissue types compared with leaves. Trichome tissue consisted of isolated trichome secretory cells. (D) Quantification of CBCA content of the developing seedlings by HPLC.

Figure 3.

Comparison of scaffolds between PK and FN assemblies. Alignments of scaffolds from PK and FN FALCON assemblies containing key cannabinoid biosynthesis enzymes are shown. Locations of exons are indicated by pink and blue lines for FN and PK, respectively. Repeat classes given are from RepeatModeler. Individual repeat types indicated were identified by manual analysis. Features of genes are further described and compared beneath the alignments. (A) Aromatic prenyltransferase (AP). (B) THCAS and CBDAS. (C) Olivetol synthase (OLS, or tetraketide synthase).