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A complete Cannabis chromosome assembly and adaptive admixture for elevated cannabidiol (CBD) content

Christopher J. GrassaiD, Jonathan P. WengeriD, Clemon J. DabneyiD, Shane G. PoplawskiiD, S. Timothy MotleyiD, Todd P. MichaeliD, Christopher J. SchwartziD, George D. WeibleniD

Abstract

Cannabis has been cultivated for millennia with distinct cultivars providing either fiber and grain or tetrahydrocannabinol. Recent demand for cannabidiol rather than tetrahydrocannabinol has favored the breeding of admixed cultivars with extremely high cannabidiol content. Despite several draft Cannabis genomes, the genomic structure of cannabinoid synthase loci has remained elusive. A genetic map derived from a tetrahydrocannabinol/cannabidiol segregating population and a complete chromosome assembly from a high-cannabidiol cultivar together resolve the linkage of cannabidiolic and tetrahydrocannabinolic acid synthase gene clusters which are associated with transposable elements. High-cannabidiol cultivars appear to have been generated by integrating hemp-type cannabidiolic acid synthase gene clusters into a background of marijuana-type cannabis. Quantitative trait locus mapping suggests that overall drug potency, however, is associated with other genomic regions needing additional study.\n\nResources available online at: http://cannabisgenome.org\n\nSummaryA complete chromosome assembly and an ultra-high-density linkage map together identify the genetic mechanism responsible for the ratio of tetrahydrocannabinol (THC) to cannabidiol (CBD) in Cannabis cultivars, allowing paradigms for the evolution and inheritance of drug potency to be evaluated.

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Claude Opus 5.5 · xhigh effort · 8 Nov 2026 · from the full text of version 4 (11 Dec 2018) · CC BY 4.0

Nanopore chromosome assembly of high-CBDA cultivar CBDRx places cannabinoid synthase genes in three linked chromosome 9 regions; CBDA synthase and the THC/CBD QTL sit in hemp-derived sequence.

Earlier Cannabis assemblies stayed in thousands of contigs and left the tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA) synthase loci unresolved, hindering study of how these genes relate to cannabinoid content and to rival explanations for the THCA/CBDA ratio (THCA synthase sequence variation versus nonfunctional CBDA synthase alleles). The authors call theirs the first chromosome-scale Cannabis assembly, built with ultra-long nanopore reads, correction-free assembly and an F2 genetic map.

Key results

  1. ShownNanopore contigs of the high-CBDA cultivar CBDRx were anchored into 10 chromosome pseudomolecules using a Skunk#1 x Carmen F2 linkage map and Hi-C. The authors present this as the first complete Cannabis chromosome assembly. They also built a PacBio assembly of an F1 plant.

    CBDRx: 34x nanopore, 746 Mbp in 1,986 contigs, N50 742 kb, longest 4.5 Mbp; F1: 5x PacBio circular consensus, 1,389 Mbp in 12,204 contigs, N50 172 kb. Map: 211,106 markers in 1,235 patterns from 96 F2 plants, 818.6 cM. 841 sequences (714,498,588 bp) anchored; BUSCO gene space 90.8%; 63% repeat-masked; 42,052 protein-coding genes. The comparison with earlier genomes is only in Table S1. · Assembly Results; Linkage mapping; Pseudomolecule generation; CBDRx chromosome assembly analysis

  2. ShownCBDRx's cannabinoid synthase copies lie in three linked regions at 25-33 Mbp, in a pericentromeric, low-recombination part of chromosome 9. There are two tandem cassette arrays nested between long terminal repeat (LTR) retrotransposons, plus one solitary copy. Each region has one complete coding sequence.

    13 copies: 7 at 25 Mbp, 5 at 29 Mbp, 1 at 30 Mbp; the arrays consist of 31-45 kb tandem repeats. LTR08 is largely confined to the 29 Mbp CBDA synthase (CBDAS) cluster, whereas LTR01 is abundant genome-wide and flanks it. Illumina read coverage supported full capture in CBDRx, against 43 of 45 copies in the F1 assembly (mostly on short contigs) and 5 of 30 in the Purple Kush assembly. · Main Text; THCAS/CBDAS and coverage analysis; Figure legends

  3. SuggestedCBDRx is mostly marijuana in ancestry. Its CBDAS region and the THC/CBD-ratio QTL sit in a hemp-derived segment of chromosome 9, while its THCA synthase (THCAS) array lies in marijuana-ancestry sequence. This fits breeders introgressing hemp CBDAS alleles into a marijuana background.

    ADMIXTURE at k=3: CBDRx is 89% marijuana and 11% hemp, with hemp ancestry mostly on chromosomes 9 and 10. About 20% of chromosome 9 is hemp-derived (AncestryHMM tracts, assuming a single hemp pulse eight generations ago). For comparison, Skunk#1 is 78% marijuana/22% naturalized and Carmen is 94% hemp/6% marijuana. · Main Text; Comparative Genomics; Figure legends

  4. SuggestedPopulation branch statistic (PBS) scans on the marijuana branch flag CBDAS but not THCAS. The authors read this as recent selection on CBDAS (for nonfunctional alleles in marijuana), and as evidence that CBDAS divergence, more than THCAS, sets the THCA/CBDA ratio.

    Extreme PBS values (against a genome-wide 99.995th-percentile line) occur at SNPs near CBDAS but not near THCAS. The only THC/CBD chemotype QTL coincides with the physical position of CBDAS. Fst: 0.229 marijuana-hemp, 0.128 marijuana-naturalized, 0.147 hemp-naturalized. PCA of 2,051 SNPs from 367 accessions separates hemp from marijuana on PC1. dN/dS ratios are cited but not reported. · Main Text; Comparative Genomics; Figure legends

  5. SuggestedThe Skunk#1 x Carmen F2 has five QTL for total cannabinoid content (potency), and none falls at a synthase cluster. So loci other than the synthase genes appear to have more influence on cannabinoid quantity.

    Composite interval mapping in WinQTLCart on 96 F2 plants, with an experiment-wise P = 0.05 threshold from 1000 permutations. The strongest potency QTL lies on chromosome 3 (not 9) and accounts for 17% of variation in cannabinoid quantity. · Main Text; QTL analysis; Figure legends

Methods, data and reproducibility

The authors sequenced the Skunk#1 and Carmen parents and 96 F2 plants (Illumina), an F1 (PacBio) and CBDRx (nanopore), and assembled the long reads without error correction (minimap2, miniasm, Racon, Pilon). They anchored the CBDRx contigs into chromosomes with a sequencing-based F2 linkage map and Hi-C, then mapped QTL for cannabinoid and biomass traits and ran admixture, ancestry-tract and PBS scans.

  • Skunk#1 (marijuana) and Carmen (hemp) parental lines, sibling-crossed for five generations; Illumina HiSeq 2500 whole-genome sequencing · Cannabis · n = 2 lines
  • Skunk#1 x Carmen F2 female plants: Illumina WGS (2x125 bp), GC cannabinoid profiles, plant height and biomass fractions · Cannabis · n = 96 F2 individuals
  • Male F1 (CO11), full sibling of the F1 that founded the mapping population; PacBio Sequel long reads · Cannabis · n = 1 plant · PRJEB29284 (ENA; genome assembly and annotation, raw-read deposit not stated)
  • High-CBDA cultivar CBDRx (15% CBDA, 0.3% THCA): 27 Gb of Oxford Nanopore MinION genomic reads · Cannabis · n = 1 plant (CBDRx:18:580) · PRJEB29284 (ENA; genome assembly and annotation, raw-read deposit not stated)
  • CBDRx leaf full-length cDNA sequenced on Oxford Nanopore GridION, for gene annotation · Cannabis · n = 4 libraries
  • Public RNA-seq libraries from the Sequence Read Archive, used for gene annotation · n = 142 libraries
  • Previously published Cannabis population genotype data combined with the new genomes for PCA, ADMIXTURE and PBS · Cannabis · n = 367 accessions; 2,051 SNPs
  • Reference CBDAS and THCAS gene sequences, used to locate synthases in the assembly by BLAST · Cannabis · AB292682 (CBDAS); AB057805 (THCAS)
Methods & tools
minimap2, miniasm, Racon, Pilon, McCortex, LB-Impute, AntMap, JoinMap 4.1, Windows QTL Cartographer 2.5, Salsa, ADMIXTURE, AncestryHMM
Reagents & environment
Versions are given only for JoinMap 4.1, WinQTLCart v2.5_011 and MapChart 2.32. Nanopore reads were base-called with Albacore using FLO-MIN106 and SQK-LSK108 settings.
Assay conditions & compute
The nanopore assembly ran on one machine with 231 Gb RAM and 56 CPUs. The MinION run took 48 h.
Reproducibility gaps
No code or scripts are linked. The CBDRx and F1 genome assemblies and annotation are in ENA study PRJEB29284 (accession only, no URL printed); a raw-read deposit is not stated. The published population data were obtained from the original authors. Most software versions are not stated.
Limitations (6)
  • Two of the three synthase-bearing contigs on chromosome 9 had no recombinants, so their order and orientation could not be resolved. They were placed next to each other by hand as the most parsimonious arrangement. · stated by the authors
  • The F1 assembly recovered 43 of 45 synthase copies, mostly as single cassettes on short contigs, so some may be incompletely assembled. · stated by the authors
  • The potency QTL point to further loci beyond the synthase genes that still need investigation. · stated by the authors
  • About 10% of linkage-map markers showed segregation distortion (Chi^2 test) and were kept in the map; the authors describe these as low in number. · stated by the authors
  • Counts and coordinates disagree across the text: 13 synthase copies (main text) vs 14 (coverage analysis); the 7-copy array at 25 Mbp vs 26 Mbp; cassettes of 31-45 kb vs 38-84 kb and 28-57 kb (Fig. 3); 1,235 vs 1,175 segregation patterns. · noted in this summary
  • The introgression account rests on a single cultivar (CBDRx) and a one-pulse, eight-generation ancestry model. The QTL come from a single 96-plant F2 cross, which limits detection of small-effect potency loci near the synthase genes. · noted in this summary

Who should read:Read this if you study Cannabis cannabinoid genetics or breeding, or assemble repeat-rich plant genomes where tandem biosynthetic gene arrays sit inside LTR retrotransposons.

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