Sequence and annotation of 42 cannabis genomes reveals extensive copy number variation in cannabinoid synthesis and pathogen resistance genes

Key results

1. ShownThree PacBio long-read assemblies of an inbred Jamaican Lion trio (mother, father, F1) reach contig N50s of 1.6 to 3.8 Mb with over 96% BUSCO (benchmarking universal single-copy orthologs) completeness, and are annotated with Iso-Seq full-length transcripts from five tissues.

   FALCON-Unzip on 84.8 to 125 Gb of long reads per genome; BUSCO 96.1%, 97.0% and 97.3% complete (father, mother, F1). Purging the maternal assembly gave an 876 Mb haploid size and 3.2 Mb N50, but 12.6% BUSCO duplicates remain. 83,464 Iso-Seq isoforms; 27,664 female and 31,108 male genes in S6 vs 27,644 and 32,108 in S20. · Sequencing, assembly and annotation; Structural variation and gene models; DNA Assembly; Gene annotation

2. ShownNormalized gene coverage over THCAS, CBDAS and cannabichromenic acid synthase (CBCAS) sorts cultivars into Type I, II and III chemotypes; the most common Bt:Bd alleles are whole-gene deletions of THCAS or CBDAS.

   39 cannabinoid synthase genes profiled for copy number variation (CNV) across 40 genomes at 52x average coverage, using unique molecular identifier (UMI) libraries (PCR vs PCR-free control coverage r = 0.999). Label HPLC chemotypes for fourteen Massachusetts samples all matched the deletion-based calls; deletion status cannot separate 0.3% from 0.5% THCA Type III plants. · Copy number variation across the genomes; Cannabinoid synthase genes; Chemotyping; Figure legends

3. SuggestedA roughly 2 Mb cassette of 8 co-oriented CBCAS genes on contig 756 is absent from many Type I plants and also carries pathogen-response genes. The authors propose that breeding hemp below 0.3% THCA could select for the deletion and so remove those genes.

   The 8 CBCAS copies are over 99.4% identical; the deleted span holds NPF3, RMT1, PIP1 and NIP1. Most Type III plants carry the contig (one exception), Type II plants are usually heterozygous, and some Type I plants (Black 84, Arcata Trainwreck) carry it. THCA synthesis by CBCAS in planta is not tested here; cited support comes from yeast work. · Cannabinoid synthase genes; Conclusions; Figure legends

4. SuggestedDeletion of a thaumatin-like protein (TLP) gene, CsTLP1, is common in cultivars reported as powdery mildew susceptible. CsTLP1 expressed in E. coli shows β-1,3-glucanase activity and inhibits Fusarium oxysporum growth on plates.

   Copy number of 23 TLPs, 35 chitinases and 24 mildew resistance locus O (MLO) genes compared with cultivator-reported resistance across 40 genomes; endochitinase CH25 and MLO absence also correlated with resistance. CsTLP1 alone did not inhibit Aspergillus flavus or Penicillium chrysogenum; with Trichoderma viride chitinase it inhibited P. chrysogenum and F. oxysporum. The mildew fungus itself was not tested. · Pathogen response genes; Anti-fungal activity; Figure legends

5. ShownContigs covered only in male genomes, intersected with male-specific Iso-Seq transcripts, place 574 genes in the non-recombining region of the Y chromosome. Whole-genome coverage also indicates an XX structure in monoecious hemp.

   40 genomes (9 male, 2 monoecious, 29 female) aligned to the paternal assembly: Y contigs have zero coverage in females and X contigs have double coverage; 118 Mb of putative Y contigs. Monoecious C3 and Fedora show an XX pattern. Y genes include FT, FY and PIN2 homologs; their role in sex determination is proposed, not tested. · Introduction; Identification of the Y chromosome; Conclusions; Figure legends