Domesticated cannabinoid synthases amid a wild mosaic cannabis pangenome

Cannabis sativa is a globally significant seed-oil, fiber, and drug-producing plant species. However, a century of prohibition has severely restricted legal breeding and germplasm resource development, leaving potential hemp-based nutritional and fiber applications unrealized. Existing cultivars are highly heterozygous and lack competitiveness in the overall fiber and grain markets, relegating hemp to less than 200,000 hectares globally1. The relaxation of drug laws in recent decades has generated widespread interest in expanding and reincorporating cannabis into agricultural systems, but progress has been impeded by the limited understanding of genomics and breeding potential. No studies to date have examined the genomic diversity and evolution of cannabis populations using haplotype-resolved, chromosome-scale assemblies from publicly available germplasm. Here we present a cannabis pangenome, constructed with 181 new and 12 previously released genomes from a total of 156 biological samples from both male (XY) and female (XX) plants, including 42 trio phased and 36 haplotype-resolved, chromosome-scale assemblies. We discovered widespread regions of the cannabis pangenome that are surprisingly diverse for a single species, with high levels of genetic and structural variation, and propose a novel population structure and hybridization history. Conversely, the cannabinoid synthase genes contain very low levels of diversity, despite being embedded within a variable region containing multiple pseudogenized paralogs and distinct transposable element arrangements. Additionally, we identified variants of acyl-lipid thioesterase (ALT) genes that are associated with fatty acid chain length variation and the production of the rare cannabinoids, tetrahydrocannabinol varin (THCV) and cannabidiol varin (CBDV). We conclude the Cannabis sativa gene pool has only been partially characterized, and that the existence of wild relatives in Asia remains likely, while its potential as a crop species remains largely unrealized.