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Abstract Cannabis sativais a globally important seed oil, fibre and drug-producing plant species. However, a century of prohibition has severely restricted development of breeding and germplasm resources, leaving potential hemp-based nutritional and fibre applications unrealized. Here we present a cannabis pangenome, constructed with 181 new and 12 previously released genomes from a total of 144 biological samples including both male (XY) and female (XX) plants. We identified 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. Across the ancient heteromorphic X and Y sex chromosomes, we observed a variable boundary at the sex-determining and pseudoautosomal regions as well as genes that exhibit male-biased expression, including genes encoding several key flowering regulators. Conversely, the cannabinoid synthase genes, which are responsible for producing cannabidiol acid and delta-9-tetrahydrocannabinolic acid, contained very low levels of diversity, despite being embedded within a variable region with multiple pseudogenized paralogues, structural variation and distinct transposable element arrangements. Additionally, we identified variants of acyl-lipid thioesterase genes that were associated with fatty acid chain length variation and the production of the rare cannabinoids, tetrahydrocannabivarin and cannabidivarin. We conclude that theC. sativagene pool remains only partially characterized, the existence of wild relatives in Asia is likely and its potential as a crop species remains largely unrealized. 

Abstract Sample preservation often impedes efforts to generate high-quality reference genomes or pangenomes for Earth’s more than 2 million plant and animal species due to nucleotide degradation. Here we compare the impacts of storage methods including solution type, temperature, and time on DNA quality and Oxford Nanopore long-read sequencing quality in 9 fish and 4 plant species. We show 95% ethanol largely protects against degradation for fish blood (22 °C, ≤6 weeks) and plant tissue (4 °C, ≤3 weeks). From this furthest storage timepoint, we assemble high-quality reference genomes of 3 fish and 2 plant species with contiguity (contig N50) and completeness (BUSCO) that achieve the Vertebrate Genome Project benchmarking standards. For epigenetic applications, we also report methylation frequency compared to liquid nitrogen control. The results presented here remove the necessity for cryogenic storage in many long read applications and provide a framework for future studies focused on sampling in remote locations, which may represent a large portion of the future sequencing of novel organisms.