Genome assemblies trace cannabinoid synthase evolution in Cannabis

Despite the therapeutic importance of cannabinoids, the scarcity of gap-free Cannabis genomes has limited the dissection of the genetic and genomic basis of cannabinoid biosynthesis and genome evolution. Here, we generated two haplotype-phased, gap-free, gold-standard assemblies and used them to investigate post-polyploid genome remodelling and refine the evolutionary history of cannabinoid synthase genes. Analyses of these gap-free assemblies recovered a γ whole-genome triplication (WGT) signal shared with core eudicots and provided no evidence for additional lineage-specific whole-genome duplication (WGD) in Cannabis. The extant Cannabis genome reflects long-term retention and divergence of a subset of γ-derived duplicates, together with subsequent non-WGD-derived gene duplications and repeat-associated remodelling that contributed substantially to present-day genome diversification. Furthermore, phylogenomic, structural and syntenic evidence indicate that a precursor duplication predating the Cannabis-Humulus split gave rise to two lineages retained in Cannabis on chromosomes 7 and 2. In Cannabis, the chromosome 7 lineage subsequently expanded and diversified into the functional cannabinoid synthase genes, whereas Humulus retained only the lineage resembling the Cannabis chromosome 2 copy. This asymmetric retention and diversification provide new insights into the origin and specialization of cannabinoid biosynthesis in Cannabis. Together, these results provide a high-quality genomic framework for understanding the history of polyploidy, the evolution of the cannabinoid synthase gene and genome diversification in Cannabis.