A trio-binning approach for genome assembly reveals extensive structural variation between two Cannabis cultivars: Punto Rojo and Cherry Pie.

With the advent of long-read DNA sequencing technologies, assembling eukaryotic genomes has become routine; however, properly phasing the maternal and paternal contributions, which is of great value for breeding programs, remains technically challenging. Here, we use the trio-binning approach to separate Oxford Nanopore reads derived from a Cannabis F1 wide cross, made between the Colombian landrace Punto Rojo and the Colorado CBD clone Cherry Pie #16. Reads were obtained from a single PromethION flow cell, generating assemblies with coverage of just 18 × per haplotype, but with good contiguity and gene completeness, demonstrating that it is a cost-effective approach for genome-wide and high-quality haplotype phasing. Evaluated through the lenses of disease resistance and secondary metabolite synthesis, both being traits of interest for the Cannabis industry, we report copy number and structural variation that, as has recently been shown for other major crops, may contribute to phenotypic variation along several relevant dimensions.