Development and validation of a minimal SNP genotyping panel for the differentiation of Cannabis sativa chemovars

Abstract

Background Due to its previously illicit nature, cannabis had not fully reaped the benefits of recent innovations in genomic plant science. However, Canada’s legalization of cannabis in 2018 triggered significant new demand for robust genotyping tools to assist breeders in meeting consumer demands. While most molecular marker-based research on cannabis has focused on screening for traits of agronomic value, more recent research has sought to use molecular markers to differentiate between cannabis cultivars. Results In this study, we have conducted whole genome sequencing of 32 cannabis chemovars, mined the sequencing data for SNPs, developed a reduced SNP genotyping panel to discriminate between sequenced chemovars, then validated the 20-SNP panel using DNA from the sequenced varieties and tested the assays on commercially available cannabis products. The assay conversion rate was high in DNA extracted from fresh plant material and middling in DNA extracted from commercial samples. However, called genotypes were internally consistent, highlighting discrepancies between genotypes predicted using sequencing data and observed using genotyping assays. The primary contributions of this work are to clearly document the process used to develop this genotyping panel and outline improvements and goals for future iterations of PCR-based, minimal SNP panels to enable efficient development genotyping tools to identify and screen cannabis varieties. Conclusions Our key recommendations are to leverage higher read length paired-end short-read technology; conduct in-depth pre- and post-processing of reads, mapping, and variant calling data; integrate trait-associated loci to develop multi-purpose panels; use iterative approaches for in vitro validation; and integrate redundancy to account for errors throughout the pipeline and the effects of irradiation sterilization on cannabis DNA extracted from commercially available dried flower samples.