A Single Nucleotide Polymorphism assay sheds light on the extent and distribution of genetic diversity, population structure and functional basis of key traits in cultivated North American Cannabis

Abstract

BackgroundThe taxonomic classification of the Cannabis genus has been delineated through three main types: sativa (long and less branched plant with long and narrow leaves), indica (short but highly branched plant with broader leaves) and ruderalis (wild type with short stature, less branching and small thick leaves). While still under discussion, particularly whether the genus is polytypic or monotypic, this broad classification reflects putative geographical origin of each group and putative chemotypic and pharmacology.MethodsHere we describe a thorough investigation of cannabis accessions using a set of 22 highly informative and polymorphic SNP markers associated with important traits such as cannabinoid and terpenoid expression as well as fibre and resin production. The assay offers insight into cannabis population structure, phylogenetic relationship, population genetics and correlation to secondary metabolite concentrations and demonstrate the utility of this assay for rapid, repeatable and cost-efficient genotyping of commercial and industrial cannabis accessions for use in product traceability, breeding programs, regulatory compliance and consumer education.ResultsThe main outcomes are the identification of 5 clusters in the sample set available, including industrial hemp, resin hemp which likely underwent a bottleneck to stabilize CBDA accumulation (Type II & III). THC resin (type I) make up the other three clusters with terpinolene (colloquial “sativa” or “NLD”), myrcene/pinene and myrcene/limonene (colloquial “indica”, “BLD”), which also putatively harbour an active CBCAS.ConclusionThe functional basis of key traits is also discussed as recently enabled by the NCBI Cannabis sativa Annotation Release 100, allowing for hypothesis testing with regards to secondary metabolite production as well as other key traits of importance for adaptable and compliant large-scale seed production under the new US Domestic Hemp Production Program.

Recent advances in Cannabis sativa genomics research

“…Key findings include: the genome‐wide distinction between drug and hemp types that resulted from selective breeding; the association between chemotypic identity and variation of loci encoding cannabinoid synthases; and errors in cultivar classification and ancestry by breeders (Van Bakel et al ., 2011; Sawler et al ., 2015; Lynch et al ., 2016; Soorni et al ., 2017). These findings are likely to facilitate the development of more accurate diagnostic systems for cannabis germplasm to assist with product compliance, traceability, provenance and consumer education (Henry et al ., 2020). Domestication and intensive breeding have narrowed the genetic and allelic diversity of cannabis gene pools (Sawler et al ., 2015; Soorni et al ., 2017).…”

“…There are only two published cannabis GWAS studies, one of which is ongoing and the other reporting marker‐trait association using a limited number of SNPs (B. J. Campbell et al ., 2019; Henry et al ., 2020; http://multihemp.eu/). The paucity of studies is likely a consequence of the previous limited accessibility of cannabis genetic diversity and insufficient marker density.…”

“…The exact number of species comprising the Cannabis genus is controversial. Some claim the genus consists of three species that display distinct phenotypic differences; namely C. sativa L., C. indica Lam (Lamarck) and C. ruderalis (Sawler et al ., 2015; Clarke & Merlin, 2016; Henry et al ., 2020). The alternative, and perhaps most accepted, viewpoint is that Cannabis is a monotypic genus consisting of a single species, Cannabis sativa L. (referred to as cannabis hereafter) (Small & Cronquist, 1976).…”

Potentials and Challenges of Genomics for Breeding Cannabis Cultivars

“…More recently, based on the latest knowledge acquired on cannabis genomics, Henry et al (2020) described the efficiency of a screening method based on KASP (Kompetitive Allele Specific PCR) technique for the identification of 22 highly informative SNPs involved in the biosynthetic pathway of cannabinoids and terpenes (important compounds for the recreational and medical cannabis industries).…”

Cannabis Seeds Authentication by Chloroplast and Nuclear DNA Analysis Coupled with High-Resolution Melting Method for Quality Control Purposes

“…Various methods of identification have been developed to authenticate medical plant species such as morphological analysis, chemical profiling, and DNA-based molecular analysis. 11 Although up to date there is no standardized method for rapid identification of cannabis, the use of internal transcribed spacer (ITS) regions has been recently reported for the identification of cannabis from seizures. 12 Forensic identification of cannabis usually consists on the detection of the presence of D-9-THC and cystolithic hairs on the leaves of the plant, 13 but genetic identification has not been taken into account yet.…”