Development and validation of genetic markers for sex and cannabinoid chemotype in <i>Cannabis sativa</i> L.

Toth, Jacob A.; Stack, George M.; Cala, Ali R.; Carlson, Craig H.; Wilk, Rebecca L.; Crawford, Jamie; Viands, D. R.; Philippe, Glenn; Smart, Christine D.; Rose, Jocelyn K. C.; Smart, Lawrence B.

doi:10.1111/gcbb.12667

Cited by 93 publications

(93 citation statements)

References 52 publications

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“…Although environmental factors play a role in determining the amount of cannabinoids present in different parts and stages of the plant (Rustichelli et al 1998), in most populations the ratio between THCA and CBDA has been found to be under genetic control (Mandolino et al 2003;Weiblen et al 2015;Toth et al 2020;Wenger et al 2020). Codominant inheritance of CBDA and THCA chemotypes is consistent with a Mendelian single-locus (de Onofri et al 2015;Weiblen et al 2015).…”

Section: Cannabinoid Oxidocyclase Gene Evolutionmentioning

confidence: 91%

Origin and evolution of the cannabinoid oxidocyclase gene family

Velzen

Schranz

2020

Preprint

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Cannabis is an ancient crop representing a rapidly increasing legal market, especially for medicinal purposes. Medicinal and psychoactive effects of Cannabis rely on specific terpenophenolic ligands named cannabinoids. Recent whole-genome sequencing efforts have uncovered variation in multiple genes encoding the final steps in cannabinoid biosynthesis. However, the origin, evolution, and phylogenetic relationships of these cannabinoid oxidocyclase genes remain unclear. To elucidate these aspects we performed comparative genomic analyses of Cannabis, related genera within the Cannabaceae family, and selected outgroup species. Results show that cannabinoid oxidocyclase genes originated in the Cannabis lineage from within a larger gene expansion in the Cannabaceae family. Localization and divergence of oxidocyclase genes in the Cannabis genome revealed two main syntenic blocks, each comprising tandemly repeated cannabinoid oxidocyclase genes. By comparing these blocks with those in genomes from closely related species we propose an evolutionary model for the origin, neofunctionalization, duplication, and diversification of cannabinoid oxidocycloase genes. Based on phylogenetic meta-analyses, we propose a comprehensive classification of three main clades and seven subclades that is intended to aid unequivocal referencing and identification of cannabinoid oxidocyclase genes. Our data suggest that cannabinoid oxidocyclase gene copy number variation may have less functional relevance than previously thought. Instead, we propose that cannabinoid phenotype is primarily determined by presence/absence of single-copy genes. Increased sampling across Cannabis’ native geographic range is likely to uncover additional cannabinoid oxidocyclase gene sequence variation.Significance statementCannabis genome sequencing efforts have revealed extensive cannabinoid oxidocyclase gene variation. However, phylogenetic relationships and evolution of these genes remains unclear. Our meta analysis of currently available data reveals that these genes comprise three main clades and seven subclades that originated through Cannabis-specific gene duplication and divergence. Our new conceptual and evolutionary framework serves as a reference for future description and functional analyses of cannabinoid oxidocyclases.

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Section: Cannabinoid Oxidocyclase Gene Evolutionmentioning

confidence: 91%

Origin and evolution of the cannabinoid oxidocyclase gene family

Velzen

Schranz

2020

Preprint

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“…Rotherham and Harbison (2011) reported a set of single nucleotide polymorphism (SNP) markers based on differences between “drug‐type” and “fiber‐type” THCAS (Kojoma et al, 2006), but the latter THCAS sequence was reclassified by Laverty et al (2019) as cannabichromenic acid synthase ( CBCAS ). These SNP markers and others derived from THCAS sequence (Staginnus et al, 2014) can distinguish THC‐type and CBD‐type plants, but they fail to differentiate THC‐type from intermediate‐type plants (Toth et al, 2020).…”

Section: Figurementioning

confidence: 99%

“…Plants that are heterozygous at the CBDAS locus have the intermediate type. The opportunity to test the validity of the model and its utility for predictive genotyping has only recently emerged with greater access to diverse populations of C. sativa (Toth et al, 2020).…”

Section: Figurementioning

confidence: 99%

Validating a predictive model of cannabinoid inheritance with feral, clinical, and industrial Cannabis sativa

Wenger

Dabney

ElSohly

et al. 2020

American J of Botany

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of America. This is an open access article under the terms of the Creat ive Commo ns Attri butio n-NonCo mmerc ial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. • 1423 Natural genetic variation affects the composition of phytochemicals in plants and can be of great economic importance. With a record of use by humans as food, fiber, and medicine spanning thousands of years (Faeti et al., 1996), recent decades of prohibition, and a rapidly changing regulatory context, Cannabis sativa L. (Cannabaceae) was until recently among the least studied agricultural crops (Small, 2016). Prominent among the traits of C. sativa are cannabinoids, a unique class of specialized metabolites synthesized and stored in glandular trichomes that are located on the floral bracts of pistillate inflorescences (Livingston et al., 2020; Fig. 1A-C). The ratio of the two most abundant cannabinoids, tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA), hereafter THC and CBD, is represented by three main classes: THC-type plants with THC:CBD ≥ 10, intermediate-type plants with THC:CBD ≈ 1, and CBD-type plants with THC:CBD ≤ 0.1 (de Meijer et al., 2003). Some authors have referred to these chemotype (chemical phenotype) classes as Type I, Type II, and Type III (Small, 2016). Here we introduce for clarity a more descriptive classification of THC-type, intermediate-type, and CBD-type plants. Descriptive terms also avoid confusion associated with recent statutory definitions of C. sativa that vary widely among political jurisdictions (e.g., "industrial hemp" and "medical marijuana"). Our findings suggest that patterns of cannabinoid inheritance render some of these popular definitions inaccurate at least from a botanical perspective.

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“…Recently, others have proposed targeted markers for the identification of fiber and resin cannabis (e.g. Cascini et al 2019 ; Hilyard et al 2019 ) as well as molecular sexing tools to differentiate feminized from regular seed stock (Toth et al 2020 ).…”

Section: Introductionmentioning

confidence: 99%

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

Henry¹,

Khatodia²,

Kapoor³

et al. 2020

J Cannabis Res

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Background The taxonomic classification of Cannabis genus has been delineated through three main types: sativa (tall and less branched plant with long and narrow leaves), indica (short and highly branched plant with broader leaves) and ruderalis (heirloom 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 origins of each group and putative chemotype and pharmacologic effect. Methods Here we describe a thorough investigation of cannabis accessions using a set of 23 highly informative and polymorphic SNP (Single Nucleotide Polymorphism) 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. We demonstrate the utility of the 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. Results We identified 5 clusters in the sample set, including industrial hemp (K5) and resin hemp, which likely underwent a bottleneck to stabilize cannabidiolic acid (CBDA) accumulation (K2, Type II & III). Tetrahydrocannabinolic acid (THCA) resin (Type I) makes up the other three clusters with terpinolene (K4 - colloquial “sativa” or “Narrow Leaflet Drug” (NLD), myrcene/pinene (K1) and myrcene/limonene/linalool (K3 - colloquial “indica”, “Broad Leaflet Drug” (BLD), which also putatively harbour an active version of the cannabichrometic acid Synthase gene (CBCAS). Conclusion The final chemical compositions of cannabis products have key traits related to their genetic identities. Our analyses in the context of the NCBI Cannabis sativa Annotation Release 100 allows for hypothesis testing with regards to secondary metabolite production. Genetic markers related to secondary metabolite production will be important in many sectors of the cannabis marketplace. For example, markers related to THC production will be important for adaptable and compliant large-scale seed production under the new US Domestic Hemp Production Program.

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Development and validation of genetic markers for sex and cannabinoid chemotype in Cannabis sativa L.

Cited by 93 publications

References 52 publications

Origin and evolution of the cannabinoid oxidocyclase gene family

Origin and evolution of the cannabinoid oxidocyclase gene family

Validating a predictive model of cannabinoid inheritance with feral, clinical, and industrial Cannabis sativa

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

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