Molecular genetics, genomics, and quantitative genetics of specialized metabolites in Cannabis sativa L.

Abstract

How genetic variation within species affects phytochemical composition is a fundamental question in the plant sciences. Insights into the inheritance of specialized metabolites can be gained through a combination of quantitative genetics, molecular marker development, and genomic analysis. Cannabis has been cultivated for over 10,000 years for pharmacological use, fiber, and grain. The distinctive aroma of Cannabis flowers is partly attributed to the specialized metabolites cannabis produces known as terpenes and terpenoids whereas the pharmacological properties are most commonly associated with cannabinoids. Cannabinoids are a class of specialized metabolites produced by cannabis which interact with specific cannabinoid receptors (CB1 and CB2) in the central nervous system. Understanding the inheritance of cannabinoid production is imperative for the development of new hemp cultivars as well as the production and saving of hemp seed. The inheritance of these economically important specialized metabolites has been poorly understood due to constraints placed on research imposed by current and past legal policy relating to cannabis. The genetic mapping population that is the focus of this dissertation exhibits variation in three main phenotypic classes of the ratio of tetrahydrocannabinol (THC) and cannabidiol (CBD), which can be grouped into (THC-type, CBD-type, and intermediate type). In Chapter 1, we tested a genetic model associating these three cannabinoid phenotypic classes with functional and non-functional alleles of cannabidiolic acid synthase (CBDAS). We characterized cannabinoid content and assayed CBDAS genotypes of >300 feral C. sativa plants in Minnesota, USA. We performed a test cross to assess CBDAS inheritance. Twenty clinical cultivars obtained blindly from the National Institute on Drug Abuse and twelve Canadian-certified grain cultivars were also examined. Frequencies of CBD-type, intermediate-type and THC-type feral plants were 0.88, 0.11, and 0.01, respectively. Genotype frequencies observed in the test cross were consistent with codominant Mendelian inheritance of the THC:CBD ratio. CBDAS genotypes blindly predicted the THC:CBD ratio among clinical cultivars, feral accessions and the same was true for industrial grain cultivars when plants exhibited >0.5% total cannabinoid content. Chapter 1 results extend the generality of the inheritance model for THC:CBD to diverse C. sativa accessions and demonstrate that CBDAS genotyping can predict the ratio in a variety of practical applications. In Chapter 2 we investigated the ancestry of a new cultivar and cannabinoid synthase genes in relation to cannabinoid inheritance. A nanopore-based assembly anchored to a high-resolution linkage map provided a chromosome-resolved genome for CBDRx, a potent CBD-type cultivar. Although CBDRx is predominantly of drug cannabis ancestry, the genome has cannabidiolic acid synthase (CBDAS) introgressed from hemp and lacks a complete sequence for tetrahydrocannabinolic acid synthase (THCAS). Three additional genomes, including one with complete THCAS, confirmed this genomic structure. Only CBDAS was expressed in CBD-type Cannabis, while both CBDAS and THCAS were expressed in a cultivar with an intermediate THC:CBD ratio. Although variation among cannabinoid synthase loci might affect the THC:CBD ratio, variability among cultivars in overall cannabinoid content (potency) was also associated with other chromosomes. Chapter 2 resulted in a high resolution genome for C. sativa. The cannabinoid abundance trait was found to be influenced by several areas throughout the genome. In Chapter 3, we focused on better understanding interactions among terpene, cannabinoid, trichome head size, and biomass traits, we phenotyped an experimental mapping population and created a high density (HD) linkage map using Illumina sequence data from 96 F2 offspring of a cross between drug type and fiber type Cannabis. The ten QTL identified for terpene, sixteen cannabinoid QTL, one trichome head size QTL and nine biomass QTL might aid in the breeding of cultivars with specific terpene and cannabinoid profiles, abundances as well as agronomic traits of interest. Chapter 3 results identified ESTs within significant QTL which identified potential genes of interest for the traits studied. This work was the first to identify a significant QTL for trichome gland size and significant QTL for the terpene ratio trait limonene:α-pinene. This work provides many avenues for further research. The identification of a significant QTL for the ratio trait limonene to α-pinene provides an avenue to research the creation and validation of a marker system to screen and select for specific terpene profiles. Significant QTL for agronomic traits were identified and thus future work can investigate more complex agronomic traits and their influence on the yields of specialized metabolites, fiber and grain. Questions that arise from this work include: which potency QTL influences total cannabinoid abundance? Can markers for the ratio of limonene to α-pinene be developed and validated in diverse populations of C. sativa? Does the inheritance of the ratio trait limonene:α-pinene display single-locus Mendelian codominance? Can a marker for trichome gland size be created, validated and accessed for its influence on cannabinoid abundance? This work lays the foundation for future research to develop and validate selectable markers to be exploited in the creation of cannabis cultivars with specific agronomic, trichome, terpene and cannabinoids traits of interest.