The Future of Cannabis Genetics Jonathan Page

Cannabis has been used for millennia Food and oil (hemp) Fibre (hemp) Drug and Medicine (“marijuana”) THC

Cannabis produces a complex suite of bioactive metabolites: The mixture is the medicine Cannabinoids psychoactivity + pain relief, appetite, antispasticity… Terpenes

How are cannabinoids synthesized? Cannabis sativa (marijuana, hemp)

Glandular trichomes on cannabis flowers

100 µm

Cannabis trichomes Subcuticular cavities Secretory disc cells

Trichome-specific gene expression analysis RNA Protein

Cannabis is the first medicinal plant with a sequenced genome Collaboration with Tim Hughes (University of Toronto) Published in Genome Biology in November 2011 Article accessed 97,925 times! Cannabis haploid genome is ~820 Mbp Genome organization 2n = 20 Nine autosomes Two sex chromosomes (X and Y) We sequenced three types: Marijuana ‘Purple Kush’ ~19% THCA Hemp ‘Finola’ Low THCA, 4% CBDA Hemp ‘USO-31’ Low to zero cannabinoids www.genomevolution.org

The cannabinoid biosynthetic pathway       

Cannabinoid pathway bifurcation: “Marijuana” vs hemp Marijuana – 18% THCA THCA THCA synthase CBGA CBDA synthase CBDA Hemp – 4% CBDA

Cannabis chemotypes result from simple genetics Chemotypes are chemical phenotypes Chemotype III (high CBD) Chemotype II (THC:CBD)  Chemotype I (high THC) 

Genetics of cannabinoid biosynthesis THCA synthase CBDA synthase Chemotype I (high THC) Chemotype II (THC:CBD mix) Chemotype III (high CBD)

Classification of Cannabis is (still!) unclear sativa indica ruderalis Cannabaceae Hemp and marijuana many strains and cultivars four chemotypes Family Genus Species? Subspecies?

Marijuana strains: Indica vs Sativa Thousands of strains exist Most strains were developed for recreational use Patients report that strains differ in their effects Patients and growers differentiate two general types: “Indica” Short plant with broad leaves Sedative and “body stone” “Sativa” Tall plant with narrow leaves Stimulating and cerebral effects

Cannabis genotyping Three main questions: What is the genetic structure of the genus Cannabis? Are hemp and marijuana genetically distinct? Are Indica and Sativa genetically distinct? Collaboration with Sean Myles (Dalhousie University) Accessed DNA samples representing diverse cannabis types: 43 hemp 81 marijuana samples Analyzed using GBS (genotyping-by-sequencing) 14,031 single-nucleotide polymorphisms (SNPs)

Cannabis genotyping: “marijuana” and hemp Sawler et al, PLOS ONE 2015

The problem of germplasm - is cannabis endangered? Genetic diversity is the basis of agricultural innovation! Sources of new genetic traits such as disease resistance BUT Cannabis is undergoing genetic reduction Drug prohibition has destroyed plants e.g. feral hemp Legal exchange of seeds and plants is difficult “Dutchification” is replacing landraces across the globe Reliance on vegetative propagation (cloning) Long-term seed viability is limited MMPR restricts access to genetics Preserving crop diversity requires seed banks (not just seed companies…), sustained funding and research efforts

The future of cannabis breeding Cannabis breeding has been a huge success: THC levels and yields have increased; CBD too But has led to a narrowing of gene pool with focus on indoor cultivation and clonal propagation High THC product with lower levels of other cannabinoids Genetic traits for consumers: Novel cannabinoid profiles for medical use New terpene profiles Sensory properties Genetic traits for growers: Disease and pest resistance Higher yield, flowering time, plant architecture

Genomics assisted breeding of cannabis X Select plants from DNA & Chemotype 100s of seeds back to breeder 100,000+ seeds Analysis & prediction DNA sequencing

Cannabis patents Q: Is patenting of cannabis strains possible in Canada? A: No. Canadian law does not allow patenting of higher life forms such as plants. Registration of new varieties through Plant Breeders Rights is possible. PBR helps “protect the variety from exploitation by others”. Varieties must be: New Distinct Uniform Stable Currently 6 registered hemp varieties and 1 registered marijuana variety

Cannabis needs for more science!

Science leadership Health Canada licensed lab Analytical testing and QC Genotyping services Developing advanced cannabis strains jpage@anandialabs.com