The long history of human use has made the exact region of origin for cannabis difficult to establish, though literature supports Northeast Asia^1,4,21. Breeding of cannabis cultivars in the hemp industry has focused on morphological improvements through established breeding programs, while marijuana, or drug-type cannabis, has primarily taken place in underground/clandestine programs through crossing landraces and/or “indica” and “sativa” lineages^22,23. The major focus of breeding was increasing the yield of THC, although other features were considered including organoleptics (aroma), morphology, color and trichome density^1,4,24. The genetic diversity between marijuana strains is lower in comparison with hemp varieties due to crossing closely related varieties^2,3. CBDA and THCA synthases are thought to be controlled by two alleles on a single locus (B), crossing of CBDA and THCA dominant strains will produce offspring with intermediate total THC:CBD ratios^5,25. With the prevalence of propagation through cuttings of mother plants, feminization of seeds and production of sensimilla, the need for male plants has decreased resulting in potential loss of genetic and phytochemical diversity¹. The “sativa” and “indica” lineages used to describe cannabis throughout the industry are based on postulation that sativa strains originated from European hemp cultivars, while indica are from potent, resinous Indian cannabis⁴ but given the use and trade of the plant in ancient times, the exact origin is unknown and these may not be distinct species²¹. Modern strains are considered dominant in either of these two “lineages” or hybrids between close relatives. These classifications focus on the pharmacological effects associated with the strains where sativa plants are considered stimulating and indica plants are associated with relaxation and sedation but this is not a botanical or chemotaxonomical classification²³. Comparisons of cannabinoid contents of these classifications have shown that the THC content can be identical between these two classification groups^3,26. Many questions remain: What is a “strain”? Does a “strain” represent a phytochemically unique variety? Are “strains” from different growers actually different? Is there a more appropriate way to classify “strains”? Are these cultivars, varietals, landraces or even species? What is the impact of domestication on the ecological fitness of the species?

Breeding closely related plants potentially leads to loss of genetic diversity within the genome²⁷. Traits signifying domestication syndrome include phenotypic changes such as increase seed size, loss of shattering, changes in reproduction, changes in secondary metabolites and loss of pest resistance compared with wild ancestors^27,28. Reviews of cannabis breeding have summarized domestication in terms of morphology, while focus on secondary metabolism has focused primarily on THC content^1,4. Recent forensic evaluations of confiscated sensimilla cannabis in the US has shown dramatic increases in total THC content over the last 30 years, from 6.3% to 11.5%^29,30 but strains with greater than 20% total THC are available in the marketplace. This artificial increase in THCA production has resulted in the loss of CBDA synthase activity in THC dominant strains. Although crossbreeding will result in THC:CBD hybrid offspring, the loss of other biosynthetic pathways is unknown due to the non-rigorous breeding programs focusing primarily on the production of a single metabolite. Our data indicate that these breeding programs have also impacted unknown related metabolites with undetermined function.

Metabolomics analysis can generate chemotaxonomic classifications of plants in addition to hypothesis generating insight of data correlations, metabolite identification and relationships that would not be possible through single metabolite evaluation^15,31. Using the correlation data and PCA loadings plots, we can hypothesize the putative identity of some of these unknown cannabinoids. For example, CMPD6 had a Pearson correlation coefficient of 0.89 with CBDA and occupies the same space within the PCA loadings plot. The UV spectra with a maximum of 224 nm identifies this compound as an acidic cannabinoid which was only detected in the presence of CBDA. Further evaluation showed that it eluted between CBDVA and CBDA, therefore is hypothesized to be CBDA-C4 with a butyl side chain on the polyketide (Table 2)³². Likewise, we putatively identified CBDA-C1, CBDM, and CBDMA among the unknowns separated by our chromatography protocol (Table 2). Due to the presence of THCA synthase in all strains, the correlation cannabinoids produced with this enzyme is less obvious. It was previously reported that low abundance cannabinoids may be regulated by upstream biosynthesis of precursor polyketides¹⁴. We found fewer unknown cannabinoids in the strains selected for higher THC content. With such strong emphasis on the synthesis of a single metabolite there is a strong possibility that other biosynthetic pathways have been lost in the process^27,28.

Several classification systems have been proposed for cannabis based on a limited number of phenotypic attributes^1,4,33. The concept of a “strain” does not reflect the crop domestication, breeding programs or plant chemistry. The strains available in the Canadian marketplace are closely related, and evaluating single metabolite classes does not provide sufficient information to understand the phytochemical diversity available. The abundance of secondary metabolites within plants does not necessarily correlate with pharmacological significance and with cannabis there is the postulated “entourage effect” describing the synergistic effects of many metabolites for anecdotal medical efficacy⁷. Domestication of the crop has limited the genetic variability in the crop and the impact on crop diversity, physiology and metabolism is not fully understood. Further research is needed to evaluate the low abundance cannabinoids for potential medicinal efficacy and to determine their roles in plant metabolism.