Cannabis sativa L. is the sole, formally recognized species within the genus Cannabis and is a member of the angiosperm family Cannabaceae (Small and Cronquist, 1976). Cannabis is diploid (Van Bakel et al., 2011), predominately dioecious, and obligate outbred (Faeti et al., 1996) and can be considered highly heterozygote (Soler et al., 2017). The extant genepool is comprised principally of domesticated or previously domesticated feral populations (Welling et al., 2016b), with intraspecific groupings based on selection of phenotypes primarily associated with seed/fiber (industrial hemp), recreational drug (marijuana) (Mandolino and Carboni, 2004) and, more recently, therapeutic end-uses (Potter, 2014).

The predominant bioactive secondary metabolites produced by Cannabis are the terpenophenolic phytocannabinoids (cannabinoids), of which >100 have been identified (ElSohly and Slade, 2005; Radwan et al., 2015). Structurally related terpenophenolic compounds also occur in other plant species such as the prenylflavonoids in Humulus lupulus (Stevens et al., 1999), a closely related species within the Cannabaceae which is thought to have diverged ∼21 MYA (Divashuk et al., 2014). However, the cannabinoids appear largely unique to Cannabis (Gertsch et al., 2010), and are formed at high concentrations within capitate stalked trichomes on the floral tissues of female inflorescences. They also accumulate within capitate-sessile trichomes and potentially bulbous trichomes on floral as well as non-floral tissues including leaves and stems (Happyana et al., 2013). Despite their relative abundance and interspersed distribution in plant tissue, the metabolic role of cannabinoids in Cannabis is largely unknown, although they may mitigate biotic stress via mitochondrial membrane dysfunction-induced necrosis in leaf cells (Morimoto et al., 2007).

Cannabinoids are produced in Cannabis in their carboxylic acid (COOH) forms and are decarboxylated to neutral cannabinoids in a non-enzymatic reaction which can be accelerated at temperatures >100°C (Dussy et al., 2005). Decarboxylation can also occur after extended periods of storage >100 days at room temperature (Hanuš et al., 2016). A notable example of this is the conversion of the non-psychoactive delta(9)-tetrahydrocannabinolic acid (THCA) to the psychoactive delta(9)-tetrahydrocannabinol (THC) (Izzo et al., 2009) upon loss of the COOH group.

The tricyclic THCA and dicyclic cannabidiolic acid (CBDA) C₅-alkyl cannabinoids are the most predominant and commonly occurring cannabinoids in Cannabis (Figure 1; Hazekamp et al., 2016). A series of C₃-alkyl cannabinoid homologs, including the tricyclic delta(9)-tetrahydrocannabivarinic acid (THCVA) and dicyclic cannabidivarinic acid (CBDVA), can also contribute significantly to the cannabinoid profiles of ecotypes from Asian (Figure 1; Hillig and Mahlberg, 2004; Welling et al., 2016a) and African provenance (Baker et al., 1983), although these compounds are typically found at low levels in contemporary domesticated forms (Swift et al., 2013; Hazekamp et al., 2016; Welling et al., 2016a). Trace amounts of other alkyl homologs have also been identified such as methyl-(C₁) (Vree et al., 1972) and butyl-(C₄) (Smith, 1997) alkyl cannabinoids, although accounts of high levels of these cannabinoids in planta are scarce.

Current understanding of the bioactivity of cannabinoids is based on their modulation of the human endocannabinoid system, a poorly defined complex ensemble of several receptors, two endogenous cannabinoid ligands N-arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerol (2-AG) as well as associated enzymatic pathways (Di Marzo and Piscitelli, 2015). The cannabinoid alkyl side chain is a critical pharmacophore (Khanolkar et al., 2000), with changes in carbon length influencing the affinity and selectivity of plant derived cannabinoids to targets of the human endocannabinoid system (Thakur et al., 2005). Indeed, recent docking studies using a 2.6-Å resolution crystal structure of the human G-protein-coupled cannabinoid type-1 receptor (CB₁R) show binding of the tricyclic core of THC with a number of transmembrane domains preceding a highly conserved membrane-proximal N-terminal region, with the alkyl side chain extending toward a Trp356^6.48 residual (Shao et al., 2016) associated with CB₁R activation (Shim et al., 2011). Subsequent partial agonist binding by THC to CB₁R stimulates mesolimbic dopamine activity (French, 1997), a mechanism believed to be partially responsible for this ligands psychoactivity.

Until recently, plant cannabinoids have primarily seen use in the context of recreational drug use of THC. However, they offer promise as novel therapeutics in a number of diverse non-communicable diseases. The company GW Pharmaceuticals, plc has developed cannabidiol (CBD) and THC containing Sativex^® (Chandra et al., 2017), a prescription medicine approved for the management of multiple sclerosis in more than 22 countries¹, as well as CBD containing Epidiolex^® which has recently been approved by the US Food and Drug Administration (FDA) for the treatment of childhood seizures associated with Lennox-Gastaut syndrome and Dravet syndrome (Chandra et al., 2017). Ananda Hemp Ltd. (a subsidiary company of Ecofibre Industries Operations Pty Ltd.) has recently launched a range of cannabinoid-based products². The C₃-alkyl cannabinoids cannabidivarin (CBDV) and delta(9)-tetrahydrocannabivarin (THCV) are also emerging as therapeutic entities. CBDV has been targeted by GW Pharmaceuticals, plc (Vemuri and Makriyannis, 2015), with phase I and II clinical trials having been initiated for the treatment of autism spectrum disorders and epilepsy, respectively. Moreover, a double-blind, placebo-controlled pilot study of 62 non-insulin treated type II diabetes subjects supports a therapeutic role for THCV in the modulation of fasting blood glucose and pancreatic β-cell function (Jadoon et al., 2016).

Current methods for the production of cannabinoid-based botanical drug products rely predominantly on clonal propagation of plants (Lata et al., 2012) due to the limited ability to predict chemical heritability in seed propagated progeny (Potter, 2014). Development of early diagnostic techniques to determine C₃-alkyl cannabinoid quality (CBDV + THCV) within the total cannabinoid fraction could assist breeders in the selection of elite alkyl cannabinoid breeding lines. While the ontogenetic variation in di-/tri-cyclic cannabinoid composition during plant development within the C₅-alkyl cannabinoid fraction has been studied (Pacifico et al., 2008; De Backer et al., 2012; Aizpurua-Olaizola et al., 2016; Richins et al., 2018), there have been limited attempts to characterize developmental changes of C₃-alkyl cannabinoid composition. Moreover, alkyl cannabinoid chemotypes have not been systematically evaluated among divergent subtaxa.

This lack of clarity in understanding the extent to which alkyl cannabinoid composition varies in planta limits the ability to use chemotypic assessment during early developmental stages as well as to predict chemotype prior to seed formation. In the present study, liquid chromatography-mass spectrometry (LC-MS) profiling of a chemotypic diversity panel with a representative range of genotypes within the Cannabis genepool was used to characterize variation in alkyl cannabinoid composition across vegetative, flowering and maturation stages. Seed-based accessions were sourced from the Ecofibre Global Germplasm Collection with priority given to accessions with provenance from Southern, Eastern and Western Asia as well as Africa to ensure adequate representation of C₃-alkyl cannabinoid chemotypes (Hillig and Mahlberg, 2004; Welling et al., 2016a; Table 1). Cluster analysis of alkyl cannabinoid fractions was performed to provide insight into the categorization and genetic regulation of alkyl cannabinoid chemotypes in Cannabis.