Abstract Cannabis (Cannabis sativa) plants produce and accumulate a terpene-rich resin in glandular trichomes, which are abundant on the surface of the female inflorescence. Bouquets of different monoterpenes and sesquiterpenes are important components of cannabis resin as they define some of the unique organoleptic properties and may also influence medicinal qualities of different cannabis strains and varieties. Transcriptome analysis of trichomes of the cannabis hemp variety ‘Finola’ revealed sequences of all stages of terpene biosynthesis. Nine cannabis terpene synthases (CsTPS) were identified in subfamilies TPS-a and TPS-b. Functional characterization identified mono- and sesqui-TPS, whose products collectively comprise most of the terpenes of ‘Finola’ resin, including major compounds such as β-myrcene, (E)-β-ocimene, (-)-limonene, (+)-α-pinene, β-caryophyllene, and α-humulene. Transcripts associated with terpene biosynthesis are highly expressed in trichomes compared to non-resin producing tissues. Knowledge of the CsTPS gene family may offer opportunities for selection and improvement of terpene profiles of interest in different cannabis strains and varieties.

Citation: Booth JK, Page JE, Bohlmann J (2017) Terpene synthases from Cannabis sativa. PLoS ONE 12(3): e0173911. https://doi.org/10.1371/journal.pone.0173911 Editor: Björn Hamberger, Michigan State University, UNITED STATES Received: January 9, 2017; Accepted: February 28, 2017; Published: March 29, 2017 Copyright: © 2017 Booth et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: GenBank accession numbers are within the paper, under the "GenBank Accessions" section. Funding: This work was supported with funds to JB from a Discovery Grant of the Natural Sciences and Engineering Research Council (NSERC) of Canada and an NSERC Graduate Scholarship to JKB. JEP contributed to the study in his academic capacity as an Adjunct Professor in the Department of Botany at the University of British Columbia. JEP is also the CEO and President of Anandia Labs Inc., which is hereby acknowledged. The funder (NSERC) provided financial support in the form of salary through a fellowship for JKB and research materials. Anandia Labs provided in-kind support in the form of plant materials. Anandia Labs did not provide financial support for this project. The funder (NSERC) and Anandia Labs did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of all authors are articulated in the ‘author contributions’ section. Competing interests: JEP is the CEO and President of Anandia Labs Inc. JB is a consultant and adviser to CannaRoyalty Corp. (since December 2016). These affiliations do not alter our adherence to PLOS ONE policies on sharing data and materials.

Introduction Cannabis sativa, referred to here as cannabis, has been used for millennia as a medicine and recreational intoxicant [1, 2]. The species Cannabis sativa comprises both marijuana and hemp [3, 4, 5]. Medicinal cannabis is highly valued for its pharmacologically active cannabinoids, a class of terpenophenolic metabolites unique to cannabis. These compounds are primarily found in the resin produced in the glandular trichomes of pistillate (female) inflorescences. Cannabis resin also contains a variety of monoterpenes and sesquiterpenes (Fig 1), which are responsible for much of the scent of cannabis flowers and contribute characteristically to the unique flavor qualities of cannabis products. Similarly, terpenes in hop (Humulus lupulus), a close relative of cannabis, are an important flavoring component in the brewing industry. Differences between the pharmaceutical properties of different cannabis strains have been attributed to interactions (or an ‘entourage effect’) between cannabinoids and terpenes [6, 7]. For example, the sesquiterpene β-caryophyllene interacts with mammalian cannabinoid receptors [8]. As a result, medicinal compositions have been proposed to incorporate blends of cannabinoids and terpenes [9]. Terpenes may contribute anxiolytic, antibacterial, anti-inflammatory, and sedative effects [6]. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 1. Glandular trichomes on the surface of pistillate inflorescences and leaves of Cannabis sativa ‘Finola’. The inflorescence (left) with a high density of glandular trichomes was at five weeks post onset of flowering. Non-inflorescence leaves (right) have lower density of glandular trichomes. Structures of representative cannabis resin components are shown in white: monoterpenes (top row), sesquiterpenes (middle row), and cannabinoids (bottom row). GBGA = cannabigerolic acid; THCA = tetrahydrocannabinolic acid; CBDA = cannabidiolic acid. https://doi.org/10.1371/journal.pone.0173911.g001 Terpene biosynthesis in plants involves two pathways to produce the general 5-carbon isoprenoid diphosphate precursors of all terpenes, the plastidial methylerythritol phosphate (MEP) pathway and the cytosolic mevalonate (MEV) pathway. These pathways ultimately control the different substrate pools available for terpene synthases (TPS). The MEP pathway is comprised of seven steps that convert pyruvate and glyceraldehyde-3-phosphate into isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) (Fig 2A). Enzymes thought to be critical for flux regulation through this pathway include the first two and final two steps: 1-deoxy-D-xylulose 5-phosphate synthase, 1-deoxy-D-xylulose 5-phosphate reductase, 4-hydroxy-3-methylbut-2-enyl diphosphate synthase, and 4-hydroxy-3-methylbut-2-enyl diphosphate reductase [10, 11]. The MEV pathway converts three units of acetyl-CoA to IPP, which is then isomerized to DMAPP by IPP isomerase. A rate-limiting step in this six-step pathway is 3-hydroxy-3-methylglutaryl-CoA reductase, which produces mevalonate [12]. IPP and DMAPP are condensed into longer-chain isoprenoid diphosphates by prenyltransferases, which include geranyl diphosphate (GPP) synthase (GPPS) and farnesyl diphosphate (FPP) synthase (FPPS). GPPS and FPPS condense one unit of IPP and one or two units of DMAPP to form 10- and 15-carbon linear trans-isoprenoid diphosphates, respectively. GPP is the 10-carbon precursor of monoterpenes and is typically derived from 5-carbon isoprenoid diphosphate units of the MEP pathway. GPP is also a building block in the biosynthesis of cannabinoids [13, 14]. FPP is the 15-carbon precursor of sesquiterpenes and is commonly produced from 5-carbon isoprenoid diphosphate units of the cytosolic mevalonate (MEV) pathway. GPPSs exist as homo- or heterodimeric enzymes. In hops, the closest known relative of cannabis, heterodimeric GPPSs can produce both GPP and the 20-carbon geranylgeranyl diphosphate (GGPP), with the ratio of large to small G(G)PPS subunits controlling the product outcome [15, 16, 17]. The linear isoprenoid diphosphates GPP and FPP are substrates for monoterpene synthases (mono-TPS) and sesquiterpene synthases (sesqui-TPS), respectively, which diversify these precursors into a large number of different mono- and sesquiterpenes. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 2. Schematic of the plastidial methylerythritol phosphate pathway (MEP) and mevalonic acid pathway (MEV) and transcript abundance in different parts of cannabis. Steps shown in bold (a) were included in the qPCR analysis (b) of relative abundance of transcripts. Letters indicate significantly different means between tissues (tested within each gene), Fisher’s LSD (alpha = 0.05). Abbreviations: Tr = trichome; Le = leaf; Sf = stamenate flower; Ro = root; Sm = stem. https://doi.org/10.1371/journal.pone.0173911.g002 TPS are typically encoded in large and diverse gene families in plants [18], where they contribute to both general and specialized metabolism. The plant TPS gene family has been annotated with six subfamilies. In angiosperms, the subfamily TPS-b is typically comprised of mono-TPS and TPS-a enzymes are often sesquiterpene synthases. TPS produce cyclic and acyclic terpenes via carbocationic intermediates, formed by divalent metal co-factor dependent elimination of the diphosphate. The reactive cationic intermediate can undergo cyclization and rearrangements until the reaction is quenched by deprotonation or water capture [19]. Many TPS form multiple products from the same substrate. The terpene composition of cannabis resin varies substantially based on genetic, environmental, and developmental factors [20, 21, 22, 23]. Concentrations and ratios of cannabinoids are relatively predictable for different strains, but terpene profiles are often unknown or unpredictable [20, 23]. To select and improve cannabis strains with desirable terpene profiles, it is necessary to identify genes responsible for terpene biosynthesis, which can be accomplished by harnessing available cannabis transcriptome and genome resources. Draft genomes and transcriptomes for the marijuana strain Purple Kush and the hemp variety ‘Finola’ have previously been published [24]. We used these resources to explore the expression of genes involved in all stages of terpene biosynthesis. We identified nine TPS gene models in the ‘Finola’ transcriptome. TPS genes and gene transcripts in the MEP and MEV pathways were highly expressed in floral trichomes. We identified biochemical functions of TPS that are highly expressed in ‘Finola’. The TPS enzymes characterized account for most of the terpenes found in ‘Finola’ resin.

Materials and methods Plant materials Cannabis seeds, ‘Finola’, were obtained from Alberta Innovates Technology Futures (www.albertatechfutures.ca). All plants were grown indoors in a growth chamber under a Health Canada license. Seeds were germinated on filter paper, then transferred to 4:1 Sunshine Mix #4 (www.Sungro.com):perlite. Daylight length was 16 h under fluorescent lights, and ambient temperature 28°C. About two weeks after germination, seedlings were transferred to larger pots. After repotting, all plants were fertilized weekly with Miracle-Gro all-purpose plant food (24-8-26) (www.miraclegro.com) according to manufacturer’s instructions. Terpene extraction Pistillate inflorescences were collected and trimmed of leaves and stems. All flowers from an individual plant were pooled. Tissue samples of ~0.2 g were weighed to determine fresh weight. Three rounds of extraction in 1 ml of pentane were performed for 1 hour each at room temperature with gentle shaking. Isobutyl benzene was added as an internal standard. After three extractions, no terpenes were identified in a fourth solvent extraction. Floral tissue was then dried overnight and weighed to determine dry weight. All three pentane extracts were combined for a total volume of 3 ml for analysis. Trichome isolation The heads of glandular trichomes were isolated from whole inflorescences as previously described [25] without XAD-4 and with the addition of 5 mM aurintricarboxilic acid in the isolation buffer. Instead of a cell disruptor, floral tissue was vortexed with glass beads in a Falcon tube to remove trichome heads. After vortexing, trichomes were separated from beads and green tissue by filtration through a 105 μm nylon mesh. Trichomes were concentrated by gentle centrifugation in ice-cold buffer. The supernatant was removed with a pipette, and the pellet of trichomes was immediately frozen in liquid nitrogen. Metabolite analysis Gas chromatography (GC) analysis of floral extracts was performed on an Agilent (www.chem.agilent.com) 7890A GC with a 7683B series autosampler and 7000A TripleQuad mass spectrometer (MS) detector at 70 eV electrospray ionization with a flow rate of 1 ml min-1 He. The column was an Agilent VF-5MS or DB-5MS (30 m, 250 μm internal diameter, 0.25 μm film). The following temperature program was used: 50°C, then increase 150°C min-1 to 320°C, hold for 5 minutes. Injection was pulsed splitless at 250°C. Compounds were identified by comparison of retention index and mass spectra to authentic standards. Standards were available for all monoterpenes and the following sesquiterpenes: β-caryophyllene, α-humulene, farnesol, valencene, germacrene D, and alloaromadendrene. Tentative identifications for all other sesquiterpenes were made by comparison of retention index and mass spectra to National Institute of Standards and Technology (NIST) MS library. Identifications of bergamotene, δ-selinene, and farnesene were strengthened by comparison to essential oils of Citrus bergamia (Bergamot) and Pimenta racemose (Bay) (www.lgbotanicals.com). TPS assay products were analyzed by the same procedure described above for plant extracts, but with the following temperature program: 50°C for 3 minutes, then increase 15°C min-1 to 280°C, hold for 2 minutes. Assay products were analyzed using Agilent HP-5 and DB-Wax columns (30 m length, 250 μm internal diameter, 0.25 μm film). For cold injection of sesqui-TPS assay products, the following program was used on a DB-Wax column: 40°C for 3 minutes, then increase 10°C min-1 to 230°C, hold for 7 minutes. Injection was at 40°C with a 1:1 split ratio. Chiral analysis of terpenes was done using a Cyclodex-B column (30 m length, 250 μm internal diameter, 0.25 μm film). Injection was pulsed splitless, with the following program: 40°C for 1 minute, then increase 5°C min-1 to 100°C, then increase 15°C min-1 to 250°C, hold for 4 minutes. Chirality was determined by retention index and comparison with authentic standards. cDNA cloning and characterization of TPS genes Total RNA was isolated from ‘Finola’ flowers, leaves, stem, and roots using Invitrogen PureLink Plant RNA reagent (www.thermofisher.com). RNA quality and concentration was measured with a Bioanalyzer 2100 RNA Nanochip assay (www.agilent.ca). cDNA was synthesized with the Superscript III reverse transcriptase kit (Thermo Fisher). Full length and N-terminally truncated cDNAs without transit peptides where applicable [26] were amplified from cDNA using gene-specific primers (S1 Table) designed from published transcriptomic data [24]. N-terminal transit peptides were predicted based on sequence alignments [27] and using the TargetP and ChloroP servers [28]. PCR amplified ‘Finola’ cDNAs were ligated into pJET vector (www.clontech.com) for sequence verification, and subcloned into expression vectors pET28b+ (www.endmillipore.ca) or pASK-IBA37 (www.iba-lifesciences.org) in the case of CsTPS5FN. High-confidence full-length TPS cDNA candidates from Purple Kush (CsTPS13PK, CsTPS30PK, and CsTPS33PK) were synthesized by GenScript (www.genscript.com) into pET28b+. For this purpose, putative TPS sequences from Purple Kush transcriptome data were verified by comparison to genomic sequences [24]. Plasmids were transformed into E. coli strain BL21DE3-C43 for heterologous protein expression, as previously described [29]. Recombinant protein was purified using the GE healthcare His SpinTrap kit (www.gelifesciences.com) according to manufacturer’s instructions. Binding buffer for purification was 20 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) (pH 7.5), 500 mM NaCl, 25 mM imidazole, and 5% glycerol. Cells were lysed in binding buffer supplemented with Roche complete protease inhibitor tablets (lifescience.roche.com) and 0.1 mg ml-1 lysozyme. Elution buffer was 20 mM HEPES (pH 7.5), 500 mM NaCl, 350 mM imidazole, and 5% glycerol. Purified protein was desalted through Sephadex into TPS assay buffer. In vitro assays were performed in 500 μl volume by incubating purified protein with isoprenoid diphosphate substrates (Sigma) as previously described [30], except that the TPS assay buffer was 25 mM HEPES (pH 7.3), 100 mM KCl, 10 mM MgCl 2 , 5% glycerol, and 5 mM DTT. Isoprenoid diphosphate substrates were dissolved in 50% methanol and added to the assay at final concentrations of 32 μM (GPP) and 26 μM (FPP). Enzyme concentrations were variable ranging from 20 to 100 μg per 500 μl assay volume. Assays were overlaid with 400 μl hexane or pentane, with 2.5 μM isobutyl benzene as internal standard. Nicotiana benthamiana transformation and transient expression The CsTPS5FN coding sequence was inserted into the Golden Gate plant expression vector pEAQ-GG, which contains a CaMV 35S promoter. This construct and the suppressor-of-silencing gene p19 were transformed into Agrobacterium tumefasciens strain AGL1. For infiltration, A. tumefasciens was grown overnight as previously described [31], then pelleted and resuspended in 10 mM 2-(N-morpholine)-ethanesulphonic acid (MES) buffer, pH 5.8, 10 mM MgCl 2 , 20 μM acetosyringone to OD 600 0.5. Equal volumes of bacteria, 25 ml each, containing TPS5 and p19 were infiltrated into the abaxial side of 4-week-old N. benthamiana plants. Infiltrated plants were grown for three days in the dark. Infiltrated leaves were harvested and ground in TPS assay buffer, and enzyme activity assays were conducted as above. RT-qPCR analysis of transcript abundance cDNA for qPCR was synthesized using the Maxima First Strand cDNA synthesis kit (Thermo Fisher) according to manufacturer’s instructions. qPCR reactions were done in 15 μl volumes with SsoFast EvaGreen supermix (Bio-Rad), 4 μl template (2 ng), and 0.3 μM primers. Primers (S2 Table) were designed using Primer3 software [32]. Reference genes were chosen by geNorm [33], analyzed with qBase+ software (www.biogazelle.com). Reference genes used for RT-qPCR of early isoprenoid biosynthesis across different plant organs were actin and CDK3. For RT-qPCR of TPS transcripts in trichomes, reference genes were CDK3 and GAPDH. RT-qPCR analyses were done with four biological and two technical replicates for the early isoprenoid biosynthetic transcripts in different organs. For TPS transcript analysis in trichomes, three biological and three technical replicates were performed. Gene expression was analyzed using qBase+. Statistical analysis was performed by ANOVA on log-transcript abundance, with Bonferroni correction. TPS gene prediction and phylogeny ‘Finola’ genome and transcriptome assemblies [24] were downloaded from the cannabis genome browser (http://genome.ccbr.utoronto.ca/cgi-bin/hgGateway). These assemblies were used as the subject of a tBLASTn search using 71 TPS genes (S3 Table) downloaded from GenBank and Phytozome. Gene and splice site prediction was performed on scaffolds containing regions with similarity to TPS sequences using the Exonerate gene prediction algorithm [34]. A preliminary Purple Kush genome assembly based on PacBio (www.pacb.com) sequencing data was also used. Predicted genes were manually curated against earlier Purple Kush sequence data, and examined to establish open reading frames, start codons, and stop codons. A maximum likelihood phylogeny was built using phylogeny.fr [35]. The alignment used for input was built using the MUSCLE algorithm with all translated amino acid sequences from the predicted TPS gene models from cannabis and the 71 published TPS sequences listed above. Alignments were curated using the Gblocks algorithm, and tree construction was performed using PhyML 3.0 with 100 bootstrap replicates.

Discussion The resin of C. sativa is rich in mono- and sesquiterpenes, which are of interest for their putative contributions to cannabis pharmacology [6]. Most studies of terpenes in cannabis have focused on phytochemical composition for forensics and breeding, while less research has gone into the molecular biology of terpene formation in cannabis. Knowledge of the genomics and gene functions of terpene biosynthesis may facilitate genetic improvement of cannabis for desirable terpene profiles. Using the hemp strain ‘Finola’ and its genome and transcriptome resources [24], we identified early isoprenoid pathway genes as well as specific CsTPS genes and their enzymes involved in the biosynthesis of nearly all of the different monoterpenes identified in extracts of the cannabis inflorescences, which are densely covered with terpene and cannabinoid accumulating glandular trichomes (Fig 1). One exception is terpinolene, for which a CsTPS has not yet been identified. The terpene profiles of cannabis can be explained by activities of both single-product and multi-product CsTPS. Individual ‘Finola’ plants showed substantial variation in their profiles of mono- and sesquiterpenes. ‘Finola’ has few monoterpene alcohols or ethers, such as linalool or geraniol, which are common in some cannabis strains. It is reasonable to expect that there are additional CsTPS not described in this work, such as a CsTPS that encodes a terpinolene synthase. Characterization of further TPS may also clarify the poor correlation between TPS and the abundance of their products shown in Fig 6A. A search of a new assembly of the Purple Kush genome, to which we recently had pre-publication access (Dr. Jonathan Page, personal communication), identified a total of 33 complete CsTPSPK gene models and additional partial sequences (Fig 7). Purple Kush is a marijuana strain which requires special research licensing to grow. Thus, characterization of this more comprehensive set of CsTPSPK will have to be completed in future work as it requires synthesized genes. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 7. Maximum likelihood phylogeny for 33 TPS translated from gene models identified in the Cannabis sativa Purple Kush genomic sequences. 41 published TPS sequences from other organisms were included for comparison. Names of cannabis genes identified in this study are in bold. Gene names from Purple Kush followed by an asterisk (*) represent biochemically characterized enzymes from Purple Kush transcriptome data. Their nearest homologue in the genome was assigned the same gene ID when the sequences had >95% amino acid identity. Branches with greater than 80% boostrap support are identified with a grey circle. https://doi.org/10.1371/journal.pone.0173911.g007 Fig 7 indicates a set of putatively orthologous CsTPSFN and CsTPSPK genes, which may contribute to overlapping terpene profiles in hemp and marijuana varieties. However, some orthologous genes may have evolved different functions in different strains, and non-orthologous CsTPS may contribute to some of the same terpene products in different cannabis strains. For example, α-pinene is a major component of strains reported as Purple Kush [37], but no obvious orthologue of the α-pinene synthase CsTPS2 as identified in the ‘Finola’ and ‘Skunk’ strains was found in the Purple Kush genome (Fig 7). Another example is the set of apparently non-orthologous single-product myrcene synthases, CsTPS3FN and CsTPS30PK identified in ‘Finola’ and Purple Kush, which only share 52.5% amino acid identity but produce the same monoterpene. Also, not all CsTPS are expected to contribute to terpene accumulation in the resin of cannabis inflorescences and some may function in a different context of the plant biology. For example, CsTPS5FN is expressed in inflorescences and the recombinant enzyme produces a mixture of monoterpenes, but does not contribute substantially to the terpene profile of the resin. This gene appears most closely related to MTS1 from hops (Fig 3) where the encoded protein was inactive in vitro [38]. Cannabis inflorescences are densely covered with glandular trichomes, which are specialized to produce and accumulate terpenes [11]. Transcripts of several CsTPS genes (Fig 6A) are abundant in trichomes isolated from mid-stage ‘Finola’ inflorescences. Transcripts associated with early isoprenoid biosynthesis and especially the MEP pathway, which feeds into both monoterpene and cannabionoid biosynthesis, were also abundant in trichomes (Fig 2B). Sesquiterpenes have been reported to be most abundant in early floral stages [39], and thus MEV pathway transcripts may be more abundant at earlier stages of flower development. Different DXS and HMGR genes were differentially expressed in roots relative to other parts of the plant. Terpenes in the roots, if present in cannabis, may contribute to defense as reported in other plant species [10]. In plants, DXS genes generally fall into two clades, of which DXS I members are generally involved in primary metabolism, and DXS II members are often induced in defense responses [40, 41, 42]. Abundance of cannabis DXS2 transcripts, which clusters with the DXS II subfamily (S1 Fig), suggests defense related terpenoids in cannabis roots and warrants future work on the cannabis root metabolome. We also observed high FPPS transcript abundance in stamenate flowers and roots, resembling a previous finding that Arabidopsis FPS1 was primarily expressed in flowers and roots compared to AtFPS2 [43]. Domestication and selective breeding can result in changes in terpene profiles and abundance. For example, domestication can lead to a decrease in the quantity or variability of terpenes [44, 45, 46]. Cannabis, especially marijuana, has been domesticated for thousands of years for increased resin volume and potency [2, 47] and as a result profiles and ecological roles of terpenes in ancestral (i.e., undomesticated) cannabis are unknown. The present study highlights the large number of CsTPS genes and the diverse products of the encoded TPS enzyme activities, which contribute to the complex terpene profiles of cannabis. The knowledge of multigene nature of the CsTPS family and the often multiple products of the encoded enzymes will be critical when selecting or breeding, or improving plants by genome editing, for particular terpene profiles for standardized cannabis varieties. While cannabinoid-free individuals have occasionally been reported [48], we are not aware of any reports in the literature of terpene-free cannabis. In this study, we observed a single monoterpene-free individual, which however still contained cannabinoids and sesquiterpenes. This observation implies that biosynthesis of the different classes of terpenoid metabolites are independently regulated. The fact that terpenes have persisted throughout domestication as a substantial and diverse component of cannabis resin highlights their significance for human preferences.

GenBank accessions GenBank accession numbers for the terpene synthases described in this paper are CsTPS1FN: KY014557, CsTPS2FN: KY014565, CsTPS3FN: KY014561, CsTPS4FN: KY014564, CsTPS5FN: KY014560, CsTPS6FN: KY014563, CsTPS7FN: KY014554, CsTPS8FN: KY014556, CsTPS9FN: KY014555, CsTPS11FN: KY014562, CsTPS12PK: KY014559, CsTPS13PK: KY014558. Accession numbers for genes in the MEP pathway are CsDXS1: KY014576, CsDXS2: KY014577, CsDXR: KY014568, CsMCT: KY014578, CsCMK: KY014575, CsHDS: KY014570, CsHDR: KY014579. Accession numbers for genes in the MEV pathway are CsHMGS: KY014582, CsHMGR1: KY014572, CsHMGR2: KY014553, CsMK: KY014574, CsPMK: KY014581, CsMPDC: KY014566, CsIDI: KY014569. Prenyltransferase accession numbers are CsGPPS.ssu1: KY014567, CsGPPS.ssu2: KY014583, CsFPPS1: KY014571, CsFPPS2: KY014580. Accession numbers for genomic regions containing putative terpene synthases from Purple Kush are CsTPS1PK: KY624372, CsTPS4PK: KY624361, CsTPS5PK: KY624374, CsTPS6PK: KY624363, CsTPS7PK: KY624368, CsTPS8PK: KY624352, CsTPS9PK: KY624366, CsTPS10PK: KY624347, CsTPS11PK: KY624348, CsTPS12PK: KY624349, CsTPS13PK: KY624350, CsTPS14PK: KY624351, CsTPS15PK: KY624353, CsTPS16PK: KY624354, CsTPS17PK: KY624355, CsTPS18PK: KY624356, CsTPS19PK: KY624357, CsTPS20PK: KY624358, CsTPS21PK: KY624360, CsTPS22PK: KY624360, CsTPS23PK: KY624362, CsTPS24PK and CsTPS25PK: KY624364, CsTPS26PK and CsTPS27PK: KY624365, CsTPS30PK: KY624367, CsTPS31PK: KY624369, CsTPS32PK: KY624370, CsTPS33PK: KY624371, CsTPS34PK: KY624373, CsTPS35PK: KY624375.

Acknowledgments We thank We thank Mr. Jan Slaski (Alberta Innovates Technology Futures) for hemp seeds; Mr. Mack Yuen (UBC), Ms. Lina Madilao (UBC), and Dr. Melissa Mageroy (UBC) for technical advice; and Dr. Justin Whitehill (UBC) and Dr. Sandra Irmisch (UBC) for comments on the manuscript.

Author Contributions Conceptualization: JB JEP JKB. Formal analysis: JKB. Funding acquisition: JB JKB. Investigation: JB JKB. Project administration: JB JKB. Resources: JB JEP. Supervision: JB JEP. Writing – original draft: JB JKB. Writing – review & editing: JB JEP JKB.