A simple Mendelian model involving codominant alleles at a single locus was proposed to explain the segregation of drug content among marijuana and hemp plants (de Meijer et al ., 2003 ; Pacifico et al ., 2006 ). Supposing that the enzymes THCA synthase and CBDA synthase are products of allelic variants at a single locus, de Meijer et al . ( 2003 ) predicted that marijuana would be homozygous for THCA synthase, hemp would be homozygous for CBDA synthase, and their hybrid would be intermediate. However, the distribution of gene sequences encoding the major cannabinoid biosynthetic enzymes suggests an alternative model involving at least two loci (Kojoma et al ., 2006 ). Linkage of separate loci for THCA and CBDA synthases with variable expression or allelic variation in enzymatic efficiency can explain the observed pattern of phenotypic segregation (van Bakel et al ., 2011 ). We evaluated these alternatives based on analyses of an F 2 population derived from a cross between inbred marijuana and hemp cultivars. Molecular cloning and phylogenetic analysis of THCA synthase and CBDA synthase homologs established a minimum number of homologs per inbred line. Genotyping, quantitative trait locus (QTL) mapping, and gene expression assays associated genomic regions and loci with phenotypes to demonstrate that despite the linkage of separate loci for THCA synthase and CBDA synthase, it is variation at the CBDA synthase locus alone that accounts for the main difference between the cannabinoid profiles of marijuana and hemp.

The primary agent of marijuana potency is concentrated in glandular trichomes on the bracts of pistillate inflorescences in the form of delta 9 ‐tetrahydrocannabinolic acid (Kim & Mahlberg, 1997 ). Cannabidiolic acid is similarly located and also of pharmacological interest (Page & Nagel, 2006 ). The decarboxylated forms of these molecules (THC and CBD) bind to endocannabinoid receptors in the human body to produce a broad array of effects that range from mild euphoria in the case of THC to anticonvulsant properties of CBD (Bostwick, 2012 ). Early reports regarded CBDA as a direct precursor to THCA (Mechoulam et al ., 1970 ; Shoyama et al ., 1975 ) but more recent work showed that CBDA and THCA are alternate products of a common precursor, cannabigerolic acid (CBGA; Taura et al ., 1995 ; Fellermeier et al ., 2001 ). THCA synthase and CBDA synthase convert CBGA to the carboxylated forms of THCA and CBDA, respectively, and share 89% nucleotide sequence identity (Sirikantaramas et al ., 2004 ). Cannabis varieties exhibit one of three major cannabinoid profiles depending on the relative abundance of THCA and CBDA. Marijuana cultivars contain high concentrations of THCA relative to CBDA, whereas hemp cultivars have relatively higher concentrations of CBDA and hybrids are intermediate (de Meijer et al ., 2003 ). Although recent work has identified the genes encoding the enzymes of the cannabinoid biosynthetic pathway (Marks et al ., 2009a ; van Bakel et al ., 2011 ; Gagne et al ., 2012 ), fundamental questions remain about the inheritance of these genes in relation to the diversity of cannabinoid phenotypes (Page & Nagel, 2006 ).

Cannabis is an economically important source of durable fibers, nutritious seeds, and psychoactive drugs with a multibillion‐dollar annual cost to law enforcement in the United States (Miron, 2008 ). The commonly cultivated forms of Cannabis known as marijuana and hemp are members of the same species that can be readily distinguished by the relative yield of tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA). Recent legislation permitting the cultivation of Cannabis for seed oil and fiber in Canada and Europe recognizes differences in cannabinoid content among varieties (Hennink, 1994 ) as the basis for separating hemp from marijuana (Mignoni, 1997 ). Although central to Cannabis regulatory policy, the hereditary basis of cannabinoid variation has remained uncertain (van Bakel et al ., 2011 ).

Real‐time quantitative PCR (RT‐qPCR) was performed with SYBR ® Green JumpStart Taq ReadyMix Capillary Formulation (Sigma‐Aldrich) on a Light Cycler (Roche) using replicate samples of the derived cDNA for analysis of expression of THCA and CBDA synthases following previously reported protocols (Marks et al ., 2009a ). Primer sets were designed to specifically amplify either gene. Primers for THCA synthase were THCAsynF: GGA CTG AAG AAA AAT GAA TTG C and THCAsynR: GGT CGT GTT GAG TGT ATA CG. Primers for CBDA synthase were CBDAsynF: GAA CTA AAG AAA AAT GAA GTG CTC AA and CBDAsynR: GTT GCA TTA TTG GGA ATA TAT TGC. Three serial dilutions of isolated PCR products synthesized using the respective primer pairs were used as templates to generate standard curves for the RT‐qPCR analysis. Sample template quantities were inferred from standard curves using Light Cycler software. Gel‐isolated RT‐qPCR products were sequenced to verify their identity.

Pistillate inflorescences bearing capitate glandular trichomes were harvested from F 2 plants after 21 d of growth under flowering conditions and total RNA was extracted from 100 mg of tissue using a Plant RNeasy Kit (Qiagen), treated with Turbo DNase (Ambion), purified, and concentrated using an RNA MinElute kit (Qiagen). One μg of total RNA from each sample served as template in synthesizing cDNA with SuperScript III First‐Strand Synthesis SuperMix (Invitrogen).

Unique sequences obtained from the mapping population were aligned with published THCA and CBDA synthase sequences (Sirikantaramas et al ., 2004 ; Kojoma et al ., 2006 ; Taura et al ., 2007 ; van Bakel et al ., 2011 ) and inspected for premature stop codons or frame shifts. Phylogenetic analysis was performed using MrBayes 3.2 (Huelsenbeck & Ronquist, 2001 ). Two analyses ran in parallel, each with six chains of one million generations and the posterior distribution was sampled every 100 generations. The first 25% of the sample was discarded as ‘burn‐in’. Clade support was assessed by posterior probability. We performed tests for evidence of selection by comparing nonsynonymous and synonymous substitution rates ( dN / dS ) using HyPhy (Kosakovsky Pond et al ., 2005 ).

Tetrahydrocannabinolic acid and CBDA synthase genes were amplified from parental marijuana and hemp plants using genomic DNA in separate 25 μl PCR reactions with specific primers for each gene as described earlier, but instead of using CBDA961Rev, we used a CBDA synthase‐specific reverse primer anchored in the 3′ end of the gene that was derived from GenBank accession AB292682 (CBDAsynR: TTA ATG ACG ATG CCG TGG). PCR products were isolated from 1% (w/v) agarose gels using the QIAEX II kit (Qiagen) and cloned into the pCR4‐TOPO vector (Invitrogen). Plasmid DNA was isolated from clones and subjected to DNA sequencing using M13 forward, M13 reverse, and internal primers. Internal primers used for sequencing THCA synthase clones were THCA583For: GTG GAG GAG GCT ATG GAG C and THCA1034Rev: CCC AAC TCA GGA AAG CTC TTG. Internal primers for CBDA synthase sequencing were CBDA694For: GGT GGT GGA GCA GAA AGC TTC and CBDA961Rev: CCA CTC CAC CAA GGA AAA C. Sequence contigs were assembled using the program SeqMan Pro (DNASTAR/Lasergene 8).

Amplified fragment length polymorphism and microsatellite genotypes for 62 F 2 individuals and the parents were used to produce a linkage group tree using JoinMap 4 (Wageningen, the Netherlands). Linkage groups were assembled from independent log‐of‐odds scores (LOD) based on G ‐tests for independence of two‐way contingency tables. Linkage groups with LOD > 3.0 and containing four or more markers were used to construct a linkage map using the function of Kosambi ( 1944 ). Cannabinoid profiles of the same 62 individual genotypes were analyzed with respect to the linkage map using Windows QTL Cartographer v.2.5 (Wang et al ., 2006 ). Significant associations between traits and linkage groups were identified using an experiment‐wise LOD threshold. Simple interval mapping was used to estimate LOD over intervals equal to the mean distance between markers for a given linkage group (i.e. intervals of 4.5 and 7.0 cM for two linkage groups with putative QTL). Significance thresholds for LOD were estimated in Win QTL Cartographer 2.5 by permutation. Experiment‐wise thresholds were estimated by 1000 permutations for a given trait over all linkage groups to obtain an α = 0.05 threshold LOD for the trait with respect to the entire map. Group‐wise thresholds were estimated in like manner, except that the permutations were performed only over the corresponding linkage group for a given trait. Results were plotted with MapChart 2.1 (Wageningen, the Netherlands).

In the case of THCA synthase, genotypes were generally determined using an agarose gel assay to visualize presence or absence of 1676 bp product amplified using THCA synthase‐specific primers derived from GenBank accession AB057805 (THCAsynF: GGA CTG AAG AAA AAT GAA TTG CTC AG and THCAsynR: GGG AAA TAT ATC TAT TTA AAG ATA ATT AAT GAT). The THCAsynR primer is selective for marijuana‐type THCA synthase and generally failed to yield products from hemp genomic DNA (J. P. Wenger, pers. obs.). Faint products observed from a subset of F 2 individuals were sequenced in order to determine the presence of marijuana‐type THCA synthase or cross‐amplification of hemp‐type THCA synthase.

In the case of CBDA synthase, c . 960 bp at the 5′ end of the gene was obtained using primers CBDAsynFor (forward; ATG AAG TGC TCA ACA TTC) and CBDA961Rev (reverse; CCA CTC CAC CAA GGA AAA C). PCR products were sequenced using the CBDAsynFor primer by the Sanger method. CBDA synthase genotypes were determined by comparison with reference sequences from marijuana (van Bakel et al ., 2011 ) and hemp (Kojoma et al ., 2006 ) in Geneious 6.1.8 software. A 4 bp deletion present in marijuana (van Bakel et al ., 2011 ) was the basis for scoring F 2 plants as homozygous for either marijuana‐type or hemp‐type CBDA synthase. Heterozygous individuals were identified when chromatograms showed multiple peaks immediately downstream of the four bp deletion.

Tetrahydrocannabinolic acid and CBDA synthase genes were amplified from genomic DNA of F 2 plants in 25 μl reactions containing 0.625 U PrimeSTAR Hot Start DNA polymerase (TaKaRa), 1× PrimeSTAR reaction buffer, 1 mM Mg 2+ , 1 μM each primer, 0.2 mM per dNTP, and c . 100 ng template. Amplification conditions for the THCA synthase gene were 94°C for 1 min, followed by 35 cycles of 94°C for 10 s, 55°C for 10 s, and 72°C for 1 min 45 s, then one cycle of 72°C for 5 min followed by a 4°C indefinite hold. Procedures for amplification of the CBDA synthase gene were identical except that cycles were reduced from 35 to 30 cycles.

Sixteen microsatellite primers previously developed for C. sativa were applied to the mapping population, including ANUCS201, ANUCS202, ANUCS204, ANUCS205, ANUCS206, ANUCS301, ANUCS302, ANUCS303, ANUCS304, ANUCS305 and ANUCS501 (Gilmore & Peakall, 2003 ) and B02, C08, C11, H09 and H11 (Alghanim & Almirall, 2003 ). PCRs were performed using 0.2–0.4 μg genomic DNA, 250 μM dNTP, 0.2 μM primer, 2.0 mM MgCl 2 , 2.0 μg μl −1 BSA, 1× PCR buffer, and 0.3 units Taq polymerase (Takara exTaq, Clontech Laboratories, Mountain View, CA, USA). One primer of each pair was end‐labeled with a fluorescent tag (Fam, Vic, or Ned; Integrated DNA Technologies, Coralville, IA, USA). PCRs consisted of a 5 min preheat at 95°C followed by two rounds of cycles. Touchdown PCR consisted of 10 cycles of 95°C denaturation for 15 s, 63°C touchdown to 53°C annealing for 30 s, and 72°C extension for 30 s. This was followed by 19 cycles of 95°C denaturation for 15 s, 52°C annealing for 30 s, 72°C extension for 1 min and one 72°C final extension for 10 min. Primers were multiplexed in combinations of ANUCS303/ANUCS204/ANUCS205, ANUCS304/ANUCS305 and ANUCS501/ANUCS201 or otherwise screened individually. Pooled PCR products and an ROX 500 fluorescent size standard were separated on an ABI377 automated sequencer (Applied Biosystems). Alleles were scored using Genotyper software (Applied Biosystems).

Approximately 100 mg of plant material was collected from dried pistillate inflorescences of parents, F 1 plants, and F 2 plants. Plant material was ground to a fine powder in liquid N 2 and genomic DNA was extracted using the Plant DNeasy Mini Kit (Qiagen). Amplification and scoring of amplified fragment length polymorphism (AFLP) markers were performed according to Datwyler & Weiblen ( 2006 ). AFLP products were analyzed on an ABI377 DNA sequencer (Applied Biosystems Inc., Grand Island, NY, USA). Markers differing between marijuana and hemp parents at a threshold of 100 relative fluorescence units were scored for 10 selective primer pairs: EcoR I‐AAC/ Mse I‐CTT (E1M8), EcoR I‐ACA/ Mse I‐CAC (E3M2), EcoR I‐ACT/ Mse I‐CAT (E6M4), EcoR I‐ACT/ Mse I‐CTT (E6M8), EcoR I‐AGC/ Mse I‐CAA (E7M1), EcoR I‐AGC/ Mse I‐CTG (E7M7), EcoR I‐AGG/ Mse I‐CAA (E8M1), EcoR I‐AGG/ Mse I‐CAT (E8M4), EcoR I‐AGG/ Mse I‐CTA (E8M5) and EcoR I‐AGG/ Mse I‐CTT (E8M8).

Dried samples of female flowers from mature plants were analyzed for content (% dry weight (DW)) of the cannabinoid compounds cannabigerol (CBG), CBDA, cannabichromene (CBC), cannabinol (CBN), tetrahydrocannabivarin (THCV) and THCA by gas chromatography (ElSohly et al ., 2000 ). THCA content expressed as a percentage of inflorescence DW was plotted against CBDA content and plants were categorized according to drug, fiber and intermediate phenotypes. The distribution of phenotypes was compared with a Mendelian expectation using a G ‐test for goodness of fit.

A hybrid cross between a single staminate hemp plant and single pistillate marijuana plant was made and segregation among F 2 progeny was examined by selfing a single F 1 plant. Cannabis is predominantly dioecious but monoecious variants are common (Hirata, 1927 ) and male sex expression may be induced in genetic females by means of hormonal treatment (Ram & Sett, 1982 ). Immature pistillate inflorescences of a clonally propagated F 1 plant were treated with AgNO 3 to induce the development of fertile staminate flowers. Pollen collected at anthesis was stained with 2,5‐diphenyl monotetrazolium bromide in 5% sucrose to test for viability (Rodriguez‐Riano & Dafni, 2000 ) and applied to pistillate inflorescences of numerous F 1 clones to produce an F 2 population.

Full‐sib inbreeding was performed in each line for multiple generations to reduce heterozygosity. Each generation, the male cohort was thinned to a single plant before anthesis. Pollen was applied by hand to flowers of a single female plant over several weeks of successive flowering and subsequent generations were established from the resulting seed. This process was repeated five times to produce near‐isogenic lines that served as parents in a hybrid cross between the drug and fiber cultivars.

Cannabis sativa L. seeds, including marijuana (Skunk #1) and hemp (Carmen) cultivars, were imported from Hortapharm BV (Amsterdam, the Netherlands) and Kenex Ltd (Ontario, Canada), respectively, under permit from the US Drug Enforcement Administration (DEA). Plants were grown in hydroponic media (Grodan) with an automated water exchange system, commercial nutrient solutions (General Hydroponics, Sebastopol, CA, USA) and high‐pressure sodium vapor lighting. An initial 4 wk of vegetative growth conditions employed an 18 : 6 h, light : dark regime and high‐N nutrient solution. During vegetative growth, the sex of individual plants was determined using a male‐linked sequence characterized amplified region (SCAR) marker (Mandolino et al ., 1999 ). Staminate and pistillate plants were subsequently maintained in separate growth chambers during 10–12 wk of flowering conditions using a 12 : 12 h, light : dark regime and high phosphorus (P) nutrient solution.

Gel purification and sequencing of qPCR products confirmed that the expressed genes of intermediate plants correspond to THCA and CBDA synthase genes of their respective marijuana and hemp parents (Fig. 3 ). Furthermore, F 2 plants with elevated CBDA content expressed the functional homolog of CBDA synthase and a relatively low level of hemp‐type THCA synthase (Kojoma et al ., 2006 ). Elevated THCA content was associated with expression of marijuana THCA synthase and extremely low levels of a nonfunctional CBDA synthase homolog.

Inflorescences of marijuana‐type and hemp‐type plants yielded significantly different quantities of cannabinoid synthase RNA transcripts as measured by RT‐qPCR (Table 2 ). Expression of THCA synthase in marijuana inflorescences was 102‐fold greater than in hemp inflorescences ( P < 0.05, t ‐test), whereas CBDA synthase expression in hemp inflorescences was 113‐fold greater than in marijuana inflorescences. Relative CBDA synthase and THCA synthase expression levels in inflorescences of F 2 plants were examined alongside variation in cannabinoid phenotype (Table 2 ). As measured by the PCR cycle during which specific products were first detected (i.e. crossing threshold), F 2 inflorescences expressing relatively more CBDA synthase were higher in CBDA than in THCA, whereas F 2 inflorescences expressing relatively more THCA synthase were higher in THCA than in CBDA. F 2 plants expressing similar levels of CBDA and THCA synthases were of intermediate phenotype.

Nonsynonymous substitutions ( dN ) were underrepresented relative to synonymous substitutions ( dS ) in the cannabinoid synthase gene family as a whole ( dN / dS = 0.531 with a 95% CI of 0.474–0.587) but this was not the case for the nonfunctional homologs. The only clade having a significantly elevated dN / dS ratio comprised the nonfunctional CBDA synthases (Fig. 3 ). These homologs shared a 4 bp deletion starting at position 153 of the gene and Skunk #1 homolog 1 also contained a premature stop codon upstream of this position.

Bayesian gene tree for tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA) synthase gene sequences inferred from a 1614 base‐pair alignment of nine unique sequences obtained from the mapping population (Genbank accession numbers KJ469374 KJ469376 and KJ469378 KJ469383 ), nine previously publishedsequences, and Genbank accession number LA634839 , the most similar sequence from the(Natsume.,). [Correction added after online publication 17 July 2015: Genbank accession numbers in the preceding sentence have been updated.] The tree was rooted with, which is the closest extant relative of(Zerega.,). Genes expressed intrichomes are underlined. Branch lengths are proportional to the number of nucleotide substitutions per site, except for the lengthy branch to, which is truncated. Genbank accession numbers for THCA synthase sequences are listed in parentheses as follows: Skunk #1 homolog 3 ( KJ469382 ), Carmen homolog 3 ( KJ469383 ), Purple Kush homlog 2 ( JH227480 ), Carmen homolog 2 ( KJ469381 ), Kojoma homolog 1 ( AB212836 ), Skunk #1 homlog 2 ( KJ469379 ), Carmen homolog 1 ( KJ469380 ), Purple Kush homolog 1 ( JH239911 ), Sirikantaramas ( AB057805 ), Kojoma homolog 2 ( AB212829 ), Skunk #1 homolog 1 ( KJ469378 ). Accession numbers for CBDA synthase are Kojoma ( AB292682 ), Carmen ( KJ469374 ), Skunk #1 homolog 1 KJ469375 ), Purple Kush homolog 1 ( JH231038 ), Purple Kush homolog 3 ( AGQN01254730 ), Purple Kush homolog 2 ( AGQN01159678 ) and Skunk #1 homolog 2 ( KJ469376 ).

Molecular cloning of THCA and CBDA synthase genes of Skunk #1 (marijuana), Carmen (hemp), as well as F 1 intermediate plants from the mapping population yielded products similar to previously published sequences. We identified nine unique sequences among 139 clones. A phylogenetic analysis including all available cannabinoid synthase sequences was rooted with the most similar sequence from Humulus sharing 74–86% nucleotide identity (Fig. 3 ). Branch lengths in Fig. 3 are proportional to the number of substitutions per site under the general time‐reversible model of molecular evolution and circles indicate Bayesian posterior probabilities (> 0.95). Skunk #1 yielded three homologs of the THCA synthase gene, including one nearly identical to the THCA synthase sequence first isolated from marijuana (Sirikantaramas et al ., 2004 ), a second that was highly similar to a sequence previously isolated from hemp (Kojoma et al ., 2006 ), and a new sequence with 92–93% nucleotide similarity to the others. Carmen yielded three homologs, including a sequence nearly identical to that previously isolated from hemp (Kojoma et al ., 2006 ), a second identical to the new sequence from Skunk #1, and a third with 94–95% nucleotide similarity to those published previously. The only CBDA synthase gene sequence obtained from Carmen was highly similar to that isolated from another hemp‐type Cannabis (Taura et al ., 2007 ), whereas two nonfunctional homologs, containing premature stop codons and frame shift mutations, were isolated from Skunk #1. Each of these homologs clustered with a nonfunctional CBDA synthase sequence from the marijuana Purple Kush genome (van Bakel et al ., 2011 ).

Analysis of cannabinoid profiles from the same 62 individuals used in mapping identified putative QTLs for cannabinoid content in association with two of the nine linkage groups. Such a small sample of genotypes would seem to be insufficient for identifying QTLs in general, but discrete cannabinoid phenotypes (Fig. 1 ) simplified the problem to such a degree that a significant QTL for the THCA/CBDA ratio could be assigned to linkage group six (Fig. 2 ). Both the group‐wise and experiment‐wise LOD thresholds ( α = 0.05) for log THCA/CBDA of 3.21 and 21.52, respectively, were surpassed by a maximum LOD of 40.44. A putative QTL for overall cannabinoid quantity located on linkage group one exceeded a group‐wise threshold of 2.18 with an LOD of 3.30 but was not significant given an experiment‐wise LOD of 3.42.

A subset of 62 plants from the F 2 population was genotyped for 103 AFLP markers, 16 microsatellite markers, CBDA synthase, and THCA synthase sequences to construct a linkage map. Nine linkage groups with LOD > 3.0 and containing at least four markers were identified (Fig. 2 ). These groups covered a genetic distance of 335.7 cM with an average between‐marker distance of 6.10 cM among 11 microsatellites, 51 AFLPs, CBDA synthase and THCA synthase.

Apart from qualitative differences in cannabinoid content, the total quantity of cannabinoids was significantly different between marijuana and hemp plants (Table 1 , P < 0.05, Tukey's honest significant difference (HSD) test). Marijuana plants averaged 4.5 times more total cannabinoids per unit inflorescence biomass than hemp plants and F 1 plants were intermediate. The overall distribution of cannabinoids in the F 2 generation indicated the independent assortment of cannabinoid quantity and quality. In particular, marijuana F 2 plants had significantly lower total cannabinoid content on average than the marijuana parent, whereas F 2 hemp plants had significantly higher content than the hemp parent (Fig. 1 ).

Delta 9 ‐tetrahydrocannabinolic acid (THCA) versus cannabidiolic acid (CBDA) content as a % of dry weight (DW) in mature pistillate inflorescences of Cannabis sativa derived from a marijuana parent and siblings, a hemp parent and siblings, the F 1 parent and siblings, and the F 2 cohort. Red arrows point to the hemp and marijuana parents; black arrows point to mean THCA content and CBDA content for marijuana‐like and hemp‐like F 2 s, respectively. The dashed line indicates the expected ratio of THCA to CBDA if the two main cannabinoid synthase enzymes were equally competitive for their common precursor, cannabigerolic acid.

Three major cannabinoid phenotypes (marijuana, hemp, and intermediate) were evident among the pistillate inflorescences of 540 genetically distinct Cannabis plants (Fig. 1 ). Plants were classified as marijuana when log(%THCA/%CBDA) ≥ 1.0, hemp when log(%THCA/%CBDA) ≤ −1.0, and intermediate when log(%THCA/%CBDA) > −1.0 and < 1.0 (Table 1 ). All F 1 plants were intermediate and segregation of cannabinoid phenotypes in the F 2 population was not significantly different from the 1 : 2 : 1 Mendelian expectation of marijuana to intermediate to hemp plants (127 : 264 : 105 plants, G = 4.16, P = 0.125).

Discussion

Cannabinoid inheritance Cannabis genotype–phenotype associations, patterns of gene expression, and the evolutionary history of the cannabinoid synthase gene family provide new insights into the genetic basis of drug content. The Mendelian pattern of inheritance observed in our mapping population (Fig. 1) is consistent with the interpretation that cannabinoid quality might be the product of a single enzymatic locus with codominant alleles. de Meijer et al. (2003) hypothesized that THCA synthase and CBDA synthase are allelic variants, whereas van Bakel et al. (2011) suggested that they could rather be linked loci. The distribution and expression of cannabinoid synthase homologs among descendants of marijuana crossed with hemp favors the alternative view (Table 2). Five generations of full‐sib inbreeding make it likely that the parental lines would be homozygous, and yet inbred Carmen and Skunk #1 yielded four and five cannabinoid synthase homologs, respectively. This observation and that of van Bakel et al. (2011) based on a draft genome point to the existence of multiple loci. However, among nine cannabinoid synthase homologs detected in our mapping population (Fig. 3), only four showed evidence of expression in RT‐qPCR assays (Table 2). Marijuana‐type plants expressed a functional THCA synthase nearly identical to that first reported by Sirikantaramas et al. (2004) and a nonfunctional CBDA synthase. Hemp‐type plants expressed functional THCA and CBDA synthases similar to those reported by Kojoma et al. (2006). It is possible that despite similar GC content and conserved primer sites among homologs of THCA synthase and among CBDA synthase homologs, qPCR bias could limit our capacity to measure the expression of other homologs. Regardless, the simultaneous presence of all four homologs in F 2 intermediate plants, as indicated by genotyping, is strong evidence of heterozygosity at separate loci for THCA synthase and CBDA synthase. Examination of the THCA/CBDA ratio in plants of intermediate phenotype suggests that THCA synthase and CBDA synthase might not be equally competitive for CBGA, the precusor that is shared by the two enzymes. All else being equal we would expect a 1 : 1 THCA/CBDA ratio in plants expressing functional copies of both enzymes, but instead we observed a ratio that is significantly skewed toward CBDA (Fig. 1). The fact that intermediate plants produce mostly CBDA despite expressing a functional THCA synthase suggests that CBDA synthase is a superior competitor for cannabigerolic acid. Colocation on the map of genes for THCA synthase and CBDA synthase in association with a QTL for the THCA/CBDA ratio (Fig. 2) is consistent with the multilocus model for the inheritance of the three qualitatively different cannabinoid phenotypes (de Meijer et al., 2003; van Bakel et al., 2011). Especially relevant to this interpretation is evidence for the expression of a nonfunctional CBDA synthase homolog in our mapping population (Table 2). If this homolog were an unlinked pseudogene, we would not expect it to segregate with cannabinoid phenotype, but the perfect association of this homolog with the marijuana phenotype suggests that it is probably an allele sharing a locus with functional CBDA synthase. Under a linked multilocus model of inheritance, plants of intermediate phenotype are expected to be heterozygous at separate loci for CBDA and THCA synthase and the genotypes of F 2 intermediates are consistent with this prediction. Expression assays of F 2 intermediates suggest that the marijuana‐type THCA synthase allele may be dominant over the hemp‐type allele, and the functional CBDA synthase allele may be dominant over the nonfunctional allele (Table 2). Thus, plants of intermediate phenotype inherit a highly expressed THCA synthase from one parent and a highly expressed CBDA synthase from the other, whereas marijuana and hemp plants inherit either a highly expressed THCA synthase or a highly expressed CBDA synthase, respectively. Genotyping also identified a single recombinant individual that was homozygous for the hemp‐type THCA synthase and homozygous for the marijuana‐type nonfunctional CBDA synthase. The marijuana phenotype exhibited by this individual suggests that the absence of functional CBDA is essential for marijuana potency.

Quality vs quantity The statistical association between cannabinoid phenotype and the location of cannabinoid synthases on the map (Fig. 2) was so strong that even a relatively small sample size was sufficient to detect it. We attribute this result to extreme divergence among parental cultivars in the THCA/CBDA ratio. This was not the case for total cannabinoid content, where a putative QTL on a different linkage group nearly approached significance in the experiment‐wise test (Fig. 2). The observation that marijuana‐like F 2 plants had reduced cannabinoid content and hemp‐like F 2 plants had elevated content relative to their respective parental lines (Fig. 1) suggests the independent assortment of drug quality and quantity. We speculate that an improved genetic map with more markers and more extensive screening of individuals is likely to provide the statistical power needed to identify QTLs for quantitative differences among cultivars in potency that is not easily explained by variation at cannabinoid synthase loci. Cannabinoid quantity varies among cultivars by four orders of magnitude (Vogelmann et al., 1987; de Meijer et al., 1992; Petri et al., 1996; Szendrei, 1997) and it is likely that other mechanisms besides the cannabinoid biosynthetic pathway are, to some extent, responsible for this variation. In particular, the size of glandular trichomes might account for substantial differences in cannabinoid quantity (Small & Naraine, 2015). The identification of genes affecting trichome development in Cannabis using homologs from model systems (Marks et al., 2009b) would therefore appear to be a promising avenue for further investigation. The high variance observed in the overall cannabinoid content of the F 1 and the parents despite five generations of full sib‐inbreeding also deserves explanation (Fig. 1). We attribute this to the fact that estimates of cannabinoid quantity were based on a single 100 mg tissue sample per plant (ElSohly et al., 2000). Cannabinoid quantity may vary among samples drawn from the same plant according to the extent to which glandular trichomes heads are intact or dislodged and whether the sample consists primarily of pistillate bracts or inflorescence bracts differing in trichome density. Variance among 100 mg samples drawn from the same genotype or even from the same inflorescence could account for much of the scatter observed in Fig. 1. The possibility of separate QTLs affecting drug quantity and quality suggest that genetic mapping and QTL analysis could aid in identifying complex mechanisms that underlie other economically important traits in Cannabis such as nutritious seed oils or properties of industrial fiber. The incomplete map presented in Fig. 2 is one linkage group short of the Cannabis haploid chromosome number (N = 10). With the addition of new markers, we expect six small linkage groups consisting of less than four markers each (not shown in Fig. 2) to associate with others and to resolve a 10th group. Next‐generation sequencing and the identification of single nucleotide polymorphisms hold promise for improving map resolution and for identifying additional QTLs.

Cannabinoid synthase evolution Phylogenetic analysis suggests that THCA and CBDA synthases arose by the duplication of an ancestral cannabinoid synthase gene (Fig. 3) and the location of QTL suggest that divergent cannabinoid phenotypes are products of subsequent sequence evolution at linked loci (Fig. 2). Gene duplications are known to result from unequal crossing‐over and the fate of duplicated regions will depend on the nature of selection (Zhang, 2005). Theoretical population genetics predicts that duplicated gene copies are likely to persist by divergence through subfunctionalization (Lynch & Force, 2000). In the case of Cannabis, we speculate that this might have been associated with the evolution of enzymatic activities yielding different secondary metabolites. The simultaneous presence of THCA and CBDA synthase‐like genes in diverse Cannabis accessions (Kojoma et al., 2006; Taura et al., 2007; van Bakel et al., 2011) suggests that multiple loci may be ancient and pervasive. Global dN/dS further suggests that amino acid replacements appear to have been selected against during the evolution of cannabinoid synthases, which is consistent with the maintenance of function (Kosakovsky Pond et al., 2005). In the case of marijuana, loss of CBDA synthase function must have occurred after an ancestral duplication because CBDA synthase and nonfunctional homologs are more closely related to each other than to THCA synthase genes (Fig. 3). Loss of function in marijuana‐type CBDA synthases can be attributed to various premature stop codons and frame shift mutations. For example, sequences in the clade including CBDA synthase Skunk #1 homolog 1 contain a premature stop codon at the 5′ end of the gene and share a 4 bp deletion with CBDA synthase Skunk #1 homolog 2. Local dN/dS is consistent with the interpretation that such mutations were selected to enhance potency (van Bakel et al., 2011) where the only branch of the gene tree exhibiting evidence of positive selection was that leading to nonfunctional CBDA homologs (Fig. 3). If nonfunctional homologs prove to be ubiquitous among drug cultivars, it is possible that selection occurred early in domestication. However, the evolution of cannabinoid synthases alone might not entirely explain recently observed increases in potency (ElSohly et al., 2000) but potentially other traits as well (see quality vs quantity). With respect to genetic diversity, intense selection by breeders for potency might also account for drug cultivars being generally less polymorphic and heterozygous than hemp cultivars (de Meijer et al., 1992; Faeti et al., 1996; Forapani et al., 2001; Datwyler & Weiblen, 2006). The available evidence suggests that there are at least two cannabinoid synthase loci and there are likely to be more. For example, we detected a THCA synthase sequence in Skunk #1 (homolog 3) which was nearly identical to that originally isolated from hemp‐type Cannabis (Kojoma et al., 2006). This homolog was not detected in a draft genome from Purple Kush (van Bakel et al., 2011) and awaits explanation. It is conceivable that additional gene duplications have occurred (Taylor & Raes, 2004). Next‐generation sequencing of diverse Cannabis cultivars and comparative genomics hold promise for further resolution of the history of duplication, divergence, and selection affecting drug content.