Inheritance and covariation of specialised metabolites among cannabis chemotypes
Matthew T. Welling


A
# Co-first authors.
§ Co-senior authors.
Handling Editor: John Wade
Abstract
The major phytocannabinoid bioactives produced by Cannabis sativa L. (cannabis) are Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD), with many minor phytocannabinoids (PCs) also thought to contribute to the pharmacological efficacy. Cannabis typically segregates into three main chemical phenotypes (chemotypes) based on their Δ9-THC/CBD ratio, a highly heritable trait determined by the segregation of two closely related berberine bridge-like enzymes that perform stereoselective oxidative cyclisation on the geranyl moiety of cannabigeroid intermediates. Apart from a small number of metabolome-wide association studies, few attempts have been made to either understand metabolite coupling among Δ9-THC/CBD chemotypes or to examine the inheritance of alternative biomarkers that could be used to discriminate chemotype. Here, we examined the metabolomes of 108 F2 segregants derived from a cross between a Δ9-THC-predominant chemotype I and a CBD-predominant chemotype III plant. Although segregation of the Δ9-THC/CBD ratio followed Mendelian genetics expectations, covariation in the inheritance of minor PCs, including cannabichromene (CBC)-types, indicated changes in cannabinoid synthase product specificity among chemotypes. In addition, several non-PC related metabolites were identified that may serve as potential biomarkers for chemotype prediction. These data have important implications for the pre-breeding and selection of cannabis chemovars and highlight the need to adopt metabolic engineering strategies to optimise PC production.
Keywords: cannabichromene, cannabidiol, Cannabis sativa L., liquid chromatography, mass spectrometry, phytocannabinoids, untargeted metabolomics, Δ9-tetrahydrocannabinol.
References
1 Small E, Cronquist A. A practical and natural taxonomy for Cannabis. Taxon 1976; 25(4): 405-435.
| Crossref | Google Scholar |
2 Welling MT, Deseo MA, Bacic A, Doblin MS. Untargeted metabolomic analyses reveal chemical complexity of dioecious Cannabis flowers. Aust J Chem 2021; 74(6): 463-479.
| Crossref | Google Scholar |
3 Grassa CJ, Weiblen GD, Wenger JP, Dabney C, Poplawski SG, Timothy Motley S, Michael TP, Schwartz CJ. A new Cannabis genome assembly associates elevated cannabidiol (CBD) with hemp introgressed into marijuana. New Phytol 2021; 230(4): 1665-1679.
| Crossref | Google Scholar | PubMed |
4 Bradshaw RHW, Coxon P, Greig JRA, Hall AR. New fossil evidence for the past cultivation and processing of hemp (Cannabis sativa L.) in eastern England. New Phytol 1981; 89(3): 503-510.
| Crossref | Google Scholar |
5 Staginnus C, Zörntlein S, de Meijer E. A PCR marker linked to a THCA synthase polymorphism is a reliable tool to discriminate potentially THC-rich plants of Cannabis sativa L. J Forensic Sci 2014; 59(4): 919-926.
| Crossref | Google Scholar | PubMed |
6 Livingston SJ, Rensing KH, Page JE, Samuels AL. A polarized supercell produces specialized metabolites in cannabis trichomes. Curr Biol 2022; 32(18): 4040-4047.e4.
| Crossref | Google Scholar | PubMed |
7 Stout JM, Boubakir Z, Ambrose SJ, Purves RW, Page JE. The hexanoyl-CoA precursor for cannabinoid biosynthesis is formed by an acyl-activating enzyme in Cannabis sativa trichomes. Plant J 2012; 71(3): 353-365.
| Crossref | Google Scholar | PubMed |
8 Welling MT, Deseo MA, Bacic A, Doblin MS. Biosynthetic origins of unusual cannabimimetic phytocannabinoids in Cannabis sativa L: a review. Phytochemistry 2022; 201: 113282.
| Crossref | Google Scholar | PubMed |
9 Zagzoog A, Mohamed KA, Kim HJJ, Kim ED, Frank CS, Black T, Jadhav PD, Holbrook LA, Laprairie RB. In vitro and in vivo pharmacological activity of minor cannabinoids isolated from Cannabis sativa. Sci Rep 2020; 10(1): 20405.
| Crossref | Google Scholar | PubMed |
10 Maione S, Piscitelli F, Gatta L, Vita D, De Petrocellis L, Palazzo E, de Novellis V, Di Marzo V. Non-psychoactive cannabinoids modulate the descending pathway of antinociception in anaesthetized rats through several mechanisms of action. Br J Pharmacol 2011; 162(3): 584-596.
| Crossref | Google Scholar | PubMed |
11 Devinsky O, Cross JH, Laux L, Marsh E, Miller I, Nabbout R, Scheffer IE, Thiele EA, Wright S, Cannabidiol in Dravet Syndrome Study Group. Trial of cannabidiol for drug-resistant seizures in the Dravet syndrome. N Engl J Med 2017; 376(21): 2011-2020.
| Crossref | Google Scholar | PubMed |
12 Beal JE, Olson R, Laubenstein L, Morales JO, Bellman P, Yangco B, Lefkowitz L, Plasse TF, Shepard KV. Dronabinol as a treatment for anorexia associated with weight loss in patients with AIDS. J Pain Symptom Manage 1995; 10(2): 89-97.
| Crossref | Google Scholar | PubMed |
13 Moreno-Sanz G, Madiedo A, Lynskey M, Brown MRD. “Flower power”: controlled inhalation of THC-predominant Cannabis flos improves health-related quality of life and symptoms of chronic pain and anxiety in eligible UK patients. Biomedicines 2022; 10(10): 2576.
| Crossref | Google Scholar | PubMed |
14 Sirikantaramas S, Morimoto S, Shoyama Y, Ishikawa Y, Wada Y, Shoyama Y, Taura F. The gene controlling marijuana psychoactivity: molecular cloning and heterologous expression of Δ1-tetrahydrocannabinolic acid synthase from Cannabis sativa L. J Biol Chem 2004; 279(38): 39767-39774.
| Crossref | Google Scholar | PubMed |
15 Taura F, Sirikantaramas S, Shoyama Y, Yoshikai K, Shoyama Y, Morimoto S. Cannabidiolic-acid synthase, the chemotype-determining enzyme in the fiber-type Cannabis sativa. FEBS Lett 2007; 581(16): 2929-2934.
| Crossref | Google Scholar | PubMed |
16 Shoyama Y, Tamada T, Kurihara K, Takeuchi A, Taura F, Arai S, Blaber M, Shoyama Y, Morimoto S, Kuroki R. Structure and function of ∆1-tetrahydrocannabinolic acid (THCA) synthase, the enzyme controlling the psychoactivity of Cannabis sativa. J Mol Biol 2012; 423(1): 96-105.
| Crossref | Google Scholar | PubMed |
17 Zirpel B, Kayser O, Stehle F. Elucidation of structure-function relationship of THCA and CBDA synthase from Cannabis sativa L. J Biotechnol 2018; 284: 17-26.
| Crossref | Google Scholar | PubMed |
18 de Meijer EP, Bagatta M, Carboni A, Crucitti P, Moliterni VM, Ranalli P, Mandolino G. The inheritance of chemical phenotype in Cannabis sativa L. Genetics 2003; 163(1): 335-346.
| Crossref | Google Scholar | PubMed |
19 de Meijer EPM, Hammond KM. The inheritance of chemical phenotype in Cannabis sativa L. (II): cannabigerol predominant plants. Euphytica 2005; 145(1): 189-198.
| Crossref | Google Scholar |
20 de Meijer EPM, Hammond KM, Sutton A. The inheritance of chemical phenotype in Cannabis sativa L. (IV): cannabinoid-free plants. Euphytica 2009; 168(1): 95-112.
| Crossref | Google Scholar |
21 Cerrato A, Citti C, Cannazza G, Capriotti AL, Cavaliere C, Grassi G, Marini F, Montone CM, Paris R, Piovesana S, Laganà A. Phytocannabinomics: untargeted metabolomics as a tool for Cannabis chemovar differentiation. Talanta 2021; 230: 122313.
| Crossref | Google Scholar | PubMed |
22 Welling MT, Liu L, Raymond CA, Ansari O, King GJ. Developmental plasticity of the major alkyl cannabinoid chemotypes in a diverse Cannabis genetic resource collection. Front Plant Sci 2018; 9: 1510.
| Crossref | Google Scholar | PubMed |
23 Aizpurua-Olaizola O, Soydaner U, Öztürk E, Schibano D, Simsir Y, Navarro P, Etxebarria N, Usobiaga A. Evolution of the cannabinoid and terpene content during the growth of Cannabis sativa plants from different chemotypes. J Nat Prod 2016; 79(2): 324-331.
| Crossref | Google Scholar | PubMed |
24 Weiblen GD, Wenger JP, Craft KJ, ElSohly MA, Mehmedic Z, Treiber EL, Marks MD. Gene duplication and divergence affecting drug content in Cannabis sativa. New Phytol 2015; 208(4): 1241-1250.
| Crossref | Google Scholar | PubMed |
25 Welling MT, Liu L, Raymond CA, Kretzschmar T, Ansari O, King GJ. Complex patterns of cannabinoid alkyl side-chain inheritance in Cannabis. Sci Rep 2019; 9(1): 11421.
| Crossref | Google Scholar | PubMed |
26 Jin D, Henry P, Shan J, Chen J. Identification of phenotypic characteristics in three chemotype categories in the genus Cannabis. Hortic Sci 2021; 56(4): 481-490.
| Crossref | Google Scholar |
27 McKernan KJ, Helbert Y, Kane LT, Ebling H, Zhang L, Liu B, Eaton Z, McLaughlin S, Kingan S, Baybayan P, Concepcion G, Jordan M, Riva A, Barbazuk W, Harkins T. Sequence and annotation of 42 cannabis genomes reveals extensive copy number variation in cannabinoid synthesis and pathogen resistance genes. BioRxiv 2020; 2020: 2020.01.03.894428 [Preprint, published 5 January 2020].
| Crossref | Google Scholar |
28 Hillig KW, Mahlberg PG. A chemotaxonomic analysis of cannabinoid variation in Cannabis (Cannabaceae). Am J Bot 2004; 91(6): 966-975.
| Crossref | Google Scholar | PubMed |
29 Jin D, Henry P, Shan J, Chen J. Identification of chemotypic markers in three chemotype categories of Cannabis using secondary metabolites profiled in inflorescences, leaves, stem bark, and roots. Front Plant Sci 2021; 12: 699530.
| Crossref | Google Scholar | PubMed |
30 Chadeau-Hyam M, Ebbels TMD, Brown IJ, Chan Q, Stamler J, Huang CC, Daviglus ML, Ueshima H, Zhao L, Holmes E, Nicholson JK, Elliott P, De Iorio M. Metabolic profiling and the metabolome-wide association study: significance level for biomarker identification. J Proteome Res 2010; 9(9): 4620-4627.
| Crossref | Google Scholar | PubMed |
31 Luo X, Reiter MA, d’Espaux L, Wong J, Denby CM, Lechner A, Zhang Y, Grzybowski AT, Harth S, Lin W, Lee H, Yu C, Shin J, Deng K, Benites VT, Wang G, Baidoo EEK, Chen Y, Dev I, Petzold CJ, Keasling JD. Complete biosynthesis of cannabinoids and their unnatural analogues in yeast. Nature 2019; 567(7746): 123-126.
| Crossref | Google Scholar | PubMed |
32 Dussy FE, Hamberg C, Luginbühl M, Schwerzmann T, Briellmann TA. Isolation of Δ9-THCA-A from hemp and analytical aspects concerning the determination of Δ9-THC in cannabis products. Forensic Sci Int 2005; 149(1): 3-10.
| Crossref | Google Scholar | PubMed |
33 de Meijer EPM, Hammond KM. The inheritance of chemical phenotype in Cannabis sativa L. (V): regulation of the propyl-/pentyl cannabinoid ratio, completion of a genetic model. Euphytica 2016; 210(2): 291-307.
| Crossref | Google Scholar |
34 de Meijer EPM, Hammond KM, Micheler M. The inheritance of chemical phenotype in Cannabis sativa L. (III): variation in cannabichromene proportion. Euphytica 2009; 165(2): 293-311.
| Crossref | Google Scholar |
35 Morimoto S, Komatsu K, Taura F, Shoyama Y. Purification and characterization of cannabichromenic acid synthase from Cannabis sativa. Phytochemistry 1998; 49(6): 1525-1529.
| Crossref | Google Scholar | PubMed |
36 Rodziewicz P, Loroch S, Marczak Ł, Sickmann A, Kayser O. Cannabinoid synthases and osmoprotective metabolites accumulate in the exudates of Cannabis sativa L. glandular trichomes. Plant Sci 2019; 284: 108-116.
| Crossref | Google Scholar | PubMed |
37 Wishart DS, Hiebert-Giesbrecht M, Inchehborouni G, Cao X, Guo AC, LeVatte MA, Torres-Calzada C, Gautam V, Johnson M, Liigand J, Wang F, Zahraei S, Bhumireddy S, Wang Y, Zheng J, Mandal R, Dyck JRB. Chemical composition of commercial cannabis. J Agric Food Chem 2024; 72(25): 14099-14113.
| Crossref | Google Scholar | PubMed |
38 ElSohly MA, Slade D. Chemical constituents of marijuana: The complex mixture of natural cannabinoids. Life Sci 2005; 78(5): 539-548.
| Crossref | Google Scholar | PubMed |
39 Schwilke EW, Schwope DM, Karschner EL, Lowe RH, Darwin WD, Kelly DL, Goodwin RS, Gorelick DA, Huestis MA. Δ9-Tetrahydrocannabinol (THC), 11-hydroxy-THC, and 11-nor-9-carboxy-THC plasma pharmacokinetics during and after continuous high-dose oral THC. Clin Chem 2009; 55(12): 2180-2189.
| Crossref | Google Scholar | PubMed |
40 Hanuš LO, Meyer SM, Muñoz E, Taglialatela-Scafati O, Appendino G. Phytocannabinoids: a unified critical inventory. Nat Prod Rep 2016; 33(12): 1357-1392.
| Crossref | Google Scholar | PubMed |
41 Tolomeo F, Russo F, Kaczorova D, Vandelli MA, Biagini G, Laganà A, Capriotti AL, Paris R, Fulvio F, Carbone L, Perrone E, Gigli G, Cannazza G, Citti C. Cis-Δ9-tetrahydrocannabinolic acid occurrence in Cannabis sativa L. J Pharm Biomed Anal 2022; 219: 114958.
| Crossref | Google Scholar | PubMed |
42 Turner CE, Elsohly MA, Boeren EG. Constituents of Cannabis sativa L. XVII. A review of the natural constituents. J Nat Prod 1980; 43(2): 169-234.
| Crossref | Google Scholar | PubMed |
43 Seo C, Jeong M, Lee S, Kim EJ, Rho S, Cho M, Lee YS, Hong J. Thermal decarboxylation of acidic cannabinoids in Cannabis species: identification of transformed cannabinoids by UHPLC-Q/TOF–MS. J Anal Sci Technol 2022; 13(1): 42.
| Crossref | Google Scholar |
44 Shani A, Mechoulam R. Cannabielsoic acids: Isolation and synthesis by a novel oxidative cyclization. Tetrahedron 1974; 30(15): 2437-2446.
| Crossref | Google Scholar |
45 Parveen I, Allen NR, Wonfor RE, Al-Fadhli AA, Forde-Thomas JE, Giles JL, Walton RT, Threadgill MD, Nash DM. Characterisation of components of an extract of hemp and preliminary assessment of anti-inflammatory activity in an ex vivo model of bovine endometritis. S Afr J Bot 2025; 180: 254-264.
| Crossref | Google Scholar |
46 Friedrich-Fiechtl J, Spiteller G. Neue cannabinoide – 1. Tetrahedron 1975; 31(6): 479-487 [In German with abstract in German and English].
| Crossref | Google Scholar |
47 Rao VK, Lewis-Bakker MM, Wasilewski E, Clarke HA, Kotra LP. Stereoisomers of cannabidiols and their pharmacological activities – a potentially novel direction for cannabinoids. Biorg Med Chem 2025; 117: 118019.
| Crossref | Google Scholar |
48 Radwan MM, Chandra S, Gul S, ElSohly MA. Cannabinoids, phenolics, terpenes and alkaloids of Cannabis. Molecules 2021; 26(9): 2774.
| Crossref | Google Scholar | PubMed |
49 Castro-Camba R, Sánchez C, Vidal N, Vielba JM. Plant development and crop yield: the role of gibberellins. Plants (Basel) 2022; 11(19): 2650.
| Crossref | Google Scholar | PubMed |
50 Alter H, Sade Y, Sood A, Carmeli-Weissberg M, Shaya F, Kamenetsky-Goldstein R, Bernstein N, Spitzer-Rimon B. Inflorescence development in female cannabis plants is mediated by photoperiod and gibberellin. Hortic Res 2024; 11(11): 245.
| Crossref | Google Scholar | PubMed |
51 Mansouri H, Asrar Z, Mehrabani M. Effects of gibberellic acid on primary terpenoids and delta-tetrahydrocannabinol in Cannabis sativa at flowering stage. J Integr Plant Biol 2009; 51(6): 553-561.
| Crossref | Google Scholar | PubMed |
52 Günther J, Halitschke R, Gershenzon J, Burow M. Heterologous expression of PtAAS1 reveals the metabolic potential of the common plant metabolite phenylacetaldehyde for auxin synthesis in planta. Physiol Plant 2023; 175(6): e14078.
| Crossref | Google Scholar | PubMed |
53 Izzo L, Castaldo L, Narváez A, Graziani G, Gaspari A, Rodríguez-Carrasco Y, Ritieni A. Analysis of phenolic compounds in commercial Cannabis sativa L. inflorescences using UHPLC-Q-Orbitrap HRMS. Molecules 2020; 25(3): 631.
| Crossref | Google Scholar | PubMed |
54 Curtasu MV, Pallesen BE, No̷rskov NP. Quantitative distribution of polyphenolic compounds during plant development in five varieties of organic hemp (Cannabis sativa L.). J Agric Food Chem 2025; 73(26): 16359-16369.
| Crossref | Google Scholar | PubMed |
55 Lynch RC, Padgitt-Cobb LK, Garfinkel AR, Knaus BJ, Hartwick NT, Allsing N, Aylward A, Bentz PC, Carey SB, Mamerto A, Kitony JK, Colt K, Murray ER, Duong T, Chen HI, Trippe A, Harkess A, Crawford S, Vining K, Michael TP. Domesticated cannabinoid synthases amid a wild mosaic cannabis pangenome. Nature 2025; 643: 1001-1010.
| Crossref | Google Scholar | PubMed |
56 Mohan Ram HY, Sett R. Induction of fertile male flowers in genetically female Cannabis sativa plants by silver nitrate and silver thiosulphate anionic complex. Theor Appl Genet 1982; 62(4): 369-375.
| Crossref | Google Scholar | PubMed |
57 Welling MT, Deseo MA, O’Brien M, Clifton J, Bacic A, Doblin MS. Metabolomic analysis of methyl jasmonate treatment on phytocannabinoid production in Cannabis sativa. Front Plant Sci 2023; 14: 1110144.
| Crossref | Google Scholar | PubMed |
58 Jost R, Berkowitz O, Pegg A, Hurgobin B, Tamiru-Oli M, Welling MT, Deseo MA, Noorda H, Brugliera F, Lewsey MG, Doblin MS, Bacic A, Whelan J. Sink strength, nutrient allocation, cannabinoid yield, and associated transcript profiles vary in two drug-type Cannabis chemovars. J Exp Bot 2025; 76(1): 152-174.
| Crossref | Google Scholar | PubMed |
59 Steel L, Welling M, Ristevski N, Johnson K, Gendall A. Comparative genomics of flowering behavior in Cannabis sativa. Front Plant Sci 2023; 14: 1227898.
| Crossref | Google Scholar | PubMed |