Sex determination in cannabis
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Sex determination in cannabis is the genetic and developmental process that establishes whether a Cannabis plant develops as male, female, or, in some cultivated forms, monoecious. Cannabis is normally dioecious: pollen-bearing (male) and seed-bearing (female) flowers form on separate individuals, and sex is set by a pair of heteromorphic sex chromosomes. Females carry two X chromosomes (XX) and males an X and a Y (XY), so the male is the heterogametic sex and the Y is transmitted only through pollen.[1]
Chromosomal sex, fixed at fertilisation, is distinct from sex expression: the male or female character of the flowers a plant actually produces. Sex expression is labile: hormonal, chemical and environmental signals can shift a plant toward the opposite sex's flowers without altering its chromosomes, and a genetically female (XX) plant induced to shed pollen transmits only X-bearing gametes.[2]
The distinction is central to cannabis cultivation and breeding. Because cannabinoids accumulate chiefly in the unpollinated female inflorescence, drug-type and cannabinoid-hemp crops are grown as all-female stands; monoecious cultivars underpin uniform fibre and seed hemp; and reliable early identification of a plant's chromosomal sex, before flowers appear, is a routine breeding and roguing task.[3] For dioecious landrace populations, the obligate outcrossing that dioecy enforces also shapes mating structure and the maintenance of genetic diversity.
Sexual system
The wild and most cultivated forms of cannabis are dioecious, and this is the ancestral condition in the family Cannabaceae.[1] A minority of hemp cultivars are monoecious, bearing both male and female flowers on one plant. Monoecy is not a separate chromosomal sex: cytogenetic and marker analysis shows monoecious cultivars carry the female XX constitution and lack a Y chromosome, so their sexual behaviour has a genetic basis laid over an otherwise female genotype.[4][5]
Sex expression in monoecious hemp is continuous, grading from wholly male to wholly female plants. Breeders score it on a scale from all-male to all-female flowering, and cultivars differ significantly in their mean position on that scale, which is one reason monoecy behaves as a heritable quantitative character, not a single-gene trait.[5] Dioecious plants can likewise produce a few flowers of the opposite sex under stress or hormonal treatment, a lability that ranges from the occasional hermaphroditic flower to the deliberate reversals used in seed production.[2] Reviews of the species treat this mixed and modifiable sexual system as the product of both genomic and environmental control.[6]
Sex chromosomes
Cannabis is diploid with 2n = 20: nine pairs of autosomes and one pair of sex chromosomes, homomorphic in females (XX) and heteromorphic in males (XY).[1][7] Cytogenetic measurement places the Y as the largest chromosome in the complement, slightly larger than the X and the largest autosome; it carries one fully heterochromatic arm and one euchromatic arm, and a subtelomeric repeat family marks the euchromatic Y arm and both arms of the X.[1]
The sex chromosome pair was localised in the genome in 2020, when segregation of X- and Y-linked transcripts across a controlled cross assigned sex to chromosome pair 1 of the reference assembly.[8] That analysis found most of the chromosome, a region of roughly 75 megabases, to be non-recombining and X-specific, with a smaller pseudoautosomal region that still recombines in males; independent sex-linked marker studies had already inferred such a pseudoautosomal region and located it to the distal euchromatic arm.[8][9]
The Y is substantially degenerated despite its size. Expression of Y-linked alleles runs at about half that of their X-linked counterparts, consistent with the loss of a large fraction of Y-linked genes and only partial dosage compensation; recombination between the X and Y was estimated to have stopped roughly 27 million years ago, and the authors suggested these may be among the oldest plant sex chromosomes dated from sequence data.[8] Y differentiation is associated with the accumulation of repetitive DNA: a LINE-like retrotransposon accumulates specifically on the sex chromosome, and further male-associated sequences correspond to copia-like retrotransposons that give intense signals on the Y.[10][11] Genomic in-situ hybridisation indicates that a male-specific region occupies most of the Y, leaving only a small pseudoautosomal segment and pointing to a long, advanced course of sex chromosome evolution.[12]
Widely used reference genome assemblies were built from female plants, so the Y chromosome is less completely represented than the rest of the genome, and a dedicated male assembly is still needed to characterise it fully.[13][14]
Genetic basis
How the sex chromosomes actually specify sex in cannabis is not settled. Two models are debated for dioecious plants generally: an active-Y system, in which a dominant, male-determining Y imposes maleness, and an X-to-autosome balance system, in which the ratio of X chromosomes to autosomes sets the sexual phenotype. Cannabis has features cited for each. The Y is required for maleness, since female and monoecious plants are alike XX and never carry a Y, which is consistent with a male-determining Y,[1][4] yet part of the hemp-genetics literature describes sex in dioecious cannabis as governed by an X-to-autosome equilibrium.[5]
The genetic architecture of sex expression is at least partly polygenic, which a purely single-locus Y model does not fully capture. Genome-wide association mapping resolves a small number of sex-determination loci of large effect, and QTL studies of dioecious and monoecious hemp likewise recover several contributing regions, implying autosomal modifiers act alongside the sex chromosomes.[7][15] The 2020 characterisation of the sex chromosomes described their location, age and degeneration but did not adjudicate between the two mechanisms.[8] Evidence from the sister genus Humulus (hop) has sharpened the question without closing it: hop uses an X-to-autosome balance system driven by the dose of an X-linked ethylene-receptor gene, which raises the possibility of a shared mechanism across the two Cannabaceae genera even though the cannabis Y is large and the hop Y small.[16]
The XY system implies a primary sex ratio near 1:1, the expectation for a cross between an XX female and an XY male. Departures from an even male-to-female ratio in cultivation arise mainly from the lability of sex expression, discussed below, rather than from distorted chromosomal segregation.[2]
Sex expression and its modification
A plant's chromosomal sex and the sex of the flowers it produces can be uncoupled. Plant hormones exert opposite, directional control over flower sex: ethylene, together with cytokinins and auxins, promotes female flowering, while gibberellins promote male flowering.[2] This lets growers reverse the apparent sex of a genetically female plant. Treatment with silver thiosulfate or silver nitrate blocks the plant's perception of ethylene, so a genetically female plant forms functional male flowers and sheds viable pollen.[2][17]
Because the masculinised plant remains chromosomally XX, its pollen carries only X chromosomes; crossing that pollen onto a normal female produces XX offspring only. This is the basis of feminised seed, which yields all-female crops without any male parent.[2][17] The commitment to a sexual pathway is made early, well before any visible floral dimorphism: differential gene expression between male and female apices is detectable from around the fourth node, and RNA-based studies find sex-biased expression already established at early vegetative stages, implicating transcription-factor families as candidate regulators.[18][19]
Molecular identification of sex
Adult male and female plants are readily told apart by their flowers, but before flowering they are not visually distinguishable, and because chemical or environmental treatment alters the flowers without altering the chromosomes, only a DNA test reports true chromosomal sex.[3] The first practical markers were male-associated sequences recovered by RAPD screening and then converted to more reproducible sequence-characterised (SCAR) markers: a male-associated band present across cultivars was stabilised into a male-specific SCAR for early identification of male plants.[20] Further male-associated markers followed, including the retrotransposon-derived MADC markers and additional male-specific loci.[11][21]
These early markers are male-associated, not strictly Y-unique, so the underlying sequences can occur at low frequency in females and the markers give occasional false positives; their frequency across accessions has itself been surveyed.[20][22] More recent methods target a gene that differs between the X and Y copies, allowing a single primer pair to distinguish XX from XY directly; one such assay resolved sex across several hundred samples of varied tissue and developmental stage at full accuracy, giving reliable identification well before flowering.[3]
Significance
Sex determination underlies several practical concerns in cannabis cultivation and breeding. Feminised seed, produced by masculinising genetic females, gives the all-female crops on which cannabinoid production depends, since cannabinoids concentrate in the unpollinated female inflorescence.[2][3] Monoecious hemp, all chromosomally female and flowering more uniformly than a dioecious stand, is bred by selecting on the quantitative degree of monoecy, a task molecular markers assist by letting breeders act on sex genotype directly.[5][7] Early molecular sexing conserves labour and growing space by allowing unwanted males to be removed at the seedling stage.[20][3]
Cannabis is often compared with its sister genus Humulus (hop): both Cannabaceae genera are dioecious with an XY male, but they differ in the size and content of the Y and, on current evidence, in how sex is specified: hop through an X-to-autosome dosage system with a small Y, cannabis through an enlarged, repeat-laden Y whose determining mechanism remains open.[16][1] For landrace conservation, the obligate outcrossing imposed by dioecy is itself consequential: it sustains heterozygosity and gene flow within a population but means a landrace cannot be maintained true from a single plant, so ex situ holdings must sample enough individuals of both sexes to keep the effective population size adequate and retain the population's diversity.
See also
References
- ↑ 1.0 1.1 1.2 1.3 1.4 1.5 Divashuk, M.G.; Alexandrov, O.S.; Razumova, O.V.; Kirov, I.V.; Karlov, G.I. (2014). "Molecular cytogenetic characterization of the dioecious Cannabis sativa with an XY chromosome sex determination system". PLoS ONE. 9 (1) e85118. doi:10.1371/journal.pone.0085118.
- ↑ 2.0 2.1 2.2 2.3 2.4 2.5 2.6 Owen, L.C.; Suchoff, D.H.; Chen, H. (2023). "A novel method for stimulating Cannabis sativa L. male flowers from female plants". Plants. 12 (19) 3371. doi:10.3390/plants12193371.
- ↑ 3.0 3.1 3.2 3.3 3.4 Riera-Begué, A.; Toscani, M.; Malik, A.; Dowling, C.A.; Schilling, S.; Melzer, R. (2025). "A simple and reliable PCR-based method to differentiate between XX and XY sex genotypes in Cannabis sativa". Planta. 262 (4) 87. doi:10.1007/s00425-025-04804-z.
- ↑ 4.0 4.1 Razumova, O.V.; Alexandrov, O.S.; Divashuk, M.G.; Sukhorada, T.I.; Karlov, G.I. (2016). "Molecular cytogenetic analysis of monoecious hemp (Cannabis sativa L.) cultivars reveals its karyotype variations and sex chromosomes constitution". Protoplasma. 253 (3): 895–901. doi:10.1007/s00709-015-0851-0.
- ↑ 5.0 5.1 5.2 5.3 Faux, A.-M.; Berhin, A.; Dauguet, N.; Bertin, P. (2014). "Sex chromosomes and quantitative sex expression in monoecious hemp (Cannabis sativa L.)". Euphytica. 196 (2): 183–197. doi:10.1007/s10681-013-1023-y.
- ↑ Baek, Y.; Vergara, D. (2025). "A review of sexual strategies in Cannabis sativa L. under genomic and environmental controls". Agrosystems, Geosciences & Environment. 8 (1) e70050. doi:10.1002/agg2.70050.
- ↑ 7.0 7.1 7.2 Petit, J.; Salentijn, E.M.J.; Paulo, M.-J.; Denneboom, C.; Trindade, L.M. (2020). "Genetic architecture of flowering time and sex determination in hemp (Cannabis sativa L.): a genome-wide association study". Frontiers in Plant Science. 11 569958. doi:10.3389/fpls.2020.569958.
- ↑ 8.0 8.1 8.2 8.3 Prentout, D.; Razumova, O.; Rhoné, B.; Badouin, H.; Henri, H.; Feng, C.; Käfer, J.; Karlov, G.; Marais, G.A.B. (2020). "An efficient RNA-seq-based segregation analysis identifies the sex chromosomes of Cannabis sativa". Genome Research. 30 (2): 164–172. doi:10.1101/gr.251207.119.
- ↑ Peil, A.; Flachowsky, H.; Schumann, E.; Weber, W.E. (2003). "Sex-linked AFLP markers indicate a pseudoautosomal region in hemp (Cannabis sativa L.)". Theoretical and Applied Genetics. 107 (1): 102–109. doi:10.1007/s00122-003-1212-5.
- ↑ Sakamoto, K.; Ohmido, N.; Fukui, K.; Kamada, H.; Satoh, S. (2000). "Site-specific accumulation of a LINE-like retrotransposon in a sex chromosome of the dioecious plant Cannabis sativa". Plant Molecular Biology. 44 (6): 723–732. doi:10.1023/A:1026574405717.
- ↑ 11.0 11.1 Sakamoto, K.; Abe, T.; Matsuyama, T.; Yoshida, S.; Ohmido, N.; Fukui, K.; Satoh, S. (2005). "RAPD markers encoding retrotransposable elements are linked to the male sex in Cannabis sativa L.". Genome. 48 (5): 931–936. doi:10.1139/g05-056.
- ↑ Razumova, O.V.; Divashuk, M.G.; Alexandrov, O.S.; Karlov, G.I. (2023). "GISH painting of the Y chromosomes suggests advanced phases of sex chromosome evolution in three dioecious Cannabaceae species (Humulus lupulus, H. japonicus, and Cannabis sativa)". Protoplasma. 260 (1): 249–256. doi:10.1007/s00709-022-01774-x.
- ↑ Grassa, C.J.; Weiblen, G.D.; Wenger, J.P.; Dabney, C.; Poplawski, S.G.; Motley, S.T.; Michael, T.P.; Schwartz, C.J. (2021). "A new Cannabis genome assembly associates elevated cannabidiol (CBD) with hemp introgressed into marijuana". New Phytologist. 230 (4): 1665–1679. doi:10.1111/nph.17243.
- ↑ Laverty, K.U.; Stout, J.M.; Sullivan, M.J.; Shah, H.; Gill, N.; Holbrook, L.; Deikus, G.; Sebra, R.; Hughes, T.R.; Page, J.E.; van Bakel, H. (2019). "A physical and genetic map of Cannabis sativa identifies extensive rearrangements at the THC/CBD acid synthase loci". Genome Research. 29 (1): 146–156. doi:10.1101/gr.242594.118.
- ↑ Faux, A.-M.; Draye, X.; Flamand, M.-C.; Occre, A.; Bertin, P. (2016). "Identification of QTLs for sex expression in dioecious and monoecious hemp (Cannabis sativa L.)". Euphytica. 209 (2): 357–376. doi:10.1007/s10681-016-1641-2.
- ↑ 16.0 16.1 Akagi, T.; Segawa, T.; Uchida, R.; Tanaka, H.; Shirasawa, K.; Yamagishi, N.; et al. (2025). "Evolution and functioning of an X–A balance sex-determining system in hops". Nature Plants. 11 (7): 1339–1352. doi:10.1038/s41477-025-02017-6.
- ↑ 17.0 17.1 Mohan Ram, H.Y.; Sett, R. (1982). "Induction of fertile male flowers in genetically female Cannabis sativa plants by silver nitrate and silver thiosulphate anionic complex". Theoretical and Applied Genetics. 62 (4): 369–375. doi:10.1007/BF00275107.
- ↑ Moliterni, V.M.C.; Cattivelli, L.; Ranalli, P.; Mandolino, G. (2004). "The sexual differentiation of Cannabis sativa L.: a morphological and molecular study". Euphytica. 140 (1–2): 95–106. doi:10.1007/s10681-004-4758-7.
- ↑ Shi, J.; Toscani, M.; Dowling, C.A.; Schilling, S.; Melzer, R. (2025). "Identification of genes associated with sex expression and sex determination in hemp (Cannabis sativa L.)". Journal of Experimental Botany. 76 (1): 175–190. doi:10.1093/jxb/erae429.
- ↑ 20.0 20.1 20.2 Mandolino, G.; Carboni, A.; Forapani, S.; Faeti, V.; Ranalli, P. (1999). "Identification of DNA markers linked to the male sex in dioecious hemp (Cannabis sativa L.)". Theoretical and Applied Genetics. 98 (1): 86–92. doi:10.1007/s001220051043.
- ↑ Törjék, O.; Bucherna, N.; Kiss, E.; Homoki, H.; Finta-Korpelová, Z.; Bócsa, I.; Nagy, I.; Heszky, L.E. (2002). "Novel male-specific molecular markers (MADC5, MADC6) in hemp". Euphytica. 127 (2): 209–218. doi:10.1023/A:1020204729122.
- ↑ Mandolino, G.; Carboni, A.; Bagatta, M.; Moliterni, V.M.C.; Ranalli, P. (2002). "Occurrence and frequency of putatively Y chromosome linked DNA markers in Cannabis sativa L.". Euphytica. 126 (2): 211–218. doi:10.1023/A:1016382128401.