Most flowering plants are bisexual, meaning flowers have both male and female reproductive organs. However, about 6% of the angiosperms develop male and female flowers on separate individuals, a phenomenon called dioecy (Renner 2014). Cannabis sativa is mainly a dioecious plant, although there are some monoecious cultivars with female and male flowers on the same individual (Moliterni et al. 2004). C. sativa is diploid with a pair of sex chromosomes; hence, sex is primarily determined by an XY chromosome system, female and monoecious plants are XX and males are XY (Moliterni et al. 2004).

C. sativa is a multipurpose crop with a wide spectrum of uses, ranging from textiles and building materials that derive from the stalks, biofuel and oil from the seeds, to medicinal and recreational purposes thanks to the cannabinoids present in the female flower’s trichomes (Schilling et al. 2020). Depending on the intended use, female plants are preferred, e.g. for pharmacological applications, or male plants, e.g. for industrial applications due to their high-quality fibre content (Salentijn et al. 2019). Adult C. sativa individuals show a high degree of dimorphism (Petit et al. 2020), which makes it easy to differentiate between male and female plants phenotypically. However, at early stages of development before the onset of flowering, this sexual dimorphism is not present (Shi et al. 2024). Thus, for research and industrial purposes, there is a need for reliable methods to sex genotype the plants before flowering.

In recent years, different methods of sex genotyping have been developed. In most cases, MADC (male-associated DNA Cannabis) sequences have been used as markers with different strategies (Mandolino et al. 1999; Techen et al. 2010; Toth et al. 2020; Torres et al. 2022), some of them with great accuracy. However, it must be taken into account that MADC sequences are only associated with male individuals, presumably because they are located on the Y chromosome. Used as PCR markers, they therefore only result in amplification of male (Y chromosomal) DNA, and an autosomal control is always needed for the female or monoecious genotypes lacking a Y chromosome. Furthermore, MADC6 and some other markers are located in retrotransposons, so they may not be present in all cultivars, potentially reducing the marker’s reliability (Toth et al. 2020). Prentout et al. 2025 tried to solve this problem by identifying Y-linked genes. However, also in this assay, autosomal controls were required. Gilchrist et al. (2023) identified SNPs between the X and the Y chromosome that can be leveraged in high resolution melting analyses to distinguish male and female individuals. This is a promising approach but requires specialised instruments and it is not clear how conserved the identified SNPs are across different C. sativa cultivars.

Hence, there is still a need to identify a universal genetic marker for sexing C. sativa seedlings. Here we present an affordable, reliable and robust PCR-based CAPS (Cleaved Amplified Polymorphic Sequence) method to differentiate between XX and XY genotypes in C. sativa across cultivars, as well as a pipeline that can be used to identify markers in other dioecious species.

At the time of this study, no male C. sativa genome assembly was publicly available. The only available genome from a male individual, ‘JL_father’ (GCA_013030025.1), was assembled at the scaffold level, making it impossible to easily identify Y chromosomal sequences.

To obtain sequences which likely originated from the Y chromosome, we previously described a computational pipeline (Shi et al. 2025). The pipeline uses RNA-seq data and hence does not rely on genome assemblies containing a Y chromosome. Briefly, we used RNA-seq samples from male and female C. sativa plants from the same developmental stage and used the following criteria to identify putative Y chromosomal transcripts.

Transcripts were required to

not map to the female CBDRx (GCA_900626175.2) genome.

The pipeline resulted in the initial identification of 379 transcripts putatively originating from the Y chromosome (Shi et al. 2025).

To build on the Shi et al. 2025 method and further reduce the inclusion of false positives, a strict TPM expression level filter (average TPM > 10 in male samples, and TPM = 0 in all female samples) was applied. This allowed us to identify 25 transcripts that originated from the Y chromosome with relatively high certainty (Supplementary Data S1). From those transcripts, the genomic sequence was retrieved through a BLAST search of the ‘JL_father’ assembly. Even though this genome is not assembled at the chromosome level, blasting the mRNA sequences of the 25 putatively Y chromosomal transcripts allowed us to identify scaffolds that are most likely part of the Y chromosome. The genomic sequences of the 25 genes were then aligned to putatively corresponding sections on the X chromosome identified through a BLAST search of the CBDRx genome assembly (GCA_900626175.2). The genes with the highest sequence divergence between X and Y that were deemed the most suitable for a PCR assay to distinguish XX and XY genotypes were FE.chrY.t5, FE.chrY.t9, FE.chrY.t122, FE.chrY.t25, FE.chrY.t42 and FE.chrY.t47 (Supplementary Data S2, transcript naming after Shi et al. 2025). Of those, we focussed on FE.chrY.t9.

FE.chrY.t9 is homologous to the PRECOCIOUS DISSOCIATION OF SISTERS 5 (PDS5) gene from Arabidopsis thaliana and was therefore renamed CsPDS5. This gene is positioned at 80.6 Mbp in chromosome X in what we believe is an ancient stratum (Toscani et al. 2025). PDS5 proteins play a crucial role in regulating genome architecture by influencing the 3D chromatin organisation; the A. thaliana PDS5 genes encode cohesin cofactors, the depletion of which compromises development, fertility and homologous recombination (HR) during DNA repair mechanisms, and it also causes subtle meiotic alterations (Pradillo et al. 2015).

Although both the X and Y chromosome versions of CsPDS5 are diverged, there is a highly conserved region, which allowed the design of a common pair of primers that amplified a 419 bp region from the X as well as from the Y chromosome. Importantly, the Y version contains an EcoRI restriction enzyme recognition inside the amplicon, which is not present in the X allele. Thus, a CAPS assay in which the PCR products are subjected to digestion with EcoRI results in a 157 bp and a 262 bp band for Y chromosome amplicons, while X chromosome amplicons remain at their original 419 bp length (Fig. 1). Hence, visualisation via an agarose gel results in only one band at 419 bp for female and monoecious (XX) samples, while the male (XY) samples display three bands, one corresponding to the amplicon derived from the X chromosome at 419 bp and two smaller bands at 157 bp and 262 bp, corresponding to the digested amplicon derived from the Y chromosome (Fig. 1). We term this assay CsPDS5-CAPS hereafter.Fig. 1

Overview over CsPDS5-CAPS for the C. sativa sex genotyping. A schematic representation of CsPDS5 with exons and introns depicted as boxes and lines, respectively (a). The direction of transcription is indicated by arrows. The principle of the CAPS PCR-based genotyping showing amplicons in green and the EcoRI digestion site in red (b). Agarose gel electrophoresis of a female (XX) and male (XY) sample pre- (c) and post- (d) digestion. White and orange arrowheads indicate undigested (419 bp) and digested (157 and 262 bp) amplicons, respectively. Size marker: GeneRuler DNA Ladder Mix (Fermentas)

The assay was initially tested using the dioecious cultivar ‘FINOLA’. Leaf samples collected at the flowering stage, when sexual dimorphism was fully apparent, were used to verify that the sex as determined by CsPDS5-CAPS agreed with the phenotypic sex. Subsequently, ‘FINOLA’ samples at different developmental stages (from germination to flowering) and from different tissues (cotyledon and leaf) were tested, and the sex as determined by CsPDS5-CAPS was compared to the phenotypic sex. In total, 312 ‘FINOLA’ samples were tested, and the CsPDS5-CAPS genotyped sex matched the phenotypic sex in all cases.

Subsequently, we assessed the performance of CsPDS5-CAPS for 14 different hemp-type cultivars (Figure 2, Table 1), six dioecious (‘FINOLA’, ‘Kompolti’, ‘CSR-1’, ‘CFX-2’, ‘Estica’ and ‘Enectarol’) and eight monoecious (‘Felina 32’, ‘Bialobrzeski’, ‘Santhica 27’, ‘Earlina 8 FC’, ‘Fedora 17’, ‘Ferimon’, ‘Futura 75’ and ‘Henola’). For all dioecious cultivars, our genotyping distinguished male and female individuals (Fig. 2a). Monoecious cultivars are typically reported to carry two X chromosomes (Faux et al. 2014) and consequently appeared genotypically female in our assay (Fig. 2b).

To test the suitability of CsPDS5-CAPS for breeding projects, we further tested the assay on progeny plants resulting from crosses between different hemp-type cultivars. For this purpose, 137 F2 individuals from a previously established ‘FINOLA’ × ‘Felina 32’ cross (Dowling et al. 2024) were tested. As ‘FINOLA’ is dioecious and ‘Felina 32’ is monoecious, the F2 population comprises male, female and monoecious individuals (Dowling et al 2024). In all 137 cases tested, our genotyping results were in agreement with the phenotypic sex (Fig. 2c). We next tested F1 individuals from five additional crosses (‘FINOLA’ × ‘Earlina 8 FC’, ‘FINOLA’ × ‘Ferimon’, ‘FINOLA’ × ‘Santhica 27’, ‘FINOLA’ × ‘Fedora 17’ and ‘Felina 32’ × ‘Estica’). Also in this case, the CsPDS5-CAPS results were in perfect agreement with the phenotypic sex.

In total, over 500 different samples were used to test our genotyping assay (Table 1). In all cases, the genetic and phenotypic sex matched, showing that the CsPDS5-CAPS is a highly reliable assay and produces robust results independently of the cultivar, crossing, developmental stage or tissue.

To improve the CsPDS5-CAPS assay, we adapted it into a high-throughput TaqMan assay. The method is based on a pair of primers that amplify a ≈200 bp fragment of the CsPDS5 gene region, in both the X and Y versions, which contains the EcoRI restriction site. A male-specific probe was designed to bind at the restriction site present in the Y chromosome, while a female-specific probe binds to the corresponding sequence in the X chromosome. Each probe is labelled with a different reporter dye: the Fem probe with VIC and the Male probe with FAM. Hence, in this TaqMan PCR setup, XX samples produce only a VIC signal, while XY samples yield both VIC and FAM signals. We validated this assay with 22 ‘FINOLA’ samples (11 male and 11 female) previously genotyped using CsPDS5-CAPS, and the results were consistent between both approaches, as shown in the allelic discrimination plot (Fig. 2d).Fig. 2

Genotyping results for different cultivars of C. sativa. CsPDS5-CAPS genotyping for female (XX) and male (XY) samples from six different dioecious cultivars (a), eight different monoecious cultivars (b) and 11 representative F2 individuals of a ‘FINOLA’ x ‘Felina 32’ cross (c). Cultivar names and phenotypic sex are depicted above the gel image (X’X’ = Monoecious (green), XX = Female (red) and XY = male (blue)). Gel images depict PCR fragments after EcoRI digestion. White and orange arrowheads indicate undigested (419 bp) and digested (157 and 262 bp) amplicons, respectively. Size marker: GeneRuler DNA Ladder Mix (Fermentas). Panel (d) shows the allelic discrimination plot of the 22 'FINOLA' samples analysed using the TaqMan SNP genotyping assay targeting the CsPDS5 polymorphism. The plot displays relative fluorescence units (RFU), with the X-axis representing the VIC signal (X allele) and the Y-axis representing the FAM signal (Y allele). Red dots correspond to XX samples, green dots to XY samples and black squares represent NTCs (no template controls)

We next analysed whether the CsPDS5 polymorphism utilised in our assay is also conserved in other hemp and marijuana cultivars that were not tested by PCR here but for which the genome sequences are available. We aligned the X and Y chromosomal versions of CsPDS5 regions from different cultivars: the marijuana cultivars ‘Ace High 3–2’ and ‘SourDiesel’, the high CBD hemp cultivars ‘Pink Pepper’ and ‘GoldenRedwood’ and a US hemp landrace variety named ‘BooneCounty’ (Lynch et al. 2025), and compared to the CsPDS5 sequence of ‘Kompolti’, for which we successfully conducted the CsPDS5-CAPS assay (Fig. 2a). The alignment shows that the CsPDS5 primer binding sites are conserved in all sequences, while the EcoRI restriction recognition site in CsPDS5 is present on all analysed Y chromosomal but absent in X chromosomal sequences (Fig. 3). This demonstrates that the selected region is extremely conserved across the different cultivars and that the assay introduced here is potentially useful for a wide variety of C. sativa cultivars and landraces.Fig. 3

CsPDS5 sequence from different cultivars of C. sativa. Alignment of three non-consecutives regions of CsPDS5. The red frame denotes the forward primer binding site (a), the EcoRI restriction site (b) and the reverse primer binding site (c). ChrX and chrY sequences from C. sativa cultivars ‘GoldenRedwood’ (GRM), ‘Ace High 3–2’ (AH3M), ‘BooneCounty’ (BCM), ‘SourDiesel’ (SODL) and ‘Kompolti’ (KOMP) cultivars, along with the X chromosome version of ‘Pink Pepper’ as reference genome cultivar. Bases conserved in the majority of sequences have a yellow background. SNPs in the primer binding sites were incorporated as degenerate bases in the primer sequences (Table 2)

Here, we described the identification of a highly conserved sex marker gene, CsPDS5, in C. sativa and the development of an affordable, robust and reliable PCR-based method that is consistent among cultivars, crosses, tissues and developmental stages.

The genotyping method consists of amplifying and digesting a region of the CsPDS5 gene, which has an enzyme restriction site on the Y version of this gene that is absent on the X version. The assay only requires the PCR reagents, a thermocycler and the EcoRI enzyme, which makes it an extremely affordable method, as only basic instruments and reagents, present in every molecular biology laboratory, are needed.

The pipeline (Fig. 4) used here to identify the sex marker genes does not require an assembled Y chromosome, typically one of the more challenging tasks in genome assembly. Instead, gene expression data from male and female individuals are used to infer Y chromosomal transcripts, and genomic data are then leveraged to identify X and Y chromosomal gene copies. This basic workflow should apply to other species (plants and animals) with sex chromosomes and may therefore serve as a blueprint to identify molecular markers for sex in a range of different species. Although we leveraged the presence of a genome assembly of a male plant to retrieve the transcripts corresponding to the Y chromosome genomic sequence, the method can be applied also to species without genome assemblies if exploiting transcript polymorphisms instead of intron polymorphisms.

It is noteworthy that male-biased expression to identify Y chromosomal genes was an important step in our pipeline as no assembled Y chromosome was available, and it was hence difficult to identify genes encoded on the Y chromosome by other means. However, if a high-quality Y and X chromosome sequence is available, then identification of gametologs that have diverged in sequence might be a more viable approach.

We have tested the CsPDS5-CAPS assay on more than 500 samples from 14 hemp-type cultivars, six crosses, different tissues and developmental stages, and in all of the cases, the genetic and phenotypic sex matched, showing an accuracy of 100%. Analyses of publicly available genomes further demonstrated the presence of the EcoRI restriction site and primer binding sites also in other hemp and marijuana cultivars, indicating that the CsPDS5 polymorphism is well conserved in C. sativa. Other C. sativa sex markers, like MADC6, are located in retrotransposons and may have experienced copy number variations and transposition (Toth et al. 2020). PDS5 genes have an essential role in the function of the cohesin complex in many eukaryotes (Panizza, et al. 2000), and thus, CsPDS5 might be a functional gene and not be subject to the same constraints as MADC6.

Also, since CsPDS5 is present on both the X and the Y chromosomes, it simultaneously provides information about XX and XY genotypes using a single primer pair, which is different from other sex genotyping methods (Techen et al. 2010; Toth et al. 2020; Torres et al. 2022; Prentout, et al. 2025).

Our assay was developed to provide a robust sex genotyping method using basic laboratory equipment. However, we further adapted the assay into a high-throughput method using a TaqMan SNP genotyping assay, which was successfully validated on 22 ‘FINOLA’ samples, which could be used in larger breeding programmes. Given the assay is based on a 3 bp difference between X and Y chromosomal CsPDS5 copies (Fig. 3), the marker is expected to be also suitable for other high-throughput methods such as PACE or amplicon sequencing.

The sex expression of C. sativa is known to be influenced by various factors, and the relative contribution of genetic vs. environmental factors for sex expression remains somewhat unclear (reviewed in Schilling et al. 2020). The observation that our molecular sex marker was in complete agreement with the phenotypic sex for hundreds of samples, including a mapping population that originated from a cross of a dioecious with a monoecious cultivar, illustrates that genetic components play a very strong role in sex determination in C. sativa. Unquestionably, external factors like the application of silver nitrate, an ethylene inhibitor that leads to the development of male flowers on female plants, can modify the sex expression (Galloch 1978; Truta, et al. 2007). However, our observations indicate that under conventional growth conditions, sex is almost solely determined by the XY sex determination system.

Below is the link to the electronic supplementary material.

Supplementary file1 (FA 41 KB) Data S1: 25 putatively Y chromosomal transcripts