Sex chromosomes in cannabis and hop were identified a century ago because of their obvious visible differences in size (heteromorphy). However, we know little about the genes they contain that control the development of the inflorescences. Here we assembled genomes, with phased sex chromosomes, for hop and cannabis. The XY chromosomes share an origin prior to the divergence between the genera around 36 MYA. Due to the inheritance patterns of the XYs, the male-specific region of the Y is highly-degenerated, with substantial gene loss, while the X shows faster rates of molecular evolution. Consistent with the hypothesis that these species lack an active-Y system, no clear sex-determining genes reside on the Y. Instead, an X-linked homolog of aminocyclopropane-1-carboxylate synthase (ACS), that is involved in the ethylene biosynthesis pathway, determines the fate of the female inflorescence. Beyond sex determination, the sex chromosomes contribute to the sexual dimorphism in ecology and physiology and have played a role in the domestication and breeding of these species.

Abstract The field of genomics has enabled extraordinary progress in horticultural crop research. However, there is still a need for cost-effective, high-resolution technologies flexible to the diversity found in emerging crops. To this end, we introduce CannSelect, a high-quality genotyping platform for Cannabis sativa. Designed for use in diversity analyses and trait mapping, probe targets were selected from four genotyped diversity panels and a curated gene list. This platform has been used to effectively map day-neutrality in a segregating population to the Autoflower1 locus with average capture efficiencies of 88.5%. With broad genome coverage, demonstrated target specificity, and reproducibility, CannSelect is expected to perform well across the diversity of C. sativa. We describe the methodology used to design CannSelect v1.0 and performance metrics for testing capture efficiency and target alignment in diverse genome assemblies. The CannSelect platform represents a robust and scalable, genome-wide genotyping tool for C. sativa researchers and breeders.

Genomic characterization of Cannabis sativa has accelerated rapidly in the last decade as sequencing costs have decreased and public and private interest in the species has increased. Here, we present seven new chromosome-level haplotype-phased genomes of C. sativa. All of these genotypes were alive at the time of publication, and several have numerous years of associated phenotype data. We performed a k-mer-based pangenome analysis to contextualize these assemblies within over 200 existing assemblies. This allowed us to identify unique haplotypes and genomic diversity among Cannabis sativa genotypes. We leveraged linkage maps constructed from F2 progeny of two of the assembled genotypes to characterize the recombination rate across the genome showing strong periphery-biased recombination. Lastly, we re-aligned a bulk segregant analysis dataset for the major-effect flowering locus Early1 to several of the new assemblies to evaluate the impact of reference bias on the mapping results and narrow the locus to a smaller region of the chromosome. These new assemblies, combined with the continued propagation of the genotypes, will contribute to the growing body of genomic resources for C. sativa to accelerate future research efforts.

Cannabinoid hemp is a dioecious crop where pistillate plants are selectively grown to maximize cannabinoid yields. Errant pollination can reduce pistillate flower yields, cannabinoid concentrations, and lead to unmarketable flowers due to the presence of undesirable seeds. We compared pollen sensitivity and agronomic performance of diploid (2n = 2x = 20 chromosomes) and triploid (2n = 3x = 30 chromosomes) cannabinoid hemp in open-field conditions. The high-CBD variety Lifter and high-CBG variety White CBG were evaluated in both their diploid and triploid counterparts in fields with and without pollen. Trials were conducted during the 2021 and 2022 field seasons in Kentucky, New York, and North Carolina (n = 6 site years). Triploids produced taller and wider plants than diploids; however, this did not result in yield differences in the pollen-free environment. In the presence of pollen, triploid Lifter and White CBG produced 87% and 77% fewer seeds than their diploid counterparts, respectively. Increased seed production in diploids also resulted in a significant reduction of seed-free biomass and cannabinoid concentrations compared to triploids. In the absence of pollen, we did not find any appreciable differences in seed-free biomass or cannabinoid concentrations between triploids and diploids. Though not completely pollen-insensitive, triploidy is an effective means to reduce seed production and improve yields and quality in cannabinoid hemp in the presence of pollen. Triploid cannabinoid hemp produces significantly less seed than diploids in the presence of pollen. Seed production in the presence of pollen reduced floral biomass and cannabinoid concentrations. Minimal biometric and yield differences were observed between triploid and diploid hemp in the absence of pollen.

Powdery mildew (PM) in Cannabis sativa is most frequently caused by the biotrophic fungus Golovinomyces ambrosiae. Based on previously characterized variation in susceptibility to PM, biparental populations were developed by crossing the most resistant cultivar evaluated, 'FL 58', with a susceptible cultivar, 'TJ's CBD'. F1 progeny were evaluated and displayed a range of susceptibility, and two were self-pollinated to generate two F2 populations. In 2021, the F2 populations (n = 706) were inoculated with PM and surveyed for disease severity. In both F2 populations, 25% of the progeny were resistant, while the remaining 75% showed a range of susceptibility. The F2 populations, as well as selected F1 progeny and the parents, were genotyped with a single-nucleotide polymorphism array, and a consensus genetic map was produced. A major effect quantitative trait locus on C. sativa chromosome 1 (Chr01) and other smaller-effect quantitative trait loci (QTL) on four other chromosomes were identified. The most associated marker on Chr01 was located near CsMLO1, a candidate susceptibility gene. Genomic DNA and cDNA sequencing of CsMLO1 revealed a 6.8-kb insertion in FL 58, relative to TJ's CBD, of which 846 bp are typically spliced into the mRNA transcript encoding a premature stop codon. Molecular marker assays were developed using CsMLO1 sequences to distinguish PM-resistant and PM-susceptible genotypes. These data support the hypothesis that a mutated MLO susceptibility gene confers resistance to PM in C. sativa and provides new genetic resources to develop resistant cultivars. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Cannabis sativa is cultivated for multiple uses including the production of cannabinoids. In developing improved production systems for high-cannabinoid cultivars, scientists and cultivators must consider the optimization of complex and interacting sets of morphological, phenological, and biochemical traits, which have historically been shaped by natural and anthropogenic selection. Determining factors that modulate cannabinoid variation within and among genotypes is fundamental to developing efficient production systems and understanding the ecological significance of cannabinoids. Thirty-two high-cannabinoid hemp cultivars were characterized for traits including flowering date and shoot-tip cannabinoid concentration. Additionally, a set of plant architecture traits, as well as wet, dry, and stripped inflorescence biomass were measured at harvest. One plant per plot was partitioned post-harvest to quantify intra-plant variation in inflorescence biomass production and cannabinoid concentration. Some cultivars showed intra-plant variation in cannabinoid concentration, while many had a consistent concentration regardless of canopy position. There was both intra- and inter-cultivar variation in architecture that correlated with intra-plant distribution of inflorescence biomass, and concentration of cannabinoids sampled from various positions within a plant. These relationships among morphological and biochemical traits will inform future decisions by cultivators, regulators, and plant breeders.

In the decades since the first cannabinoids were identified by scientists, research has focused almost exclusively on the function and capacity of cannabinoids as medicines and intoxicants for humans and other vertebrates. Very little is known about the adaptive value of cannabinoid production, though several hypotheses have been proposed including protection from ultraviolet radiation, pathogens, and herbivores. To test the prediction that genotypes with greater concentrations of cannabinoids will have reduced herbivory, a segregating F2 population of Cannabis sativa was leveraged to conduct lab- and field-based bioassays investigating the function of cannabinoids in mediating interactions with chewing herbivores. In the field, foliar cannabinoid concentration was inversely correlated with chewing herbivore damage. On detached leaves, Trichoplusia ni larvae consumed less leaf area and grew less when feeding on leaves with greater concentrations of cannabinoids. Scanning electron and light microscopy were used to characterize variation in glandular trichome morphology. Cannabinoid-free genotypes had trichomes that appeared collapsed. To isolate cannabinoids from confounding factors, artificial insect diet was amended with cannabinoids in a range of physiologically relevant concentrations. Larvae grew less and had lower rates of survival as cannabinoid concentration increased. These results support the hypothesis that cannabinoids function in defense against chewing herbivores.

Hemp (Cannabis sativa L.) research and commercial production has recently experienced a global revival motivated by passage of laws reversing long-standing prohibitions and by expansion in markets. Collaborative research has been initiated in response to renewed interest in hemp production, such as the American Society for Horticultural Science Hemp Research and Extension Professional Interest Group (ASHS Hemp). Collaborators new to this crop have identified a lack of standard definitions, descriptions, and procedures for cohesive study specific to hemp production. Standards are necessary for synthesis of data gathered across research and industry programs. ASHS Hemp convened a workshop of hemp researchers and industry representatives to establish consensus on a minimum set of standards for research data and industry assessments. The resulting morphology and physiology standards developed at the workshop are presented here with a focus on plant height, flowering time, and crop quality. Plant height was defined as the vertical distance between the root crown at the soil surface and the stem node (or tip) of the apical meristem of the tallest branch. Plant height was importantly distinguished from stem length and canopy height, which may differ based on pruning and management of the plant. Flowering time was defined to indicate date of initiation of inflorescence development as the earliest day terminal flowering clusters appear visually. Flowering time was distinguished from solitary flowering behavior and floral maturity. Crop quality was determined to be a feature that should be established first by industry based on market standards and then subsequently adopted by researchers targeting outcomes in specific areas. A standard moisture content for dry flower, seed, and straw must be established. A moisture content of 10% to 12% was identified as a current standard for floral yield, whereas 8% was identified as a moisture content standard for seed crops. Bast-to-hurd ratio and decortication efficiency were fiber quality metrics identified for minimum standards, and thousand seed weight, protein content, oil content, and oil composition were considered for minimum seed quality standards. The hemp research community is well positioned to standardize genomic references and establish best management practices for production targets. These efforts would be assisted by the adoption of the proposed standard definitions, descriptions, and procedures decided by consensus at the ASHS Hemp 2022 workshop.

Flowering time is an important trait for all major market classes of hemp (Cannabis sativa), affecting yields and quality of grain, fiber, and cannabinoids. C. sativa is usually considered a short-day plant, flowering once night length reaches a critical threshold. Variations in flowering time within and across cultivars in outdoor grown populations have been previously identified, likely corresponding to genetic differences in this critical night length. Further, some C. sativa are photoperiod insensitive, colloquially referred to as “autoflowering.” This trait has anecdotally been described as a simple recessive trait with major impacts on phenology and yield. In this work, the locus responsible for the “autoflower” trait (Autoflower1), as well as a major-effect flowering time locus, Early1, were mapped using bulked segregant analysis. Breeder-friendly high-throughput molecular marker assays were subsequently developed for both loci. Also detailed are the flowering responses of diverse cultivars grown in continuous light and the result of crossing two photoperiod insensitive cultivars of differing pedigree.

Cannabis sativa is cultivated for multiple uses including the production of cannabinoids. In developing improved production systems for high-cannabinoid cultivars, scientists and cultivators must consider the optimization of complex and interacting sets of morphological, phenological, and biochemical traits, which have historically been shaped by natural and anthropogenic selection. Determining factors that modulate cannabinoid variation within and among genotypes is fundamental to developing efficient production systems and understanding the ecological significance of cannabinoids. Thirty-two high-cannabinoid hemp cultivars were characterized for traits including flowering date and shoot-tip cannabinoid concentration.Additionally, a set of plant architecture traits, as well as wet, dry, and stripped inflorescence biomass were measured at harvest. One plant per plot was partitioned post-harvest to quantify intra-plant variation in inflorescence biomass production and cannabinoid concentration. Some cultivars showed intra-plant variation in cannabinoid concentration, while many had a consistent concentration regardless of canopy position. There was both intra- and inter-cultivar variation in architecture that correlated with intra-plant distribution of inflorescence biomass, and concentration of cannabinoids sampled from various positions within a plant. These relationships among morphological and biochemical traits will inform future decisions by cultivators, regulators, and plant breeders as well as our broader understanding of intra-plant variation of specialized biochemicals.Highlight In-season hemp plant architecture measurements can predict post-harvest traits related to the distribution of biomass and concentration of cannabinoids.### Competing Interest StatementThe authors have declared no competing interest.* (THC) : Δ9□tetrahydrocannabinol (CBD) : Tetrahydrocannabinolic acid (THCA) Cannabidiol (CBDA) : Cannabidiolic acid (CBCA) : Cannabichromenic acid (CBC) : Cannabichromene (CBGA) : Cannabigerolic acid (CBG) : Cannabigerol (CBN) : Cannabinol (THCV) : Tetrahydrocannabivarin (THCVA) : Tetrahydrocannabivarinic acid (CBDV) : Cannabidivarin (CBDVA) : Cannabidivarinic acid (CBL) : Cannabicyclol (CBLA) : Cannabicyclolic acid (Δ8-THC) : Δ8□tetrahydrocannabinol (MCD) : Maximum canopy diameter (MCDH) : Height at maximum canopy diameter

After decades of prohibition of the cultivation and breeding of hemp ( Cannabis sativa < 0.3% ∆9-tetrahydrocannabinol [THC]), there is untapped potential for genetic improvement of this crop to provide food, feed, fiber, and medicinal compounds. Successful breeding efforts will require the development and characterization of germplasm resources, optimization of crossing methods, better understanding of sex determination, high-throughput phenotyping platforms, and deployment of genomic tools for rapid selection. This review provides a brief overview of these topics and some key opportunities for genetic improvement of hemp to support an emerging industry utilizing this newly legalized crop.

Hemp ( Cannabis sativa ) is a burgeoning crop, but research‐based information about genetic and environmental effects of cannabinoid production is limited and will be essential for expanded cultivation. There are limited data available about the effect of environmental stressors on cannabinoid content, particularly for tetrahydrocannabinol (THC) in high‐cannabidiol (CBD) hemp. To address this, five stress treatments were applied in a replicated field trial with three high‐CBD hemp cultivars and cannabinoid content was assayed over a 3‐week time‐course spanning floral maturation. Cannabinoid production in terminal inflorescence shoot tip samples of three cultivars was measured under stress imposed by flooding, ethephon, powdery mildew, herbicide, and physical wounding in a split plot design. The treatments had limited effects on cannabinoid levels, with the exception of herbicide treatment which resulted in decreased cannabinoid content. Notably, there was no evidence that any of these stresses caused THC concentration or the ratio of THC to CBD to increase at harvest.

Plants face a constant struggle to acquire nutrients and defend themselves against herbivores. Mycorrhizae are fungal mutualists that provide nutrients that can increase plant growth and alter resistance to herbivores. The beneficial effects of mycorrhizae for nutrient acquisition can depend on the quantity and type of soil nutrients available, with plants usually benefiting more in terms of growth from mycorrhizae when nutrients are limited. However, it is unclear how the addition of different nutrients might shift mycorrhizal-conferred resistance to herbivores by changing defensive secondary chemistry and nutrient availability. We conducted two concurrent greenhouse experiments to test how three levels of fertilizers (low, medium and high) and three types of fertilizers (organic, organically derived and inorganic) altered mycorrhizae-conferred resistance to herbivores in tomato plants. In addition, we looked at whether mycorrhizae-conferred resistance was driven by plant secondary metabolites or the nutrient content of the leaves. Association with mycorrhizae was associated with an increase in biomass at low levels of fertilization and decreased biomass at high levels of fertilization. Interestingly, mycorrhizae increased resistance to herbivores at medium levels of fertilization, but had no effect at low and high levels of fertilization. Mycorrhizae improved resistance most strongly when plants were fertilized with a phosphorus rich, organically derived fertilizer. In both experiments, increased resistance was correlated with changes in the plant's foliar nitrogen content. Synthesis and applications. Our study supports the potential for mycorrhizae to improve either crop growth or pest resistance under lower fertilizer conditions. However, mycorrhizae did not provide both growth and resistance benefits under any treatment. While mycorrhizae have the potential to benefit crops in in lower input systems, it may be challenging to maximize both growth and resistance benefits.

Given the dramatic rise in high-cannabinoid hemp (Cannabis sativa L.) production in the last decade, there is an increasingly urgent need to characterize available germplasm and develop knowledge to accelerate the breeding of uniform and stable cultivars. Despite persistent cultivation of hemp cultivars for grain and or fiber around the world, the diversity and genetic underpinning of key traits for breeding and cultivation are poorly understood. For 30 high-cannabidiol hemp cultivars replicated on two field sites, we sought to evaluate yield, agronomic performance, and disease resistance while also conducting a detailed study of cannabinoid accumulation over the course of floral maturation. We observed significant variation in both within and among cultivars. During the growing season, the plants clustered into five groups by growth rate and varied in flowering time from photoperiod insensitive to photoperiod sensitive with very short critical photoperiods. Based on the observed ratio of total potential cannabidiol (CBD) to total potential tetrahydrocannabinol (THC), there was segregation for cannabinoid chemotype in some seeded cultivar populations. Analysis of cannabichromene (CBC) production revealed that some cultivars had a discretely lower CBD:CBC ratio than the others. There was a continuous range of powdery mildew severity by cultivar, with one that had little to no observed powdery mildew suggesting it might have genetic resistance. Biomass production at harvest was strongly influenced by location and cultivar, and there was variation by cultivar in the relative cannabinoid production in shoot tip samples compared with whole plant samples. While our results provide preliminary guidance regarding relative performance of current cultivars, our analyses indicate a need for additional hemp breeding to provide stable, uniform, and legally compliant cultivars with improved disease resistance and flowering times optimized for the latitudes of different growing locations.

The breeding of hybrid cultivars of hemp (Cannabis sativa L.) is not well described, especially the segregation and inheritance of traits that are important for yield. A total of 23 families were produced from genetically diverse parents to investigate the inheritance of morphological traits and their association with biomass accumulation and cannabinoid yield. In addition, a novel classification method for canopy architecture was developed. The strong linear relationship between wet and dry biomass provided an accurate estimate of final dry stripped floral biomass. Of all field and aerial measurements, basal stem diameter was determined to be the single best selection criterion for final dry stripped floral biomass yield. Along with stem diameter, canopy architecture and stem growth predictors described the majority of the explainable variation of biomass yield. Within-family variance for morphological and cannabinoid measurements reflected the heterozygosity of the parents. While selfed populations suffered from inbreeding depression, hybrid development in hemp will require at least one inbred parent to achieve uniform growth and biomass yield. Nevertheless, floral phenology remains a confounding factor in selection because of its underlying influence on biomass production, highlighting the need to understand the genetic basis for flowering time in the breeding of uniform cultivars.