Background: Cannabis sativa is a primarily dioecious angiosperm that exhibits sexual developmental plasticity. Developmental genes for staminate male flowers have yet to be elucidated; however, there are regions of male-associated DNA from Cannabis (MADC) that correlate with the formation of pollen producing staminate flowers. MADC2 is an example of a PCR-based genetic marker that has been shown to produce a 390-bp amplicon that correlates with the expression of male phenotypes. We demonstrate applications of a cost-effective high-throughput male genotyping assay and other genotyping applications of male identification in Cannabis sativa. Methods: In this study, we assessed data from 8200 leaf samples analyzed for real-time quantitative polymerase chain reaction (qPCR) detection of MADC2 in a commercial testing application offered through Steep Hill Laboratories. Through validation, collaborative research projects, and follow-up retest analysis, we observed a > 98.5% accuracy of detection of MADC2 by qPCR. We also carried out assay development for high-resolution melting analysis (HRM), loop-mediated isothermal amplification (LAMP), and TwistDx recombinase amplification (RPA) assays using MADC2 for male identification. Results: We demonstrate a robust high-throughput duplex TaqMan qPCR assay for identification of male-specific genomic signatures using a novel MADC2 qPCR probe. The qPCR cycle quotient (Cq) value representative of MADC2 detection in 3156 males and the detection of tissue control cannabinoid synthesis for 8200 samples and the absence of MADC2 detection in 5047 non-males demonstrate a robust high-throughput real-time genotyping assay for Cannabis. Furthermore, we also demonstrated the viability of using nearby regions to MADC2 with novel primers as alternative assays. Finally, we also show proof of concept of several additional commercially viable sex determination methodologies for Cannabis sativa. Discussion: In industrial applications, males are desirable for their more rapid growth and higher quality fiber quality, as well as their ability to pollinate female plants and produce grain. In medicinal applications, female cultivars are more desirable for their ability to produce large amounts of secondary metabolites, specifically the cannabinoids, terpenes, and flavonoids that have various medicinal and recreational properties. In previous studies, traditional PCR and non-high-throughput methods have been reported for the detection of male cannabis, and in our study, we present multiple methodologies that can be carried out in high-throughput commercial cannabis testing. Conclusion: With these markers developed for high-throughput testing assays, the Cannabis industry will be able to easily screen and select for the desired sex of a given cultivar depending on the application.

We are working toward a complete functional and genomic characterization of the cannabinoid synthase family in Cannabis (itself part of the larger Berberine Bridge Enzyme family). This clade, which is unique to Cannabis , includes four main subclades that appear to have arisen by a series of gene duplications. We have functionally characterized three cannabinoid synthases, in addition to the three already characterized, so that now all four subclades contain at least one characterized enzyme. The previously uncharacterized Clade C enzymes have relatively low activity and produce CBCA as their primary product. In addition, we report genomic characterization to better understand the structure and high level of copy number variation in this family. We report a pattern of shared regions immediately upstream of the cannabinoid synthase genes that suggests a specific sequence of breakpoints, and hence gene duplication events. We present a model of gene family expansion to account for the observed data, along with evidence that this expansion occurred under selective pressure. This work adds to our understanding of both the high level of variability in this family and the origin of THCA in type III plants lacking a functional THCAS gene.

Hop Latent Viroid (HLVd) is a significant threat to cannabis cultivation, impacting yield and quality. This study investigated the efficacy of the combination of meristem tip culture with thermotherapy in eliminating HLVd from thirteen cannabis cultivars. HLVd disease eradication was achieved in five cultivars, with varying degrees of success depending on the variety. The treatment’s effects were reproducible and persisted for several months. Long-read Nanopore cDNA sequencing of HLVd-positive and -negative samples from the cultivar ‘Annalee’ identified differentially regulated genes potentially involved in HLVd pathogenesis. These genes are implicated in cell wall architecture, cell signaling and defense, and growth-related synthesis, providing insights into HLVd’s molecular mechanisms. This study demonstrates the potential of thermotherapy for viroid clearance in Cannabis sativa, contributing to the understanding of HLVd pathology and mitigation strategies for large-scale cultivation.

Cannabichromene (CBC) is a nonpsychoactive cannabinoid with pharmaceutical potential that is attracting significant interest for its potential therapeutic applications. However, the factors governing CBC production in Cannabis sativa remain largely unknown. This study aimed to investigate the variation in CBC accumulation within different Type III (cannabidiol dominant) C. sativa cultivars. We hypothesized that distinct subpopulations with differing CBC production exist within these cultivars. Quantitative analysis was performed on a diverse collection of Type III C. sativa cultivars to determine CBC levels. We assessed CBC accumulation across generations and environments for select accessions. Temporal changes in CBC during flowering and its distribution in floral and leaf tissues were also examined. Our analysis revealed distinct chemotypic subpopulations exhibiting varying CBC production levels. Notably, certain cultivars consistently displayed high CBC levels across generations and diverse growing conditions, suggesting a strong genetic influence. The observed intra‐varietal variation and the stability of high‐CBC phenotypes indicate a significant genetic component in CBC biosynthesis. Understanding this complex interplay of genetic and environmental factors has important implications for breeding programs focused on enhancing CBC production in C. sativa .