Reginald Gaudino

The chemical phenotype (chemotype) of Cannabis sativa is defined by the ratio of cannabidiolic acid (CBDA) to Δ9‐tetrahydrocannabinolic acid (THCA). Although the Mendelian segregation of these traits suggests a single‐locus biallelic system, recent sequencing and phylogenetic evidence indicate they are encoded by two distinct, tightly linked genes. The precise genomic architecture of this region, known as the B locus, has remained poorly defined. In this study, we analyzed recently released high‐quality Cannabis reference genomes to resolve the structure of the B locus. Our results demonstrate that this region functions as a supergene, characterized by suppressed recombination that facilitates Mendelian‐like switching between phenotypic states. Comparative genomic analysis reveals substantial structural polymorphism within the locus, including significant variations in gene copy number and large‐scale insertions/deletions (indels). Furthermore, we functionally characterized three previously unstudied members of the cannabinoid oxidocyclase family. We find that these enzymes primarily catalyze the production of cannabichromenic acid (CBCA), reinforcing the model that cannabinoid profile is dictated specifically by the presence and expression of THCAS or CBDAS. Finally, we mapped the expression profile of the entire berberine bridge enzyme (BBE) family, identifying widespread expression across plant tissues, including in glandular trichomes. Collectively, these findings resolve the genomic architecture of the B locus, clarify the enzymatic basis of cannabinoid profile determination, and establish a framework for understanding the evolutionary maintenance of chemotype diversity in C. sativa .

Rationale Cannabichromene (CBC) is a non-psychoactive cannabinoid attracting significant interest for its potential therapeutic applications. However, the factors governing CBC production in Cannabis sativa remain largely unknown. Objectives This study aimed to investigate the variation in CBC accumulation within different Type III (CBD-dominant) C. sativa cultivars. We hypothesized that distinct subpopulations with differing CBC production exist within these cultivars. Methods 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. Results 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. Conclusions 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 Cannabis sativa.

Background: The endocannabinoid system (ECS) is a complex signaling network that regulates diverse physiological processes, including pain, mood, metabolism, and immune response, through coordinated interactions among receptors, enzymes, and lipid-derived ligands. Despite extensive research on individual ECS components, the systems-level organization and network resilience of the ECS remain underexplored. Here, we present a systems-level analysis of the ECS that integrates protein–protein and protein–chemical interactions into a unified network framework. Methods: We constructed integrated ECS networks that combine protein–protein and protein–chemical interactions, utilizing data from multiple public databases. Network analyses were performed in Python using NetworkX to assess molecular connectivity and interaction topology. We utilized centrality measures to identify major hubs, employed community detection algorithms to examine the clustering of nodes, and performed targeted perturbations by sequentially removing the top-ranked nodes based on degree and betweenness centrality to assess network robustness. Results: Centrality analyses identified the primary cannabinoid receptors, cannabinoid receptor 1 (CNR1) and cannabinoid receptor 2 (CNR2), as major hubs with extensive connectivity to endogenous and exogenous ligands. Non-canonical receptors, including transient receptor potential vanilloid 1 (TRPV1) and G-protein coupled receptor 55 (GPR55), also emerged as highly ranked nodes across multiple centrality measures, underscoring their integrative roles within the ECS signaling pathway. Community detection revealed biologically meaningful modules centered around receptor and metabolic clusters, with CNR1, CNR2, anandamide (AEA), 2-arachidonoylglycerol (2-AG), and major phytocannabinoids maintaining key network connectivity. Perturbation analyses demonstrated that removal of top hubs, particularly CNR1, caused pronounced losses in edge connectivity and disrupted signaling pathways among cannabinoids. However, the redistribution of influence toward CNR2 and GPR55 under multi-node removal conditions revealed compensatory plasticity and resilience within the ECS network. Conclusion: This systems-level study highlights the hierarchical and robust architecture of the ECS. The identification of hub nodes, functional communities, and compensatory mechanisms provides insight into how the ECS maintains signaling integrity in the face of perturbation. These findings establish a network-based framework for studying cannabinoid biology and may inform future therapeutic strategies targeting the ECS and its interacting molecular pathways.

Rationale Cannabichromene (CBC) is a non-psychoactive cannabinoid attracting significant interest for its potential therapeutic applications. However, the factors governing CBC production in Cannabis sativa remain largely unknown. Objectives This study aimed to investigate the variation in CBC accumulation within different Type III (CBD-dominant) C. sativa cultivars. We hypothesized that distinct subpopulations with differing CBC production exist within these cultivars. Methods 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. Results 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. Conclusions 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 Cannabis sativa.

Rationale Cannabichromene (CBC) is a non-psychoactive cannabinoid attracting significant interest for its potential therapeutic applications. However, the factors governing CBC production in Cannabis sativa remain largely unknown. Objectives This study aimed to investigate the variation in CBC accumulation within different Type III (CBD-dominant) C. sativa cultivars. We hypothesized that distinct subpopulations with differing CBC production exist within these cultivars. Methods 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. Results 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. Conclusions 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 Cannabis sativa.