Network ontology transcript annotation identifies genetic signals underlying sex determination.

Cannabis sativa L. (marijuana, hemp, cannabis) is an angiosperm species currently evolving sex chromosomes. Genetic mechanisms, primarily an XY chromosome system, dictate cannabis sex expression in dioecious populations. However, sexual expression is also governed by the interplay of hormone regulatory gene networks, influenced by both genetic and environmental factors. Within the species, some populations exhibit dioecy, monoecy, or a gradient of both. Dioecious individuals produce exclusively male or female flowers, while monoecious plants bear both male and female flowers. Remarkably, through interruption of phytohormone signal transduction via abiotic stressors, genetically male or female cannabis are able to produce flowers of the opposite sex. Previous transcriptomic analysis have identified genes associated with masculinization through the application of phytohormone signal disruption using silver thiosulfate treatment. We analyzed transcriptomic data from cannabis treated with colloidal silver to similarly induce masculinization. Using Nota (Network ontology transcript annotation), a multilayer network analysis (Random-Walk-with-Restart) tool, we identified candidate genes involved in sex-determination. Nota and a companion program Jack facilitate multi-layer network analyses, enabling discovery and annotation of gene-trait associations. Our findings highlight Nota’s robust application to enrich the genetic architecture of complex traits, particularly in non-model systems like cannabis, and complex traits such as sex determination. Our analyses identified genes associated with cell wall morphogenesis and embryogenic tissue homeostasis, indicating that silver ion treatment perturbs phytohormone signal transduction through metal ion imbalance. In this reproductive strategy, cannabis is able to make use of its widely investigated sex-determining genetic architecture to navigate transient changes in co-expression and cross-cellular signaling driving embryogenic cell wall re-patterning.