Multiplex high resolution melting using MADC2 and SCAR markers for sex identification of Cannabis sativa L. cultivation

Abstract This study introduces a multiplex high-resolution melting (mHRM) assay for efficient sex identification of Cannabis sativa L. using three MADC markers and nine SCAR markers. This method enables accurate and rapid determination of plant sex at early developmental stages, which is crucial for optimizing cannabis breeding programs. The optimal detection limit for bulk analysis was found to be a 1:10 male-to-female ratio. Among the markers tested, mHRM using the combination of MADC2 and Cs_197 provided both high accuracy and speed, leading to a reliable and cost-effective alternative to traditional sex identification techniques. The feasibility of the method was validated through seedling test, confirming its effectiveness in accurately identifying sex and detecting male contamination in pooled bulk samples. This study demonstrates that mHRM can provide a reliable solution for large-scale cannabis cultivation, where early sex determination is essential for maximizing yields. Furthermore, this approach contributes to the growing demand for molecular tools in plant breeding and genetic studies. The application of mHRM in cannabis sex determination offers significant potential in both industrial and research settings.