Promising high fidelity genetic markers for sexing <i>Cannabis sativa</i> seedlings

The uses of Cannabis sativa, a dioecious species with an XY sex chromosome system, are varying from fiber and oil to cannabinoids, among others. In most cases, males are undesirable, and the sexual dimorphism at immature plants is too subtle for reliable phenotypic sexing, making genetic approaches promising. In this technical note, we present a multiplex PCR mix that includes 2 markers of Y-specific coding regions and 1 autosomal control marker. This PCR mix, tested across 12 hemp-type cultivars, encompassing approximately 200 individuals, achieved a 99.5% true positive rate in identifying the sex of C. sativa samples. Despite being tested on a limited number of germplasm, we believe that these markers offer a promising solution for sexing a larger diversity of C. sativa seedlings.