Development of Sequence Characterized Amplified Region Marker for Early Sex Determination of Cannabis sativa L. using Multiplex-Polymerase Chain Reaction

BACKGROUND: Cannabis are typically dioecious, with female and male reproductive parts located on separate plants. Female cannabis plants are preferred for the production of cannabinoids, terpenes, and other valuable compounds synthesized within the female floral tissue. Female and male plants look identical during seedling and vegetative phases, and only after transition to flowering phase can male, female, and monoecious plants be distinguished. Early identification of the sex of cannabis plant can conserve time and money by avoiding the undesired sex of the plant. The objective of the present study was to provide a sex determination method for cannabis plants using the sequence characterized amplified region (SCAR) marker, and set multiplex-polymerase chain reaction (PCR) conditions for more accurate early sex determination of cannabis to overcome the disadvantages of SCAR markers. METHODS AND RESULTS: Cannabis seeds were collected and cultivated for several purposes. Leaf samples were harvested and stored during vegetative growth phase. In silico mapping of three male-specific sequences (MADC3, MADC4, and MADC6) revealed through random amplified polymorphic DNA, showed that MADC3 has a very similar sequence with the female cannabis genome. Primers were produced based on the sequences (SCAR1, SCAR2, and SCAR3, respectively) and subjected to gradient PCR. Only SCAR3 produced a male-specific band with an annealing temperature of over 61.4℃. PCR was performed on cannabis plants produced for several purposes by mixing SCAR3 and internal standard 3 (IS3). Result showed that all cannabis plants produced an IS3 band of 197 bp, but only male cannabis plants produced a SCAR3 band of 118 bp. CONCLUSIONS: Multiplex-PCR primer produced a male-specific band of 119 bp in cannabis bred for several purposes, and the presence of the 197 bp cannabis common band confirmed that PCR proceeded normally for all samples. The multiplex-PCR primer is greatly beneficial because the IS3 band is a specific sequence found only in cannabis, making it possible to differentiate cannabis from other plants in addition to determining the sex from with a single PCR run.