Rapid Specific PCR Detection Based on THCAS and CBDAS for the Prediction of Cannabis sativa Chemotypes: Drug, Fiber, and Intermediate

<i>Cannabis sativa</i> L. is divided into three main groups: drug-type, intermediate-type, and fiber-type. The presence of tetrahydrocannabinol (THC) exceeding 0.2-0.3% in drug-type and intermediate <i>Cannabis</i> that utilized for recreational and medicinal purposes renders them illegal due to potential mental health implications. Fiber-type contains high cannabidiol (CBD) and low THC, making it suitable for household use such as textiles and animal feed. Accurate classification is essential to prevent misuse of the plant. High-performance thin-layer chromatography (HPTLC) and ultra-performance liquid chromatography (UPLC), used respectively for the qualitative and quantitative analyses of THC and CBD particularly in female inflorescences, categorized 85 samples of 46 cultivars used in this study into three distinct chemotypes. While chemotype analysis of a very specific organ of the plants accurately identifies <i>Cannabis</i> groups, it requires time-consuming plant development to maturity. Genotype analysis targeting tetrahydrocannabinolic acid synthase (<i>THCAS</i>) and cannabidiolic acid synthase (<i>CBDAS</i>) genes offers a faster alternative for classifying <i>Cannabis</i> types, allowing for sample determination from any part at any developmental stage of the plant. DNA sequencing allowed a phylogenetic analysis based on these genes, classifying all 85 samples of 46 cultivars into the same three groups identified by chemotype analysis. This study is the first to successfully examine the relationship between chemotype and genotype in 85 samples of 46 cultivars. Rapid identification of <i>Cannabis</i> types through genotype analysis lays the groundwork for future development of detection kits.