The development of two fast genotyping assays for the differentiation of hemp from marijuana.

The legalization of hemp cultivation in the United States has raised the need for reliable methods to distinguish between legal hemp and illegal marijuana. Genetic analysis has emerged as a powerful tool, surpassing traditional chemical methods in specific aspects, such as analyzing trace amounts, aged samples, and different parts of the sample. Genetic differences in cannabinoid synthase genes offer promise for precise crop type determination, particularly focusing on genes like tetrahydrocannabinolic acid synthase (THCAS), cannabidiolic acid synthase (CBDAS), and cannabichromenic acid synthase (CBCAS). However, previous research faced several challenges in developing discriminatory genetic markers, including limited sample sizes, high similarity between the synthase genes, and the presence of pseudo synthase genes. A comprehensive study using Next-Generation Sequencing (NGS) introduced a differentiation flowchart based on THCAS, CBDAS, and THCAS pseudogenes. To bridge the gap between NGS and the practical requirements of crime laboratories, two rapid genotyping assays were developed: a CE-based SNaPshot™ assay and a TaqMan™ real-time PCR assay. While the SNaPshot™ assay effectively differentiated various hemp and marijuana types, differentiation was limited with marijuana samples containing THC% close to the 0.3% legal threshold (0.3%-1%). The TaqMan™ qPCR SNP genotyping assay provided quicker results, making it an efficient choice for crime laboratories. However, this method had the same limitations as the SNaPshot™ assay with addtional challenges in differentiating edible hemp seed samples, and it did not provide additional CBD information. The study also highlighted the influence of two variants of one THCAS pseudogene on chemotype determination, emphasizing the necessity for precise genetic analysis for accurate categorization of cannabis varieties.