Investigation of chloroplast regions rps16 and clpP for determination of Cannabis sativa crop type and biogeographical origin

Abstract

Cannabis sativa can be classified as either hemp (a legal crop containing less than 0.3% delta-9-tetrahydrocannabinol, THC) or marijuana (an illegal drug containing more than 0.3% THC). Despite its legalization in 33 states for medicinal or recreational use, marijuana remains the most commonly used illicit drug in the USA, and it is heavily trafficked into and within the country. Discriminating between marijuana and hemp is critical to the legal process. Genetic analysis provides a means of analyzing samples unsuitable for chemical analysis, and in addition to discriminating between crop types, DNA may be able to determine the biogeographical origin of samples. In addition, the sharing of rare haplotypes between different seizures may be useful for linking cases and providing investigative leads to law enforcement. This study evaluates the potential of two highly polymorphic regions of the chloroplast genome of C. sativa, rps16 and clpP, to be used for determination of crop type and biogeographical origin. Custom fragment analysis and SNaPshot™ assays were developed to genotype nine polymorphic loci in hemp samples from the USA and Canada, marijuana samples from USA-Mexico and Chile, and medical marijuana samples from Chile. Haplotype analysis revealed eight haplotypes. Only Canadian hemp could be completely differentiated from the other sample groups by haplotype. Phylogenetic analysis and principal component analysis suggested a closer relationship among USA-Mexico marijuana, Chilean marijuana and medical marijuana, and USA hemp. Genotyping additional polymorphisms in future studies is expected to reveal further differences between these sample groups.