The multitude of different uses for the Cannabis sativa plant has seen it spread across the world in the forms of a medicinal drug, useful fiber, and recreational drug. The popularity of cannabis as an illicit drug has led to the widespread trafficking of cannabis across borders and has drawn the attention of law enforcement for decades. An issue that persists even with increasing legalization and regulation of cannabis markets around the world. One of the methods proposed for combatting this trafficking is the forensic genotyping of seized cannabis plants to discover their genetic and geographical origins.



DNA profiling of cannabis plants can reveal how genetically related two plant samples are. Cannabis plants that are clonally propagated will have completely identical DNA profiles, allowing them to be matched as direct relatives. Cannabis plants that are sexually propagated are open to more genetic variation as a result of inbreeding during their cultivation. If this variation can be linked to a geographical area, then this could give insight into the plant's history and possible distribution.





Methods for the forensic genetic analysis of C. sativa



Autosomal DNA and organelle markers are thought to be key to studying cannabis genetics. Both mitochondrial DNA (mtDNA) and chloroplast DNA (cpDNA) are inherited maternally and generally have low mutation rates, so they could be used to discriminate between samples from different backgrounds. Maps of both the mitochondrial and chloroplast genomes are also both available in the literature for comparison to regions of mtDNA and cpDNA obtained from samples.



Recently, a collaboration between researchers at Sam Houston State University and the U.S. Department of Homeland Security worked with a DNA database containing 510 samples of cannabis plant matter from different sources in order to genotype autosomal and organelle DNA. The samples were from a variety of locations, with the majority being seized at the US-Mexico border with others coming from northeast Brazil and southern Chile. As well as cannabis plant matter, three types of hemp seed from America were also tested. The flower, leaf, stem and seed samples were also compared separately to examine the abundance of cpDNA in each tissue.



The autosomal genotyping was accomplished using a previously developed 13-autosomal short tandem repeat (STR) multiplex. Similarly, the organelle typing utilized a modified version of this to genotype five chloroplast and two mitochondrial markers from a subset of the original 510 sample database. For organelle typing, the researchers also developed an assay for real-time polymerase chain reaction (RT-PCR) quantitation of the cpDNA using a synthetic DNA standard.



The RT-PCR quantitation was also validated in the course of the study. The limit of detection of the assay was found to be 0.02 pg/μL, below which the linearity of the standard curve was observed to be below an R2 estimation of 0.99, which was defined as the limit of acceptance. Due to the likelihood of seized cannabis containing contaminating DNA from other plant or human sources, the RT-PCR primers were selected to promote binding to cpDNA regions that are specific to cannabis plants, namely Cscp001. Tests on non-cannabis species showed that any cross-reactivity was below the limit of detection, supporting the specificity of Cspc001. This is the first reported instance of cannabis organelle typing being successfully carried out using RT-PCR quantitation.





Statistical analysis of the database



Of the 510 samples in the study, 83% returned full STR profiles with the others returning only partial profiles due to mixtures or low template DNA in the original samples. From the full STR profiles, 356 distinguishable genotypes and 25 identical genotypes were identified. With the duplicate genotypes most likely due to the sampling of the same plant twice for tissue subsampling.



The individual study of the various plant tissue types showed that cannabis seeds and cannabis leaf held the greatest concentration of cpDNA and that this had a significant effect on the amount of cpDNA that could be effectively extracted for analysis.



Phylogenetic analysis examining the autosomal genotypes of the sample groups found that within the US-Mexico border seizure samples all samples showed a degree of relation to each other. In addition to this, each of the four geographical regions the samples came from could be distinguished using phylogenetic analysis as each geographical region displayed a statistically significant difference in genetics. There was also a stark genetic difference between the hemp samples and the US-Mexico, Brazil and Chile samples, and the Chilean samples appear to be a genetic admixture of the US-Mexico and Brazilian populations.



Given the results of the investigation into genetic relatedness and the fact that cannabis mtDNA and cpDNA is inherited uniparentally, it was also predicted that there may be some haplotype sharing between the populations. Haplotypes are groups of alleles that can be inherited together from a single parent, and as a result, can pass through many generations of reproduction with little change to their sequence. The detection of shared haplotypes can possibly indicate that samples belong to the same “family tree” and share a genetic ancestor from a past generation. This potential haplotype sharing was confirmed by the subsampling of mtDNA and cpDNA typing as only five distinguishable haplotypes were observed across the populations. Similarly to the autosomal genotypes, the hemp samples were easily distinguishable from the other populations as the hemp samples contained a distinct haplotype.





The consequences for American cannabis research



While the genetic relatedness and population substructure of samples from the US-Mexico border, Brazil and Chile may not have led to any particularly surprising results, the importance of this study goes beyond the confirmation of predicted substructures. The development of the first real-time PCR quantitation method alone could be of importance for law enforcement and cannabis researchers alike. In addition to this the study also presents a US DNA database for the nuclear, chloroplast and mitochondrial DNA of cannabis samples, which could be of great use to future cannabis genetics researchers.