Nuclear, chloroplast, and mitochondrial data of a US cannabis DNA database

• Published in: International journal of legal medicine Vol. 132; no. 3; pp. 713 - 725
• Main Authors: Houston, Rachel, Birck, Matthew, LaRue, Bobby, Hughes-Stamm, Sheree, Gangitano, David
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.05.2018; Springer Nature B.V
• Subjects: Cannabis; Cannabis - genetics; Cell Nucleus - genetics; Chloroplasts; Chloroplasts - genetics; Control methods; Databases, Nucleic Acid; Deoxyribonucleic acid; DNA; DNA Fingerprinting; DNA, Mitochondrial; Drug Trafficking; Forensic Medicine; Genotype; Haplotypes; Hemp; Humans; Marijuana; Markers; Medical Law; Medicine & Public Health; Microsatellite Repeats; Original Article; Phylogenetics; Phylogeny; Plants (botany); Polymorphism, Single Nucleotide; Population; Population genetics; Quality control; Seeds; Seizing; Seizures; Brazil; Mexico; Chile; United States > US; Short tandem repeats; Mitochondrial DNA; Forensic plant science; Chloroplast DNA; DNA database; Cannabis sativa
• ISSN: 0937-9827; 1437-1596
• DOI: 10.1007/s00414-018-1798-4

As Cannabis sativa (marijuana) is a controlled substance in many parts of the world, the ability to track biogeographical origin of cannabis could provide law enforcement with investigative leads regarding its trade and distribution. Population substructure and inbreeding may cause cannabis plants to become more genetically related. This genetic relatedness can be helpful for intelligence purposes. Analysis of autosomal, chloroplast, and mitochondrial DNA allows for not only prediction of biogeographical origin of a plant but also discrimination between individual plants. A previously validated, 13-autosomal STR multiplex was used to genotype 510 samples. Samples were analyzed from four different sites: 21 seizures at the US–Mexico border, Northeastern Brazil, hemp seeds purchased in the US, and the Araucania area of Chile. In addition, a previously reported multi-loci system was modified and optimized to genotype five chloroplast and two mitochondrial markers. For this purpose, two methods were designed: a homopolymeric STR pentaplex and a SNP triplex with one chloroplast (Cscp001) marker shared by both methods for quality control. For successful mitochondrial and chloroplast typing, a novel real-time PCR quantitation method was developed and validated to accurately estimate the quantity of the chloroplast DNA (cpDNA) using a synthetic DNA standard. Moreover, a sequenced allelic ladder was also designed for accurate genotyping of the homopolymeric STR pentaplex. For autosomal typing, 356 unique profiles were generated from the 425 samples that yielded full STR profiles and 25 identical genotypes within seizures were observed. Phylogenetic analysis and case-to-case pairwise comparisons of 21 seizures at the US–Mexico border, using the Fixation Index ( F ST ) as genetic distance, revealed the genetic association of nine seizures that formed a reference population. For mitochondrial and chloroplast typing, subsampling was performed, and 134 samples were genotyped. Complete haplotypes (STRs and SNPs) were observed for 127 samples. As expected, extensive haplotype sharing was observed; five distinguishable haplotypes were detected. In the reference population, the same haplotype was observed 39 times and two unique haplotypes were also detected. Haplotype sharing was observed between the US border seizures, Brazil, and Chile, while the hemp samples generated a distinct haplotype. Phylogenetic analysis of the four populations was performed, and results revealed that both autosomal and lineage markers could discern population substructure.