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

Abstract

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 ) 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.

Tandem repeats lead to sequence assembly errors and impose multi-level challenges for genome and protein databases

“…Other specialized databases have been developed recently in this regard (e.g. (122–126)), starting off from human-centered research questions and expanding to examples of many other species, such as the tobacco plant (127), Trichophytum rubrum , a fungus causing skin disease (128), or the Cannabis plant to characterize the origin of hemp seeds (US Cannabis DNA database; (129)). Despite this diversity, the majority of these databases rely on the results of well-established automated bioinformatic approaches such as the Tandem Repeats Finder (TRF) program (130) or RepeatMasker (118) to characterize repeat content.…”

Massively parallel sequencing of 12 autosomal STRs in Cannabis sativa

“…DNA extraction was performed according to Houston et al. . DNA concentrations were estimated using the Qubit™ dsDNA HS Assay Kit (Thermo Fisher Scientific) on the Qubit® 2.0 Fluorometer (Thermo Fisher Scientific) .…”

“…Additionally, Canadian-grown hemp seeds (N = 3) were purchased from three brands: Navitas TM Organics, Badia Spices Inc., and Manitoba Harvest Hemp Food. DNA extraction was performed according to Houston et al [8]. DNA concentrations were estimated using the Qubit TM dsDNA HS Assay Kit (Thermo Fisher Scientific) on the Qubit R 2.0 Fluorometer (Thermo Fisher Scientific) [21].…”

“…MPS technology has been successfully tested in the fields of medicine, microbiology, environmental, and forensic science , and offers an invaluable opportunity to expand its applications to the field of forensic plant science, specifically the genetic identification of Cannabis sativa samples. Previous studies have shown the value of STR typing for the genetic identification of marijuana . As with human identification, CE of STR markers is the gold standard for the DNA‐based identification of marijuana for forensic or intelligence purposes.…”

Interpol review of controlled substances 2016–2019

“…Marijuana (Genetic and/or Proteomic Analyses):2016 13-loci STR multiplex method to accurately genotype 199 Cannabis sativa samples from 11 U.S. Customs and Border Protection seizures [ 698 ]; loop-mediated isothermal amplification DNA -based assay for the identification of c. sativa [ 699 ]; rbcL gene for genetic discrimination of seized c. sativa [ 700 ]; comparison of three DNA markers for the detection of male genotype in industrial hemp and medicinal cannabis plants [ 701 ]; review of genetic and genomic tools for cannabis sativa [ 702 ]; RNA-Seq analysis for phenotypic differentiation of fiber-type and seed-type cannabis [ 703 ]; analysis of the nuclear genomic diversity among 340 Cannabis varieties, including fiber and seed oil hemp, high cannabinoid drug-types, and feral population [ 704 ]; analysis of DNA extracted from seed embryos used for individualization of four hemp and six marijuana varieties [ 705 ]; review of genetic resources available for phenotype tracking of cannabis [ 706 ]; 2017 high resolution melting SNP protocol for differentiation of drug and non-drug cannabis plants [ 707 ]; genotyping of 154 individual plants from 20 cultivars for identification of genetic clusters [ 708 ]; restriction fragment length polymorphism of the THCA synthase gene for differentiation of Cannabis subspecies [ 709 ]; 13 loci STR system for forensic DNA profiling of marijuana samples (validated according to relevant ISFG and SWGDAM guidelines) [ 710 ]; loop-mediated isothermal amplification assay for identifying the drug-type strains of C. sativa [ 711 ]; detection and partial characterization of a 16srix-c phytoplasma associated with hemp witches’-broom [ 712 ]; genetic determination of Cannabis sativa var. indica based on six genomic SSRs for genotyping 154 individual plants from 22 cultivars to determine intra-varietal diversity [ 708 ]; 2018 Phylogenetic analysis of samples 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, to determine population substructure using autosomal and lineage markers [ 713 ]; genetic characterization of hemp [ 714 ]; method for extracting DNA from cannabis resin based on the evaluation of relative PCR amplification ability [ 715 ];…”