RESUMO
Cannabis sativa can be classified in two main types, according to psychotropic cannabinoid ∆9-tetrahydrocannabinol (∆9-THC) content: the drug-type and the fiber-type. According to the European Monitoring Center for Drugs and Drug Addiction, most of the European Union countries consider the possession of cannabis, for personal use, a minor offense with possibility of incarceration. Despite of the model of legal supply (i.e., Spanish cannabis clubs, Netherlands coffee shops) or medical use (i.e., Italy), cannabis remains the most used and trafficked illicit plant in the European Union. Differentiating cannabis crops or tracing the biogeographical origin is crucial for law enforcement purposes. Chloroplast DNA (cpDNA) markers may assist to determine biogeographic origin and to differentiate hemp from marijuana. This research aims: to identify and to evaluate nine C. sativa cpDNA polymorphic SNP sites to differentiate crop type and to provide information about its biogeographical origin. Five SNaPshot™ assays for nine chloroplast markers were developed and conducted in marijuana samples seized in Chile, the USA-Mexico border and Spain, and hemp samples grown in Spain and in Italy. The SNapShot™ assays were tested on 122 cannabis samples, which included 16 blind samples, and were able to differentiate marijuana crop type from hemp crop type in all samples. Using phylogenetic analysis, genetic differences were observed between marijuana and hemp samples. Moreover, principal component analysis (PCA) supported the relationship among hemp samples, as well as for USA-Mexico border, Spanish, and Chilean marijuana samples. Genetic differences between groups based on the biogeographical origin and their crop type were observed. Increasing the number of genetic markers, including the most recently studied ones, and expanding the sample database will provide more accurate information about crop differentiation and biogeographical origin.
Assuntos
Cannabis , DNA de Cloroplastos , Polimorfismo de Nucleotídeo Único , Cannabis/genética , Marcadores Genéticos , DNA de Cloroplastos/genética , México , Reação em Cadeia da Polimerase , Europa (Continente) , Itália , Chile , EspanhaRESUMO
Mitoviruses were initially known for their presence in the mitochondria of fungi and were considered exclusive to these organisms. However, recent studies have shown that they are also present in a large number of plant species. Despite the potential impact that mitoviruses might have on the mitochondria of plant cells, there is a lack of information about these ancient RNA viruses, especially within the Cannabaceae family. Cannabis sativa has been in the spotlight in recent years due to the growing industrial applications of plant derivatives, such as fiber and secondary metabolites. Given the importance of Cannabis in today's agriculture, our study aimed to expand the knowledge frontier of Mitoviruses in C. sativa by increasing the number of reference genomes of CasaMV1 available in public databases and representing a larger number of crops in countries where its industrial-scale growth is legalized. To achieve this goal, we used transcriptomics to sequence the first mitoviral genomes of Colombian crops and analyzed RNA-seq datasets available in the SRA databank. Additionally, the evolutionary analysis performed using the mitovirus genomes revealed two main lineages of CasaMV1, termed CasaMV1_L1 and CasaMV1_L2. These mitoviral lineages showed strong clustering based on the geographic location of the crops and differential expression intensities.
Assuntos
Cannabis , Vírus de RNA , Cannabis/genética , Filogenia , Vírus de RNA/genética , Mitocôndrias/genética , FungosRESUMO
Background: Cannabis plants and their seed have been used in many cultures as a source of medicine and feeding during history. Today, there is an increasing demand for cannabis seeds for medical use. Moreover, a seed sales market with no legal regulations has also grown. This may pose some issues if a quality control is not set in place. Identification of cannabis strains is important for quality control purposes in a nonregulated growing market and in cases of illegal traffic and medical use. Owing to the high price as a pharmacological drug, commercial products of cannabis plants and seeds for medical users are often subjected to adulterations, either when packing or distributing certified seeds in the market. Materials and Methods: Cannabis commercial seeds and cannabis seeds for medical use were analyzed with high-resolution melting (HRM) analysis using barcoding markers. Humulus lupulus L. plants from a local market were used as outgroup control. DNA barcoding uses specific regions of the genome to identify differences in the genetic sequence of conserved regions such as internal transcribed spacer (ITS) and rbcL. DNA barcoding data can be generated with real-time polymerase chain reaction combined with HRM analysis to distinguish specific conserved DNA regions of closely related species. HRM analysis is the method of choice for rapid analysis of sequence variation. Results: The melting temperature (Tm) of homogeneous packages was consistent with single genotypes. However, packages containing contaminating seeds showed Tm differences of 0.2°C on average. Conclusions: An effective, rapid, and low-cost method based on ITS nuclear DNA and on chloroplast rbcL regions for screening and detection of contamination in commercial cannabis seeds was developed and applied for the analysis of different samples. This approach can be used as a quality control tool for cannabis seeds or other plant material.
Assuntos
Cannabis , Código de Barras de DNA Taxonômico , Cannabis/genética , Cloroplastos/genética , Código de Barras de DNA Taxonômico/métodos , DNA Intergênico , DNA de Plantas/genética , Controle de Qualidade , Sementes/genéticaRESUMO
Cannabis sativa (marijuana and hemp) is one of the most controversial crops worldwide. In the USA, the state-specific legalization of marijuana and recently legalized hemp pose a problem for law enforcement. This study seeks to utilize chloroplast hSTRs, INDEL, and SNPs markers to develop genotyping methods to aid in the differentiation of legal hemp from illicit marijuana and also for tracking the flow of trafficked marijuana. Three polymorphic regions: trnK-matK-trnK, ycf3, and accD-psal, of the C. sativa chloroplast genome were evaluated in order to distinguish crop type and biogeographic origin. A total of nine polymorphic sites were genotyped from five distinct populations (hemp from the USA and Canada, marijuana from Chile and USA-Mexico, and medical marijuana from Chile) with a custom fragment and SNaPshotTM assay. The study also combined genotype results from the same sample set using 21 additional polymorphic markers from previous studies. The effectiveness of these multi-locus assays to distinguish sample groups was assessed using haplotype analysis, phylogenetic analysis, pairwise comparisons, and principal component analysis. Results indicated a clear separation of Canadian hemp using only the nine polymorphic sites developed in this study. The additional 21 markers were able to separate US hemp from both marijuana groups to a significant level (p < 0.05) when assessing average Fixation Indices (FST). This study demonstrated the applicability of these organelle markers for the determination of crop type and biogeographic origin of C. sativa. However, a more extensive database is needed to evaluate the true discriminatory power of these markers.
Assuntos
Cannabis/classificação , Cannabis/genética , Código de Barras de DNA Taxonômico/métodos , DNA de Cloroplastos/genética , Genoma de Cloroplastos , Técnicas de Genotipagem , Haplótipos , Canadá , Chile , Marcadores Genéticos , Genótipo , Mutação INDEL , México , Repetições de Microssatélites , Filogenia , Filogeografia , Polimorfismo de Nucleotídeo Único , Análise de Componente Principal , Estados UnidosRESUMO
Cannabis sativa L. is a plant cultivated worldwide as a source of fiber, medicine, and intoxicant. Traditionally, C. sativa is divided into two main types: fiber type (hemp) and drug type. Drug-type C. sativa differs from hemp by the presence of a high quantity of the psychoactive drug, Δ9-tetrahydrocannabinol (Δ9 THC). Cannabis sativa is the most commonly used used illicit controlled substance in Chile. Chile is the third greatest consumer of Cannabis in South America. The objective of this study was to determine the genetic composition of ten drug seizures of Cannabis spp. in the south of Chile using a high resolution melting (HRM) strategy combined with a barcoding marker, ITS. C. sativa samples were selected from previously processed more than a thousand crime cases at the, Araucania region crime lab, National Dept. of Health. Ten cases were selected. Sample collection was based on the following: a) dry and fresh samples with no evidence of decomposition or degradation, b) defined plant fragments such as flowers and leaves from individual plants and, c) samples with different content of THC, CBN and CBD. Five sub samples were randomly selected from each case (N=50). The commercial Silver Haze strain was used as a control. Two real-time PCR and HRM analyses were conducted. The first analysis was performed with a representative sample of each of the 10 cases studied. Then a second assay was performed with all subsamples of cases 1, 5 and 8. Results showed that real-time PCR combined with HRM analysis using ITS allowed to determine the genetic composition of cannabis in all cases studied. The derivative of melting and the analysis of the shape of the curve and the peak of Tm, showed that three groups can be clearly distinguished. A first group exhibited a peak of Tm close to 87.4°C and includes cases 7 and 8. A second group had a peak of Tm close to 87.6°C and includes case 5. A third group displayed a peak of Tm close to 87.9°C and includes case 1, 6 and Silver Haze strain. A second experiment was performed using subsamples of cases 1, 5 and 8. Case 1 displayed a unique composition of the drug suggesting that this seizure contained cannabis clonally propagated. In case 5, two genotypes were present, therefore this could be associated with two strain or two different origin. Case 8, was composed of a mixture of cannabis strains indicating the presence of various crop type and/or different biogeographic origin. In general, our results suggested genetically homogeneous seizures from Araucanía Region. The high latitude (37° 35' and 39° 37' South latitude) and the natural geographic borders that surround southern Chile helps the control of cannabis traffic into the country. Finally, HRM analysis coupled with the barcode ITS demonstrated to be a rapid and low-cost screening method.
Assuntos
Cannabis/genética , Código de Barras de DNA Taxonômico , DNA Intergênico/genética , Chile , DNA de Plantas/genética , Tráfico de Drogas , Genética Forense/métodos , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Temperatura de TransiçãoRESUMO
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.
Assuntos
Cannabis/classificação , Cannabis/genética , Cloroplastos/genética , Produtos Agrícolas , Código de Barras de DNA Taxonômico/métodos , Genética Forense/métodos , Canadá , Cannabis/química , Chile , DNA de Plantas , Loci Gênicos , Genoma de Planta/genética , Genótipo , Haplótipos , México , FilogeniaRESUMO
According to the Brazilian Federal Police (BFP), the Brazilian Cannabis sativa illicit market is mainly supplied by drugs originated from Paraguay and Northeastern Brazil (Marijuana Polygon region). These two known routes, the increasing indoor cultivations (supported by online market), and drugs from Uruguay are also in BFP's sight. Forensic tools to aid police intelligence were published in the past years. In genetics, microsatellites have gained attention due to their individualization capability. This study aims to evaluate the effectiveness and efficiency of two STR multiplex systems previously proposed in 94 Cannabis sativa samples seized in Brazil. Principal coordinate analyses (PCoA), forensic parameters, and genetic structure analysis were executed. Both panels were effective in individualizing and origin discriminating all samples, and the system proposed in 2015 demonstrated better results. For this marker set, the probability of identity for a random individual is approximately one in 65 billion; also, the PCoA shows a clear genetic distinction among samples according to its origin. Bayesian inference populational structure analysis indicated a significant genetic diversity among seizure groups, matching with its origin. Overall, the STR multiplex systems were able to achieve its purpose in individualizing and differentiating, according to geographic region, Brazilian Cannabis sp. samples.
Assuntos
Cannabis/genética , Loci Gênicos , Genótipo , Repetições de Microssatélites , Reação em Cadeia da Polimerase Multiplex , Brasil , Tráfico de Drogas/prevenção & controle , Genética Forense , Estruturas Genéticas , Humanos , Aplicação da Lei/métodos , Análise de Componente PrincipalRESUMO
DNA is one of the fastest growing tools in forensic sciences, increasing reliability in forensic reports and judgments. The use of DNA has increased in different areas of the forensic sciences, such as investigation of plant species, where plastid DNA has been used to elucidate and generate evidence in cases of traceability of genetically modified and controlled plants. Even with several advances and the practice of using DNA in forensic investigations, there are just few studies related to the identification of genetic tools for the characterization of drug and nondrug-types of Cannabis. Herein, the whole plastomes of two drug-type Cannabis are presented and have their structures compared with other Cannabis plastomes deposited in the GenBank, focusing in the forensic use of plastome sequences. The plastomes of Cannabis sativa "Brazuka" and of the hybrid Cannabis AK Royal Automatic presented general structure that does not differs from the reported for other C. sativa cultivars. A phylogenomic analyses grouped C. sativa "Brazuka" with the nondrug C. sativa cultivars, while the hybrid Cannabis AK Royal Automatic placed isolated, basal to this group. This suggests that the analysis of plastomes is useful toward genetic identification of hybrids in relation to C. sativa.
Assuntos
Cannabis/genética , Genomas de Plastídeos , Plastídeos/genética , DNA de Plantas , Bases de Dados de Ácidos Nucleicos , Ciências Forenses , Sequenciamento de Nucleotídeos em Larga Escala , Filogenia , Análise de Sequência de DNARESUMO
It is known that Cannabis in Brazil could either originate from Paraguay or be cultivated in Brazil. While consumer markets in the North and Northeast regions are maintained by national production, the rest of the country is supplied with Cannabis from Paraguay. However, the Brazilian Federal Police (BFP) has exponentially increased the seizure number of Cannabis seeds sent by mail. For this reason, the aim of the study was to assess the 13-loci short tandem repeat (STR) multiplex system proposed by Houston et al. (2015) to evaluate the power of such markers in individualization and origin differentiation of Cannabis sativa samples seized in Brazil by the BFP. To do so, 72 Cannabis samples seized in Brazil by BFP were analyzed. The principal coordinate analysis (PCoA) and probability identity (PI) analysis were computed. Additionally, the Cannabis samples' genotypes were subjected to comparison by Kruskal-Wallis H, followed by a multiple discriminant analysis (MDA). All samples analyzed revealed a distinct genetic profile. PCoA clearly discriminated the seizure sets based on their geographic origin. A combination of seven loci was enough to differentiate samples' genotypes, and the PI for a random sample is approximately one in 50 billion. The Cannabis samples were 100% correct as classified by Kruskal-Wallis H, followed by an MDA. The results of this study demonstrate that the 13-loci STR multiplex system successfully achieved the aim of sample individualization and origin differentiation and suggest that it could be a useful tool to help BFP intelligence in tracing back-trade routes.
Assuntos
Cannabis/genética , Tráfico de Drogas , Genótipo , Repetições de Microssatélites , Brasil , Impressões Digitais de DNA , DNA de Plantas/análise , Loci Gênicos , Humanos , Reação em Cadeia da Polimerase MultiplexRESUMO
Cannabis sativa, known by the common name marijuana, is the psychoactive drug most widely distributed in the world. Identification of Cannabis cultivars may be useful for association to illegal crops, which may reveal trafficking routes and related criminal groups. This study provides evidence for the performance of a segment of the rbcL gene, through genetic signature, as a tool for identification for C. sativa samples apprehended by the Rio de Janeiro Police, Brazil. The PCR amplified and further sequenced the fragment of approximately 561 bp of 24 samples of C. sativa rbcL gene and showed the same nucleotide sequences, suggesting a possible genetic similarity or identical varieties. Comparing with other Cannabaceae family sequences, we have found 99% of similarity between the Rio de Janeiro sequence and three other C. sativa rbcL genes. These findings suggest that the fragment utilized at this study is efficient in identifying C. sativa samples, therefore, useful in genetic discrimination of samples seized in forensic cases.