Characterization and evaluation of nine Cannabis sativa chloroplast SNP markers for crop type determination and biogeographical origin on European samples.

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.

Evaluation of the trnK-matK-trnK, ycf3, and accD-psal chloroplast regions to differentiate crop type and biogeographical origin of Cannabis sativa.

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.

ITS barcoding using high resolution melting analysis of Cannabis sativa drug seizures in Chile: A forensic application.

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.

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

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.

Psychedelic fungus (Psilocybe sp.) authentication in a case of illegal drug traffic: sporological, molecular analysis and identification of the psychoactive substance.

In nature, there are >200 species of fungi with hallucinogenic properties. These fungi are classified as Psilocybe, Gymnopilus, and Panaeolus which contain active principles with hallucinogenic properties such as ibotenic acid, psilocybin, psilocin, or baeocystin. In Chile, fungi seizures are mainly of mature specimens or spores. However, clandestine laboratories have been found that process fungus samples at the mycelium stage. In this transient stage of growth (mycelium), traditional taxonomic identification is not feasible, making it necessary to develop a new method of study. Currently, DNA analysis is the only reliable method that can be used as an identification tool for the purposes of supporting evidence, due to the high variability of DNA between species. One way to identify the species of a distinctive DNA fragment is to study PCR products analyzed by real time PCR and sequencing. One of the most popular sequencing methods of forensic interest at the generic and intra-generic levels in plants is internal transcribed spacer (ITS). With real time PCR it is possible to distinguish PCR products by differential analysis of their melting temperature (Tm) curves. This paper describes morphological, chemical, and genetic analysis of mycelia of psychedelic fungi collected from a clandestine laboratory. The fungus species were identified using scanning electron microscopy (SEM), mass spectrometry, HRM analysis, and ITS sequencing. The sporological studies showed a generally smooth surface and oval shape, with maximum length 10.1 µm and width 6.4 µm. The alkaloid Psilocyn was identified by mass spectrometry, while HRM analysis and ITS sequencing identified the species as Psilocybe cubensis. A genetic match was confirmed between the HRM curves obtained from the mycelia (evidence) and biological tissue extracted from the fruiting bodies. Mycelia recovered from the evidence and fruiting bodies (control) were genetically indistinguishable.

Yield and quality of sweet corn at different plant populations / Produtividade e qualidade do milho doce em diferentes populações de plantas

The objective of this study was to evaluate the plant population density effect upon yield components and the chemical composition of grains in two sweet corn hybrids in Winter-Spring cultivation. The experimental design used was the complete randomized blocks with four replications. The treatments were based on the combination between two sweet corn hybrids (Tropical Plus e RB-6324) and five plant populations (40,000, 55.000, 70,000, 85,000 and 100,000 plants ha-1), in a factorial scheme 2 x 5, evaluated in two winter-spring growing seasons (2009 and 2010). The increment in the population of 40,000 to 100,000 plants ha-1 resulted in higher husked commercial corn ear productivity in both hybrids, Tropical Plus and RB-6324. The increase of plant population reduced the length and diameter of corn ears in Tropical Plus and RB-6324. The mass of the corn ears from Tropical Plus also reduced; however, it did not affect the commercial standard and industrial use. In larger plant populations the hybrid RB-6324 produced corn ears with grains that were sweeter and with lower starch content. The hybrid Tropical Plus presented higher mass, length, productivity, sugar content and lower diameter of corn ears when compared to RB-6324.

O objetivo do presente estudo foi avaliar o efeito da população de plantas sobre os componentes de produção e a composição química de grãos de dois híbridos de milho doce no cultivo de Inverno Primavera. O delineamento experimental utilizado foi o de blocos completos casualizados com quatro repetições. Os tratamentos constituíram-se da combinação entre híbridos de milho doce (Tropical Plus e RB-6324) e populações de plantas (40.000, 55.000, 70.000, 85.000 e 100.000 plantas ha-1), no esquema fatorial 2 x 5, avaliados em dois anos agrícolas (2009 e 2010), em cultivo de Inverno-Primavera. O incremento na população de plantas de 40.000 até 100.000 plantas ha-1 resultou em maiores produtividades de espigas comerciais despalhadas dos híbridos Tropical Plus e RB-6324. O aumento na população de plantas reduziu o comprimento e o diâmetro de espigas de Tropical Plus e RB-6324, e a massa de espigas de Tropical Plus, mas não afetou o padrão comercial e de uso industrial das mesmas. Em populações de plantas mais elevadas o híbrido RB-6324 produziu espigas com grãos mais adocicados e com menor conteúdo de amido. O híbrido Tropical Plus apresentou maiores massa, comprimento, produtividade, conteúdo de açúcares totais, e menor diâmetro de espigas do que o RB-6324.

Yield and quality of sweet corn at different plant populations / Produtividade e qualidade do milho doce em diferentes populações de plantas

The objective of this study was to evaluate the plant population density effect upon yield components and the chemical composition of grains in two sweet corn hybrids in Winter-Spring cultivation. The experimental design used was the complete randomized blocks with four replications. The treatments were based on the combination between two sweet corn hybrids (Tropical Plus e RB-6324) and five plant populations (40,000, 55.000, 70,000, 85,000 and 100,000 plants ha-1), in a factorial scheme 2 x 5, evaluated in two winter-spring growing seasons (2009 and 2010). The increment in the population of 40,000 to 100,000 plants ha-1 resulted in higher husked commercial corn ear productivity in both hybrids, Tropical Plus and RB-6324. The increase of plant population reduced the length and diameter of corn ears in Tropical Plus and RB-6324. The mass of the corn ears from Tropical Plus also reduced; however, it did not affect the commercial standard and industrial use. In larger plant populations the hybrid RB-6324 produced corn ears with grains that were sweeter and with lower starch content. The hybrid Tropical Plus presented higher mass, length, productivity, sugar content and lower diameter of corn ears when compared to RB-6324.

O objetivo do presente estudo foi avaliar o efeito da população de plantas sobre os componentes de produção e a composição química de grãos de dois híbridos de milho doce no cultivo de Inverno Primavera. O delineamento experimental utilizado foi o de blocos completos casualizados com quatro repetições. Os tratamentos constituíram-se da combinação entre híbridos de milho doce (Tropical Plus e RB-6324) e populações de plantas (40.000, 55.000, 70.000, 85.000 e 100.000 plantas ha-1), no esquema fatorial 2 x 5, avaliados em dois anos agrícolas (2009 e 2010), em cultivo de Inverno-Primavera. O incremento na população de plantas de 40.000 até 100.000 plantas ha-1 resultou em maiores produtividades de espigas comerciais despalhadas dos híbridos Tropical Plus e RB-6324. O aumento na população de plantas reduziu o comprimento e o diâmetro de espigas de Tropical Plus e RB-6324, e a massa de espigas de Tropical Plus, mas não afetou o padrão comercial e de uso industrial das mesmas. Em populações de plantas mais elevadas o híbrido RB-6324 produziu espigas com grãos mais adocicados e com menor conteúdo de amido. O híbrido Tropical Plus apresentou maiores massa, comprimento, produtividade, conteúdo de açúcares totais, e menor diâmetro de espigas do que o RB-6324.

Plant Science research productivity in Chile during the past 20 years

The purpose of this study was to carry out a bibliometric analysis of the articles published in Chile during 1987-2006 that were categorized into the Plant Science discipline by ISI. The data was extracted from the Web of Science datábase, obtaining a total of 911 articles for analysis. Almost half of the articles were the outcome of an international collaboration, the United States being the country with more collaborative links with Chile within this discipline. A list was made with the 10 most-cited articles and 10 most frequently used journals. The analysis showed that almost 50 percent of the articles were concentrated in 10 different journals. The number of articles produced each year revealed a steady growth in scientific production since 1987. This is probably related to the progressive interaction between industry and public-research institutions.