Cannabis Sativa Monograph Pdf To Excel
Materials and MethodsPlant materialPsychoactive Cannabis samples used in this study were seized from 29 different locations (C1-C29) representing geographically distinct and problematic areas of Turkey (western and eastern parts). Some of the materials, used in this study, were previously also utilized for discriminating drug type Cannabis from hemp types.
All the information relevant to the seized samples and accessions are shown in. Provincial locations of the seized Cannabis accessions are shown on a map of Turkey in. Ten seeds were planted from each accession to produce material for DNA extraction. Plants were grown in a fully automated greenhouse.DNA extraction from leafLeaves collected from three week old seedlings were shock-frozen in liquid nitrogen and stored at -80ºC until DNA isolations were performed.
DNAs were extracted individually from a total of 290 samples. Total DNAs of the samples were extracted using a standard 2X CTAB protocol with minor modifications. For each accession, 100 mg of leaf sample from 10 different plants were used and DNAs were isolated individually. After concentrations were determined by an Eppendorf BioPhotometer, sample DNAs were diluted to the working concentration of 20 ng/µL.PCR amplification of the DNA with RAPD primersIn this study, 22 arbitrary RAPD primers that gave the most informative patterns (in terms of repeatability, scorability and the ability to distinguish between individuals) were selected for identification.
Each reaction contained 2.5 mM MgCl 2; 10 mM Tris-HCl (pH 8.8); 50 mM KCl; 0.8% Nonidet P40; 200 mM of each of the dNTPs; 0.5 µM primer; 20 ng DNA template and 0.3 units of Taq DNA Polymerase (Bioron) in a final reaction volume of 25 µl. After a pre-denaturation step of 3 min at 94ºC, amplification reactions were optimized for every individual primer and optimization was usually started by cycling the reaction 45 times at 94ºC for 1 min, at annealing temperature for 50 sec and 72ºC for 1 min in Eppendorf Mastercycler gradient thermocycler.
A final extension was allowed for 10 min at 72ºC. Upon completion of the reaction, amplified products were loaded onto a 2.0% agarose/1x Tris-Borate EDTA gel and electrophoresed at 4 V/cm.In the RAPD assay, two sets of PCR amplifications were carried out. In the first set (SET1), each accession was represented by DNA of randomly selected an individual plant in that accession. In the second set (SET2), pooled DNA from all ten individuals in each accession was used (the one individual used in the first set was also included).Every primer was amplified for the two sets simultaneously.
Then the resultant products were run in agarose gel and evaluated. Amplifications were repeated at least twice (in different time periods) for each primer, using the same reagents and procedure.Data collection and statistical analysisEach DNA fragment generated was treated as a separate character and scored as a discrete variable, using 1 to indicate presence, and 0 for absence.
Accordingly, a rectangular binary data matrix was obtained and statistical analysis was performed using the NTSYS-pc version 2.1 statistical package. A pairwise similarity matrix was generated using simple matching coefficient (by means of SIMQUAL procedure of NTSYS-pc) and principal coordinate analysis (PCoA) was performed using a batch mode of NTSYS-pc both for SET1 and SET2. Then, cluster analysis was performed (by means of SAHN procedure of NTSYS-pc) via unweighted pair-group method using arithmetic average (UPGMA) to develop a dendrogram both for SET1 and SET2. To estimate the strength of the grouping, generated by cluster analysis, bootstrap analysis was performed with 2000 replications using the winboot computer program. Also, a matrix comparison of Mantel Z test , for the correspondence of the similarity matrices of SET1 and SET2, was performed (by means of MXCOMP procedure of NTSYS-pc) for the null hypothesis that there is no association between the similarity matrices of SET1 and SET2. To obtain significance level, 5000 permutations were performed.
In addition, a genetic similarity matrix was calculated according to.An analysis of molecular variance (AMOVA) was performed using GENALEX 6 in SET2 to partition the total molecular variance between and within populations (clusters). Significance level was detected via permutation test (n = 1000).
Cannabis clusters were defined according to the PCoA results. Cannabis accessions that do not cluster closely with any of the clusters were removed from data set before AMOVA. ResultsRAPD amplification and analysisThe RAPD markers, used in the analysis of SET1 and SET2 allowed reproducible and informative polymorphisms (pictures of gels that were run at different time were given in for illustration purpose). Selected primers yielded a total of 264 bands in SET1, and 241 bands in SET2, 93% and 92% of which were polymorphic, respectively. However, using bulked samples resulted in much more consistent and reliable amplifications. The RAPD B1 primer was the one that gave the highest number of polymorphisms in SET1 and SET2. In the accession C20, that is the accession seized from Hatay, 119 markers were missing.
This was due to lack of material to produce DNA from that accession.Results from statistical analysis of genotypic dataResults from PCoA of RAPD markers by using SET2 showed that Cannabis accessions are basically separated into two main groups by PCo axis 1. In fact, this separation was in agreement with the geographical regions of Turkey. The first group (group 1) was made up of Cannabis plants, which were seized from mostly western part (costal regions, namely Mediterranean, Aegean, and Marmara) of Turkey and there were 18 accessions. The second one (group 2) was made up of Cannabis plants that were seized from mostly eastern part of Turkey and there were 8 accessions. Accessions C21 (Gaziantep21), C7 (Kocaeli7), and C20 (Hatay20) were not attributable to any group and we named them as outliers.
Compared to group 1, group 2 showed more variation.The genetic relationships among Cannabis accessions, in SET2, were presented in a dendrogram. Results of analysis of SET2 via cluster analysis and PCoA were slightly different. Group 1 Cannabis accessions in PCoA, were clearly grouped as one major branch with a similarity of 81% based on simple matching similarity index. However, part of Cannabis accessions in group 2 (Elazig23, Malatya24, Trabzon28, and Rize25) were attached to the cluster of group 1, and then the rest of group 2 were attached. Accession Gaziantep21, which was seen to be an outlier in the PCoA, took place in this group. Overall similarity, based on simple matching similarity index, was 71%.Unlike SET2, there was no separation in the seized Cannabis accessions in SET1 via either PCoA or cluster analysis.
Therefore, no result from PCoA or cluster analysis is presented in here from SET1.Results from matrix comparison via Mantel Z test showed that there is a statistically significant association between the similarity matrices of SET1 and SET2 (P. DiscussionThis study was conducted on two SET of Cannabis materials, by using RAPD markers, which were analyzed simultaneously. The degrees of polymorphism of the markers found were 93% for SET1 and 92% for SET2. Reported the degree of polymorphism of RAPD markers for different hemp varieties in a range from 31.1% to 97.1%, which includes our findings.Clusters were observed between the seized Cannabis accessions in SET2 via both cluster analysis and PCoA but not in SET1. A weak correlation, resulted from matrix comparison via the Mantel test between similarity matrices of SET1 and SET2, might be interpreted as a reason for not having similar results from SET1 and SET2. This might be due to the template effect, where amplification of faint fragments failed in bulked samples.
Hence, more consistent bands were amplified and the specific accessions were better represented.In the PCoA of SET2, all accessions, except accessions Gaziantep21, Hatay20, and Kocaeli7, were clearly separated into two main groups. The first group was made up of Cannabis plants, which were seized from mostly western parts of Turkey, while the second group was made up of Cannabis plants that were seized from mostly eastern parts of Turkey. Visual examination of distribution of Cannabis accessions on the first two principal coordinates indicates that there is more variation in group 2 compared to group 1. Averaged genetic similarity, which were found to be 0.263 and 0.220 for group 1 and group 2 respectively, supported visual examination. Findings from AMOVA supported these results by having estimated variances as 18.58 (with 17 degrees of freedom) and 26.92 (with 7 degrees of freedom) for group 1 and group 2, respectively. Results from AMOVA indicated that 20.23% of the genetic variation was attributable to differences among accessions groups while 79.77% of the genetic variation was attributable to between accessions within accessions groups. Identified geographic sources of seized drugs by using AFLP markers.
In that study, 27.2% of the genetic variation from AMOVA was due to differences between drug and hemp lines, while 20.9% differences among hemp varieties. Our among group variation is comparable with their among hemp varieties variation. In a comparison study of six hemp types Cannabis varieties with RAPD markers, it is reported that the proportion of among-cultivars variance changed dramatically ranging from 12.8% up to 76%. The later was observed between two highly selected, divergent cultivars.Results from UPGMA for group 2 was slightly different compared to PCoA, although accessions of the group 1 were clustered together in UPGMA.
PCoA is a translation of similarities/dissimilarities between objects into the actual distances between objects in multidimensional space. On the other hand, in cluster analysis, once a group or cluster is formed from two or more objects, that group can not be broken later in the process. As a result, the dendrogram is not a representation of all pairwise dissimilarities between objects.Examination of UPGMA Dendrogram accession by accession would give information about relatedness in individual level. For example, 0.91 genetic relatedness was found between accessions C3 and C11, and C5 and C15 with a bootstrapping P value of 58.6% and 50.2%, respectively. These results might lead us to speculation that there might be relationship between the individual illicit Cannabis growers of accessions C3 and C11, and C5 and C15.
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Cannabis Sativa Monograph Pdf To Excel Free
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