Evaluation Of Molecular Markers In Identification Of Poisonous Plants

Background:Poisoning by poisonous plants is the most popular and prominent public health problem because of the accessibility and traditional medical custom. However, in most conditions, obtaining integral biological evidence which favored to determine cause is unable, so the traditional techniques—morphological marker, cytological marker and biochemical marker have limitations, techniques to control poisoning by poisonous plants are very meager. Molecular marker's advantages make it more useful to identifying poisonous plants. The popular techniques are RAPD, ISSR and DNA barcoding.Objective:To assess the feasibility of molecular techniques in emergency poisonous plants poisoning events by using RAPD, ISSR and DNA barcoding to identify the common poisonous plants in China. Evaluate the timeliness, facticity, repeatability and operability of identifying plants from genus taxonomic categories of the three techniques.Methods:Choose 19 species of common poisonous plants of Ranunculaceae and Euphorbiaceaec in China as objects. Select dry leave or fresh leave as samples, using improved CTAB method to extract DNA. RAPD:random primers with clear brands and good repeatability are used for experiments and statistical analysis, NYSTS and SPSS are used for clustering analysis and drawing phylogenetic tree. Calculate accuracy rate with comparing to morphological taxonomic categories.3 person counting brands simultaneously to evaluate influence of subjectivity. All the experiments repeat 3 times to compute repetitive rate. ISSR:Primers with clear brands and good repeatability are used for experiments and statistical analysis, NYSTS and SPSS are used for clustering analysis and drawing phylogenetic tree. Calculate accuracy rate with comparing to morphological taxonomic categories.3 person counting brands simultaneously to evaluate influence of subjectivity. All the experiments repeat 3 times to compute repetitive rate. DNA barcoding:4 primers which recommend by the Consortium for the Barcode of Life (CBOL) are used for experiments and statistical analysis, PAUP and MEGA are used for clustering analysis and drawing phylogenetic tree. Calculate accurate rate with comparing to morphological taxonomic categories. Count the rate of PCR and sequencing and obtain the optimal segment. All the experiments repeat 3 times to compute repetitive rate.Results:1. Total DNA can be extracted from the 19 poisonous plants by CTAB method. The concentration of DNA is above 100ng/μL and with little RNA and protein.2. RAPD:Obtain 3 primers and the percentages of polymorphism are all 100.00%. Present low ability to the samples identification. Subjective influence rate is 43.75%-76.92%. Repetitive rate is 30.77%-47.06%.3. ISSR:Obtain 9 primers and the percentages of polymorphism are all 100.00%. The best primer is 873, its accurate rate is 68.42%, subjective influence rate is 26.09%, repetitive rate is 45.00%.4. DNA barcoding:Optimal segment is trnH-psbA which has obvious barcoding gap, accurate rate is 100.00%, PCR and sequencing rates are 100.00%.5. Evaluation:Timeliness:RAPD and ISSR can accomplish identification in 8.50-9.00 hours and DNA barcoding needs 42.17-54.17 hours. Facticity:RAPD can not identify objective samples, ISSR has the accurate rate at 68.42%, and DNA barcoding can handle the identification with accurate rate at 100.00%. Repeatability:RAPD and ISSR both have high subjective influence rate and low repetitive rate. trnH-psbA of DNA barcoding presents good repeatability. Operability:DNA barcoding needs more PCR product whereas RAPD and ISSR need little, RAPD and ISSR have harsher require of experiment condition whereas DNA barcoding has no such request. RAPD and ISSR can achieve in a single laboratory with low cost whereas DNA barcoding can not.DNA barcoding has an authoritative database GenBank to align on line whereas RAPA and ISSR have not.Conclusion:According to timeliness RAPD and ISSR are better than DNA barcoding, in the aspect of facitity and repeatability, DNA barcoding is much better than RAPD and ISSR, the limitation of operation is more easily to solve. DNA barcoding can be a useful tool in analysis of cause in emergency poisoning scene.