Several Major Asteraceae Invasive Species Genetic Analysis By ISSR And AFLP

Invasive plants are the non-native plants which migrate to a new environment through natural and human factors.Once migrated,they destroy the ecology environment and harm to human body health.As a result,they make damage and make a huge loss to agriculture,forestry and animal husbandry.Since the Asteraceae plants have many different kinds with complex shapes and are adaptable to the environment also,they occupy the most proportion in all invasive plants.DNA from Asteraceae plants were extracted by CTAB method and detected through agarose gel electrophoresis.The figures of images of agarose gel electrophoresis showed total DNA extractions were tidy,clear and no-impurity.The measurements of Asteraceae DNA absorbance were detected by UV spectrophotometry.The values of OD₂30?OD₂60 and OD₂80 were obtained.We compared the values of OD₂30?OD₂60 and OD₂80.The ratio of A₂60nm/A₂30nm and A₂60nm/A₂80nm are between 1.8 and 2.0.High-purity and good-quality Asteraceae plant DNA were extracted and received.The reactions were made using the extracted DNA and the ninth set of universal primers for selective amplification.Universal primers for amplification were published by the Columbia University.The bands using primer UBC840?primer UBC880,primer UBC807?primer UBC844?primer UBC866,primer UBC810.primer UBC886.primer UBC836 and primer UBC879 were detected that which were high resolution,good polymorphism?more bright.The single factor experiments of the Asteraceae showed those DNA can be amplified at various concentrations.The results of amplification bands have been measured and analyzed.The optimum concentration of primer is 0.2 mol/L.The optimal concentration of Mg²⁺ is 2.5 mmol/L.The optimum concentration of dNTPs is 0.2 mmol/L and the concentration of DNA is 1 ng/?L.The preliminary results of single factor experiments were showed that the amount of DNA,the concentration of primer,the concentration of Mg²⁺ and the concentration of dNTPs have great influence on the ISSR-PCR reaction.The 4 conditions were accepted as the 4 factors in the orthogonal test.The orthogonal test of 4 factors and 4 levels was caried out.The optimal reaction system as follows:in 20 ?L system,the optimum concentration of primer is 0.2 mol/L.The optimal concentration of Mg²⁺ is 2.5 mmol/L.The optimum concentration of dNTPs is 0.2 mmol/L and the concentration of DNA is 1 ng/?L.The calculation of R value range can be revealed the influence degree of different factors on the experiment.The larger the value of R is,the more significant influence of the factors on the experiment.The influence of various factors on the ISSR-PCR were as follows:Mg²⁺>Primer>dNTPs>DNA.4275 DNA bands were amplified by ISSR markers,most bands with molecular weight of 166-1000bp,59 DNA bands were obtained by each primer combinations.The genetic similarity coefficients were obtained by ISSR bands based on the statistics.The similar coefficient between 0.84-0.87 was obtained and the average similarity coefficient was 0.8519.The similarity coefficient of E.adenophorum and E odoratum was 0.8664 which was larger than others.The similarity coefficient showed that they were the closest relationship.The similarity coefficient of Erigeron Philadelphicus and Conyza Canadensis was smallest.The similarity coefficient showed that they were the the biggest difference.The AFLP markers were amplified 1944 DNA bands.27 DNA bands were obtained by each primer pairs.Eight species of Asteraceae plants similarity coefficient were between 0.69 and 0.74.The average similarity coefficient was 0.694187.Just like ISSR results the similarity coefficient of E.adenophorum and E.odoratum was 0.7346.It was proved that the closest genetic relationship and the smallest difference between E.adenophorum and E.odoratum.The similarity coefficient of the Bidens tripartita and Dahlia pinnata was the smallest.The results showed that the 8 plants had the same relationship when the similarity coefficient of 0.85 and 0.71 were as the threshold.The UPGMA genetic clustering map was constructed.UPGMA genetic map of AFLP markers was obtained after restriction enzyme,enzyme linked,pre-amplification.and selective amplification.It showed that the E.adenophorum and the E.odoratum are very similar in homology.The similarity coefficient of ISSR and AFLP are 0.8664 and 0.7346 respectively.The differences sequences of E.adenophorum and E.odoratum were obtained after polyacrylamide gel electrophoresis,choosing different bands,gel recovery and sequencing.The relationships among the Asteraceae plants were determined by ISSR and AFLP.The theoretical basis for the identification and prevention of Asteraceae invasive species were provided in this paper.In summary,we constructed the ISSR and AFLP molecular fingerprints of the Asteraceae such as E.adenophorum and E.odrata in this thesis.Quarantine weeds in the Eupatorium like E.adenophorum and E.odorata are the main objects in this study.Also,we contrasted E.adenophorum and E.odorata with some homology plants which are of the same family but different genera,such as Solidago canadensis L,Erigeron Philadelphicus L,Conyza Canadensis L,Eupatorium fortuneei Turcz,Bidens tripartita L,Dahlia pinnata Cav.Nine groups of DNA amplification primers,which had the best results,were chosen from 44 groups to build the optimization system of ISSR by orthogonal experimental.Therefore,clear molecular fingerprints of E.adenophorum and E.odorata were taken in ISSR,which have 127 special sites from E.adenophorum,134 special sites from E.odorata and 59 sites from both of them.And 8 groups of primers,which had the best amplification results,were chosen from 64 groups,which have 43 special sites from E.adenophorum,62 special sites from E.odorata and 12 sites from both.DNA fragments of E.adenophorum and E.odorata which express specifically were chosen through the analysis of ISSR and AFLP above.Then,gel of DNA fragments were recovered and sequenced.The program of DNA amplification reactions with designed specific primers was set in a 94? pre-denaturation and 28 cycles with 94? denaturation for 30s,55? annealing for 30s,72? extension for 60s.4 DNA bands of E.adenophorum amplificated specifically were obtained:21-880EA?558 bp?,22-880EA?560 bp?,26-810EA?540 bp?and 32-807EA?258 bp?.Also,5 DNA bands of E.odoratum amplificated specifically were obtained:18-879EO?362 bp?,20-879EO?493 bp??29-807EO?338 bp?,34-807EO?340 bp?and 37-879EO?340 bp?.As a result in agarose gel electrophoresis,we could firmly believe that tissue or samples of E.adenophorum or E.odoratum do exist in the sample when 3 or more specific bands were amplificated twice or more times consistently in the plant sample,which could build specific and rapid methods of molecular biology detection for invasive plants like E.adenophorum and E.odoratum.