Studies On The Genomic Diversity Of Germplasm Resources And Molecular Marker Linked To Ralstonia Solanacearum Resistance Gene In Tobacco

Tobacco, originated from Southern America, is an important economic plant in theworld. It plays an important role in the economic construction of China, which has largestoutput and consumption of tobacco in the world. The initiatory research about molecularmarker technology and genetic diversity of tobacco has made some achievements since themid-1990's, but still it is behind the researches done into other plants, comparativelyspeaking, only the RAPD marker is used frequently while other markers are rarely used. Inthis study, the AFLP and RAPD technology which are used to analyze tobacco genome DNAare established firstly, then the genetic relationship of tobacco germplasms is researched byusing the established methods, and the molecular marker linked to resistance gene ofRalstonia solanacearum is analysised by RAPD, based on the BSA. The main results are asfollows:1. Two methods—CTAB method and SDS method are tested in isolation of tobaccogenomic DNA. The results show that the value of OD₂₆₀/OD₂₈₀ for the DNA samplesobtained with SDS method is around 1.6, hence the degree of purity of DNA extracted byusing SDS method is lower than that extracted by using the CTAB method, the value ofOD₂₆₀/OD₂₈₀ for the DNA samples obtained with CTAB method is over 1.8, the molecularweight of DNA is more than 21 kb, the DNA were completely digestible with EcoRâ… /Mseâ… and a clear AFLP figure is obtained. So the high-quality genomic DNA suitable fortobacco AFLP analysis can be obtained by CTAB method if the process is reasonable.2. The factors influencing RAPD Analysis, including the concentration of DNAtemplate, Mg²⁺, dNTP, primers, Taq polymerase, Thermal cycles and annealingtemperature in tobacco are studied. An optimal PCR system for RAPD in tobacco is found: in 25μl reaction solution, contained 40ng DNA template, 0.4μmol/L random primers,2.5mmol/L Mg²⁺, 0.2mmol/L dNTP, 1U Taq polymerase. The amplification program isdevised: 94℃for 5min;denaturing at 94℃for 1min; primer annealing at 38℃for 1min,extension at 72℃for 1.5min, 39 cycles; at last extension at 72℃for 5min.3. The key factors influencing AFLP analysis are studied during the AFLP process byusing four kinds of tobacco germplasms. An optimized system of silver-staining isestablished for tobacco AFLP analysis. The ascertained factors in the system are that usageof DNA template is 400ng, Mse I/EcoR I is 4U respectively, T4 DNA ligase is 2.8U andaccomplishment of restriction and ligation, is in the same system with the ligationtemperature of 37℃, reactive time of 7hs. In addition, the effects of pre-amplification andselective amplification and silver-stained are tested, the conclusion is the following thatfingerprinting map of tobacco germplasms is constructed with primer E-ACT/M-CAC andthat there are more amplified bands with high polymorphism and good quality.4. The genomic DNA of 48 tobacco germplasms are tested with RAPD using 28arbitrary 10-mer primers which are screened from 200 primers, altogether 184 RAPD bandsobtained, among them 86 are polymorphic bands, the average bands and polymorphic bandsare 6.5 and 3 are produced by one primer, the percentage of polymorphic bands is46.7ï¼…. And then they are amllified by 4 AFLP selective primers, altogether 321 AFLP bandsare obtained, among them 174 are polymorphic bands, the average bands and polymorphicbands are 80.2 and 43.5 are produced by one primer, the percentage of polymorphic bands is54.2ï¼…. According to the total of bands, polymorphic bands and percentage of polymorphicbands, the AFLP silver staining technology is an effective means for researching geneticdiversity and system evolvement of tobacco, due to the high performance and reproduction.5. UPGMA cluster analysis based on two molecular markers data divide 48 varietiesinto two groups: N.tobacum L group and N.rustica L.group. N.tobacum L. group can beclassified into four sub groups,the genetic distance of 48 cultivars is between 1.41～11.0,this cluster result basically accord with theoretic expectation case. The results show thatthe AFLP approach is highly efficient in generating accurate information on geneticbackgroud, relationship and evolution of tobacco cultivars than RAPD method.6. 57 grown varieties of tobacco are tested for resistance to Ralstonia Solanacearum byartificial inoculation on seedling in greenhouse. The result show that the resistance exists remarkably among various varieties, three varieties(TI448A, D101, G80) are of highresistance, 17 varieties(NC82, Zhongyan 90, etc.) show medium resistance, most varietiesshow susceptible, there is no immune resistance.7. A cross between Ralstonia solanacearum resistant variety "Ti448A" and susceptiblevariety "honghua da jinyuan" is made for probing into the genetic resistant law to Ralstoniasolanacearum in tobacco. Resistance of 2 parents, F₁ population, F₂ population and BCF₁population are identified and investigated by artificial inoculation on seedling in greenhouse.Results show that the resistance gene of"Ti448A" is controlled by a single dominant gene.8. DNA of F2 population is extracted to develop to resistant and sensitive poolrespectively based on the BSA. By RAPD, about 200 random primers are used to amplifythe two types of gene pools. Only primer S503 is found to be polymorphic with a fragmentof 750bp in the two parents, F1 and F2 Population. So, the specific fragment S503-750 is aRAPD marker linked to resistance gene. By analysizing the software JOINMAP(version 1.4),the recombination is 5.984ï¼…and the linking distance is 6.261cM.