Construction Of Genetic Linkage Map And Mapping QTL Of Nicotine Content In Burley Tobacco

Nicotine is an important chemical composition in tobacco,and its content is closely related to tobacco quality and availability.To find molecular markers linked to nicotine content through quantitative trait loci(QTL) mapping could be helpful for molecular marker-assisted breeding,and is also very useful for further understanding the mechanism of nicotine synthesis in tobacco.Based on developing DH genetic population and constructing genetic linkage map,QTL controlling nicotine content,total nitrogen and other major quality traits,and plant height and other agronomic traits were mapped and analyzed in the experiment.The main results were reviewed as follows:1.Optimizing disinfection method in tobacco anther cultureThirteen different disinfection types were compared in the experiment,results showed that 10%saturated bleaching powder solution dealing with bud and anther presented lower ratio pollution and higher germination ratio than 70%alcohol solution.Among the thirteen different disinfection types,10%saturated bleaching powder solution dealing with bud and anther for 10 min and then rinsed by steriled water three times could be the best approach.2.Developing two DH genetic populationsF₁ individuals derived from two crosses of Barley cultivar B37×LAB21 and B37×B67 were used to construct DH populations with anther culture,followed by chromosome double with colchicines.Burley cultivar B37 shows high nicotine content and middle resistance to black shank and Burley cultivars LAB21 presents low nicotine content and B67 is susceptible to black shank.At last,two DH populations,one with 106 lines related to nicotine content,and another with 86 lines related to black shank resistance,were developed.During the period of population development,counting the number of chloroplast in stoma guard cell in leave and flow-cytometric method were used to identify chromosome ploidy of the regenerated plants.The result shows that counting chloroplast was a simple and economical method in identifying the chromosome ploidy. Comparatively,the flow-cytometric method was more efficient and accurate.3.Constructing one genetic linkage map in burley tobaccoWith the DH population for nicotine content,625 pairs of SRAP,825 pairs of SM primers(AFLP),2,100 pairs of AFLP methylation primers were screened for the polymorphism between the parents,and 183 polymorphism markers were obtained.A map containing 18 linkage groups and 92 markers was constructed with Mapmaker / EXP, its total length covered 1502.9 cM with an average of 16.3 cM between two markers. 4.mapping QTL for nicotine content,and other quality traits,agronomical traitsThe correlation analysis on nicotine content with other quality traits and agronomic traits based on the two-year field test showed that the correlation between nicotine content and reduced sugar,potassium achieved significant level at 5%,while relation coefficient between the nicotine content and nitrogen was the largest,which reached exremely significant level at 0.1%.However,there were no significant correlations between nicotine content and agronomic traits.With QTL mapping software,Windows QTL Cartographer,v2.5,analysis of single marker(ASM) and composite interval mapping(CIM) were employed to identify locus related to nicotine content and other traits at the significance lever of P＜0.01.A total of 21 genetic loci related to quality traits were detected by ASM,including 8 loci for nicotine content,7 for total nitrogen,4 for total sugar,2 for potassium.15 sites related to agronomic traits were detected also,which were 5 for total leaves,3 for plant height,3 for lumbar width,2 for lumbar length,1 for laimosphere and pitch,respectively.A total of seven QTL,BTNic1,BTNic2,BTNic3,BTNic4,BTNicS,BTNic6 and BTNic7,for nicotine content in leaves were detected based on the analysis of nicotine content in two year field trials.With the nicotine content in leaves from different parts and sampling time in 2006,four QTL,BTNic1,BTNic2,BTNic4 and BTNicS,controlling nicotine content were detected by CIM,while two of them could be detected repeatly. BTNicl was located on 1^st linkage group flanked by PM43P06a and PM44P22,which is adjacent to PM44P22 at 0.69 cM,and explained 16.7%of nicotine content variance with an additive effect of 56.47%.BTNic2 was located on the 10^th linkage group flanked by PM38P32 and PM07P28a,which is nearly to PM2P10,and explained 17.0%of nicotine content variance with an additive effect of 46.8%.Three QTL controlling nicotine content was detected based on the data nicotine content gotten in 2007.In addition,2 QTL controlling total nitrogen content,4 QTL controlling total sugar were also be detected. While the QTL peaks of nicotine content and total nitrogen was in the same interval.QTL analysis on agronomic traits was measured in 2006,one QTL related the plant height,and stem thickness,pitch and lumbar leaf length were detected,respectively.Three QTL related to total leave number were also detected at the LOD of 2.5.