| Tobacco bacterial wilt(TBW)is the main disease which affects tobacco growth and resulted in the reduction of yield and quality in the area of southern China.TBW resistance and some agronomic traits are considered as the important indicators to evaluate the growth,yield and quality in tobacco.As everyone knows,TBW resistance and most of agronomic traits are quantitative traits which controlled by multiple genes and easily affected by environment.Development of molecular markers closely linked to TBW resistance and agronomic traits could speed up the breeding process of superior varieties by using marker-assisted selection(MAS)techniques.Therefore,an tobacco F7 recombinant inbred line(RIL)populations derived from the cross of “118-3” and “Yueyan 98” was used to construct the genetic map by using SSR markers in the current study,the quantitative trait loci(QTL)of TBW resistance and some agronomic traits were detected.The major results are as follows:1.Establishment of a rapid and simplified silver staining method for detection of SSR markers in polyacrylamide gels.Plyacrylamide gel electrophoresis combined silver staining technique is one of the effective and high resolution method to detect DNA fragments with several bases differences.However,the traditional silver staining method has several weaknesses such complicated operation,time-consuming,more reagents required,and it is not suitable for our large scales of genotyping.Based on the previous studies,a improved silver staining method was established with only two major steps of the staining and development.The improved method only required three kinds of reagents(silver nitrate,formaldehyde and sodium hydroxide)and took6 to 7 minutes.In addition,the improved method detected the SSR amplification products with the excellent characteristics of intensity,contrast,sensitivity and reproducibility,and it has been successfully used for detection of SSR polymorphisms in the current study.These results indicate that the improved method is suitable for the genotyping of large scale of SSR markers in tobacco.2.Construction of a genetic map by using a tobacco RIL population derived from the cross of “118-3” and “Yueyan 98”.214 SSRs presented polymorphic between two parents of the RIL population were screened from 1,405 SSRs,and these polymorphic SSRs were used to genotype the RILs.Based on the genotyping data,a genetic map oftobacco covering a total length of 3,126.10 cM with 24 linkage groups was constructed by software of MapMaker 2.0.The length of linkage groups ranged from48.51 to 292.04 cM,with an average of 130.25 cM.The map contained 426 SSR alleleic variations,and the number of allelic loci in the linkage groups ranged from 6to 38,with an average distance of 7.34 cM.This genetic map could be used for QTL mapping of TBW resistance and some agronomic traits.3.Identification of the QTLs for TBW resistance and some agronomic traits.The RIL population was planted in the experimental station of nanxiong tobacco science research institute of Guangdong province in 2015 and 2016.Thirteen of traits,which were TBW disease index,stem girth,pitch,SPAD value,length and width as well as weight of the largest upper leaf,length and width as well as weight of the largest middle leaf,length and width as well as weight of the largest lower leaf,were investigated.Based on the phenotpying and genotyping data of RILs,the QTLs for 13 traits were analyzed with multiple QTL mapping method(MQM)by using MapQTL5.0 software,and 14 QTLs were identified totally.Among these QTLs,2 QTLs were for the TBW resistance,5 QTLs were for the stem girth,2 QTLs were for the weight of the largest middle leaves,and five traits including the length of the largest midde leaf,the weight of largest lower leaf,the length of the largest upper leaf,the width of the largest lower leaf and the pitch had one individual QTL.In addition,several QTLs were overlapped in the some areas of the genetic map. |
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