Dynamic QTL Mapping For Bacterial Wilt Resistance In Tobacco

Tobacco bacterial wilt(TBW)caused by Ralstonia solanacearum is one of the soil-borne bacteriosis,which significantly affects yield and quality of tobacco.The most effective way to prevent TBW is genetic improvement and breeding resistant varieties.However,the current research basis of quantitative trait locus(QTL)mapping for TBW is relative weak,the resistant sources is insufficient,the QTL analysis is for unconditional QTLs which depends only on the accumulated effect of the TBW-resistant phenotype performance during whole period,and should not fully reflect the conditional QTLs detected by the net genetic effect in consecutive time intervals.Therefore,it is necessary to enhance the utilization strength of new resistant sources,and analyze the dynamic(conditional and unconditional)QTLs of resistance for better understanding inheritance of resistance.It could provide novel resistance genes and fundamental basis for tobacco TBW breeding program.In the present study,a recombinant inbred line(RIL)population constructed by the cross of a high TBW-resistant landrace Dayemihe and a TWB-susceptible variety Changbohuang was utilized for QTL analysis.Based on the phenotypic data of each RIL at different periods,dynamic QTLs for TBW resistance were detected and genetic characteristics of TBW resistance were dissected.The results will provide a basis for resistance gene cloning and marker-assisted selection in breeding program.The major results are as follows:1.The SSR and InDel markers polymorphic among tobacco parents were developed by using restriction-site associated DNA(RAD-seq)technique.Two parental genotypes,Dayemihe and Changbohuang,were sequenced by RAD-seq on the Illumina MiSeq PE300 platform.46,602,052 and 42,946,668 high quality clean reads were obtained in Dayemihe and Changbohuang,respectively.Through sequences analysis,26,344 SSR loci and 160,910 InDel loci were detected in the parents.1,103 SSRs and 14,344 InDels were identified to be putative polymorphisms between parents based on the sequence analysis.50 SSRs and 50 InDels were randomly selected to validate their polymorphisms in 4 tobacco genotypes,the resultshowed that the polymorphic rates for SSRs and InDels between the two parental genotypes were 64% and 50%,respectively.These results indicate RAD-seq could be used to identify a large number of SSR and InDel loci and develop polymorphic SSR and InDel markers as well,and the result could provide basis for the development and application of SSR and InDel markers.2.A genetic map was constructed by using SSR and InDel markers.183 RILs derived from the cross of TBW-resistant landrace Dayemihe and TBW-susceptible variety Changbohuang were used as mapping population.The polymorphisms for1,450 SSRs and 300 InDels were screened in the two parents,the polymorphic markers were selected to genotype the RIL population.Based on the genotyping data,a genetic map consisting of 27 linkage groups and 598 markers was constructed by using JoinMap 3.0 software.The total length of the map was 2235.15 cM and the length for linkage groups varied from 33.25 cM to 140.40 cM.The number of markers on each linkage group ranged from 5 to 47,with an average distance of 3.74 cM.This map was used to map dynamic QTLs of TBW resistance.3.According to the field data of TBW resistance in RIL population at different stages of disease development,dynamic QTLs for TBW were mapped.Combination of the constructed genetic map and TBW disease index of each RIL at different stages in 2016 and 2017,dynamic QTLs for TBW resistance were analyzed by using multiple QTL mapping method,and 9 unconditional QTLs and 13 condition QTLs were detected in both 2016 and 2017 at the level of LOD > 2.5.Among the 9unconditional QTLs,3 and 7 QTLs were detected in 2016 and 2017,respectively;and one of them was detected in both two years.These QTLs could explain 6.5% to16.4% of the phenotypic variation.Among the 13 conditional QTLs,8 and 5 QTLs were detected in 2016 and 2017,respectively.These QTLs could explain 6.8% to10.9% of the phenotypic variation.In addition,qBWR2-1 and qBWR7-1 were detected in both unconditional and conditional analysis in 2016,respectively;and qBWR21-1 and qBWR24-1 were detected in unconditional and conditional analysis in 2017,respectively.The results showed that the number and genetic effects of QTLs for TBW resistance had large changes during the invasion period of bacterial wilt,itcould suggest that the genes controlling quantitative trait of TBW resistance exist spatiotemporal expression patterns at different stages of disease development.