Joint Linkage And Linkage Disequilibrium Mapping For Tobacco Bacterial Wilt Resistance

Tobacco bacterial wilt?TBW?is one of the main diseases affecting tobacco yield and quality,and its occurrence often causes great economic losses.Breeding variety resistant to TBW is the best measure to reduce the detriment of tobacco,and marker-assisted selection?MAS?could speed up the breeding process.Therefore,understanding the genetic characteristics of TBW resistance and exploring the excellent alleles closely related to TBW resistance are very important for improving TBW resistance through MAS.At present,the genetic analysis of TBW resistance mainly adopts the method of either linkage analysis or association analysis.However,the TBW ressitance belongs to quantitative trait inheritance,the detectable information is limited if the genetic analysis is performed by only one method of either linkage analysis or association analysis.In the current study,linkage analysis and association analysis were carried out with a population of 133 F₇ single-grain recombinant inbred lines?RILs?and a 94-accessions natural population,respectively;and joint linkage-linkage disequilibrium mapping was also performed to dissect the TBW resistant genetics.The main results are as follows:1.The additive main effects and multiplicative interaction?AMMI?models of the disease indexes in RIL and natural populations were analyzed by R language for acquiring the phenotypic effects of both genes and environment.The results showed that environmental factors had the greatest influence on the TBW resistance of two populations,followed by genotypes,and the least effect was the interaction between genes and environment.The stability parameters of each line or each accession to TBW resistance was evaluated,the result generally showed that the stablility of RIL population to TBW resistance was higher than that of natural population.For RIL population,the stability parameters of RILs differed significantly in the range from0.007 to 0.798 with an average of 0.510,in which line 48?0.007?was the stablest one.The stability parameters of accessions in natural population varied relatively little,from 0.532 to 0.619 with an average of 0.605,in which Fengzi 6?0.532?was the stablest accession.2.133 RILs were genotyped by 546 molecular markers including 253 SSRs and293 InDels,and a genetic linkage map was constructed with 463 markers which include 192 SSRs and 271 InDels.The total length of the genetic map covered1230.73 cM in the genome,and the average distance between markers was 2.66 cM.The QTL mapping for TBW resistance was performed based on the TBW disease index data of RIL population in four environments,and four QTLs,which were qTBWR-6-E4,qTBWR-11-E3,qTBWR-17-E1 and qTBWR-18-E2,were detected.The LOD values of these QTLs were all greater than 3,and the explanation rates of phenotypic variation for TBW disease index were all over 10%.These QTLs were located in LG6,LG11,LG17 and LG18,respectively.The results enriched the QTL loci for TBW resistance and could provide a theoretical basis for MAS breeding in tobacco.3.The natural population with 94 tobacco accessions was genotyped by using546 molecular markers,and population structure and linkage disequilibrium-based association analysis were carried out for detection of molecular markers related to TBW resistance.The results showed that 546 markers produced 1503 allelic variations in 94 tobacco accessions,with an average of 2.75 varients per marker.The polymorphic information content?PIC?values of these markers ranged from 0.1007 to0.8760,with an average of 0.4584.Based on the information of the allele variations,376 haplotypes were constructed and 94 tobacco accessions were divided into two groups,indicating the structure of the natural population was simple and would be helpful for reduction of pseudo-association.The association analysis of two-years'field data was performed with 376 haplotypes or 1503 varients,respectively.8haplotypes and 17 alleles were identified to be associated with TBW disease index and explained from 8.21%to 11.42%and from 7.91%to 23.31%of the variation of TBW disease index,respectively.4.Joint linkage-linkage disequilibrium mapping was conducted by using RIL population and natural population.In the parallel mapping,we found that the distance between haplotype Hap227 and QTL qTBWR-18-E2 in LG18 was only 1.14 cM,while the distance between haplotype Hap363 and QTL qTBWR-17-E1 in LG17 was3.15 cM.These two regions may have genes related to TBW resistance.In the integrated mapping,8 haplotypes and 10 alleles were detected to be associated with TBW resistance.The explanation rates of phenotypic variation for the TBW disease index were from 4.95%to 7.06%and from 3.49%to 6.27%,respectively.In addition,two variant loci?also haplotype Hap'159?of the left-wing marker RAD-66 in QTL qTBWR-6-E4 detected in linkage analysis were identified to be associated with disease resistance in the integrated mapping analysis;haplotypes Hap'545,Hap'546and variants S'1 and S'3 detected in association analysis were also identified to be associated with disease resistance in the integrated mapping analysis.5.Four haplotypes?Hap'159,Hap'545,Hap'546,Hap 227?and two variant loci?S'1 and S'3?,which were identified to be related to TBW resistance by at least two of three analysis methods?linkage analysis,association analysis and integrated mapping analysis?,were selected to test their contribution effects for TBW resistance.The results showed that Hap'159 and Hap'546?also haplotype Hap'363?had reduced effects on the TBW disease index,while Hap'545,Hap 227,S'1 and S'3 had additive effects on the TBW disease index.Using all these haplotypes and variant loci to evaluate tobacco materials,the reduction effect could reach-12.91,which was much higher than the average effect value of single haplotype or locus or other combinations among them,and all the carrier materials showed resistance.A recombinant inbred line 106 with high resistance and stability was found by the analysis of combining molecular markers and stability parameters,this line was 0.094in stability parameter and 5.56 in the average disease index under two–year multi environments.The results could provide reference for MAS of TBW resistance.