Molecular Markers Development And Mapping Of Tobacco Bacterial Wilt Resistance

Tobacco bacterial wilt is a soil-borne bacterial disease caused by Ralstonia solanacearum.It is also a catastrophic disease affecting global tobacco production.It has many hosts,is widely distributed,and is difficult to control.Breeding resistant varieties is the most scientific and effective way to control the disease.In this study,the strains collected from 5 regions of Hunan,Yunnan,Shandong,Chongqing and Fujian were identified from three aspects:morphological characteristics,biochemical types and evolutionary types;a set of identification systems for tobacco bacterial wilt inoculation was established and optimized;Using a natural population composed of 219 flue-cured tobacco varieties,the genome-wide association analysis was used to locate the segments that were significantly associated with tobacco bacterial wilt resistance variation in the tobacco genome,and predicted candidate genes for disease resistance;through 254 F_2:3 family populations Laboratory artificial inoculation identification and simplified genome re-sequencing were performed at the seedling stage,a linked genetic map was constructed,and disease-resistant QTL mapping was carried out.The main findings are as follows:1.Taking Ralstonia solanacearum strains from 5 regions as the test object,the other 4 strains except HN strain were found to be strong pathogenic strains with strong fluidity and a central reddish edge and milky white,which were identified by biochemical type.It was found that the test strains belonged to the common biochemical typeⅢin China,and the test strains were found to belong to the evolution typeⅠthrough the evolutionary classification framework,and were divided into sequence variants 15,17 and 34.Yunnan strains belonging to the domestic common sequence variant 17 were selected YN-TC-2 was used as the test strain.2.Based on the method of bacterial root irrigation and improvement,the optimal inoculation concentration of the bacterial solution was confirmed to be 1.5×10⁸ cfu/mL by the gradient inoculation test.Under this concentration,the most significant difference was found in the resistance between the resistant control FDSHB and the susceptible control HHDJY.A fast,stable,and efficient identification system for tobacco root wilt disease soil root irrigating bacteria thermostatic water bath was established.3.Using simplified genome resequencing,219 tobacco germplasm materials were subjected to high-throughput population SNP locus typing,and information on 384,904 available SNP loci was obtained.The genomic linkage disequilibrium（LD）of the test material was calculated and evaluated.The average LD attenuation distance of flue-cured tobacco varieties is 256 kb.A field disease nursery was used to investigate the disease index of bacterial wilt under natural disease conditions.Through genome-wide association analysis,a segment significantly associated with tobacco bacterial wilt resistance variation was located in the tobacco genome.The peak value SNP is located at 23,977,382 bp on chromosome 17（C17＿23,977,382）.Through gene annotation in this segment,4 candidate disease resistance genes were predicted.4.Taking FDSHB as the disease-resistant parent and HHDJY as the susceptible parent,an F_2:3population with 254 families was constructed.The population was subjected to artificial inoculation identification and simplified genome resequencing of tobacco bacterial wilt at the seedling stage,and a linked genetic map with 349 upper markers was constructed,and 2 resistant QTL segments were detected,namely qBTW-5-1 and qBTW-12-1,which can explain 6.7%and 7.1%of phenotypic variation,respectively.