Preliminary Screening Of Candidate QTL Genes For Resistance To Brown Spot In Tobacco

Tobacco brown spot is a fungal disease that resulted in the decline of yield and quality of flue-cured tobacco.It is the most economical and effective method to control brown spot disease by exploitation and utilization of resistance genes and breeding the resistant cultivars for tobacco.Using cross combination from JYH and NC82,research group has detected the scope of major effect QTL on chromosome 24.On this foundation,BC₃F₂ population derived from a cross of JYH and NC82 was developed.The scope of major effect QTL related with resistance to brown spot disease was narrowed down using 26 SNP markers by Mass ARRAY method.On this basis,combined with bioinformatics methods and RT-PCR technique,candidate genes were preliminary studied at DNA level and transcriptional level.VIGS system and CRISPR system were constructed for gene function verification.Through the above test methods,the results are as follows:1.Using SNP site Mass Array mass spectrometry,26 SNP loci were designed in the target area.The BC₃F₂ generation population of 255 individual individuals derived from JYH and NC82 was obtained after crossbreeding,and then the genetic map within the target area was con structed.The phenotypic analysis showed that the resistance to brown spot was significantly different between the two parents.The average disease index of the BC₃F₂ population was 3.43.Combined with genotypic and phenotypic data,the main effect QTL of tobacco brown spot resistance was located between the target interval M29^M28,and the interval genetic distance was 1.6 cM.2.According to the published tobacco genome database?https://solgenomics.net/?and NCBI software,the information of 31 genes in the target area is successfully constructed.Based on test analysis,gene Nitab4.5₀000264g0150.1?Nitab4.5₀000264g0 130.1?Nitab4.5₀000264g0170.1?Nitab4.5₀002011g0070.1?Nitab4.5₀000264g0180.1 have obvious sequence differences.3.After inoculation,the change of gene expression in the target area was detected.Whether it is disease resistant variety or susceptible variety,the expression of gene Nitab4.5₀000264g0050.1?Nitab4.5₀000264g0040.1?Nitab4.5₀000264g0130.1?Nitab4.5₀000264g0070.1 appear uptrend at all time points.The gene expression of the disease resistant material was significantly higher than that of the susceptible material at all time points,and the response to the disease course was carried out after the inoculation of brown spot disease.Nitab4.5₀000264g0050.1 is the calmodulin binding protein gene and Nitab4.5₀000264g0130.1 is the glutathione peroxidase gene,which is closely related to the resistance of plants.4.VIGS technology was used to screen and identify 31 genes in the target area,and the system of VIGS technology for the study of brown spot disease was constructed.The results showed that the target gene was effectively silenced.In addition to a small number of spots in tobacco leaves after the gene Nitab4.5₀000264g0130.1 was silenced,the phenotype of brown spot disease was not obvious when other genes were silenced.