Analysis Resistance Mechanism Of The Tobacco Germplasm Y18 To TMV,CMV And PVY

Virus disease is one of the major diseases of the tobacco（Nicotiana tabacum）.It is of the great practical and theoretical significance to study the mechanisms of plant resistance to viral diseases and to explore and utilize superior resistance genes.Tobacco virus diseases include tobacco mosaic virus disease,cucumber mosaic virus disease,potato virus Y and so on.The N gene from N.glutinosa is the first cloned plant resistance gene（R gene）and has been broadly used in tobacco breeding.e IF4E,encoding a eukaryotic translation initiation factor,is the most widely used recessive resistance gene in tobacco anti-PVY breeding.The multiplication and movement of potato virus Y in the cell will be limited when the gene loses its function,and thus increase the plant’s PVY resistance.However,the resistance genes to CMV have been rarely reported in tobacco.In this thesis,an F₂population was constructed from the tobacco germplasm Y18,containing the N gene fragment and had both resistance to the three virus diseases,and the susceptible parent K326 to map the chromosomal location of the N gene based on bulked segregant analysis（BSA）and SNP genotyping.Then the N gene loss-of-function mutant,generated by CRISPR/Cas9 system,was used to analyze the function of N gene-mediated resistance to TMV and CMV.Finally,Y18×K326 F_2:3pedigree populations were identified for PVY resistance.The main findings were as follows:1.Based on the results of F₂phenotype identification,BSA resistant and susceptible gene pools were constructed and the genotype were analyzed using SNP micro-arrays.Among them,2,437 SNPs were detected between the two parents,while a total of 1,321SNPs were detected between the resistant and susceptible pools.There were 291 SNPs among two parents and the BSA pools.The genomic data of Honghuadajinyuan in China Tobacco Genome Database were used to analyze the chromosome segments enriched in SNP loci.Six genes were annotated with TMV resistance-related gene in the SNP enriched region of chromosome.SNP loci were used to develop SNP genotyping markers based on Sanger sequencing.The genetic distance of Y18 resistant genes was calculated using Jion Map4.0 mapping software,and finely determined using QTL Ici Mapping software to determine the molecular marker P75574-P75993.Interval,narrow section is 419 Kb.2.The N gene in Y18 was knocked out using CRISPR-Cas9 technique,and one homozygous T₂mutant line were obtained.One adenine was inserted at the editing site between G and C,resulting in the change of the encoding protein sequence of the N gene.The identification of TMV and CMV resistance of T₂generation plants showed that the mutants had obvious TMV symptoms,indicating that the TMV resistance function mediated by N gene was lost.After inoculation of CMV,the incidence and disease index of the mutants were significantly higher than that of the wild type Y18,indicating that the N gene might be involved in the resistant pathway of tobacco to CMV.3.Based on the eIF4E gene sequence,a molecular marker for identifying PVY resistance in tobacco was developed,and the PVY resistance of some tobacco germplasm was identified by the marker.Combined with the results of the disease identification,6tobacco germplasm,including Y18,showing PVY resistance were identified e IF4E gene missing,while 5 other tobacco germplasm containing e IF4E were also observed PVY resistance.These results indicated that other types of PVY resistant sources may be existence in tobacco germplasm.4.To analyze the effects of N gene in PVY resistance of tobacco,120 F_2:3lines were identified PVY resistance and whether it contains N gene or e IF4E.The results showed that 24 lines without e IF4E（of which 20 lines contained the N gene,4 lines were absent of the N gene）were resistant to PVY;96 lines containing the e IF4E gene（68 lines containing the N gene,28 lines without N gene）were more susceptible to PVY.Correlative analysis was performed using the R language Cor.test function.It was found that the absent of e IF4E was significantly positively correlated with the PVY resistance（p<2.2e-16,R=0.87）,while N gene molecular markers was negatively correlated with PVY resistance（p=0,007665 R=-0.24231）,indicating that Y18 resistance to PVY was due to deletion of the e IF4E gene,and the N gene did not mediate PVY resistance.