Mapping,Cloning And Functional Characterization Of Botrytis Cinerea Resistance Genes Based On GWAS In Tomato

In this study, 300 sequenced tomato materials were inoculated with gray mold repeatedly, and we statistic the size of lesion, perform GWAS with sequencing SNP data. Then, we screened out several candidate genes and transformed those genes for phenotype identification. Finally, we selected two genes for functional identification further.1. Leaves of wild-type and genetically modified materials were inoculated with gray mold, and screened out some genes which are resistant or susceptible to B.C.2. T1 and T2 overexpression transgenic materials were inoculated with gray mold for several times in different seasons, the results indicated that the phenotype of resistance were stable for GW13 and GW9. Compared to the control, GW13 transgenic plants are more resistant, whereas GW9 transgenic plants are more susceptible. Except the resistance to Botrytis cinerea is different from wild type, there are no other obvious phenotypes between overexpression transgenic materials and the control.3. SNPs analysis. There is a SNP difference in open reading frame sequence of GW13 between resistant and susceptible plants, whereas GW9 have several SNPs difference in the promoter. There may be cause the resistance difference, but it still need to be confirmed further.4. The expression level of disease related marker genes have been studied, the results showed that they are differently regulated in GW9 and GW13 transgenic plants. In GW9 transgenic materials, several genes related to ROS were positive regulated. In GW13 transgenic materials, ERF1 b and PI-I、PI-II, which are marker genes of ethylene and JA signaling, were positive regulated respectively. While NPR1, the SA signaling marker gene, were down regulated.5. The expression of GW9 and GW13 are induced by Botrytis cinerea. The results indicate that the expression levels of these two genes were improved in tomato material TS-179,which is resistant to B.C. Wherease they were down regulated in tomato material TS-47,which is susceptive to B.C.6. The expression level of GW9 and GW13 in mutant of ET and ABA were down regulated, the results indicated that they may be in relation with hormone signaling pathways.7. We found two genes that interact with GW9 through yeast two-hybrid method: SRC2 and CHP-rich zinc protein. Those two genes have been studied extensively in relation to stress resistance.8. GW9 genetically modified materials were inoculated with gray mold for DAB staining before and after the treatment, results show that the ability to clear ROS in GW9 overexpression transgenic plants significantly weaker than the controls.There are still a lot of things should be done to analyse the function of GW9 and GW13 genes.