Mining And Analysis Of Late Blight Resistance Genes Of Potato Cultivar ’Cooperation-88’

Late blight is a devastating disease of potato production caused by Phytophthora infestans,a pathogenic blight fungus that originated from central Mexico.The fungus is an oomycete of the genus Phytophthora（Oomycota）.The global economic losses caused by this disease amount to billions of euros per year.The tetraploid potato cultivar’ Cooperation-88’ is a staple cultivar in the southern part of China,which has medium to high resistance to late blight.,however,the specific mechanism and gene composition of this variety for late blight resistance are not known.In this study,the genetic composition of late blight resistance genes in ’Cooperation88’ was initially elucidated through effector recognition identification and genomewide screening of genes encoding core NBS-LRR-like proteins in three diploid’Cooperation-88’materials.The main findings are as follows:1.Mining of unknown late blight resistance genes in ’Cooperation-88’ based on effector identification.Using PVX-agroinfection method,we found that P.infestans effectors PITG₀9160 and PITG₀7555 could be recognized by ’Cooperation-88’ and stimulate allergic response.According to the "gene-for-gene" hypothesis,it was inferred that ’Cooperation-88’ contains unknown late blight resistance genes that can recognize the effectors PITG₀9160 and PITG₀7555.2.Screening for genes encoding the core NBS-LRR-like structural domain protein of ’Cooperation-88’.In combination with three diploid ’Cooperation-88’ materials（19C291,19C-375,19C-471）obtained by solitary reproduction induction in the early stage of the group,the proteins with NBS-LRR conserved structural domains were scanned genome-wide by whole-genome sequencing and assembly of these three materials.The results showed that 19C-291 contained 3607,19C-375 contained 4510,and 19C-471 contained 6304 genes encoding NBS-LRR type proteins.The gene sequence similarity analysis revealed that the three materials contained core 202 genes encoding NBS-LRR type proteins.3.Differential expression analysis of the genes encoding 202 NBS-LRR type proteins in ’Cooperation-88’ candidates.By inoculating three diploid ’Cooperation-88’with P.infestans and using the known expression pattern of the late blight resistance gene Rpi-R2 as the screening condition,the results showed that Rpi-R2 was expressed at the highest level 12 h after inoculation.Primers were designed for the genes encoding 202 candidate NBS-LRR type proteins using Primer3.0 online website,and real-time fluorescent quantitative PCR（qRT-PCR）was performed using cDNA at 12 h postinoculation as the template.Finally,19NBS-LRR genes were screened for up-regulated expression in potato late blight infestation,and these 19 genes were analyzed for gene annotation.By transient expression of the effector PITG₀9160 and PITG₀7555 in three ’Cooperative 88’ diploids inoculated,39 and 43 up-regulated expression genes were finally screened,respectively,and two genes were found to be both effectorinduced up-regulated expression and disease-causing epidemic mold-induced upregulated expression by comparison with disease-causing epidemic mold-induced upregulated expression genes.In summary,by identifying the P.infestans effectors PITG₀9160 and PITG₀7555,it was inferred that ’Cooperation-88’ contains unknown late blight resistance genes corresponding to these two effectors.In addition,by screening and identifying ’Cooperation-88’ containing 202 core NBS-LRR genes,and using qRT-PCR to detect the expression pattern of these genes induced by P.infestans,19 genes were found to be up-regulated.It was inferred that these 19 genes could be new late blight resistance genes of ’Cooperation-88’.Meanwhile,the up-regulated expression genes were screened by overexpressing effector PITG₀9160 and PITG₀7555,and finally two genes were screened to be both up-regulated expression induced by effectors and up-regulated expression induced by PITG₀9160,PITG₀7555 and P.infestans infection.The results of the study are beneficial to expand the knowledge of plantpathogen interactions and provide new to disease-resistant related genetic resources for potato breeding against late blight.