Evaluation Of Potato Germplasm For Late Blight Resistance And Transcriptome Analysis Of Qingshu9 In Response To Phytophthora Infestans Infection

As one of the main food crops in China,potato has great potential in contributing food security.Due to the rapid virulence variation of Phytophthora infestans,the causal agent of late blight of potato,loss of genotype-specific disease resistance is common and the frequent late blight threatens worldwide sustainable potato production.Breeding for disease resistant varieties is the most effective strategy to control the disease.In the breeding for disease resistance program,use of disease resistance resources,especially the broad-spectrum,durable resistance genes,and development of varieties with pyramided multiple resistance genes.In this study,the selective sweep analysis was employed to examine genetic differences between resistant and susceptible populations of 1229 progenies derived from 36 color potato crosses.In addition,we employed Agrobacterium tumefaciens-mediated transient gene expression to investigate composition of disease resistance genes in the dominant potato varieties Qingshu 9 and Qingshu 2,by examining their recognition of known avirulence RXLR effector genes of P.infesrtans.Finally,we employed RNA-seq technology and performed transcriptome analyses of Qingshu 9 for expression of genes responsive to the infection of P.infestans at different time points.The main findings are as follows:1.Evaluation of potato germplasm for late blight resistance and selection elimination analyses.By evaluation of 24 potato lines for their performance of late blight resistance in the field and the controlled conditions,6 displayed highly resistant(HR),3 were resistant(R),3 were moderately resistant(MR),5 were susceptible(S),and 6 were highly susceptible(HS).A total of 1229 progenies derived from 36 crosses of color potatoes were evaluated for their field performance of late blight disease resistance.Disease resistant and susceptible pools were constructed using 25 progenies each that showed HR and HS,respectively,and SLAF sequencing technology was employed to identify SNP loci in the HR population.A total of 48859 high-confidence SNP loci,evenly distributed on potato 12 chromosomes,were obtained.The genetic diversity,phylogenetic relationship,and population structure of the potato HR populations were complicated than the HS populations.Selective sweep analysis was performed based on the developed polymorphic SNP loci to identify candidate genes related to late blight resistance.Based on the population segregation and gene polymorphism,a total of 39 candidate genes were annotated.The selected sweep site region genes were annotated and analyzed in the GO database,COG database,and KEGG database.2.Analysis for composition of resistance genes of the dominant potato varieties Qingshu 9 and Qingshu 2.Using Agrobacterium tumefaciens-mediated transient expression assay,the recognition of 9 known P.infestans avirulence effector genes by Qingshu 2 was determined.The results showed that Qingshu 2 could recognize Avr2,Avr3 a,Avr3b,Avr4,Avr Smira2 and Avrvnt1,as indicated by the recognition-triggered hypersensitive necrosis.PCR amplification of gene-specific molecular markers suggested that Qingshu 2 contains multiple disease resistance genes,including R3 a,R3b,R10,Rpi-Smira1,Rpi-blb2,Rpi-abpt,Rpi-RD and Rpi-phu1,and Qingshu 9 may contain broad-spectrum disease resistance genes highly homologous to RB,Rpi-sto1 and Rpi-pta1,as well as R3 a,R10,Rpi-blb2,Rpi-blb3,Rpi-abpt,and Rpi-RD.3.Transcriptome analyses of genes in Qingshu 9 responsive to infection by P.infestans at different time points of 0h,24 h,48h and 72 h post-inoculation,were conducted by using high-throughput sequencing-based RNA-seq technology.By k-mean cluster analysis of differentially expressed genes(DEGs)between the sequencing samples,genes with different expression patterns were identified.The expression level of DEGs was extremely high for the subcluster?1 and subcluster?4 genes at 24 h post-inoculation,suggesting importance of this infection stage in defense response of Qingshu 9.Functional annotation analyses showed that genes involved in plant-pathogen interaction,biosynthesis,metabolic pathways,secondary metabolism,and photosynthesis,were significantly enriched.In addition,many genes related to biosynthesis of zeatin and lignin were enriched,both are important factors related to disease resistance.Analysis of transcription factors showed an enrichment of 980 transcription factor genes(TFs),belonging to 68 TFs family.The number of AP2-EREBP and MYB families responsive to P.infestans was the most,with 86 and 74 genes,respectively,which was accounted for 6.94% and 7.55% of the total annotated TFs,respectively.The P.infestans infection-responsive TFs also included members of b HLH,WRKY,NAC,b ZIP families.The expression of 9 randomly selected DEGs with significant differential expression in the disease resistance pathways,including PR1,WRKY transcription factor gene,and salicylic acid-inducing gene 19,was validated by q RT-PCR assay with completely consistent results,demonstrating the reliability of the transcriptome analysis.