QTL Analysis Of Downy Mildew Resistance And Early Transcriptome Analysis In Cucumber Line PI197088 During Pseudoperonospora Cubensis Infection

Cucucmber is one of the major vegetables grown in facilities,and Downy mildew（DM）,is one of the most common leaf diseases.DM is caused by the obligate biotrophic oomycete Pseudoperonospora cubensis[（Berkeley&M.A.Curtis）Rostoyzev],is one of the major cucumber foliar diseases in the world.Currently,there are still no effective cucumber cultivars show a high level of resistance to the new strain on the market.Finding new resisitance genes,enriching cucumber resistance resources,and breeding new resistant cucumber is the most cost-effective method of DM prevention.Studying the inheritance pattern of DM resistance,QTL mapping and regulation of gene regulation,which provided the foundation for resistant breeding program.Based on the screening results of resistance to downy mildew in cucumber by the partner,this article first determined the disease resistant materials,and then the inheritance laws of cucumber downy mildew resistance and QTL mapping of resistance genes was studied by using different genetic generations.Using next-generation sequencing technology and bioinformatics methods,transcriptome analysis was performed to clarify PI 197088 response to Ps.cubensis infection during the early stage.This study lay a foundation for a understanding the interaction mechanism between cucumber and downy mildew,excavating cucumber downy mildew resistance genes and breeding new varieties of cucumber.The main findings are as follows:1.Cucumber germplasm screening of resistance to downy mildewBased on the works of the research group,we obtained 6 candidate materials,and the resistance of the seedling stage and the adult stage was identified.The results showed that the resistance level of seedling stage and adult stage were consistent.PI 197088 and PI 330628exhibited high resistant characteristic,and 9930 and CCMC exhibited high susceptible under the different circumstances.Consider other indicators,PI 197088 and CCMC were selected for the resistance and ssusceptable germplasm in this study.2.Inheritance of downy mildew resistance in cucumber PI197088Four generation of P₁,P₂,F₁ and F₂ derived from a crossing between PI197088（resistance）and CCMC（susceptable）.Joint analysis of multiple generation was applied for inheritance of downy mildew resistance in cucumber in the present study.The results showed that the resistance of cucumber fitted into major gene+polygene model.The best model were E-4 model（two pair of equal additive effects major gene+additive-dominance effects polygene）and E-1 model（two pair of additive-dominant-epistasis major gene+additive-dominance effect polygene）in two experiments,respectively.The heritability of the major gene was 66.68%and 80.17%in E-4 and E-1,respectively.Therefore,major gene should be high priority in the breeding of resistance to cucumber downy mildew.3.QTL analysis of downy mildew resistance in PI 197088More than 2,000 SSR primers were used to analysis the polymorphic between the parents（PI 197088 and CCMC）,and 420 pairs of primers with polymorphism between the parents were obtained.Using these polymorphic primers,183 F₂ mapping populations were analyzed.The genetic map contains 141 SSR markers,covered 7 chromosomes of cucumber and the total length is 635.25 c M.Using these 183 F₃ families,seven（3 indoor,2 greenhouse,and 2field）downy mildew inoculation experiments were performed to evaluated the DM resistance of the 183 F₃ families.QTL mapping were performed by Using Win QTL Cart 2.5 and R/qtl software.In combination with three environments and three QTL detection methods,five QTL loci related to downy mildew resistance were identified on chromosomes 1,3,4,and 5（dm1.1,dm3.1,dm4.1,dm5.1 and dm5.2）.Among them,dm4.1,explained 27%of phenotypic variation（R²）,is a main effect QTL and the confidence interval is 13.8 c M.Additionally,dm1.1 and dm5.2 showed moderate effects,R² is 10.62%and 12.46%,respectively.While dm3.1 and dm5.1 were minor-effect QTLs,and R² is 7.0%and 7.6%,respectively.4.High-throughput sequencing analysis of the cucumber gene expression during the early stage of Ps.cubensis infectionTranscriptome sequencing of the downy mildew-resistant cucumber line PI 197088within 24 h（0h,6h,24h）after infection by Ps.cubensis using time-course RNA-sequencing.The gene regulation mechanism of resistant cucumber was studied by analysis of gene expression pattern and function enrichment.On the first day of inoculation,the interaction between cucumber and Ps.cubensis had been initiated.At three points,we obtained 19602,19325,and 19414 cucumber expression genes,respectively.The number of genes in low abundance expression was significantly higher than high abundance expression（RPKM>1000）.There were 4072 differentially expressed genes（DEGs）in pair-wise comparisons between time points.Functional enrichment analysis revealed a higher proportion of DEGs involved in metabolic process and cellular process;cell and cell part;and catalytic activity and binding.Most DEGs were associated with metabolic pathways,biosynthesis of secondary metabolites,plant hormone signal transduction,and plant-pathogen interaction pathway.5.The screening of resistant-related genes in cucumber PI 197088 to downy mildewWe collected QTL studies of downy mildew resistance in PI 197088 and our research,which were used for Meta-analysis by Bio Mercator4.2 software.According to the gene function annotation of the consistent QTL（Meta-QTLs）region,the candidate gene of cucumber resistant-downy mildew disease was discovered by combining the expression pattern of transcriptome.We obtained 11 Meta-QTLs from 29 qualified QTLs,mainly located on chromosomes 2,3,4,5 and 6.According to the physical location of each Meta-QTLs,combined with gene expression,SNP analysis,gene annotation and function analysis,we found 26 genes in Meta-QTLs,which were selected as candidate downy mildew-resistant genes in PI 197088 at the early stage of Ps.cubensis infection.