QTL Mapping And Functional Verification Of Candidate Genes For White Rot Disease Resistance To Grape

Grape white rot is a fungal disease caused by Coniella diplodiella（Speg.）Sacc,which leads to a significant decrease in grape yield.Currently,fungicides are mainly used for the prevention and control of grape white rot disease in production.However,this method not only harms the environment and food safety but also increases cultivation costs.Therefore,breeding resistant varieties is the optimal way to prevent and control white rot disease.Grapevines have characteristics such as a long juvenile period and high heterozygosity,which makes traditional breeding methods time-consuming and inefficient.Utilizing molecular biology techniques to explore grape disease resistance genes can improve the efficiency of targeted breeding and provide theoretical support for the utilization of disease-resistant germplasm and genes.In this study,the‘Manicure Finger（V.vinifera L）’and the‘0940（V.davidii）’and their hybrid offspring（F₁）were used as research materials.The disease resistance traits of the parents and offspring were observed and identified for three consecutive years,and a high-density genetic map was constructed using whole-genome resequencing technology to locate QTLs for grape resistance to white rot.The QTL for disease resistance was located on chromosome 3.Finally,one candidate gene PR1 was screened from the QTL localization interval by combining transcriptome data,and its biological function was verified.The main results are listed below:1.The resistance level standard for white rot disease was determined by white rot resistance determinations in 2019,2020 and 2021,which showed that‘Manicure Finger’was located at level 4（moderately susceptible）and‘0940（V.davidii Foex.）’at level 1（highly resistant）;the F1 population was distributed in different white rot resistance levels,indicating that white rot disease resistance is a quantitative trait controlled by multiple genes.2.Genotyping of‘Manicure Finger’and‘0940’and the F₁ population by whole-genome resequencing,and construction of a genetic map.The genetic map contained 9337SNP markers with an average marker density of 0.3 c M.Combined with the results of the three-year phenotypic identification of the F₁ population,a stable QTL locus Rcd1（resistance to C.diplodiella 1）was identified on chromosome 3,which explained up to17.9%of the phenotypic variation,and an SNP marker significantly associated with white rot resistance was found.Functional annotation of genes in the QTL regions revealed the presence of 40 disease resistance genes within these QTL loci.In addition,several QTL loci with low reproducibility were found on chromosomes 8,12 and 18.3.The transcriptome results of‘Manicure Finger’and‘0940’after inoculation with C.diplodiella showed that triterpenoid biosynthesis,plant-pathogen interactions,flavonoid and flavonol biosynthesis,keratin and wax biosynthesis,and stilbenoid biological pathways play key roles in the defense mechanism of‘0940’against white rot.Using weighted gene co-expression network analysis（WGCNA）,125 candidate genes were identified as having important roles in C.diplodiella resistance in‘0940’,of which only pathogenesis-related protein 1（PR1,Gene ID:100246419）was located in the locus Rcd1,which is presumed to be a key gene for white rot resistance in grapes.4.PCR amplification was used to clone the coding sequence（CDS）and promoter sequences of the PR1 gene from‘0940’and‘Manicure Finger’grape cultivars.The results revealed that the CDS sequences of Vv PR1 and Vd PR1 were completely identical,and their promoter sequences showed 96.7%similarity.The q RT-PCR analysis revealed significant differences in the expression pattern of the PR1 gene between‘Manicure Finger’and‘0940’after 24h and 48h inoculation with C.diplodiella.The expression of the PR1 gene was significantly higher in‘0940’than in‘Manicure Finger’.GUS staining analysis of the PR1 promoter activity in the two cultivars revealed that the promoter activity of‘0940’was stronger.5.In vitro,purification of Vd PR1 fusion protein revealed its ability to inhibit the growth of C.diplodiella mycelium.The plant expression vector p CAMBIA1302-Vd PR1was constructed,and Vd PR1 was transferred into the leaves of the susceptible variety’Jingxiu’by vacuum infiltration and was found to significantly improve the resistance of this grape leaf to C.diplodiella.In summary,The results of three years of white rot resistance identification in the F₁population,combined with the high-density genetic map,led to the detection of one stable white rot resistance QTL locus,distributed on chromosome 3.This QTL explained up to17.9%of the phenotypic variation.Based on the QTL localization and RNA-seq results after parental inoculation with C.diplodiella,the PR1 gene was identified as a key gene involved in white rot resistance.Through functional analysis of the PR1 gene,it was found that the stronger promoter activity of the PR1 gene in‘0940’might be responsible for the higher expression of the PR1 gene in this grape than in‘Manicure Finger’.Furthermore,antimicrobial assays and overexpression experiments demonstrated that the PR1 protein inhibits the growth of the C.diplodiella,and overexpression of the PR1 gene in the susceptible cultivar‘Jingxiu’enhanced its resistance to white rot disease in leaves.