Identification, Inheritance Analysis And Mapping Of Major Qtls For Gummy Stem Blight Resistance In Cucumis Sativus-Hystrix Introgression Line

Didymella bryoniae (Auersw.) is a phytopathogenic fungus that causes gummy stem blight (GSB) disease in cucurbits. It infects all parts of cucurbits including stems, leaves, flowers, fruits, seeds and roots of cucurbits. Globally, GSB is currently ranked highly as an important fungal disease due to substantial economic losses it causes in the cucurbit production. Under field conditions, D. bryoniae and other pathogenic fungi co-infect the host plant thus making identification and management of the disease difficult. Previous reports had revealed absence of effective GSB resistant cucumber cultivars. Due to the narrow genetic base of cucumber, the studies of GSB resistant genes are quite limited. Wild species of Cucumis are rich sources of useful genes for the improvement of cultivated cucumber. They exhibit good resistance to GSB and hence exploring and transferring these valuable genes into elite lines would enhance cucumber GSB resistance improvement.Five different pathogenic fungi were isolated and purified from infected cucumber plants. Morphological, pathogenic, and molecular characterization confirmed their specific generic taxonomic units. GSB resistance screening was executed on53introgression line (IL) derived from the progeny of Cucumis hystrix x C. sativus and10cucumber cultivars by using D. bryoniae strain A1.4ILs and3cultivars exhibited consistent variations with respect to resistance D. bryoniae were used in a half7×7diallel analysis. Segregating F2populations derived a across between GSB resistant IL HH1-8-1-2and susceptible CL ’8419’were used to study the inheritance and quantitative trait loci (QTL) analysis of GSB resistance. Annotation and function prediction of candidate genes were also conducted to explore the molecular mechanism of GSB in cucumber. The main results are as follows:1. Morphological, pathogenic, and molecular characterization of Didymella bryoniae and co-infecting fungal pathogensFive different pathogenic fungi were isolated from infected cucumber plants and those associated with disease symptoms were named as:Nl, Q1,3-16,8102and HH. Strain A1and Nl were certified to be the most virulent pathogen on cucumber cultivar,’P02’, By comparing conidial morphology of these isolates with known D. bryoniae control strains A1, TMK-2and A, we could identify N1as D. bryoniae. Internal transcribed spacer (ITS)1and ITS2sequences analysis further confirmed that N1and the control strains A1and TMK-2belong to D. bryoniae. To assign the other four pathogenic isolates, we blasted the ITS sequence to NCBI database and obtained56homologous sequences. Phylogenetic analysis placed all these sequences into six distinct groups. Based on identity of the56known sequences, we could assign N1, A1and TMK-2to D. bryoniae; A to Phoma sp; HH to Alternaria sp;8102to Corynespora cassiicola;3-16to Arthrinium sp; Q1to Plectosphaerella cucumerina.Simple PCR markers that could specifically amplify D. bryoniae, Alternaria sp, C. cassiicola, Arthrinium sp, and P. cucumerina were generated to improve the efficiency of management of these diseases. Via ITS sequence analysis, the A strain could be distinguished from the rest D. dryoniae strains owing to two base deletions. Information on the dominant pathogens responsible for confusing GSB epidemics in Nanjing’s cucumber fields will be useful to target breeding and disease control strategies.2. Identification of cucumber introgression lines with resistance to gummy stem blightScreening for resistance gummy stem blight (GSB) was achieved by inoculating selected plants at seedling stage.4introgression lines (HH1-8-1-2, HH1-8-1-16, HH1-8-5and BC1F5CT-01-2) and1cucumber cultivar (’Jinyou No.10’) were identified to resistant while1introgression line (HH1-8-57) and3cucumber cultivars (’8419’,’Xintaimici’and ’Jinchun No.4’) were identified as stable susceptible resources to GSB. No correlation was found between stem and leaf GSB resistance in cucumber while the resistance of ILs under different seasons was stable. The characterization of agronomic characters showed that the fruit thorn colour of HH1-8-1-2was brown falling GSB resistant IL HH1-8-1-2between the black and white fruit thorn colours of wild and cucumber cultivars, respectively. This also confirmed that HH1-8-1-2originated from wide hybridization.3. Combining ability and inheritance analysis of resistance to gummy stem blight in Cucumis sativus-hystrixThe analysis of general (GCA) and specific combining ability (SCA) of resistance to GSB showed highly significant GCA and non-significant SCA effects indicating the importance of additive genetic components in controlling GSB resistance. The lines of HH1-8-1-2, HH1-8-57and’8419’combined well with other parents as indicated by GCA. CC3, CC2had significant SCA when crossed with HH1-8-57. The comparison of the crosses with low GSB infection and GCA effects of their parents showed that such crosses involved at least one parent with high or average negative GCA effect. The results revealed that it is feasible to use highly or moderately resistant parents in conventional breeding methods to achieve genetic improvement of GSB resistance in cucumber.The results of inheritance analysis showed that broad-sense heritability of resistance to GSB were73%of F2(HH1-8-1-2x’8419’self-crossed),75%of F2’(’8419’xHHl-8-1-2self-crossed) in2009fall and49.9%of102064F2(HHl-8-1-2x’8419’self-crossed) in2010spring. According to nearly bimodal distribution of disease rating with one peak toward HH1-8-1-2in F2populations, the inheritance model of GSB resistance in cucumber HH1-8-1-2is controlled by major gene plus polygene.4. Genetic mapping of major QTLs for gummy stem blight resistance in Cucumis sativus-hystrix introgression linesSix quantitative trait loci (QTL) on chromosomes one, four and six were identified for GSB resistance by screening individual chromosome regions from two introgression distribution maps published previously and named as:GSB1a, GSBlb, GSB4a, GSB4b, GSB6a and GSB6b.Two genetic maps were constructed to identify QTLs in target locus by using microsatellite (simple sequence repeat, SSR) markers. The linkage group1of chr4and group2of chr6covers12cM and44cM with an average genetic distance of1.5cM and2.44cM, respectively. The positions of main effect QTLs to GSB resistance on chr4were SSR26165-SSR04534, SSR04649-SSR17459and SSR17459-SSR04454. The positions of main effect QTLs to GSB resistance on chr6were SSR21115-SSR19755and SSR19755-SSR12510.5. Annotation and function prediction of gummy stem blight resistance gene in Cucumis sativus-hystrix introgression linesAccording to the results of genetic and physical mapping of significant markers linked to GSB, GSB locus were delimited to3.569Mbp and1.299Mbp genomic DNA regions in chromosome4and6, respectively. Annotation and function prediction of genes in target region speculated33and24candidate genes, respectively, in which the potential candidate of GSB gene might be included. This work provides a foundation for map-based cloning of the GSB gene and understanding the molecular mechanism of the gummy stem blight in cucumber.