Genetic Reseach On Disease Resistance To Cladosporium Cucumerinum And Differential Expressed Analysis Of Related Resistance Genes In Cucumber

Scab (Cladosporium cucumerinum) is an important disease in cucumber all over the world and causes enormous loss in production. Single disease resistant gene source has hidden danger for crop production. Therefore, screening different disease resistant materials from germplasm resources is benefit for the sustainable development of breeding and production. Obtaining molecular marker or genes linked with economic traits is the foundation of molecular breeding. Suppression subtractive hybridization (SSH) technique has been widely applied for the plant growth and development, gene clone of adversity and disease resistance related genes.In this study, on the basis of the resistant identification to scab with the method of artificial inoculation at seedling stage, HX1 (HR), HX5 (MR) and HX8 (S) were selected and the six generations of two combinations (HX1×HX8, HX5×HX8) were constructed to study their inheritance of scab resistance. Based on the reported Ccu gene mapping and nearly 2000 SSR primers developed by whole genome sequencing data, validation of SSR markers linked with resistance genes to scab for HX1 and new SSR marker selection for HX5 were studied. cDNA-SSH library at the early stage of inoculation with Cladosporium cucumerinum was constructed using SSH technique. Lots of ESTs related with scab resistance were obtained and their functions were analyzed in order to understand the molecular mechanism of resistance to scab in transcriptional level in cucumber. Main conclusions are as followed.1 Through identification of resistance to scab with the method of artificial inoculation at seedling stage, 4 resistance materials, 24 moderate resistance (MR) materials were obtained among 117 materials. The accessions of moderated resistance materials took 20.51%. Through repeat identification,1 high resistant materials HX1 (Cucumis sativus var. sativus, DI=5%), 1 moderate resistant material HX5 (C. sativus var. xishuangbannesis, DI=38.7%) and 1 susceptible material HX8 (C. sativus var. sativus,DI=80%) were selected.2 The inheritance of HX1 accord with a mix genetic model of two major genes with additive-dominance-epistatic effects plus poly-genes with additive-dominance effects (E₁ model) and that of HX5 accord with poly-gene (C model). The additive effect of two major genes was larger than dominant effect in HX1. Heritability of the major genes in F2, B1 and B2 were estimated to be 96.91%, 98.19% and 72.51% respectively. Poly-gene heritability of three generations was all 0. Major gene heritability was important in three generations. For resistant line HX5, the heritability was mainly dominant effect of poly-gene. The result of inheritance analysis according to quantitative trait was nearly the same.3 Based on the reported mapping result of Ccu gene in SSR linkage map, it was verified that the linkage distance between SSR06576/SSR17631 and scab resistant gene were 3.7cM and 1.5cM respectively in combination HX1×HX8. It is expressed that the obvious disease resistant gene of HX1 might be the same with Ccu gene or their sequence might be near in genome.4 Disease identification and molecular determination of F2 population of combination HX5×HX8 were performed and SSR13511 linked with resistant related genes to scab were obtained. The linkage distance was 29.8cM. Based on whole genome sequencing information, SSR13511 was checked at No.4 chromosome in cucumber.5 Based on above analysis, it was concluded preliminarily that there was at least 1 gene related with disease resistant to scab in our domestic cucumber germplasm, which was different from the single dominant Ccu gene resistant to scab mainly originated from foreign countries. There might be above two kinds of disease resistant genes in breeding line HX1.6 Through microscope observation, it showed that no obvious difference existed in 3 materials at the early stage of infection. Variance analysis showed that there was significant difference of the spore germination rate at different observation time and no obvious difference among 3 materials. The spore germination rate at 72h after inoculation was significantly higher than that at other time points.7 A forward and reverse cDNA-SSH library of HX1 seedling leaves inoculated with Cladosporium cucumernium were built. 200 positive clones longer than 200bp were selected to sequence, and finally 198 ESTs with high quality was got. After repeated and redundant sequences removed, 105 ESTs including 50 singletons and 55 contigs were obtained. Protein homology search in non-redundant protein database revealed that 88 ESTs were highly homologous with known protein, accounting for 83.8% of all EST sequences, of which only 2 ESTs were unknown function protein. The functions of these ESTs involved in specifically expressed genes under certain stress, energy and basic metabolism, signal transduction pathway and so on.