Identification And Cloning Of Genes For Downy Mildew Resistance In Cucumber

Downy mildew(DM)is one of the important leafy diseases in cucumber,which has caused the damages of cucumber production over 70 countries since it was found in 1868.Till now some researches on the genes of DM resistance have been carried out and some progress has been made,however,the results have limited utilization in practice.Due to research genetic materials used in different research groups the locations of QTL for DM were reported on chrl,chr5 and chr6.However,the molecular markers linked to the resistance genes were still unsatisfied for use in breeding.In addition,fine mapping and map-based cloning of disease-resistant gene and QTLs has proceeded slowly.Up to now,there have been no reports of cloning cucumber downy mildew resistant gene and QTLs,and their function verification,which can’t satisfy the needs of marker assisted selection in practice in breeding and germplasm innovation.How to further exploit the cucumber downy mildew resistance gene and speed up molecular breeding for cucumber disease resistance,is becoming an urgent topic for cucumber disease resistance breeding.In order to speed up the identification of DM related genes the current study was carried out in two ways.One way was to find resistant genes for DM.An inbred line K8 with DM resistance and an inbred line K18 with DM susceptibility selected from Xintai Mici were used to create a RIL population of 86 lines,which were employed for the current study.The plants of the population were evaluated for DM resistance on the growth stages of seedling and adult,respectively.A genetic map with SSR markers was made.The correlation analysis of cucumber downy mildew resistance between seedling and adult stages had completed.QTL analysis for DM resistance was conducted and candidate genes were exploited.A total of 11 candidate genes related to DM resistance were cloned and their sequences were compared between parents.The other way was to find susceptible gene(S-gene)for DM.Candidate genes for cucumber DM susceptibility were identified with the comparison of homologous gene sequences of DM S-genes from other species and with phylogenetic analysis.Candidate S-genes were cloned from a group of 15 cucumber entries of various genetic background and DM resistance.The difference of sequences was screened.The first level of protein structures from gene sequences of both natural and mutation lines were predicted.Whether the amino acid changes due to the mutation was evaluated.The bioinformatics of the candidate genes was also studied with the prediction of gene structure and domain,comparison with the amino acid sequences of homologous S-genes of known function from Arobidopsis,and with the prediction of the second-level and third-level structures of proteins.The results are listed as following:1.A total of 2112 pairs of SSR primers were employed to test the parental lines for polymorphism and 112 were polymorphic,which were used on the RIL population for mapping analysis.A genetic map was constructed by Joinmap program with 7 linkage groups of total length of 513.2cM and 86 SSR markers with average distance of 5,97cM.The 7 linkage groups are alined with the 7 cucumber chromosomes.2.QTLs for DM resistance were analysized by using MapQTL 4.0 with phenotypic data of the RIL population tested on three different growth stages in 2015.Two QTLs(denoted as dmQTL5.1 and dmQTL5.2)on chr5 were detected in three tests.dmQTL5.1 was located between the markers SSR16110 and SSR 11012,and its peak point was nearby marker SSR22518.Its LOD was 14.17,which explained 53.6%of phenotypic variation of DM resistance.dmQTL5.2 was located between the markers SSR26904 and SSR14194.Its LOD was 8.85,which explained 31.1%of phenotypic variation of DM resistance.With the DM test on seedling stage one QTL,dmQTL6.1,was also found on chr6.Its LOD was 1.72,which explained 9.4%of phenotypic variation of DM resistance.3.The downy mildew resistance in seedling stage showed correlation with downy mildew resistance in adult stage in spring and autumn.The correlation coefficient were significantly with 0,61542 and 0.67648,respectively.4.By the help of bioinformatics the genes and their function were predicted in the region of 4319kb between SSR16110 and SSR11012 for major QTL dmQTL5.1.A total of 397 candidate genes were estimated,among which DM resistant related genes could translated into 11 LRR-rich resistant proteins,10 transcription factor-related proteins,20 zinc finger proteins and one stress regulating protein.5.The 11 candidate genes for LRR-rich proteins were cloned and their sequences were analysized.The results indicated that only two genes,Csa5M464830.1 and Csa5M495930.1,had sequence variation between parental lines,leading to the change of amino sequences.A large difference for Csa5M464830.1 was found in K8,leading to the rear part of the gene without translation of amino acid and lacking of protein struction domain,which cause the loss of function of related protein.6.The cloning and alignment analysis of gene CsaDMR6-2,a candidate homologous gene to DMR6 in Arabidopsis.The identification of CsaDMR6-2 in the 15 cucumber entries of different genetic background indicated that the DM resistant materials of European type had single-base mutation,which affect the primary,secondary and tertiary protein structures.However,the functional analysis of this gene will be performed in the future.