Downy Mildew Resistance In Cucumber Research Articles

Sigma factor binding protein 1 (CsSIB1) is a putative candidate of the major-effect QTL dm5.3 for downy mildew resistance in cucumber (Cucumis sativus).

The dm5.3 major-effect QTL in cucumber encodes a homolog of Arabidopsis sigma factor binding protein 1 (CsSIB1). CsSIB1 positively regulates defense responses against downy mildew in cucumber through the salicylic acid (SA) biosynthesis/signaling pathway. Downy mildew (DM) caused by the oomycete pathogen Pseudoperonospora cubensis is an important disease of cucumber and other cucurbits. Our knowledge on molecular mechanisms of DM resistance is still limited. In this study, we reported identification and functional characterization of the candidate gene for the major-effect QTL, dm5.3 for DM resistance originated from PI 197088. The dm5.3 QTL was Modelized through marker-assisted development of near isogenic lines (NILs). NIL-derived segregating populations were used for fine mapping which narrowed the dm5.3 locus down to a 144kb region. Based on multiple lines of evidence, we show that CsSIB1 (CsGy5G027140) that encodes the VQ motif-containing sigma factor binding protein 1 as the most likely candidate for dm5.3. Local association analysis identified a haplotype consisting of 7 SNPs inside the coding and promoter region of CsSIB1 that was associated with DM resistance. Expression of CsSIB1 was up-regulated with P. cubensis infection. Transcriptome profiling of NILs in response to P. cubensis inoculation revealed key players and associated gene networks in which increased expression of CsSIB1 antagonistically promoted salicylic acid (SA) but suppressed jasmonic acid (JA) biosynthesis/signaling pathways. Our work provides novel insights into the function of CsSIB1/dm5.3 as a disease resistance (R) gene. The roles of sigma factor binding protein genes in pathogen defense in cucumber were also discussed.

Transcriptome profiling reveals response genes for downy mildew resistance in cucumber.

We discovered a potential defense pathway of cucumber to downy mildew. The signaling that activates the pathways of ROS and lignin accumulation may play an important role in the defense response. Many resistance genes were identified by transcriptome analysis. Downy mildew (DM), caused by Pseudoperonospora cubensis, is one of the most destructive diseases and causes severe yield losses of cucumber. However, the genes and pathways involved in regulating DM resistance were still poorly understood. In our study, we observed that the highly sensitive inbred line 53 (IL53) exhibited more severe disease symptoms than the highly resistant inbred line 51 (IL51) under P. cubensis infection. Furthermore, lignin, limiting the germination and extension of P. cubensis, and H2O2, as a signaling molecule during the resistant process, were both shown to increase, indicating that the signaling that activates these pathways might be responsible for the resistance divergence between IL51 and IL53. Transcriptome analysis, using the resistant and susceptible pools in F2 populations with IL51 and IL53 as parents, showed that a series of differentially expressed genes was involved in multiple functions of defense response: pathogen-associated molecular pattern recognition, signal transduction, reactive oxygen species and lignin accumulation, and transcription regulators. Combining physiological data with transcriptomes, we predicted a potential molecular mechanism of cucumber resistance to DM. Our research provided a foundation for further studies on the mechanism of cucumber resistance to DM.

Identification of Novel Loci and Candidate Genes for Cucumber Downy Mildew Resistance Using GWAS.

Downy mildew (DM) is one of the most serious diseases in cucumber. Multiple quantitative trait loci (QTLs) for DM resistance have been detected in a limited number of cucumber accessions. In this study we applied genome-wide association analysis (GWAS) to detected genetic loci for DM resistance in a core germplasm (CG) of cucumber lines that represent diverse origins and ecotypes. Phenotypic data on responses to DM infection were collected in four field trials across three years, 2014, 2015, and 2016. With the resequencing data of these CG lines, GWAS for DM resistance was performed and detected 18 loci that were distributed on all the seven cucumber chromosomes. Of these 18 loci, only six (dmG1.4, dmG4.1, dmG4.3, dmG5.2, dmG7.1, and dmG7.2) were detected in two experiments, and were considered as loci with a stable effect on DM resistance. Further, 16 out of the 18 loci colocalized with the QTLs that were reported in previous studies and two loci, dmG2.1 and dmG7.1, were novel ones identified only in this study. Based on the annotation of homologous genes in Arabidopsis and pairwise LD correlation analysis, several candidate genes were identified as potential causal genes underlying the stable and novel loci, including Csa1G575030 for dmG1.4, Csa2G060360 for dmG2.1, Csa4G064680 for dmG4.1, Csa5G606470 for dmG5.2, and Csa7G004020 for dmG7.1. This study shows that the CG germplasm is a very valuable resource carrying known and novel QTLs for DM resistance. The potential of using these CG lines for future allele-mining of candidate genes was discussed in the context of breeding cucumber with resistance to DM.

QTL identification for downy mildew resistance in cucumber using genetic linkage map based on SSR markers

Fourteen cucumber lines were tested for genetic homozygosity and performed pairwise comparison to identify a pair with the highest DNA polymorphic level. Cucumber accessions CSL0067 and CSL0139 were selected to generate 315 F2 populations. The genetic linkage map based on 66 polymorphic SSR markers was constructed. It composed of eight linkage groups (LGs) spanning 474.4 cM. Downy mildew disease reaction was evaluated in cotyledons, first and second true leaf on 7, 10, and 14 day after inoculation. The results showed that downy mildew resistance was controlled by multiple recessive genes. The susceptible to resistant ratio of F2 progenies fit 9:7 susceptible/resistant segregation types corresponding to duplicate recessive epistasis. Fourteen QTLs were detected. The phenotypic variance ranged from 5.0 to 12.5%, while LOD values ranged from 3.538 to 9.165. Two major QTLs and two QTL hotspots were identified. Moreover, the additive effects data explained that these QTL reduced downy mildew susceptibility.

Analysis of QTL DM4.1 for Downy Mildew Resistance in Cucumber Reveals Multiple subQTL: A Novel RLK as Candidate Gene for the Most Important subQTL.

One of the biggest problems in cucumber cultivation is cucurbit downy mildew (DM), caused by the obligate biotroph Pseudoperonospora cubensis. Whereas DM in cucumber was previously efficiently controlled by the dm-1 gene from Indian cucumber accession PI 197087, this resistance was broken by new DM strains, prompting the search for novel sources of resistance. A promising source of resistance is the wild cucumber accession PI 197088. It was previously shown that DM resistance in this genotype inherits polygenically. In this paper, we put the focus on one of the QTL, DM4.1 that is located on chromosome 4. QTL DM4.1 was shown to consist of three subQTL: DM4.1.1 affected pathogen-induced necrosis, DM4.1.2 was shown to have an additive effect on sporulation, and DM4.1.3 had a recessive effect on chlorosis as well as an effect on sporulation. Near-isogenic lines (NILs) were produced by introgressing the subQTLs into a susceptible cucumber line (HS279) with good horticultural traits. Transcriptomic analysis revealed that many genes in general, and defense pathway genes in particular, were differentially expressed in NIL DM4.1.1/.2 compared to NIL DM4.1.3 and the susceptible parent HS279. This indicates that the resistance from subQTL DM4.1.1 and/or subQTL DM4.1.2 likely involves defense signaling pathways, whereas resistance due to subQTL DM4.1.3 is more likely to be independent of known defense pathways. Based on fine-mapping data, we identified the RLK gene CsLRK10L2 as a likely candidate for subQTL DM4.1.2, as this gene was found to have a loss-of-function mutation in the susceptible parent HS279, and was strongly upregulated by P. cubensis inoculation in NIL DM4.1.1/.2. Heterologous expression of this gene triggered necrosis, providing further evidence that this gene is indeed causal for subQTL DM4.1.2.

Identification of candidate genes for quantitative downy mildew resistance in cucumber – a review

Cucurbit downy mildew (DM), caused by the obligate biotrophic oomycete Pseudoperonospora cubensis, is a major foliar disease of cucumber. Cucumber accession PI197088 was previously shown to be one of the most promising donors for DM resistance. The resistance in PI197088 is controlled by multiple quantitative trait loci (QTL), each with a relatively small effect. We developed a cucumber line with a 12 Mb introgression from PI197088 in a susceptible background. This introgression line was partially resistant to DM. Subsequently, we combined fine mapping data with RNAseq and whole genome resequencing to identify candidate genes for the resistance conferred by this introgression. Interestingly, fine-mapping data suggested that the partial resistance is caused by a combination of multiple genetically linked genes, leading to three subQTL, each with a different effect on the disease phenotype. In one of these subQTL we identified several Receptor Like Kinase genes (RLK). A novel RLK gene appeared to be present in resistant genotypes. In susceptible genotypes, including the reference genotype “Chinese Long 9930”, this novel gene has a 551 base pair deletion, and was therefore not correctly predicted during the annotation of the cucumber genome. In another subQTL, we found a loss-of-function mutation in the novel susceptibility gene (S-gene) CsAAP2A, encoding an amino acid transporter. In the resistant genotype a non-functional allele of this gene is present due to integration of a transposable element in its coding sequence.

CsIVP functions in vasculature development and downy mildew resistance in cucumber.

Domesticated crops with high yield and quality are frequently susceptible to pathogen attack, whereas enhancement of disease resistance generally compromises crop yield. The underlying mechanisms of how plant development and disease resistance are coordinately programed remain elusive. Here, we showed that the basic Helix-Loop-Helix (bHLH) transcription factor Cucumis sativus Irregular Vasculature Patterning (CsIVP) was highly expressed in cucumber vascular tissues. Knockdown of CsIVP caused severe vasculature disorganization and abnormal organ morphogenesis. CsIVP directly binds to vascular-related regulators YABBY5 (CsYAB5), BREVIPEDICELLUS (CsBP), and AUXIN/INDOLEACETIC ACIDS4 (CsAUX4) and promotes their expression. Knockdown of CsYAB5 resulted in similar phenotypes as CsIVP-RNA interference (RNAi) plants, including disturbed vascular configuration and abnormal organ morphology. Meanwhile, CsIVP-RNAi plants were more resistant to downy mildew and accumulated more salicylic acid (SA). CsIVP physically interacts with NIM1-INTERACTING1 (CsNIMIN1), a negative regulator in the SA signaling pathway. Thus, CsIVP is a novel vasculature regulator functioning in CsYAB5-mediated organ morphogenesis and SA-mediated downy mildew resistance in cucumber.

CsWRKY50 mediates defense responses to Pseudoperonospora cubensis infection in Cucumis sativus

The cucumber (Cucumis sativus L.), an economically important vegetable crop, is often infected by Pseudoperonospora cubensis (P. cubensis), which results in inhibited growth and reduced yield. WRKY transcription factors (TFs) play critical roles in plant disease resistance. However, little is known about the function of WRKY TFs in cucumber downy mildew resistance. In this study, we reported that CsWRKY50, a cucumber WRKY subgroup Ⅱc TF localized in the nucleus, plays an important role in cucumber defense responses to downy mildew. In addition, several putative cis-acting elements involved in abiotic stress responsiveness were also identified in the CsWRKY50 promoter. Expression analysis revealed that CsWRKY50 can be induced by P. cubensis infection, abiotic stress and diverse signaling molecules. The overexpression of CsWRKY50 in cucumber enhanced the resistance of the plant to the fungal pathogen P. cubensis. In addition, less ROS accumulated in 35S:CsWRKY50 transgenic plants infected by the pathogen due to the higher expression levels of antioxidant enzymes. Importantly, after P. cubensis infection, the transcript levels of several hormone-related defense genes were also upregulated in transgenic plants, including SA- and JA-responsive genes and SA-synthesis genes. Collectively, our results indicate that CsWRKY50 positively regulates cucumber disease resistance to P. cubensis via multiple signaling pathways.

QTL Analysis for Downy Mildew Resistance in Cucumber Inbred Line PI 197088.

Downy mildew (DM), caused by Pseudoperonospora cubensis, is one of the major foliar diseases prevailing in cucumber-growing areas. The mechanism of DM resistance in cucumber, particularly the plant introduction (PI) 197088 from India, is presently unclear. Quantitative trait locus (QTL) mapping is an efficient approach to studying DM resistance genes in cucumber. In this study, we performed QTL mapping for DM resistance in PI 197088 with 183 F2-derived F3 (F2:3) families from the cross between PI 197088 (DM resistant) and Changchunmici (DM susceptible). A linkage map was constructed using 141 simple sequence repeat markers. Phenotypic data were collected from seven independent experiments. In total, five QTL were detected on chromosomes 1, 3, 4, and 5 with DM resistance contributed by PI 197088. The QTL on chromosome 4, dm4.1, was reproducibly detected in all indoor experiments, which could explain 27% of the phenotypic variance detected. Additionally, dm1.1 and dm5.2 showed moderate effects, while dm3.1 and dm5.1 were minor-effect QTL. This study revealed the unique genetic architecture of DM resistance in PI 197088, which may provide important guidance for efficient use in cucumber breeding for DM resistance.

QTL mapping for downy mildew resistance in cucumber via bulked segregant analysis using next-generation sequencing and conventional methods.

QTL mapping using NGS-assisted BSA was successfully applied to an F 2 population for downy mildew resistance in cucumber. QTLs detected by NGS-assisted BSA were confirmed by conventional QTL analysis. Downy mildew (DM), caused by Pseudoperonospora cubensis, is one of the most destructive foliar diseases in cucumber. QTL mapping is a fundamental approach for understanding the genetic inheritance of DM resistance in cucumber. Recently, many studies have reported that a combination of bulked segregant analysis (BSA) and next-generation sequencing (NGS) can be a rapid and cost-effective way of mapping QTLs. In this study, we applied NGS-assisted BSA to QTL mapping of DM resistance in cucumber and confirmed the results by conventional QTL analysis. By sequencing two DNA pools each consisting of ten individuals showing high resistance and susceptibility to DM from a F2 population, we identified single nucleotide polymorphisms (SNPs) between the two pools. We employed a statistical method for QTL mapping based on these SNPs. Five QTLs, dm2.2, dm4.1, dm5.1, dm5.2, and dm6.1, were detected and dm2.2 showed the largest effect on DM resistance. Conventional QTL analysis using the F2 confirmed dm2.2 (R 2=10.8-24%) and dm5.2 (R 2=14-27.2%) as major QTLs and dm4.1 (R 2=8%) as two minor QTLs, but could not detect dm5.1 and dm6.1. A new QTL on chromosome 2, dm2.1 (R 2=28.2%) was detected by the conventional QTL method using an F3 population. This study demonstrated the effectiveness of NGS-assisted BSA for mapping QTLs conferring DM resistance in cucumber and revealed the unique genetic inheritance of DM resistance in this population through two distinct major QTLs on chromosome 2 that mainly harbor DM resistance.

QTL mapping for downy mildew resistance in cucumber inbred line WI7120 (PI 330628).

Host resistance in WI7120 cucumber to prevailing downy mildew pathogen field populations is conferred by two major-effect, one moderate-effect and two minor-effect QTL. Downy mildew (DM) caused by the obligate oomycete Pseudoperonospora cubensis is the most devastating fungal disease of cucumber worldwide. The molecular mechanism of DM resistance in cucumber is poorly understood, and use of marker-assisted breeding for DM resistance is not widely available. Here, we reported QTL mapping results for DM resistance with 243 F2:3 families from the cross between DM-resistant inbred line WI7120 (PI 330628) and susceptible '9930'. A linkage map was developed with 348 SSR and SNP markers. Phenotyping of DM inoculation responses were conducted in four field trails in 2years at three locations. Four QTL, dm2.1, dm4.1, dm5.1, and dm6.1 were consistently and reliably detected across at least three of the four environments which together could explain 62-76% phenotypic variations (R (2)). Among them, dm4.1 and dm5.1 were major-effect QTL (R (2)=15-30%) with only additive effects; dm2.1 (R (2)=5-15%) and dm6.1 (R (2)=4-8%) had moderate and minor effects, respectively. Epistatic effects were detected for dm2.1 and dm6.1 with both dm4.1 and dm5.1. One additional minor-effect QTL, dm6.2 (R (2)=3-5%) was only detectable with the chlorosis rating criterion. All alleles contributing to DM resistance were from WI7120. This study revealed two novel QTL for DM resistance and the unique genetic architecture of DM resistance in WI7120 conferring high level resistance to prevailing DM populations in multiple countries. The effects of disease rating scales, rating time and criteria, population size in phenotyping DM resistance on the power of QTL detection, and the use of DM resistance in WI7120 in cucumber breeding were discussed.

Identification of quantitative trait loci for downy mildew resistance in cucumber (Cucumis sativus L.)

Downy mildew, caused by Pseudoperonospora cubensis (Berk. & Curt.) Rostov, is one of the most economically important foliar diseases in cucumber (Cucumis sativus L.). Cucumber line CS-PMR1, derived from self-pollination of USDA Plant Introduction 197088, has a high level of resistance to downy mildew and is considered to be promising breeding material. In this study, we performed quantitative trait locus (QTL) analysis for downy mildew resistance using 111 recombinant inbred lines (RILs) derived from a cross between CS-PMR1 and the old Japanese cultivar Santou, which exhibits moderate resistance. The resistance of the RILs and their parents was evaluated by diverse methods using different plant organs (cotyledons, true leaves), stages (seedlings and adult plants), and evaluation criteria (lesion expansion and extent of sporulation). The high resistance of CS-PMR1 was associated with many QTLs with relatively small effects, whereas the moderate resistance of Santou was associated with one major QTL and possibly two others with relatively small effects. In all assays, the major QTL at which the Santou allele was associated with increased resistance had the largest effect. This QTL allele from Santou and several of the most effective QTL alleles identified in CS-PMR1 should be highest priority for selection to efficiently breed new cultivars that carry adequate levels of downy mildew resistance.

QTL Mapping of Downy Mildew Resistance in an Introgression Line Derived from Interspecific Hybridization Between Cucumber and Cucumis hystrix

AbstractDowny mildew (DM), caused by Pseudoperonospora cubensis (Berk. & M.A. Curtis) Rostovzev, is a worldwide major disease of cucumbers (Cucumis sativus L.). By screening 10 introgression lines (ILs) derived from interspecific hybridization between cucumber and the wild Cucumis, C. hystrix, through a whole plant assay, one introgression line (IL52) was identified with high DM‐resistance. IL52 was further used as a resistant parent to make an F2 population with ‘changchunmici’ (susceptible parent). The F2 population (300 plants) was investigated for DM‐yellowing, DM‐necrosis and DM‐resistance in the adult stage. A genetic map spanning 642.5 cM with 104 markers was constructed and used for QTL analysis from the population. Three QTL regions were identified on chromosome 5 and chromosome 6. By interval mapping analysis, two QTLs for DM‐resistance were determined on chromosome 5 (DM_5.1 and DM_5.2), which explained 17.9% and 14.2% of the variation, respectively. QTLs for DM‐yellowing were in the same regions as DM‐resistance. For DM‐necrosis, by interval mapping analysis, one QTL was determined on chromosome 5 (Necr_5.1) that explained 18.3% of the variation and one on chromosome 6 (Necr_6.1) that explained 13.9% of the variation. Our results indicated that the identification of molecular markers linked to the QTLs could be further applied for marker‐assisted selection (MAS) of downy mildew resistance in cucumber.

A Heat Shock Protein Gene, CsHsp45.9, Involved in the Response to Diverse Stresses in Cucumber

Heat shock proteins (Hsps) are a family of highly conserved proteins present in all organisms. They mediate a range of cytoprotective functions as molecular chaperones and are recently reported to regulate the immune response. Using suppression subtractive hybridization, we isolated and characterized a cucumber cDNA, designated CsHsp45.9, which encodes a putative heat shock protein of 45.9 kDa protein, containing three conserved DnaJ domains belonging to the Type I Hsp40 family. Real-time quantitative RT-PCR analysis revealed that CsHsp45.9 was significantly induced in cucumber leaves inoculated with downy mildew (Pseudoperonospora cubensis) in this incompatible interaction. Gene expression was also strongly up-regulated by various abiotic stresses. CsHsp45.9 was mainly expressed in flowers with a flower-specific, stamen- and pistil-predominant expression pattern. This suggests that CsHsp45.9 harbors broad-spectrum responses to both biotic and abiotic stresses and may play a role in downy mildew resistance in cucumber.

Dominant variance has an important role in downy mildew resistance in cucumber

Downy mildew inflicts severe damage on cucurbits in humid areas of production throughout the world. The genetics of resistance to downy mildew (Pseudoperonospora cubensis) in the cucumber (Cucumis sativus L.) was studied by the means of a half diallel table between 6 inbred lines. Decomposition of dominance variation indicated that the dominance effect was not unidirectional and that dominant genes were not uniformly distributed among the parents. These facts were confirmed and using a further detailed Hayman’s graphical analysis. Narrow-sense heritability estimates were 0.42 so the breeders are able to use selection for this trait. In other hands the heterosis in some crosses was high so breeders are able to use specific cross to produce hybrid with high level resistance.