SNP marker related to resistance to cucumber powdery mildew and application thereof

Abstract

The application discloses a SNP marker related to cucumber powdery mildew resistance and application thereof, relates to the field of genetic engineering and molecular biology. The application discloses a SNP molecular marker related to cucumber powdery mildew resistance, which is located at 6,284,831 bp of the first chromosome of a cucumber Chinese long genome v3; meanwhile, the SNP site is located at the 341th base of a gene; the cucumber with the SNP site base G is a high-resistance powdery mildew variety; and the cucumber with the SNP site base A is a high-susceptibility powdery mildew variety. CsAAH The gene is a negative regulation factor of the cucumber powdery mildew resistance. CsAAH The gene and the SNP site can be used for the detection of the cucumber powdery mildew resistance and molecular breeding. CsAAH CsAAH CsAAH CsAAH CsAA The gene and the SNP site can be used for the detection of the cucumber powdery mildew resistance and molecular breeding.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and molecular biology, and in particular to a SNP marker associated with resistance to cucumber powdery mildew and its application. Background Technology

[0002] cucumber( Cucumis sativus Powdery mildew (L.) is an important vegetable crop widely cultivated in my country and worldwide. It is one of the most common and damaging diseases in cucumber production, especially prevalent under greenhouse cultivation conditions. The pathogen is mainly... Podosphaera xanthii After the disease occurs, a white powdery fungal layer forms on the leaf surface, causing the leaves to yellow, age, and lose photosynthetic capacity. In severe cases, it can lead to reduced yield, decreased quality, or even premature crop failure.

[0003] Currently, the control of cucumber powdery mildew still relies mainly on chemical agents. However, this method has the following problems: long-term application of pesticides can easily lead to increased drug resistance in pathogens; the risk of pesticide residues and environmental pollution is high; production costs are increased; and it is not conducive to green agriculture and sustainable production. Therefore, obtaining stable disease-resistant varieties from a breeding perspective is a more economical, environmentally friendly, and suitable solution for industrial promotion.

[0004] Although some studies have reported some QTLs, resistance loci, and candidate genes for cucumber powdery mildew resistance, the following problems still exist: many loci are only located in large intervals and have not been precisely mapped to functional genes; the number of functional variation loci that can be directly used for molecular marker-assisted breeding is limited; the sources of resistance vary among different materials, and the molecular basis of resistance in many superior germplasms has not yet been explored; and there are still relatively few regulatory factors or susceptibility factors that can be directly used as gene editing targets.

[0005] Allantoate amidohydrolase (AAH) is a key enzyme in the purine degradation pathway and participates in allantoic acid metabolism. Previous plant studies have shown that allantoin / allantoic acid metabolism not only participates in nitrogen reuse but may also affect plant stress responses, redox homeostasis, and hormone signaling processes. However, whether AAH-like genes are involved in the regulation of powdery mildew in cucumber has not been clearly reported before.

[0006] Therefore, discovering new disease resistance sites and functional genes, and developing molecular markers that can be directly used for breeding, is of great value. Summary of the Invention

[0007] The purpose of this invention is to provide a SNP marker related to powdery mildew resistance in cucumber and its application, thereby addressing the problems existing in the prior art. This invention identifies stably inherited major loci of powdery mildew resistance in cucumber powdery mildew-resistant materials. Verification has been performed. CsAAH Genes are negative regulators of cucumber powdery mildew resistance. CsAAHThere is an SNP site at the 341st base of the gene. CsAAH Genes and SNP loci can be used for cucumber powdery mildew resistance detection and molecular breeding.

[0008] To achieve the above objectives, the present invention provides the following solution: This invention provides a SNP molecular marker associated with resistance to powdery mildew in cucumbers. The SNP molecular marker is located at 6,284,831 bp on chromosome 1 of the cucumber Chinese long genome v3. Cucumbers with a G base at the SNP site are highly resistant to powdery mildew, while cucumbers with an A base at the SNP site are highly susceptible to powdery mildew.

[0009] The present invention also provides a primer pair for detecting SNP molecular markers associated with cucumber powdery mildew resistance, the primer pair comprising an upstream primer with a nucleotide sequence as shown in SEQ ID NO.3 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.4.

[0010] The present invention also provides the application of the above-mentioned SNP molecular markers in the detection of cucumber powdery mildew resistance.

[0011] This invention also provides the application of the above-mentioned SNP molecular markers in cucumber-assisted breeding.

[0012] The present invention also provides the application of the above primer pairs in the preparation of detection products for cucumber powdery mildew resistance.

[0013] This invention also provides cucumber CsAAH The application of genes in assisted breeding, through the suppression of... CsAAH Gene expression enhances cucumber resistance to powdery mildew; CsAAH The nucleotide sequence of the gene is shown in SEQ ID NO.1; cucumber CsAAH The gene contains the above-mentioned SNP molecular markers; CsAAH Cucumbers with a G at position 341 of their gene are highly resistant to powdery mildew. CsAAH Cucumbers with an A at the 341st base of the gene are highly susceptible to powdery mildew.

[0014] This invention also provides a method for improving cucumber powdery mildew resistance by interfering with... CsAAH Gene expression enhances cucumber's resistance to powdery mildew.

[0015] The present invention also provides a detection product for cucumber powdery mildew resistance, comprising the above-mentioned primer pair.

[0016] This invention also provides a method for identifying resistance to powdery mildew in cucumbers, comprising the following steps: (1) Extract genomic DNA from cucumbers to be tested; (2) Using the cucumber genomic DNA to be tested as a template, PCR amplification was performed using the primer pair shown in SEQ ID NO.3 and SEQ ID NO.4; (3) Obtain the genotype through sequencing to determine the disease resistance of the cucumber to be tested; CsAAH Cucumbers with a base G at position 341 of the gene are highly resistant to powdery mildew, while cucumbers with a base A at position 341 are highly susceptible to powdery mildew.

[0017] The present invention also provides the application of the above-mentioned identification method in the breeding of cucumbers resistant to powdery mildew.

[0018] The present invention discloses the following technical effects: No information about this has been found in the existing technology. CsAAH / AAH Public reports on the role of gene-like organisms in cucumber powdery mildew resistance.

[0019] This invention is the first to link AAH-related genes with cucumber powdery mildew resistance. CsAAH The gene has clear breeding value. It can be used to develop SNP molecular markers for MAS breeding, and also as a target for RNAi or CRISPR gene editing to create new disease-resistant materials; the SNP molecular marker can be used to detect cucumber powdery mildew resistance. The RNAi silencing results of this invention directly prove... CsAAH Decreased gene expression enhances disease resistance. CsAAH Genes are negative regulators of cucumber powdery mildew resistance. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The results of genome-wide association analysis based on BSA-seq using the ΔSNP-index are shown; the horizontal axis represents the position of each chromosome in cucumber, and the vertical axis represents the ΔSNP-index value; the area shown in the red box is the significant association interval detected on chromosome 1. Figure 2 The following are the location results obtained from linkage analysis using molecular markers: A: Schematic diagram of chromosome 1 and marker distribution; B: LOD value distribution diagram of the corresponding segments; Figure 3 Genotypic analysis results for fine mapping of recombinant single plants after expanding the F2 population; Figure 4 Candidate gene CsaV3_1G010130 ( CsAAH Gene structure analysis and sequence difference analysis between resistant and susceptible parents; Figure 5 After inoculation with powdery mildew CsAAH Expression changes in the resistant parent CR125 and the susceptible parent CS055; Figure 6 Comparison of local three-dimensional structures of CsAAH proteins derived from the resistant parent CR125 and the susceptible parent CS055; the dashed circle indicates the region of local conformational difference corresponding to the substitution of amino acid at position 114; Figure 7 Powdery mildew inoculation treatment and uninoculated control conditions CsAAH Temporal representation analysis; Figure 8 for CsAAH Analysis of expression patterns in different tissues of cucumber (root, stem, leaf, female flower, male flower and ovary); Figure 9 Green fluorescence channel, red channel, bright field, and overlay diagrams for subcellular localization of CsAAH protein; A: Fluorescence distribution of the GFP empty vector control in tobacco leaf epidermal cells; B: Fluorescence distribution of the CsAAH-GFP fusion protein; Scale bar 20 μm; Figure 10 Phylogenetic tree analysis results of CsAAH and other plant AAH homologs; Figure 11 For wild-type plants (WT), empty vector transformed plants (EV) and CsAAH - Phenotypic comparison of RNAi plants (represented by RNAi-3) after inoculation with powdery mildew; Figure 12 Statistical results of disease grading / disease index after inoculation of powdery mildew in WT, EV and RNAi plants; Figure 13 In WT, EV and RNAi plants CsAAH Expression level detection results. Detailed Implementation

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0024] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0025] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0026] The terms “include,” “including,” “have,” and “contain” used in this article are all open-ended terms, meaning that they include but are not limited to.

[0027] This invention identifies stable, heritable major loci of powdery mildew resistance in novel cucumber powdery mildew-resistant materials; precisely locates the resistance loci to the smallest possible interval; identifies candidate genes truly related to resistance within the located interval; clarifies whether the gene participates in the regulation of cucumber powdery mildew resistance through expression analysis, localization analysis, and functional verification; and develops markers based on these genes for cucumber powdery mildew resistance detection and molecular breeding.

[0028] Experimental materials used in this invention A. Two cucumber inbred line materials CR125: Shows stable resistance to powdery mildew; CS055: Shows susceptibility to powdery mildew.

[0029] B. Pathogenic materials Samples were collected from naturally infected cucumber leaves, pathogen DNA was extracted, and the ITS sequence was amplified. Sequencing results were compared with database sequences, showing similarities to... Podosphaera xanthii Sequence identity reached 100%. The strain used was ultimately confirmed as: Podosphaera xanthiiRace 1 (published literature on this strain: Cui HN, Fan C, Ding Z, Wang XZ, Tang LL, Bi YD, Luan FS, Gao P. CmPMRl and CmPMrs are responsible for resistance to powdery mildew caused by Podosphaera xanthii race 1 in Melon. Theoretical and Applied Genetics, 2022, 135 (4): 1209-1222). This pathogen has shown stability in continuous identification over many years and is used for disease resistance identification in this invention.

[0030] Example 1 I. Parental whole-genome resequencing and SNP molecular markers This invention uses the disease-resistant inbred line CR125 and the disease-susceptible inbred line CS055 as parents to perform hybridization and construct segregating populations. Genetic populations are constructed using these two materials, including: F1 population; F2 population; and F3 population. 2：3 Family lineage. The F2 population consisted of approximately 300 plants in 2023 and expanded to 1,300 plants in 2024 for fine-tuning.

[0031] Inoculate with powdery mildew during the seedling stage Podosphaera xanthii Race 1: Investigate disease manifestations after inoculation. On the 7th day after powdery mildew inoculation, investigate the disease status of the plant leaves.

[0032] Grade 0: No visible lesions on the leaves; Grade 1: Lesions cover ≤5% of the leaf area; Grade 3: Lesions cover 6%-10% of the leaf area; Level 5: Lesions cover 11%-25% of the leaf area; Level 7: Lesions cover 26%-50% of the leaf area; Level 9: Lesions cover more than 50% of the leaf area, with large areas of the leaf covered with a powdery white layer or showing obvious yellowing and necrosis.

[0033] Results: CR125 consistently showed resistance; CS055 consistently showed susceptibility; all F1 cells showed susceptibility, indicating that resistance was not dominant but possibly recessive; the segregation ratio of resistance to susceptibility in the F2 population was 1:3; 2:3 The family segregation results also support a single-gene recessive inheritance pattern.

[0034] Therefore, it can be concluded that CR125 is effective against powdery mildew. Podosphaera xanthii Resistance in race 1 is controlled by a single recessive nuclear gene.

[0035] II. Preliminary Localization by BSA-seq From the F2 population, phenotypically extreme disease-resistant and susceptible plants were screened to construct disease-resistant and susceptible pools, respectively. High-throughput sequencing was performed on both pools, and the sequences were aligned to the cucumber reference genome. Genome-wide differences were analyzed using SNP-index and ΔSNP-index. Results are shown below. Figure 1 .

[0036] like Figure 1 As shown, a significant association peak exists on cucumber chromosome 1, with a candidate interval of 4.78 Mb-6.94 Mb; the interval size is approximately 2.16 Mb. This indicates that the main site controlling CR125 resistance to powdery mildew is located in this interval on chromosome 1. The area shown in the red box is the significant association interval detected on chromosome 1, indicating that this region is closely related to cucumber powdery mildew resistance.

[0037] III. Precise Positioning 1. Tag Development Molecular markers were developed based on sequence polymorphisms between parents within candidate intervals, with priority given to the development of CAPS markers for genotyping in the F2 population.

[0038] 2. Initially narrowing the interval like Figure 2 As shown, through population linkage analysis, the resistance loci were narrowed down to the markers SNP8 and SNP9; the corresponding physical interval is approximately 6.22Mb-6.29Mb.

[0039] 3. Expand the target audience and further define the target audience. The F2 population was expanded to 1300 plants, and recombinants were screened using flanking markers, yielding 32 recombinant individuals. New internal markers were then developed within this interval, and the results were analyzed in conjunction with the disease phenotype of each recombinant. Results are shown below. Figure 3 .

[0040] IV. Candidate Gene Screening Within a fine-mapping region of 21.46 kb, a total of 3 genes were predicted: 1. CsaV3_1G010110, encoding F-box domain-containing protein; 2. CsaV3_1G010120, encoding C-type lectin receptor-like tyrosine-proteinkinase; 3. CsaV3_1G010130, encoding allantoate amidohydrolase-related protein; Sequence comparisons between the three genes and their parents revealed that only CsaV3_1G010130 showed a non-synonymous SNP in its coding region between the resistant parent CR125 and the susceptible parent CS055; the other two genes showed no significant coding differences directly related to disease resistance. Therefore, CsaV3_1G010130 was selected as a candidate gene. This gene was named... :CsAAH .

[0041] five, CsAAH Basic genetic information Based on the cloning and sequence analysis results: Gene name: CsAAH ; Corresponding gene number: CsaV3_1G010130; Encoding region length: 1266bp; Protein length: 421 aa; Protein annotation: allantoate amidohydrolase-related protein.

[0042] Between CR125 and CS055 CsAAH A non-synonymous SNP exists in the coding region, leading to an amino acid substitution. Since this variation occurs in the only candidate gene, it is presumed to be directly related to the disease resistance phenotype. Candidate gene CsaV3_1G010130 ( CsAAH The gene structure and sequence differences between resistant and susceptible parents are shown in the figure. Figure 4 .

[0043] CsAAH The nucleotide sequence of the gene coding region (5'-3'):

[0044] CsAAH The amino acid sequence corresponding to the gene: MVSDAARYLERTFLSPASIKASFLLQKWMEDAGLRTWVDCMGNLHGRTEGRNASAEALLIGSHLDTVVDAGKFDGALGIISAISALKVLNMNGKLEELKRPIEVIA FSDEEGVRFQSTFLGSAAIAGILPVSSLEISDKSGITIKDVIKESGVQITEENLLQLKYDRKSVWGYVEVHIEQGPVLEWSGFPLGVVRGIAGQTRLKVTVRGSQG HAGTVPMPMRQDPMAASAELIVQLEKLCKQPESYLSFDGHCTDSTLKSSLSTSLVCTVGEISTWPSASNVIPGQVTFTVDLRTIDDIGREAVIYEFSNQVHNICSSR SVSCNIERKHDANAIISNSELSSQLKSAASTALKKMVGEIQEEVPVLMSGAGHDAMAMSHLTKVGMLFVRCRGGVSHSPAEHVLDDDIWAAGLAVLEFLENHL (SEQ ID NO.2).

[0045] like Figure 4 As shown, the upper part is CsAAH A schematic diagram of the gene structure; the middle section shows the nucleotide sequence alignment results of the coding region between the resistant parent CR125 and the susceptible parent CS055; the bottom section shows the corresponding amino acid sequence alignment results. The results indicate that... CsAAH A G / A nonsynonymous SNP exists at position 341 of the coding region, leading to an R / G substitution at position 114. CsAAH At position 341 of the gene, a G is changed to an A, and at position 114, an R is changed to a G. This SNP molecular marker is located at 6,284,831 bp on chromosome 1 of the cucumber Chinese long genome v3 (http: / / cucurbitgenomics.net / CuGenDB / ftp / genome / cucumber / Chinese_long / v3 / ). CsAAH At position 341 of the coding region, the resistant parent CR125 carries the G allele, while the susceptible parent CS055 carries the A allele. This locus can be used as a genotyping site for cucumber powdery mildew resistance: plants carrying the G allele typically exhibit higher resistance to powdery mildew; plants carrying the A allele typically exhibit higher susceptibility to powdery mildew. Therefore, it can be concluded that... CsAAHThe allele with G at the 341st base in the coding region is associated with high resistance to powdery mildew, and the individual carries the G allele at this location; the allele with A at the 341st base is associated with high susceptibility to powdery mildew, and the individual carries the A allele at this location.

[0046] The primers used to detect SNP molecular markers associated with cucumber powdery mildew resistance are: Upstream primer: 5'-ATGGTGAGTGATGCTGCTCG-3' (SEQ ID NO.3); Downstream primer: 5'-TTACAGATGGTTTTCTAGAA-3' (SEQ ID NO.4).

[0047] qRT-PCR reaction system (total volume 20 μL): 2×PCR Master Mix: 10.0 μL; upstream primer (10 μmol / L): 0.5 μL; downstream primer (10 μmol / L): 0.5 μL; genomic DNA: 1.0 μL; nuclease-free water: 8.0 μL.

[0048] PCR reaction program: 95℃ pre-denaturation for 3 min; 35 cycles: 95℃ for 30 s, 55℃ for 30 s, 72℃ for 30 s; 72℃ final extension for 5 min; store at 4℃.

[0049] The purified PCR product was recovered and sent for sequencing to detect SNPs.

[0050] Example 2 CsAAH Response to powdery mildew infection CsAAH Expression analysis: In order to judge CsAAH Whether to respond to powdery mildew infection, and whether to inoculate the resistant parent CR125 and the susceptible parent CS055 with powdery mildew. Podosphaera xanthii qRT-PCR analysis was performed on leaves at different time points after race 1. Following powdery mildew inoculation... CsAAH The expression changes in the resistant parent CR125 and the susceptible parent CS055 are shown in [the table]. Figure 5 The x-axis represents post-inoculation time (0h, 24h, 48h, 72h), and the y-axis represents relative expression levels. A comparison of the local three-dimensional structures of CsAAH proteins derived from the resistant parent CR125 and the susceptible parent CS055 is shown in [reference needed]. Figure 6 The time-series expression analysis results of CsAAH under powdery mildew inoculation treatment and uninoculated control conditions are shown in the figure. Figure 7 The PCR primer sequences are as follows: Upstream primer: 5'-TGCTGCTATTGCTGGTAT-3' (SEQ ID NO.5); Downstream primer: 5'-AAACAGATTTGCGGTCAT-3' (SEQ ID NO.6).

[0051] The qRT-PCR reaction system (total volume 20 μL) includes: 2×SYBR Green qPCR Master Mix: 10 μL; upstream primer (10 μM): 0.4 μL; downstream primer (10 μM): 0.4 μL; diluted cDNA template: 2.0 μL; ddH2O: 7.2 μL; PCR reaction procedure: 95℃ pre-denaturation for 30s; 95℃ for 5s, 60℃ for 30s, 40 cycles; followed by melting curve analysis: 95℃ for 15s, 60℃ for 1min; then slowly increase the temperature to 95℃ and continuously collect fluorescence signals.

[0052] like Figure 5 As shown, after vaccination CsAAH Expression was observed in both parents; however, at all detection time points, the expression level in CR125 was consistently lower than that in CS055; overall, CsAAH The trend was downward after vaccination; like Figure 6 As shown, a comparison of the local three-dimensional structures of CsAAH proteins derived from the resistant parent CR125 and the susceptible parent CS055 reveals that the dashed circle indicates the region of local conformational difference corresponding to the substitution of amino acid at position 114, suggesting that this non-synonymous SNP may affect the local structure and function of the protein.

[0053] like Figure 7 As shown, after inoculation with powdery mildew CsAAH The expression level gradually decreased over time, while the control group remained relatively stable overall.

[0054] like Figure 8 Tissue expression analysis showed that the gene was expressed in roots, stems, leaves, female flowers, male flowers, and ovaries, with the highest expression level in leaves.

[0055] visible, CsAAH It participates in the leaf response to powdery mildew pathogens; lower levels of this pathogen are found in disease-resistant materials. CsAAH The expression level is related to the formation of disease resistance; this gene is a negative regulator of disease resistance.

[0056] Example 3 Transient expression of CsAAH in tobacco leaves A 35S::CsAAH-GFP fusion expression vector was constructed and transiently expressed in tobacco leaves. The steps are as follows: (1) Using CR125 leaf cDNA as a template, the CsAAH open reading frame (ORF) sequence was amplified, and the stop codon was removed during amplification.

[0057] PCR reaction system (50 μL): 2× high-fidelity PCR Mix: 25 μL; upstream primer (10 μM): 2 μL; downstream primer (10 μM): 2 μL; cDNA template: 2 μL; ddH2O: 19 μL.

[0058] PCR program: 98℃ for 2 min; 35 cycles: 98℃ for 10 s, 58℃ for 15 s, 72℃ for 1 min and 30 s; 72℃ for 5 min.

[0059] (2) Vector ligation. The amplified product was purified and ligated into the plant expression vector pCAMBIA1300-35S-GFP. After ligation, it was transformed into Escherichia coli DH5α. Single clones were picked, and the correct insertion and reading frame were verified by bacterial PCR and sequencing.

[0060] (3) Agrobacterium transformation The verified recombinant plasmid was transformed into Agrobacterium tumefaciens GV3101. Positive single clones were picked, inoculated into LB liquid medium containing the appropriate antibiotic, and cultured at 28°C with shaking at 200 rpm until OD. 600 ≈0.8-1.0. After centrifugation, the bacterial cells were resuspended in the infection solution, which consisted of: 10 mM MgCl2, 10 mM MES (pH 5.6), and 100-200 μM Macetosyringone (AS). The bacterial concentration was adjusted to OD0.05. 600 =0.6-0.8, incubate at room temperature for 2-3 hours for induction.

[0061] (4) Transient expression of tobacco Select 4-6 week old Nicotiana benthamiana For healthy plant leaves, inject Agrobacterium tumefaciens solution into the underside of the leaves using a 1mL needleless syringe.

[0062] Experimental setup: Treatment group: 35S::CsAAH-GFP; Control group: 35S::GFP empty vector. Infected plants were cultured at 22℃–25℃ under 16h light / 8h dark conditions for 48–72h.

[0063] (5) Confocal microscopy After infection, leaves were collected, and the GFP signal was observed using a laser confocal microscope. Key technical parameters are as follows: GFP excitation wavelength: 488nm; GFP emission wavelength: 500–530 nm; 100–200 μM acetosyringone (AS). Subcellular localization results of CsAAH protein are shown in [Figure number missing]. Figure 8 .

[0064] (6) Results and Analysis The fluorescence distribution of the GFP empty vector control in tobacco leaf epidermal cells is as follows: Figure 9 As shown in Figure A, the fluorescence distribution of the CsAAH-GFP fusion protein is as follows: Figure 9 As shown in Figure B, the columns represent the green fluorescence channel, red channel, bright field, and overlay image, respectively. The results show that the empty GFP control is widely distributed in the cell membrane, cytoplasm, and nucleus; the green fluorescence of the 35S::CsAAH-GFP fusion protein is mainly concentrated in the periphery of the cell, consistent with the plasma membrane distribution characteristics, indicating that the CsAAH protein is mainly located in the plasma membrane. The empty GFP is widely distributed in the cell; the CsAAH-GFP fluorescence signal is mainly located in the plasma membrane. This suggests that the CsAAH protein may participate in the powdery mildew infection process through membrane-related signaling or metabolic response pathways.

[0065] Phylogenetic tree analysis results of CsAAH homologs with other plant AAH proteins are shown below. Figure 10 .like Figure 10 As shown, CsAAH clusters with AAH homologous proteins in cucurbit crops, indicating that it has high homology and conservation with known plant AAH proteins.

[0066] Example 4 cucumber CsAAH Establishment and functional analysis of Agrobacterium-mediated RNAi silencing system Select CsAAH RNAi vectors were constructed using specific fragments and transformed into cucumbers via Agrobacterium-mediated transformation, yielding multiple independent RNAi lines. These lines were then subjected to treatment with powdery mildew. Podosphaera xanthii Race 1 vaccination identification and testing CsAAH Level of expression.

[0067] according to CsAAH The coding region sequence was selected, and a 234 bp gene-specific segment was chosen as the RNAi target fragment. BLAST analysis confirmed that this fragment had no highly homologous contiguous sequences with other genes in the cucumber genome, thus reducing the risk of off-target effects.

[0068] The nucleotide sequence of the RNAi target fragment is as follows: CTTTCTTCTTCAAAAATGGATGGGTCGATTAAGAACGTGGGTCGACTGCATGGGCAATCTACATGGTCGAACTGAGGGAAGGAATGCAAGTGCTGAAGCATTACTGATTGGTTCTCATTTGGATACCGTTGTTGATGCTGGAAAATTTGATGGTGCATTAGGCATCATTTCTGCTATCTCTGCTTTAAAAGTTCTTAATATGAATGGGAAGTTAGAGGAACTAAAGAG (SEQ ID NO.7).

[0069] 1. Amplification using cucumber leaf cDNA as a template CsAAH Specific fragments. The PCR system and PCR procedure are the same as in Example 2.

[0070] 2. Constructing pFGC1008-CsAAH-RNAi The target fragment was cloned into the pFGC1008 vector flanking an intron in both forward and reverse directions, forming a hairpin RNA structure named pFGC1008-CsAAH-RNAi. Validation was performed by bacterial PCR, double enzyme digestion, and Sanger sequencing.

[0071] 3. Agrobacterium-mediated cucumber transformation (1) Transformed strains The recombinant vector was introduced into EHA105.

[0072] (2) Receptor material Cotyledonary nodes of aseptically germinating cucumber seedlings were used as transformation recipients.

[0073] Experimental groups: WT was the wild-type control, EV was the empty vector transformation control, and RNAi was the target gene interference experimental group.

[0074] (3) Infection conditions Agrobacterium cultured to OD 600 =0.5-0.8; the infection solution contains 100μM AS; soak the explants for 10-15 min; after wiping off the surface bacterial solution, co-culture for 2-3 days.

[0075] (4) Screening and regeneration Explants were transferred to an induction medium containing screening and antibacterial antibiotics to induce callus and shoot differentiation, followed by rooting to obtain regenerated plants.

[0076] 4. Positive identification of genetically modified organisms Genomic DNA was extracted from regenerated plants, and transgenic positive plants were identified by PCR using vector-specific primers or target fragment primers. This invention uses vector-specific primers located in the CaMV 35S promoter region and gene-specific primers located within the CsAAH-RNAi target fragment for PCR amplification. The primer sequences used are as follows: F: 5'-GTCCCTACAAATGCCATCA-3' (SEQ ID NO. 8); R: 5'-TTCCTCTAACTTCCCATTCATATTAAG-3' (SEQ ID NO. 9).

[0077] The 25 μL system consisted of: 2×Taq PCR Master Mix: 12.5 μL; 35S-F (10 μmol / L): 1.0 μL; CsAAH-RNAi-R (10 μmol / L): 1.0 μL; genomic DNA template: 1.0 μL; and ddH2O: 9.5 μL.

[0078] Reaction program: 95℃ pre-denaturation for 3 min; 35 cycles: 95℃ for 30 s, 58℃ for 30 s, 72℃ for 30 s; final extension at 72℃ for 5 min; store at 4℃.

[0079] PCR products were detected by 1.5% agarose gel electrophoresis. The final RNAi line was obtained.

[0080] 5. RNAi plant expression detection Leaf samples from WT, EV and RNAi plants were collected, and RNA was extracted and analyzed by qRT-PCR before (0h) and 72h after powdery mildew inoculation.

[0081] Results: RNAi plants CsAAH The expression level was significantly lower than that of WT and EV; the degree of downregulation varied among different RNAi lines; and the downregulation effect was stable in representative lines.

[0082] 6. Inoculation and resistance identification for powdery mildew Inoculate WT, EV, and RNAi plants at the three-leaf stage. Podosphaera xanthii Race 1. Maintain suitable conditions for disease development, and investigate disease incidence and calculate the disease index on day 7 post-inoculation. A comparison of phenotypes after inoculation of wild-type plants (WT), empty vector-transformed plants (EV), and CsAAH-RNAi plants (represented by RNAi-3) with powdery mildew is shown in [reference needed]. Figure 11 The statistical results of disease grading / disease index of WT, EV and RNAi plants after inoculation with powdery mildew are shown in [the table below]. Figure 12 In WT, EV and RNAi plants CsAAH The expression level detection results are shown in Figure 13 .

[0083] like Figure 11 As shown, WT and EV plants exhibited a distinct powdery mildew layer on their leaf surfaces; RNAi plants showed a significant reduction in lesions and a significantly lower disease severity compared to WT and EV; there was no significant difference between WT and EV; Figure 12 As shown, the disease index of RNAi strains was significantly lower than that of WT and EV.

[0084] like Figure 13 As shown, in RNAi strains CsAAH Expression levels decreased significantly. Compared with wild-type (WT) and empty vector control (EV), RNAi lines showed a significant reduction in lesions and a significant decrease in disease index after inoculation; indicating that when RNAi is expressed... CsAAH When gene expression is suppressed, cucumbers become more resistant to powdery mildew.

[0085] In conclusion, CsAAH Genes are negative regulators of cucumber powdery mildew resistance.

[0086] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A SNP molecular marker associated with resistance to cucumber powdery mildew, characterized in that, The SNP molecular marker is located at 6,284,831 bp on chromosome 1 of the cucumber Chinese long genome v3; cucumbers with a base of G at the SNP site are highly resistant to powdery mildew; cucumbers with a base of A at the SNP site are highly susceptible to powdery mildew.

2. A primer pair for detecting SNP molecular markers associated with cucumber powdery mildew resistance, characterized in that, The primer pair includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.3 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.

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3. The application of the SNP molecular marker as described in claim 1 in detecting cucumber powdery mildew resistance.

4. An application of the SNP molecular marker as described in claim 1 in cucumber-assisted breeding.

5. The application of the primer pair as described in claim 2 in the preparation of a detection product for cucumber powdery mildew resistance.

6. Cucumber CsAAH The application of genes in assisted breeding is characterized by, By inhibiting CsAAH Gene expression enhances cucumber resistance to powdery mildew; CsAAH The nucleotide sequence of the gene is shown in SEQ ID NO.1; cucumber CsAAH The gene contains the SNP molecular marker as described in claim 1; CsAAH Cucumbers with a G at position 341 of their gene are highly resistant to powdery mildew. CsAAH Cucumbers with an A at the 341st base of the gene are highly susceptible to powdery mildew.

7. A method for improving cucumber powdery mildew resistance, characterized in that, Through interference CsAAH Gene expression enhances cucumber's resistance to powdery mildew.

8. A test product for cucumber powdery mildew resistance, characterized in that, Includes the primer pair as described in claim 2.

9. A method for identifying cucumber powdery mildew resistance, characterized in that, Includes the following steps: (1) Extract genomic DNA from cucumbers to be tested; (2) Using the cucumber genomic DNA to be tested as a template, PCR amplification was performed using the primer pair shown in SEQ ID NO.3 and SEQ ID NO.4; (3) Obtain the genotype through sequencing to determine the disease resistance of the cucumber to be tested; CsAAH Cucumbers with a base G at position 341 of the gene are highly resistant to powdery mildew, while cucumbers with a base A at position 341 are highly susceptible to powdery mildew.

10. The application of the identification method according to claim 9 in cucumber powdery mildew resistance breeding.

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