DNA methylation molecular marker based on cucumber CsDHAR gene and application

Abstract

The invention discloses a DNA methylation molecular marker based on a cucumber CsDHAR gene and application, and relates to the technical field of molecular markers and crop genetic breeding. The nucleotide sequence of the molecular marker is as shown in SEQ ID NO: 1; the primer pair is used for amplifying and detecting the molecular marker, the nucleotide sequence of a forward primer of the primer pair is as shown in SEQ ID NO: 2, and the nucleotide sequence of a reverse primer of the primer pair is as shown in SEQ ID NO: 3; the molecular marker and the corresponding amplification primer can be applied to aphid-resistant cucumber germplasm screening, so that a solid theoretical basis and a genetic basis are provided for breeding aphid-resistant cucumbers, the screening rate of aphid-resistant excellent varieties can be effectively increased, and the progress of aphid-resistant breeding of cucumbers is promoted.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular markers and crop genetic breeding, and particularly relates to a DNA methylation molecular marker based on a cucumber CsDHAR gene and application. BACKGROUND

[0002] Cucumber (Cucumis sativus L.) belongs to the Cucurbitaceae family and is an economically important crop as a kind of annual herbaceous plant, which enjoys a significant industrial position at home and abroad.

[0003] The cotton aphid, also known as the melon aphid or lice, is an insect of the order Homoptera and the family Aphididae. They are important pests of cotton and many other plants, and are widely distributed around the world. Cotton aphids mainly feed on the sap of young leaves by piercing and sucking, which causes leaf curling, plant dwarfing, bud and boll shedding, and poor cotton quality. They not only harm cotton, but also harm melons, soybeans, jute, potatoes, sweet potatoes, and many other plants. Cotton aphids have very strong reproductive ability, usually reproducing a generation in ten to fifteen days in cooler temperatures, and only four to five days in warm weather. A single aphid can produce 60 to 70 offspring in its lifetime.

[0004] DNA methylation is a key epigenetic regulatory mechanism in plants, which affects gene expression and chromosome structure by adding methyl groups to cytosine bases in DNA. In plants, DNA methylation occurs not only in CpG sequences, but also commonly in non-CpG sequences such as CHG and CHH (H represents A, C or T). This methylation pattern is crucial for plant growth and development, response to environmental changes, genomic imprinting, and silencing of transposable elements. Dynamic changes in plant DNA methylation help regulate the plasticity of the genome, enabling plants to adapt to different environmental stresses and life cycle stages.

[0005] With the development of molecular biology technology, molecular marker assisted breeding has become an important means of crop resistance breeding. However, most studies focus on markers related to gene sequence variation, and the role of epigenetic regulation in cucumber aphid resistance is not paid enough attention, especially the specific mechanism of DNA methylation in cucumber when encountering aphid stress has not been elucidated. As an important epigenetic modification method, DNA methylation can change the methylation level of the gene promoter region, affect the binding efficiency of transcription factors and promoters, and then regulate gene expression. This mechanism is widely used in plant response to biological stress, but it has not been deeply studied in cucumber resistance to aphids. The prior art has not yet determined the key methylation regulation genes related to aphid resistance in cucumber, and there is a lack of DNA methylation molecular markers that can be directly used for aphid resistance breeding. In view of this, it has become an urgent need in the current technology to develop a DNA methylation molecular marker that can assist in cucumber aphid resistance breeding, in order to cultivate varieties with excellent genetic characteristics and solve the problems of complicated process, low efficiency and limited accuracy in traditional cucumber aphid screening and identification process. SUMMARY

[0006] The present application aims to at least solve one of the problems in the prior art or related art.

[0007] To this end, the first aspect of the present application provides a DNA methylation molecular marker based on cucumber CsDHAR gene, the nucleotide sequence of the molecular marker is shown as SEQ ID NO: 1.

[0008] Further, the methylation level of the molecular marker is negatively correlated with the aphid resistance of cucumber.

[0009] The second aspect of the present application provides a primer pair for amplifying and detecting the DNA methylation molecular marker based on cucumber CsDHAR gene, in the primer pair, the nucleotide sequence of the forward primer is shown as SEQ ID NO: 2, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO: 3.

[0010] The third aspect of the present application provides the application of the DNA methylation molecular marker based on cucumber CsDHAR gene or the primer pair in the screening of aphid-resistant cucumber germplasm.

[0011] Further, the screening method comprises the following steps: extracting the cucumber genomic DNA to be tested; purifying the extracted genomic DNA after bisulfite conversion; using the primer pair to perform PCR amplification with the purified bisulfite-modified DNA as a template; Sequencing and analyzing the PCR amplification product, screening the high aphid resistance cucumber varieties according to the methylation level of the molecular marker.

[0012] Further, the reaction system of the bisulfite conversion is: DNA 6-10ul, CT Conversion Mix 120-150ul, ddH2O 12-15ul, total system 138-175ul; the procedure of the bisulfite conversion is: ①98℃ 10min; ②64℃ 40min; ③98℃ 5min; ④64℃ 40min; ⑤98℃ 5min; ⑥64℃ 40min; ⑦4℃ Hold.

[0013] Further, the purification adopts the column purification method, adsorbs, washes, desulfonates and elutes the reaction liquid after the bisulfite conversion, and finally obtains the purified bisulfite modified DNA.

[0014] Further, the system of the PCR amplification is: bisulfite modified DNA 2-5ul, 2x EpiArt HSTaq Master Mix 25-30ul, forward and reverse primers each 2-3ul, ddH2O 19-20ul, total system 50-61ul; the procedure of the PCR amplification is: ①95℃ 3min; ②95℃ 15sec, 50℃ 15sec, 72℃ 15sec, 33 cycles; ③72℃ 5min.

[0015] The fourth aspect of the application provides an application of the DNA methylation molecular marker based on the cucumber CsDHAR gene or the primer pair, including one or more of the following: (1) the application in detecting or identifying aphid-resistant cucumber varieties; (2) the application in screening aphid-resistant cucumber breeding parents; (3) the application in improving the breeding rate of aphid-resistant excellent cucumber varieties; (4) the application in predicting the influence of the methylation level of CsDHAR promoter on transcription factor binding.

[0016] The fifth aspect of the application provides a kit for screening aphid-resistant cucumber germplasm, which comprises the primer pair.

[0017] Compared with the prior art, the application at least includes the following beneficial effects: This invention successfully developed a novel DNA methylation molecular marker corresponding to the cucumber CsDHAR gene. The DNA methylation level of this molecular marker is negatively correlated with aphid resistance in cucumbers; that is, the lower the methylation level, the stronger the aphid resistance. Based on this discovery, specific primer pairs were designed to effectively and rapidly amplify and detect this molecular marker, thereby efficiently screening for superior aphid-resistant varieties and improving the accuracy of selection in segregating generations. This molecular marker and its corresponding amplification primers can be applied to the screening process of aphid-resistant cucumber germplasm, providing not only a solid theoretical basis and genetic foundation for breeding aphid-resistant cucumbers, but also effectively accelerating the screening rate of superior aphid-resistant varieties and promoting the progress of aphid-resistant cucumber breeding. Attached Figure Description

[0018] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the scope of this application. In the drawings: Figure 1 The expression level of CsDHAR after insect inoculation in the cucumber resistant variety provided in Example 1 of this invention; Figure 2 The DNA methylation level after inoculation with CsDHAR in the cucumber resistant variety provided in Example 1 of this invention; Figure 3 The PCR amplification results of the CsDHAR gene DNA methylation molecular marker provided in Example 2 of this invention are shown below; wherein, 1-1 to 1-3 are resistant varieties 9LH; 2-1 to 2-3 are resistant varieties GF-1; 3-1 to 3-3 are intermediate varieties GLH; 4-1 to 4-3 are intermediate varieties HZLZ; 5-1 to 5-3 are susceptible varieties JY; and 6-1 to 6-3 are susceptible varieties YZ2332. Figure 4 The DNA methylation levels of the CsDHAR gene in different aphid-resistant cucumber germplasms provided in Example 2 of this invention after insect inoculation; Figure 5 The results of screening different aphid-resistant cucumber germplasms provided in Comparative Example 1 of this invention using traditional cucumber aphid resistance identification methods are presented. Detailed Implementation

[0019] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0020] In a first aspect, this invention provides a DNA methylation molecular marker csdhar based on the cucumber CsDHAR gene, the nucleotide sequence of which is shown in SEQ ID NO:1. The methylation level of the DNA methylation molecular marker csdhar based on the cucumber CsDHAR gene is negatively correlated with the aphid resistance of cucumber.

[0021] This invention successfully developed a novel DNA methylation molecular marker, csdhar, corresponding to the cucumber CsDHAR gene. Studies have shown that the lower the DNA methylation level of csdhar, the stronger the cucumber's resistance to aphids.

[0022] To identify key genes for aphid resistance in cucumbers, this invention involved infecting the aphid-susceptible variety DRM2-OE and the aphid-resistant variety DRM2-CR with aphids. Through combined whole-genome sulfite sequencing (WGBS) and transcriptome analysis, differentially methylated regions (DMRs), DMR-related genes (DMR genes), differentially expressed genes (DEGs), and differentially methylated genes (DMR-DEGs) were systematically screened. Key findings: The CsDHAR gene was expressed as DMR-DEG in the aphid-resistant material DRM2-CR, while no induced differential methylation or expression was observed in the aphid-susceptible material DRM2-OE, confirming its close association with aphid resistance. The nucleotide sequence of the CsDHAR gene is shown in SEQ ID NO:4.

[0023] In a second aspect, the present invention provides a primer pair for amplifying and detecting DNA methylation molecular markers based on the cucumber CsDHAR gene, wherein the nucleotide sequence of the forward primer is shown in SEQ ID NO:2 and the nucleotide sequence of the reverse primer is shown in SEQ ID NO:3.

[0024] This invention utilizes molecular marker technology to design specific primers for the precise detection of DNA methylation levels of the cucumber CsDHAR gene molecular marker csdhar. This enables efficient and rapid amplification and detection of the molecular marker, thereby efficiently screening for superior aphid-resistant varieties and improving the accuracy of selection in segregating generations.

[0025] A third aspect of this invention provides the application of a DNA methylation molecular marker based on the cucumber CsDHAR gene or the aforementioned primer pair in the screening of aphid-resistant cucumber germplasm.

[0026] To verify the practicality of this molecular marker, the DNA methylation molecular marker based on the cucumber CsDHAR gene and the above primer pair were applied to the screening of cucumber germplasms with different resistances. A detection system based on sulfite treatment, marker-specific primer PCR amplification and Sanger sequencing was established, which effectively verified the association between the methylation level of the CsDHAR gene and the aphid resistance of the germplasm.

[0027] The results showed that the methylation level of the DNA methylation molecular marker csdhar of the cucumber CsDHAR gene was associated with aphid resistance in cucumbers: the aphid resistance of different cucumber germplasms was negatively correlated with the DNA methylation level of this molecular marker—the lower the methylation level, the stronger the aphid resistance of the cucumber. Furthermore, the detection results of this embodiment were consistent with those of traditional aphid resistance identification methods, indicating that the provided molecular marker can be effectively used for the breeding of aphid-resistant cucumber germplasms.

[0028] In some embodiments, the filtering method includes the following steps: Extract genomic DNA from cucumbers to be tested; The extracted genomic DNA was purified by sulfite conversion; PCR amplification was performed using purified sulfite-modified DNA as a template and the primer pairs used. The PCR amplification products were sequenced and analyzed, and cucumber varieties with high aphid resistance were screened based on the DNA methylation level of molecular markers.

[0029] The screening method provided in this invention directly detects epigenetic loci that are clearly negatively correlated with aphid resistance, fundamentally avoiding environmental interference from traditional methods and ensuring stable and reliable results. The standardized molecular experimental procedure also allows the results to be reproduced in different laboratories. This method drastically reduces the screening cycle from weeks to days, significantly lowering time and labor costs. Furthermore, its broad applicability supports multiple scenarios, from initial screening of germplasm resources to early selection in breeding, as well as batch testing, providing an efficient and reliable technical solution for large-scale aphid-resistant breeding.

[0030] In some embodiments, the reaction system for sulfite conversion is: DNA 6 μl-10 μl, CT Conversion Mix 120 μl-150 μl, ddH2O 12 μl-15 μl, total system 138 μl-175 μl; the conversion program is: ① 98℃ for 10 min; ② 64℃ for 40 min; ③ 98℃ for 5 min; ④ 64℃ for 40 min; ⑤ 98℃ for 5 min; ⑥ 64℃ for 40 min; ⑦ 4℃ Hold.

[0031] The sulfite conversion reaction system provided in this invention, through multiple cycles of "98℃ pre-denaturation" and "64℃ conversion", can fully open the DNA double strand, ensuring that sulfite completely converts unmethylated cytosine into uracil, thereby avoiding misjudgment of methylation level due to incomplete conversion; at the same time, by strictly controlling the high temperature duration and optimizing the conversion temperature, DNA degradation and breakage are effectively reduced, ensuring the template quality for subsequent PCR amplification.

[0032] In some embodiments, purification is performed using a column purification method, in which the reaction solution after sulfite conversion is adsorbed, washed, desulfonated, and eluted to finally obtain purified sulfite-modified DNA.

[0033] The column purification method used in this invention can efficiently remove reaction residues and obtain high-purity DNA that meets the requirements of subsequent PCR and sequencing. This method integrates the desulfonation step into the purification process, which not only simplifies the operation but also avoids DNA damage and amplification inhibition that may result from separate treatments. Simultaneously, its strong adsorption capacity for modified DNA ensures a recovery rate far exceeding that of the traditional phenol-chloroform extraction method, making it particularly suitable for trace samples. Furthermore, the entire process requires no toxic organic solvents, improving experimental safety.

[0034] In some embodiments, the PCR amplification system is as follows: 2 μl-5 μl of sulfite-modified DNA, 25 μl-30 μl of 2×EpiArtHS Taq Master Mix, 2 μl-3 μl each of forward and reverse primers, 19 μl-20 μl of ddH2O, and a total system of 50 μl-61 μl; the amplification program is as follows: ① 95℃ for 3 min; ② 95℃ for 15 sec, 50℃ for 15 sec, 72℃ for 15 sec, 33 cycles; ③ 72℃ for 5 min.

[0035] The amplification system set up in this embodiment of the invention ensures the specificity and accuracy of the target fragment amplification through thorough pre-denaturation at 95℃, an annealing temperature of 50℃ highly matched to the primer Tm value, and an extension at 72℃ optimal for Taq enzyme. Since DNA is easily degraded after sulfite modification, the time consumed in each step is short, effectively reducing damage to the template from high temperatures. Simultaneously, the 33 cycles and the final end-completion step ensure sufficient and complete product while avoiding the accumulation of non-specific products. This standardized process ensures the universality and reproducibility of the method across different cucumber germplasms, providing high-quality amplified products for subsequent sequencing.

[0036] A fourth aspect of this invention provides the application of a DNA methylation molecular marker or primer pair based on the cucumber CsDHAR gene, including one or more of the following: (1) Application in detecting or identifying aphid-resistant cucumber varieties; (2) Application in screening aphid-resistant cucumber breeding parents; (3) Application in improving the breeding rate of superior cucumber aphid-resistant varieties; (4) Application in predicting the effect of CsDHAR promoter methylation level on transcription factor binding.

[0037] The DNA methylation molecular marker provided in this invention, as a novel epigenetic marker, not only provides a solid theoretical basis and genetic foundation for breeding aphid-resistant cucumbers, but also, when applied to the process of aphid-resistant germplasm selection, can effectively accelerate the screening rate of superior aphid-resistant varieties and promote the progress of aphid-resistant cucumber breeding.

[0038] A fifth aspect of the present invention provides a kit for screening aphid-resistant cucumber germplasm, comprising the primer pairs described above.

[0039] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Experimental methods not specifically described in the examples are generally performed under standard conditions or as recommended by the manufacturer.

[0040] Example 1: Cucumber transcriptome and DNA methylation sequencing The study used the resistant cucumber variety DRM2-CR and the susceptible variety DRM2-OE, with 6 replicates of each variety. The cucumbers were cultured in the same environment until the leaf-bud stage. Forty 2-3 instar cucumber aphids were selected and inoculated onto the leaves. After 72 hours, the aphids were removed, and leaf samples were taken for subsequent experiments to extract DNA and RNA. Transcriptome and DNA methylation sequencing were performed according to the following steps.

[0041] Transcriptome sequencing was performed by Wuhan Bena Technology Co., Ltd., and the process is as follows: S1: Transcriptome sequencing of cucumber varieties susceptible to and resistant to aphids (1) RNA was extracted from insect-infested DRM2-OE, insect-infested DRM2-CR, non-insect-infested DRM2-OE, and non-insect-infested DRM2-CR using the RNA Easy Fast Plant Tissue RNA Rapid Extraction Kit (Tiangen).

[0042] (2) The deionized total RNA sample was denatured at an appropriate temperature to open its secondary structure, and the mRNA was enriched using oligo (dT) magnetic beads.

[0043] (3) Add a fragmentation reagent to the mRNA obtained in (2) and react at an appropriate temperature for a certain time to fragment the mRNA.

[0044] (4) Add the pre-prepared one-strand synthesis reaction system to the fragmented mRNA, synthesize one-strand cDNA on the PCR instrument according to the corresponding program, prepare the two-strand synthesis reaction system, react at the appropriate temperature for a certain time, and synthesize two-strand cDNA.

[0045] (5) Prepare the reaction system, react at an appropriate temperature for a certain time to repair the ends of the double-stranded cDNA, and add an A base to the 3' end. Prepare the adapter ligation reaction system, react at an appropriate temperature for a certain time to ligate the adapter to the cDNA.

[0046] (6) Prepare the PCR reaction system and set the reaction program to amplify the ligation product.

[0047] (7) After denaturing the PCR product into single strands, prepare a cyclization reaction system, mix thoroughly, react at a suitable temperature for a certain period of time to obtain a single-stranded circular product. After digesting the uncyclized linear DNA molecules, the final library is obtained.

[0048] (8) Use Agilent 2100 Bioanalyzer to detect the fragment size and concentration of the library.

[0049] (9) After the library passes the test, sequencing is performed using the BGI T7 high-throughput sequencing platform.

[0050] S2: DNA methylation sequencing of cucumber varieties susceptible to and resistant to aphids. (1) DNA extraction was performed using the rapid plant genomic DNA extraction system (Tiangen).

[0051] (2) After the sample passes the test, 100 ng of genomic DNA and 0.5 ng of unmethylated lambda DNA are mixed using a Covaris S220 ultrasonic disruptor and then broken into 200-400 bp fragments.

[0052] (3) After fragmentation, use EZ DNA Methylation-Gold TM The kit (Zymo Research) converts unmethylated cytosine to uracil, followed by small fragment library construction (Benagen, WuHan, China).

[0053] (4) Library testing was performed using the 5400 Fragment Analyzer System. Samples that passed the library testing were sequenced at both ends on the Illumina Novaseq (Illumina, CA, USA) sequencing platform using the PE150 sequencing strategy.

[0054] The obtained raw transcriptome sequencing data were subjected to differential expression analysis using DESeq2 (v1.36.0). By comparing the highly susceptible cucumber aphid variety DRM2-CR and the highly resistant variety DRM2-OE, differentially expressed genes (DEGs) with significantly upregulated or downregulated expression levels were identified. The screening criteria were |Log2(FoldChange)|≥1 and padj<0.05.

[0055] The raw DNA methylation sequencing data were quality assessed using FastQC (fastqc_v0.11.5), and then filtered using fastp software (fastp0.23.1). The filtered data became the clean data, which was then re-assessed using FastQC and used for subsequent analysis. The clean data was then aligned to a reference genome using Bismark (v0.16.3; Krueger F, 2011). During alignment, the reference genome was first converted (C-to-T, G-to-A), and an index was generated using bowtie2 (Langmead B, 2012). Simultaneously, the sequenced reads were also converted and directly aligned to the reference genome. The best unique alignment was then re-aligned to the original reference genome to obtain the methylation status and location of all C sites. When reads align to the same position on the reference genome, they are considered repetitive sequences. After removing repetitive sequences, sequencing depth and genome coverage are calculated. Methylation analysis results are extracted (bismark_methylation_extractor, --no_overlap) and converted to BigWig format. IGV is used to visualize the methylation analysis. Based on the C-site conversion in lambda DNA, the BS conversion rate during library construction is evaluated. For each identified methylation site, its methylation level (ML) is calculated using the following formula:

[0056] ML represents the methylation level, and mC represents the number of reads that support the site being methylated C.

[0057] Differentially methylated regions (DMRs) were then analyzed using DSS (v2.12.0). Based on their distribution across the genome, DMRs were divided into coding regions (from TSS to TES) and promoter regions (2kb upstream of TSS). GO (Gene Ontology) enrichment analysis of DMR-related genes was performed using the R package GOseq, with a p-value less than 0.05 considered significant enrichment. KEGG pathway enrichment analysis of DMR-related genes was then performed using KOBAS software.

[0058] Based on transcriptomic and DNA methylation data analysis of the cucumber aphid-resistant variety DRM2-CR and the susceptible variety DRM2-OE, such as... Figure 1 As shown, the expression of the CsDHAR gene was significantly upregulated in DRM2-CR compared to DRM2-OE after insect inoculation; Figure 2 As shown, the CsDHAR gene is a differentially expressed gene (DMR-DEG) associated with differential methylation in the cucumber aphid-resistant material DRM2-CR, while in the cucumber aphid-susceptible material DRM2-OE, this gene was not induced to undergo differential methylation or expression. Therefore, a DNA methylation molecular marker csdhar located in the CsDHAR gene was designed. The nucleotide sequence of the CsDHAR gene DNA methylation molecular marker is shown in SEQ ID NO:1. Combined transcriptome and DNA methylation data analysis demonstrated that the DNA methylation level of the cucumber CsDHAR gene DNA methylation molecular marker csdhar is related to cucumber aphid resistance. A negative correlation was found between the aphid resistance of different cucumber varieties and the DNA methylation level of the molecular marker csdhar; that is, the lower the DNA methylation level of the molecular marker csdhar, the higher the aphid resistance of the cucumber.

[0059] Example 2: Application of DNA methylation molecular markers in screening cucumber aphid-resistant germplasm. S1: Extracting genomic DNA from cucumbers to be tested Cucumber varieties (9LH, GF-1, GLH, HZLZ, JY, YZ2332) for aphid resistance testing were selected. Three biological replicates were taken for each variety and cultured in the same environment until the leaf-bud stage. Forty 2nd-3rd instar cucumber aphids were then inoculated onto the leaves, and the aphids were removed after 72 hours. Genomic DNA was extracted from the leaves using a rapid plant genomic DNA extraction system (Tiangen), following the kit instructions. Specific steps are as follows: (1) Take 100 mg of cucumber leaves and grind them thoroughly with liquid nitrogen. Add 400 μl of buffer FP1 and 6 μl of RNase A (10 mg / ml), vortex for 1 min, and let stand at room temperature for 10 min.

[0060] (2) Add 130 μl of buffer FP2, mix thoroughly, and vortex for 1 min.

[0061] (3) Centrifuge at 12,000 rpm for 5 min and transfer the supernatant to a new centrifuge tube.

[0062] (4) Centrifuge the supernatant again at 12,000 rpm for 5 min, and transfer the supernatant to a new centrifuge tube.

[0063] (5) Add 0.7 times the volume of isopropanol to the supernatant and mix thoroughly. At this time, flocculent genomic DNA will appear. Centrifuge at 12,000 rpm for 2 min, discard the supernatant and keep the precipitate.

[0064] (6) Add 600 μl of 70% ethanol, vortex for 5 seconds, centrifuge at 12,000 rpm for 2 minutes, and discard the supernatant.

[0065] (7) Repeat step (6).

[0066] (8) Open the lid and invert it, place it at room temperature for 10 minutes to completely dry the remaining ethanol.

[0067] Add an appropriate amount of elution buffer TE, and dissolve the DNA in a 65°C water bath for 60 minutes, inverting and mixing several times during the process to aid dissolution. The final DNA solution is then obtained. The concentration and purity of the extracted DNA are determined, and the solution is stored at -20°C.

[0068] S2: Purification of DNA after sulfite conversion The extracted DNA was treated with DNA Methylation Bisulfite Kit (Novizan) according to the kit instructions. The specific steps are as follows: (1) Add 7 μl of DNA (1000 ng / μl), 130 μl of CT Conversion Mix, and 13 μl of ddH2O to a 200 μl sterile PCR tube.

[0069] (2) Invert the tube and blow it to mix it. After a short centrifugation, collect the reaction liquid to the bottom of the tube.

[0070] (3) Place the PCR tube in the PCR instrument and the amplification program is shown in Table 1.

[0071] Table 1. Amplification program for DNA transformation reaction

[0072] (4) Place the EpiArt DNA Columns adsorption column into the Collection Tube.

[0073] (5) Add 600 μl of E-Binding Buffer to the adsorption column, and then add the converted reaction product to the adsorption column. Gently invert the column 10 times to mix the reaction solution with the E-Binding Buffer completely.

[0074] (6) Centrifuge at 12,000 rpm for 60 seconds. Discard the filtrate and return the adsorption column to the collection tube.

[0075] (7) Add 500 μl of E-Wash Buffer (with added ethanol) to the adsorption column and centrifuge at 12,000 rpm for 60 seconds. Discard the filtrate and return the adsorption column to the collection tube.

[0076] (8) Add 500 μl of E-Desulphonation Buffer to the adsorption column and allow it to stand at room temperature for 15 min. Centrifuge at 12,000 rpm for 60 sec. Discard the filtrate and return the adsorption column to the collection tube.

[0077] (9) Add 500 μl of E-Wash Buffer (with added ethanol) to the adsorption column and centrifuge at 12,000 rpm for 60 seconds. Discard the filtrate and return the adsorption column to the collection tube.

[0078] (10) Repeat step (9).

[0079] (11) Centrifuge the empty column at 12,000 rpm for 2 min.

[0080] (12) Transfer the adsorption column to a new 1.5 ml centrifuge tube, open the cap and let it air dry completely for 2 min. Add 10-20 μl of E-Elution Buffer to the center of the adsorption column membrane. Let it stand at room temperature for 1 min, centrifuge at 12,000 rpm for 2 min, and collect the DNA filtrate. Discard the adsorption column and store the sulfite-modified genomic DNA at -20℃.

[0081] S3: PCR amplification (1) Design primers. Primer information is shown in Table 2.

[0082] Table 2 Information on methylation amplification primers

[0083] (2) Add 2 μl of sulfite-modified genomic DNA, 25 μl of 2×EpiArt HS TaqMaster Mix, 2 μl each of forward and reverse primers, and 19 μl of ddH2O to a sterile PCR tube.

[0084] (3) Invert the tube and blow it to mix it. After a short centrifugation, collect the reaction liquid to the bottom of the tube.

[0085] (4) Place the PCR tube in the PCR instrument and the amplification program is shown in Table 3.

[0086] Table 3 PCR amplification program

[0087] (5) Perform gel electrophoresis on the PCR products, such as Figure 3 As shown, the target fragment matches the expected length. The target fragment is recovered to obtain the molecularly labeled product.

[0088] (6) Ligate the recovered target fragment with the 5×TA / Blunt-Zero Cloning Mix vector to prepare the reaction system: 1 μl of 5×TA / Blunt-Zero Cloning Mix, 1 μl of PCR purified product, and 3 μl of ddH2O. Gently tap the bottom of the tube to mix, then centrifuge briefly at low speed to collect all liquid at the bottom of the centrifuge tube. Incubate at room temperature for 5 min. After the reaction is complete, place the centrifuge tube on ice.

[0089] (7) Remove Fast-T1 competent cells from -70℃ and quickly place them on ice to thaw. Add the ligation product, gently tap the tube wall to mix, and incubate on ice for 30 min. After heat shock in a 42℃ water bath for 30 sec, quickly place them on ice for 2 min. Add 900 μl of LB liquid medium to the centrifuge tube, mix well, and incubate at 37℃ and 200 rpm for 1 h. Centrifuge at 5,000 rpm for 3 min, discard 900 μl of supernatant, resuspend the bacterial culture in the remaining medium, and spread evenly on LB solid medium plates containing ampicillin. Incubate the plates upright at 37℃ for 10 min until the bacterial culture is completely absorbed, then invert the plates and incubate overnight.

[0090] (8) Pick 5 single clones and mix them with 10 μl of ddH2O as template bacterial solution. Add 10 μl of 2×Rapid Taq Master Mix, 2 μl of M13 Primer Mix, 2 μl of bacterial solution, and 6 μl of ddH2O to a sterile PCR tube. Place the PCR tube in the PCR instrument and follow the reaction program shown in Table 4.

[0091] Table 4 PCR reaction procedure

[0092] S4: Sequencing and analyzing data This embodiment selected various cucumber materials (9LH, GF-1, GLH, HZLZ, JY, YZ2332) for aphid resistance screening and identification. The determined sequences were compared in SnapGene, and the correctly aligned results were statistically analyzed using GraphPad Prism for methylation. The results were then presented graphically using TBtools. Figure 4 As shown, the DNA methylation levels of the CsDHAR gene DNA methylation marker csdhar in cucumbers were selected and ranked from high to low. Based on the conclusion obtained from this invention that there is a negative correlation between the aphid resistance of different cucumber varieties and the DNA methylation level of the CsDHAR gene DNA methylation marker csdhar, cucumber varieties were selected and ranked from low to high based on their aphid resistance levels. A higher proportion of white cucumbers corresponds to a lower methylation level; a higher proportion of black cucumbers corresponds to a higher methylation level. Therefore, the resistant varieties are 9LH and GF-1, the intermediate varieties are GLH and HZLZ, and the susceptible varieties are JY and YZ2332.

[0093] Comparative Example 1: Traditional methods for identifying cucumber aphid resistance Comparative Example 1 selected various cucumber materials (9LH, GF-1, GLH, HZLZ, JY, YZ2332) for aphid resistance screening and identification. The traditional cucumber aphid resistance identification method, namely a 3-day simplified life table screening, was used for verification. The specific screening steps were as follows: (1) Select the cucumber varieties that need to be identified for aphid resistance, and culture each variety in 3 biological replicates in the same environment until the two-leaf-one-heart stage.

[0094] (2) Select 40 relatively active 3-4 instar cucumber aphids and place them directly on the true leaves of the cucumber, while covering the entire cucumber seedling with a breathable net bag.

[0095] (3) Count the number of aphids on cucumber leaves 3 days after inoculation.

[0096] (4) Using a 3-day simplified life table, the aphid resistance of the cucumber varieties to be identified was determined. The selected cucumber varieties were ranked from lowest to highest aphid resistance level based on the number of aphids on the leaves. For example... Figure 5 As shown, the resistant varieties are 9LH and GF-1, the intermediate varieties are GLH and HZLZ, and the susceptible varieties are JY and YZ2332.

[0097] Analysis of Example 2 and Comparative Example 1 shows that this invention uses the DNA methylation level of specific CSDH molecular markers to screen cucumber varieties for aphid resistance, and sorts the screening results from low to high. The results obtained by this method are highly consistent with traditional screening methods, fully demonstrating the effectiveness and reliability of the molecular markers amplified by the primer pairs provided by this invention in the breeding of cucumber aphid-resistant germplasm. Furthermore, compared to traditional methods, the method of this invention is faster and more efficient, providing a new technical means for aphid-resistant cucumber breeding.

[0098] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A DNA methylation molecular marker based on the cucumber CsDHAR gene, characterized in that, The nucleotide sequence of the molecular marker is shown in SEQ ID NO:

1.

2. The DNA methylation molecular marker based on the cucumber CsDHAR gene according to claim 1, characterized in that, The methylation level of the molecular markers was negatively correlated with the aphid resistance of cucumbers.

3. A primer pair for amplifying and detecting the DNA methylation molecular marker based on the cucumber CsDHAR gene as described in claim 1 or 2, characterized in that, In the primer pair, the nucleotide sequence of the forward primer is shown in SEQ ID NO:2, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO:

3.

4. The application of the DNA methylation molecular marker based on the cucumber CsDHAR gene as described in claim 1 or 2, or the primer pair as described in claim 3, in the screening of aphid-resistant cucumber germplasm.

5. The application according to claim 4, characterized in that, The screening method includes the following steps: Extract genomic DNA from cucumbers to be tested; The extracted genomic DNA was purified by sulfite conversion; Using purified sulfite-modified DNA as a template, PCR amplification was performed using the primer pair described in claim 3. Sequencing and analyzing PCR amplification products, and screening cucumber varieties with high aphid resistance based on the methylation level of the molecular markers described in claim 1 or 2.

6. The application according to claim 5, characterized in that, The reaction system for sulfite conversion was as follows: DNA 6 μl-10 μl, CT Conversion Mix 120 μl-150 μl, ddH2O 12 μl-15 μl, total system volume 138 μl-175 μl; the sulfite conversion procedure was as follows: ① 98℃ for 10 min; ② 64℃ for 40 min; ③ 98℃ for 5 min; ④ 64℃ for 40 min; ⑤ 98℃ for 5 min; ⑥ 64℃ for 40 min; ⑦ Hold at 4℃.

7. The application according to claim 5, characterized in that, The purification process employs a column purification method, in which the reaction solution after sulfite conversion is adsorbed, washed, desulfonated, and eluted to finally obtain purified sulfite-modified DNA.

8. The application according to claim 5, characterized in that, The PCR amplification system consisted of: 2-5 μl of sulfite-modified DNA, 25-30 μl of 2×EpiArt HS Taq Master Mix, 2-3 μl each of forward and reverse primers, and 19-20 μl of ddH2O, for a total volume of 50-61 μl. The PCR amplification program was as follows: ① 95℃ for 3 min; ② 95℃ for 15 sec, 50℃ for 15 sec, 72℃ for 15 sec, 33 cycles; ③ 72℃ for 5 min.

9. The application of the DNA methylation molecular marker based on the cucumber CsDHAR gene as described in claim 1 or 2, or the primer pair as described in claim 3, characterized in that, Includes one or more of the following: (1) Application in detecting or identifying aphid-resistant cucumber varieties; (2) Application in screening aphid-resistant cucumber breeding parents; (3) Application in improving the breeding rate of superior cucumber aphid-resistant varieties; (4) Application in predicting the effect of CsDHAR promoter methylation level on transcription factor binding.

10. A kit for screening aphid-resistant cucumber germplasm, characterized in that, It includes the primer pair as described in claim 3.

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