Functional molecular marker for detecting wheat powdery mildew resistance gene WTK4 and application of functional molecular marker

Abstract

The invention belongs to the field of plant biotechnology and wheat molecular breeding, and discloses a functional molecular marker for detecting wheat powdery mildew resistance gene WTK4 and application of the functional molecular marker, the wheat powdery mildew resistance gene WTK4 is located on a short arm of an aegilops tauschii 7D chromosome, and a WTK4 homologous gene in a Chinese spring reference genome RefSeq v2.0 version is located on a long arm of a 4A chromosome. Differential sites of the functional molecular marker are located in a WTK4 gene, and the polymorphism is 105bp insertion / deletion (Indel). The molecular marker provided by the invention is combined with a PCR (Polymerase Chain Reaction) method, so that whether the powdery mildew-resistant gene WTK4 exists in a material or not can be accurately detected; the material containing the WTK4 gene can be divided into an aegilops tauschii disease-resistant genotype and a Chinese spring susceptible genotype according to the size of an amplified band. The method promotes the utilization of the wheat powdery mildew resistance gene WTK4, can be applied to molecular marker-assisted selective breeding, and accelerates the breeding process of wheat disease-resistant varieties.

Description

Technical Field

[0001] The present invention belongs to but is not limited to the technical fields of plant biotechnology and wheat molecular breeding, and in particular relates to a functional molecular marker for detecting a wheat powdery mildew resistance gene WTK4 and an application thereof. Background Art

[0002] Wheat powdery mildew is a fungal disease of wheat caused by Blumeria graminis f.sp.tritici (Bgt) of the Poaceae family. It is also one of the most common and important diseases in wheat production in my country. In severe cases, it can cause a 30%-50% reduction in wheat production, seriously endangering my country's wheat production. Due to the large-scale planting of wheat varieties with single resistance in my country for a long time, wheat powdery mildew has been selected, resulting in the gradual loss of resistance of most resistant varieties in my country. Screening and breeding of disease-resistant wheat varieties is the most basic and effective method for preventing and controlling wheat powdery mildew.

[0003] With the advancement of sequencing technology and the development of molecular biotechnology, although more than 100 (Pm1-Pm69) powdery mildew resistance genes or QTLs have been located in wheat and its related species, most of the resistance gene resources have not been fully applied and promoted. Through traditional breeding methods, it is difficult to apply excellent disease resistance genes in wheat related species to wheat breeding. Compared with conventional breeding methods, molecular marker-assisted selection technology has many advantages in the process of screening and breeding disease-resistant wheat varieties, such as clear purpose, shortening the breeding process, and improving selection efficiency. However, most of the molecular markers used for disease resistance sites are currently linked molecular markers. There is a certain distance between the marker and the disease resistance site. Wheat varieties with different genetic backgrounds may have recombination. Tracking only by linked molecular markers will lead to the loss of the target disease resistance site. Compared with traditional linked molecular markers, gene-specific molecular markers co-segregate with the target gene within the gene, and there is no recombination phenomenon. Molecular marker detection is not affected by material type and genetic background. The detection results are highly reliable, which greatly improves the transfer and utilization of disease resistance genes. WTK4 is a powdery mildew resistance gene from Aegilops tauschii. It has broad-spectrum resistance and maintains resistance in hexaploid synthetic wheat. It is an excellent powdery mildew resistance gene resource for wheat. Currently, WTK4 has not been used in wheat varieties, and molecular markers for tracking the WTK4 gene have not been reported.

[0004] In view of the above analysis, the technical problems that need to be solved urgently in the prior art are:

[0005] How to efficiently and accurately detect and track WTK4 is of great significance to wheat powdery mildew resistance breeding, and the development of WTK4 gene-specific functional molecular markers is particularly urgent. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention provides a functional molecular marker for detecting wheat powdery mildew resistance gene WTK4 and application thereof.

[0007] The present invention is achieved in this way. A functional molecular marker for detecting the wheat powdery mildew resistance gene WTK4 is characterized in that the Indel molecular marker is WTK4-Indel, which is developed based on the difference in the sequence of the WTK4 gene in Aegilops tauschii CIae8 and the WTK4 homologous gene in Chinese spring, and a 105bp insertion / deletion site at the 866bp position of the third intron region. A WTK4 gene-specific Indel molecular marker is developed, and the primer nucleotide sequence of the Indel molecular marker includes an upstream primer WTK4-Indel-F and a downstream primer WTK4-Indel-R.

[0008] Furthermore, the nucleotide sequences of the upstream and downstream primers for detecting the functional molecular marker of the wheat powdery mildew resistance gene WTK4 are as follows:

[0009] WTK4-Indel-F:5'-GTGGGTTTTTGGAGCAGTGG-3'

[0010] WTK4-Indel-R: 5'-AATAACGCACAACCATAGGCA-3'

[0011] Another object of the present invention is to provide a gene functional molecular marker for detecting wheat powdery mildew resistance gene WTK4 for use in identifying wheat powdery mildew resistance. The marker can specifically track the WTK4 gene and can distinguish the WTK4 genotype into a Chinese spring susceptible genotype and a knotweed resistant genotype.

[0012] Further, the above molecular markers are used to detect the DNA of the wheat to be tested. If the PCR product shows a specific band of 375 bp, it indicates that the sample to be tested contains the WTK4 gene of the Aegilops tauschii genotype and has powdery mildew resistance. If the PCR product expands a specific band of 480 bp, it indicates that the wheat to be tested is the WTK4 gene of the Chinese Spring genotype and does not have powdery mildew resistance.

[0013] Furthermore, the detection method comprises the following steps:

[0014] S1: Extract the DNA of the sample to be tested using the CTAB method;

[0015] S2: PCR amplification using detection primer pairs;

[0016] S3: Perform electrophoresis analysis on the PCR products.

[0017] S4: In the above identification method, the PCR reaction system and amplification conditions of step S2 are as follows:

[0018] Reaction system: Each 10 μL includes: 50 ng / μL genomic DNA 1.0 μL, 5 μL PCR Master Mix,

[0019] Amplification conditions: pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 15 seconds, annealing at 55°C for 15 seconds, extension at 72°C for 15 seconds, for 32 cycles; extension at 72°C for 5 minutes; storage at 4°C.

[0020] Furthermore, the PCR products were separated by electrophoresis using 1% agarose gel, and the bands were read using a gel imager.

[0021] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0022] First, the efficient use of wheat powdery mildew resistance gene resources is the key to breeding disease-resistant varieties. The molecular marker developed by the present invention is based on the sequence of the cloned gene WTK4 and is a gene-specific molecular marker. Compared with traditional linked molecular markers, gene-specific molecular markers co-segregate with the target gene within the gene and do not have recombination. Molecular marker detection is not affected by material type and genetic background, and the detection result has high credibility, which greatly improves the transfer and utilization of gene WTK4.

[0023] The WTK4 functional marker was used to amplify 385 wheat natural populations. Only 7 wheat varieties contained WTK4, and the amplified band type was consistent with the amplified band type of Chinese Spring. The powdery mildew resistance identification results showed that these 7 common wheat varieties were highly susceptible to powdery mildew. The marker was amplified in 94 Aegilops tauschii lines, and the band amplification was consistent with the size of CIae8. The powdery mildew resistance identification results showed that these 12 Aegilops tauschii lines were highly resistant to wheat powdery mildew. This shows that the frequency of wheat powdery mildew resistance gene WTK4 in wheat germplasm resources is low and cannot be used in wheat breeding. It is an excellent disease resistance gene resource for wheat. The marker can specifically track the WTK4 gene and is a specific functional molecular marker. It can accurately and quickly identify whether the material contains the WTK4 gene, and can judge the powdery mildew resistance of the material by the amplification type, which accelerates the efficient use of gene WTK4.

[0024] Second, the technical solution of the present invention fills the technical gap in the industry at home and abroad:

[0025] WTK4 is a gene from Aegilops tauschii that confers resistance to wheat powdery mildew. It has broad-spectrum resistance and is an excellent resistance gene resource for wheat. It has not yet been used in wheat disease-resistant breeding. The developed WTK4 gene-specific molecular marker can accurately identify the powdery mildew resistance of the WTK4 gene and material, greatly improving the utilization efficiency of WTK4 in future wheat disease-resistant breeding and filling the industry gap of the lack of WTK4 molecular marker identification at home and abroad.

[0026] Third, the technical solution of the present invention significantly solves several key problems of the prior art in disease-resistant breeding by developing a method for detecting wheat powdery mildew resistance genes based on the specific molecular marker WTK4-Indel. Traditional disease resistance identification methods mostly rely on long-term phenotypic observation and control of environmental conditions, which is time-consuming and labor-intensive, and has significant uncertainty and inefficiency. The present invention directly identifies whether wheat carries the disease resistance gene WTK4 at the molecular level through molecular detection of genomic DNA, thereby improving detection speed, accuracy and consistency.

[0027] Technically, the present invention solves the problem of difficulty in judging due to the polymorphism of powdery mildew resistance traits in existing breeding detection. Through the specific WTK4-Indel molecular marker, the present invention can accurately distinguish wheat plants carrying disease-resistant genotypes (Aegilops genotype) and susceptible genotypes (Chinese Spring genotype), avoiding the complex phenotypic measurement process that traditional detection relies on. In addition, the marker detection method of the present invention does not rely on environmental conditions and growth period restrictions for breeding materials, and therefore can be widely used in early disease-resistant breeding screening, solving the problems of difficult early judgment and low breeding efficiency in the breeding process.

[0028] In terms of industrial application, the present invention greatly shortens the cycle of disease-resistant breeding, allowing breeding institutions to screen a large amount of materials in a short period of time, quickly lock in disease-resistant genotype plants, and significantly improve the efficiency and success rate of disease-resistant breeding. At the same time, the use of the labeling method of the present invention can provide disease-resistant genotype data at the molecular level, facilitate breeding records and the preservation and utilization of disease-resistant gene resources, and reduce manpower and resource consumption. In the rapid promotion of disease-resistant wheat, this technical solution supports the screening of large-scale germplasm resources and provides a stable and reliable technical guarantee for the industrial production of wheat resistant to powdery mildew. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a schematic diagram of the Indel site of the WTK4 gene sequence in Aegilops tauschii and Chinese spring provided in an embodiment of the present invention: Figure 1 A is a schematic diagram of the gene structure of WTK4. Figure 1 B is the comparison of three Indel sites between CIae8 and Chinese Spring. Figure 1C is the amino acid sequence alignment of CIae8 and WTK4 from Chinese spring, where DD-7D-WTK4 and CS-4A-WTK4 represent the homologous protein sequences of CIae8 and WTK4 from Chinese spring, respectively;

[0030] Figure 2 These are some of the detection results of the functional molecular marker WTK4-Indel developed in the embodiment of the present invention in the natural population of Aegilops tauschii / wheat: Figure 2 A is the detection result of molecular markers in the natural population of Aegilops tauschii. Figure 2 B is the detection result of molecular markers in natural wheat populations, where CS, N7AT7B, N7BT7A, N7DT7B and N4AT4B represent Chinese Spring and Chinese Spring tetrasomic materials lacking chromosomes 7A, 7B, 7D and 4A, respectively, and Fielder, AY61 and PI574467 represent common wheat and Aegilops tauschii without WTK4 gene, respectively;

[0031] Figure 3 The molecular markers provided in the embodiments of the present invention detected the sequencing results of Aegilops tauschii and wheat containing the WTK4 gene and the powdery mildew resistance identification results: Figure 3 A is the sequence alignment result of WTK4 gene in 13 accessions of Aegilops tauschii and 8 accessions of Triticum aestivum. Figure 3 B shows the results of identification of powdery mildew resistance of 13 Aegilops tauschii and 8 Triticum aestivum containing the WTK4 gene at the seedling stage, wherein the number below WTK4 indicates the position in the coding sequence, and Aet and Ta represent Aegilops tauschii and Triticum aestivum, respectively. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0033] The WTK4-Indel functional molecular marker of the present invention is based on the specific differences between the powdery mildew resistance gene WTK4 of wheat and Aegilops tauschii, especially the 105bp Indel site of the gene in the third intron region. Through sequence analysis of the WTK4 gene, specific primers WTK4-Indel-F and WTK4-Indel-R were designed. This pair of primers can specifically identify and amplify the WTK4 gene, and then detect whether the powdery mildew resistance gene WTK4 exists in wheat plants. The design of this molecular marker effectively makes up for the limitation of relying on phenotypic identification in traditional breeding, and provides a fast and accurate means of disease resistance gene detection.

[0034] The working principle of WTK4-Indel marker detection is based on PCR amplification. By using WTK4-Indel-F and WTK4-Indel-R to amplify plant genomic DNA, PCR products of different sizes are generated. In the electrophoresis test, if the WTK4 gene sequence carries a disease-resistant type (Aegilops genotype), the product is 375bp; if it is a susceptible type (Chinese Spring genotype), the product is 480bp. Through this difference, the genotype can be intuitively identified and whether the plant has the ability to resist powdery mildew can be determined. It is suitable for genetic screening of Aegilops tauschii and common wheat varieties.

[0035] The WTK4-Indel molecular marker of the present invention has important application value in molecular marker-assisted selection breeding. It can be used for early screening of disease-resistant wheat varieties, avoiding the complexity and timeliness of traditional disease resistance testing. By performing PCR amplification and electrophoresis detection on breeding materials, the disease-resistant genotype of the plant is quickly determined, and disease-resistant materials containing the WTK4 gene are selected, which accelerates the breeding process and improves breeding efficiency.

[0036] In addition, this marker can also be extended to other related plant materials in specific applications, providing a universal method for disease resistance gene detection. By adapting the WTK4-Indel marker, researchers can quickly obtain disease resistance genotype information of plants in various wheat lines and different growth stages, effectively assist in the transmission and stability evaluation of disease resistance genes, and achieve early selection and promotion of disease-resistant varieties.

[0037] The embodiment of the present invention provides a functional molecular marker WTK4-Indel for detecting the powdery mildew resistance gene WTK4 of wheat. The marker detects the 105 bp Indel site in the third intron region based on the difference in the WTK4 gene sequence between Aegilops tauschii and Chinese spring. For Aegilops tauschii, there is no 105 bp insertion at the 855th to 856th nucleotides of the third intron of the WTK4 gene, and its nucleotide sequence is shown in SEQ ID NO.1. There is a 105 bp insertion in the homologous gene of WTK4 in Chinese spring, and the sequence is shown in SEQ ID NO.2 (Table 1; Figure 1 A).

[0038] Example 1 Obtaining the functional molecular marker WTK4-Indel of the wheat powdery mildew resistance gene WTK4

[0039] 1. Development of WTK4-Indel, a functional molecular marker of WTK4

[0040] The present invention first locates the wheat powdery mildew resistance gene of the Aegilops tauschii strain CIae8 and finds that it overlaps with the location interval of the cloned wheat powdery mildew resistance gene WTK4. Through functional verification, it is proved that the powdery mildew resistance locus of the Aegilops tauschii strain CIae8 on 7DS is WTK4. Using the WheatOmics 1.0 website, a whole genome comparison of WTK4 was performed in the wheat crop reference genome database. Only the homologous gene of WTK4 exists on the 4A short arm of Chinese Spring. In addition to containing a large number of SNP sites, the main difference between the homologous gene TraesCS4A02G481300 in Chinese Spring and WTK4 is that there are two Indel sites in the third intron region ( Figure 1 B). The cDNA sequence similarity between the two is 98.8%, and there are 20 SNP sites and a 6 bp insertion in TraesCS4A02G481300 in Chinese Spring. The amino acid sequence similarity between the two is 98.34%, with 10 amino acid differences and 2 amino acid insertions ( Figure 1 C).

[0041] According to the difference in WTK4 gene sequences between Chinese Spring and CIae8, the embodiment of the present invention designed an Indel marker WTK4-Indel at the insertion site using Primer 5.0 software for the 105 bp Indel site in the third intron region.

[0042] Table 1 Typing information of developed functional molecular markers

[0043]

[0044] Primer sequences for developed functional molecular markers

[0045] The sequence of the forward primer WTK4-Indel-F is:

[0046] 5'-GTGGGTTTTTGGAGCAGTGG-3'

[0047] The sequence of the reverse primer WTK4-Indel-R is:

[0048] 5'-AATAACGCACAACCATAGGCA-3'.

[0049] 2. Verification of WTK4-Indel, a functional molecular marker of WTK4

[0050] The CTAB method was used to extract genomic DNA of the samples to be tested, including Aegilops tauschii materials: CIae8, PI574467, AL8 / 78, AY61, and common wheat materials: Fielder, Chinese Spring (CS), and Chinese Spring tetrasomic materials (N7AT7B, N7BT7A, N7DT7B, and N4AT4B).

[0051] PCR amplification system: Each 10 μL includes: 50 ng / μL genomic DNA 1.0 μL, 5 μL PCR Master Mix,

[0052] PCR amplification conditions: pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 15 seconds, annealing at 55°C for 15 seconds, extension at 72°C for 15 seconds, for 32 cycles; extension at 72°C for 5 minutes; storage at 4°C.

[0053] The PCR products were separated by 1% agarose gel electrophoresis and the bands were read by gel imager.

[0054] The results of marker detection showed that the amplified band size in CIae8 was 375 bp, the amplified band size in Chinese Spring was 480 bp, and there was no amplified band in the sequenced varieties PI574467, AL8 / 78, AY61, and Fielder that did not contain the WTK4 gene ( Figure 1 ). The results of amplification of the marker in the oligome-tetrasomal group of the seventh part of the Chinese Spring showed that there were amplified bands in N7AT7B, N7BT7A and N7DT7B, and the band size was consistent with that of the Chinese Spring, indicating that there were no homologous genes of WTK4 on 7A, 7B and 7D of the Chinese Spring. The marker did not amplify the band in N4AT4B of the Chinese Spring, further proving that the homologous gene of WTK4 in the Chinese Spring is located on chromosome 4A, which is consistent with the genome alignment information of the Chinese Spring ( Figure 2 ).

[0055] The molecular marker designed in the embodiment of the present invention can specifically track the WTK4 gene, that is, the primer set is used to PCR amplify and detect the plant DNA. If the amplification product is a 375bp specific band, the WTK4 carried in the material is of the Aegilops tauschii type; if the amplification product is a 480bp specific band, the WTK4 carried in the material is of the Chinese Spring type; if there is no amplification product, the wheat does not carry the WTK4 gene.

[0056] Example 2 Application of the functional molecular marker of wheat powdery mildew resistance gene WTK4 in powdery mildew resistance of natural Aegilops tauschii / wheat population.

[0057] In order to evaluate the effect of WTK4 gene on powdery mildew resistance, the presence of WTK4 gene in a natural population consisting of 385 wheat varieties and 94 Aegilops tauschii lines was detected, the powdery mildew resistance of the materials containing WTK4 gene was identified at the seedling stage, and the WTK4 gene sequence was cloned and compared for the materials containing WTK4 gene.

[0058] 1. Identification of WTK4 functional molecular markers in natural populations

[0059] DNA of 385 wheat varieties and 94 Aegilops tauschii lines were extracted by CTAB method and identified by using the molecular marker WTK4-Indel developed by the present invention.

[0060] The PCR reaction system and product detection were the same as those in Example 1.

[0061] If the amplified product is a 375bp specific band, the WTK4 carried in the material is a Tauschia genotype. If the amplified product is a 480bp specific band, the WTK4 carried in the material is a Chinese Spring genotype. If there is no amplified product, the wheat does not carry the WTK4 gene.

[0062] The results showed that among the 94 Aegilops tauschii lines, 12 of them amplified a specific band of 375 bp, which was consistent with the size of CIae8 ( Figure 2 A). Among the 385 natural wheat populations, only 7 wheat varieties amplified a specific band of 480 bp in size, and the amplified band was consistent with the size of Chinese Spring ( Figure 2 B).

[0063] 2. Cloning and sequence analysis of WTK4 gene

[0064] The WTK4 gene was cloned in 7 common wheat varieties with amplified bands of the same size as Chinese Spring and 12 Aegilops tauschii lines with amplified bands of the same size as CIae8, and sequence comparison and analysis were performed using DNAMAN software.

[0065] Sequence analysis revealed that the 12 Aegilops tauschii lines containing WTK4 had a 100% similarity to the WTK4 gene in CIae8 and all belonged to the beaded variety of Aegilops tauschii ( Figure 3 A). The sequences of WTK4 in 7 common wheat samples were 100% similar to the homologous gene TraesCS4A02G481300 in Chinese Spring, and most of the common wheat samples containing WTK4 gene belonged to local varieties of wheat ( Figure 3 A).

[0066] The results showed that the developed gene functional molecular marker can not only specifically detect germplasm resources containing the WTK4 gene in natural populations, but also distinguish the materials containing the WTK4 gene into Aegilops tauschii genotype and Chinese Spring genotype according to the size of the amplified band.

[0067] 3. Identification of powdery mildew resistance in natural populations

[0068] Powdery mildew resistance was identified in a natural population consisting of 385 wheat varieties and 94 Aegilops tauschii lines. The test materials were planted in a sterile greenhouse with a humidity of 65%-75%, a light of 14h / 22℃, and a dark of 10h / 20℃. When they grew to two leaves and one heart, they were inoculated with physiological race E26. The powdery mildew phenotype was investigated when the powdery mildew spores were fully developed on the susceptible control (approximately 7 days after inoculation). The disease level was divided according to the 0-4 level standard at the seedling stage, that is:

[0069] IT=0: immune, no allergic spots and spores;

[0070] 0: Highly resistant, with allergic spots on the leaves;

[0071] 1: Highly resistant, with a few obvious powdery mildew spores on the leaves;

[0072] 2: Moderately resistant, with powdery mildew conidia piles on the leaves;

[0073] 3: moderate, with large numbers of conidia on the leaves;

[0074] 4: Highly susceptible, with leaves covered with a large number of conidia.

[0075] The results showed that 8 common wheat varieties containing the WTK4 gene, including China Spring, were highly susceptible to powdery mildew, and 13 Aegilops tauschii lines, including CIae8, were highly resistant to wheat powdery mildew ( Figure 3 B) The results show that the developed WTK4 functional molecular marker can not only specifically screen out materials containing the WTK4 gene, but also divide them into disease-resistant Aegilops tauschii genotypes and disease-susceptible Chinese Spring genotypes. The molecular marker can be applied to molecular marker-assisted selection breeding to accelerate the transfer of the WTK4 gene into wheat varieties.

[0076] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. A functional molecular marker WTK4-Indel for detecting the wheat powdery mildew resistance gene WTK4, characterized in that: The marker is based on the difference in WTK4 gene sequences between Aegilops tauschii and Chinese spring, and detects the 105bp Indel site in the third intron region to develop a WTK4 gene-specific Indel molecular marker. The primer nucleotide sequence of the Indel molecular marker includes an upstream primer WTK4-Indel-F and a downstream primer WTK4-Indel-R.

2. The functional molecular marker WTK4-Indel for detecting the wheat powdery mildew resistance gene WTK4 according to claim 1, wherein the marker comprises an upstream primer WTK4-Indel-F and a downstream primer WTK4-Indel-R, The nucleotide sequences of the upstream and downstream primers are as follows: WTK4-Indel-F:5'-GTGGGTTTTTGGAGCAGTGG-3' WTK4-Indel-R: 5'-AATAACGCACAACCATAGGCA-3'.

3. Use of the functional molecular marker WTK4-Indel of the wheat powdery mildew resistance gene WTK4 as claimed in claim 1 or 2 in identifying the WTK4 gene in Aegilops tauschii and wheat varieties / lines.

4. Use of the functional molecular marker WTK4-Indel of the wheat powdery mildew resistance gene WTK4 as claimed in claim 1 or 2 in molecular marker-assisted selection breeding.

5. The use according to any one of claims 3 or 4, characterized in that: The wheat powdery mildew resistance gene WTK4 is amplified using the functional molecular marker WTK4-Indel, which is specific to the wheat powdery mildew resistance gene WTK4 as described in claim 1 or 2. The amplified product is subjected to electrophoresis and gel detection. When a specific band of 375bp or 480bp marked by the WTK4-Indel appears, it indicates that the wheat powdery mildew resistance gene WTK4 is present in the plant. If there is no amplified band, it indicates that the plant does not contain the WTK4 gene.

6. The use according to any one of claims 3 or 4, characterized in that The plant genomic DNA of Aegilops tauschii or common wheat varieties / lines is amplified using the functional molecular marker WTK4-Indel specific to the wheat powdery mildew resistance gene WTK4 as described in claim 2. If the amplified product is a 375bp specific band, the WTK4 carried is a disease-resistant genotype of Aegilops tauschii. If the amplified product is a 480bp specific band, the WTK4 carried is a Chinese spring disease-susceptible genotype. If there is no amplified product, the WTK4 gene is not carried.

7. A method for detecting whether a plant material contains a WTK4 gene, characterized in that: The molecular marker WTK4-Indel as described in claim 1 or 2 is used to amplify genomic DNA.

8. The method according to claim 7, characterized in that The functional molecular marker WTK4-Indel specific to the wheat powdery mildew resistance gene WTK4 as described in claim 2 is used to amplify the plant genomic DNA of Aegilops tauschii or common wheat varieties / lines. If the amplification product is a 375bp specific band, the WTK4 carried in the wheat is an Aegilops tauschii disease-resistant genotype. If the amplification product is a 480bp specific band, the WTK4 carried in the wheat is a Chinese spring disease-susceptible genotype. If there is no amplification product, the wheat does not carry the WTK4 gene.

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