Wheat Stripe Rust Resistance Loci and Developed KASP Markers

Abstract

The wheat stripe rust resistance site of the present application and the KASP marker developed therefor belong to the technical field of molecular biology and crop breeding. The technical problem to be solved in the present application is: how to screen wheat with stripe rust resistance. In order to solve this technical problem, the present application provides the application of a substance for detecting the polymorphism or genotype of the AX-111915032 site in the identification or auxiliary identification of wheat stripe rust resistance. The substance for detecting the polymorphism or genotype of the AX-111915032 site includes a primer composition composed of single-stranded DNA shown in SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3. The molecular markers (SNP sites) provided in the present application and the corresponding primers thereto can be used for molecular marker-assisted selection breeding, which is helpful for the screening of wheat stripe rust resistance germplasm resources and provides material reserves and technical support for the genetic improvement of wheat stripe rust resistance traits.

Description

Technical Field

[0001] The present application belongs to the technical field of molecular biology and crop breeding, and specifically relates to a wheat stripe rust resistance locus and a KASP marker developed therefor. Background Art

[0002] Wheat stripe rust is an airborne fungal disease caused by Puccinia striiformis West.f.sp.tritici (Pst), which mainly infects wheat leaves, but can also infect almost all above-ground vegetative organs such as wheat sheaths, spikes, stems, husks and awns. Controlling wheat stripe rust has always been a difficult task. Chemical control can reduce the losses caused by stripe rust, but it is costly and long-term use can cause environmental pollution. Therefore, breeding varieties resistant to wheat stripe rust is considered to be the most economical, safe and effective strategy.

[0003] In recent years, 89 stripe rust resistance genes have been reported to be distributed on 20 chromosomes other than chromosome 1A. In addition, more than 300 quantitative trait loci (QTLs) for stripe rust resistance have been identified, with the most resistance loci in the B genome. Due to the rapid mutation of wheat stripe rust fungi, resistance genes are prone to lose resistance, and some genes may be linked to undesirable traits. Therefore, the number of effective genes that can be used to improve wheat stripe rust resistance is relatively limited. Therefore, finding new stripe rust resistance genes (QTLs) and developing molecular markers associated with them is of great significance for breeding. At present, KASP (Kompetitive Allele Specific PCR) marker technology has been widely used in SNP site detection in various crops such as wheat, rice, and corn, eliminating the electrophoresis step and achieving high-throughput genotyping. Using the genotype data generated by the wheat SNP chip, combined with QTL positioning and genome-wide association studies (GWAS), the relevant SNPs can be converted into KASP markers and directly used in molecular marker-assisted breeding to improve breeding efficiency.

[0004] Lantian 25 was developed by the Wheat Research Institute of Gansu Academy of Agricultural Sciences. It is immune to mixed bacteria of stripe rust in the seedling stage and to mixed bacteria of Tiaozhong 29, Tiaozhong 31, Shui 4, Shui 14, Shui 7, Tiaozhong 32 and in the adult stage. A recombinant inbred line (RIL) population including 237 families was constructed using Lantian 25 and Huixianhong. The QTL analysis of stripe rust resistance of the RIL population was carried out using the genetic map constructed using the 50K chip. A QTL that is stable under multiple environmental conditions was detected, located on chromosome 2D and named QYR.gaas-2D. It is closely linked to AX-111915032 (12.7Mb) and can explain 4.9-8.5% of the phenotypic variation. The KASP marker Kasp-2D-YR developed based on it can be used for molecular marker detection, providing a new tool for wheat stripe rust resistance breeding. Summary of the invention

[0005] The technical problem to be solved by the present application is: how to identify or assist in identifying wheat stripe disease resistance.

[0006] In order to solve the above technical problems, the present application provides an application, which can be an application of a substance for detecting the polymorphism or genotype of the AX-111915032 site in the following A1)-A6):

[0007] A1) Identify or assist in identifying wheat stripe rust resistance;

[0008] A2) preparing products for identifying or assisting in identifying wheat stripe rust resistance;

[0009] A3) Screening or assisting in the screening of wheat varieties resistant to stripe rust;

[0010] A4) preparing a product for screening or assisting in screening wheat varieties resistant to stripe rust;

[0011] A5) Wheat breeding and / or assisted breeding;

[0012] A6) preparing wheat breeding and / or breeding-assisted products;

[0013] The AX-111915032 site is a SNP site in the wheat genome, which is the 37th nucleotide of SEQ ID No. 4, and its nucleotide type is G or A.

[0014] In the present application, the wheat breeding assessment index may include wheat stripe rust resistance.

[0015] In the present application, the purpose of the breeding includes cultivating wheat with high resistance to wheat stripe rust (higher resistance to wheat stripe rust than that of the parent).

[0016] The genotype of the AX-111915032 site may be a GG genotype, an AA genotype or an AG genotype.

[0017] The GG genotype indicates that the nucleotide type at the AX-111915032 site in the wheat genome is the homozygous type of G; the AA genotype indicates that the nucleotide type at the AX-111915032 site in the wheat genome is the homozygous type of A; the AG genotype indicates that the nucleotide type at the AX-111915032 site in the wheat genome is the heterozygous type of G and A.

[0018] Furthermore, in the application, the substance for detecting the polymorphism or genotype of the AX-111915032 site is: a primer combination for amplifying a wheat genomic DNA fragment including the AX-111915032 site.

[0019] Furthermore, in the application, the primer composition may be composed of a single-stranded DNA having a nucleotide sequence of positions 22-44 of SEQ ID No.1, a single-stranded DNA having a nucleotide sequence of positions 22-45 of SEQ ID No.2, and a single-stranded DNA having a nucleotide sequence of SEQ ID No.3.

[0020] Furthermore, in the application, the primer composition may be composed of the single-stranded DNA shown in SEQ ID No.1, the single-stranded DNA shown in SEQ ID No.2, and the single-stranded DNA shown in SEQ ID No.3.

[0021] The present application also provides the above-mentioned primer composition.

[0022] In the present application, the PCR primers in the primer composition may be labeled or not labeled with a marker. The marker refers to any atom or molecule that can be used to provide a detectable effect and can be connected to a nucleic acid. Markers include, but are not limited to, dyes; radioactive labels, such as 32p; binding moieties, such as biotin; haptens, such as digoxin (DIG); luminescent, phosphorescent or fluorescent moieties; and fluorescent dyes alone or in combination with moieties that can inhibit or move the emission spectrum by fluorescence resonance energy transfer (FRET). The marker can provide a signal that can be detected by fluorescence, radioactivity, colorimetry, weight determination, X-ray diffraction or absorption, magnetism, enzyme activity, etc. The marker can be a charged moiety (positive or negative charge) or, alternatively, can be charge neutral. The marker can include a nucleic acid or protein sequence or a combination thereof, as long as the sequence containing the marker is detectable. In some embodiments, the nucleic acid is directly detected (e.g., directly read the sequence) without a marker.

[0023] Furthermore, the primer combination can be used to identify or assist in identifying wheat stripe rust resistance.

[0024] The present application also provides a reagent or a kit containing the above composition.

[0025] The present application also provides a DNA molecule, wherein the DNA molecule is a DNA molecule whose nucleotide sequence is shown in SEQ ID No.4.

[0026] The present application also provides a method for identifying or assisting in identifying wheat stripe rust resistance, the method comprising using a substance for detecting the polymorphism or genotype of the AX-111915032 locus to detect the genotype of the AX-111915032 locus, and identifying or assisting in identifying wheat stripe rust resistance according to the genotype of the AX-111915032 locus of the wheat to be tested;

[0027] The AX-111915032 site is a SNP site in the wheat genome, which is the 37th nucleotide of SEQ ID No. 4, and its nucleotide type is G or A.

[0028] Furthermore, in the method described above, the method for detecting the genotype of the above-mentioned AX-111915032 site of the wheat to be tested includes using the genomic DNA of the wheat to be identified as a template, performing PCR amplification using the above-mentioned primer combination to obtain a PCR product; and determining the genotype of the AX-111915032 site based on the sequencing result or fluorescence signal of the PCR product.

[0029] Furthermore, the method specifically comprises the following steps:

[0030] S1) extracting genomic DNA of wheat to be tested;

[0031] S2) using the genomic DNA extracted in S1) as a template and the primer combination as an amplification primer to perform PCR amplification to obtain a PCR amplification product;

[0032] S3) judging or assisting in judging the stripe rust resistance of the tested wheat according to the genotype of the AX-109906455 locus of the PCR amplification product;

[0033] Furthermore, in the above method, in step S3), the genotype of the AX-109906455 site can be determined based on the sequencing result or fluorescence color development of the PCR product.

[0034] Furthermore, in the method described above, the primer composition is a primer composition consisting of a single-stranded DNA having a nucleotide sequence of positions 22-44 of SEQ ID No.1, a single-stranded DNA having a nucleotide sequence of positions 22-45 of SEQ ID No.2, and a single-stranded DNA having a nucleotide sequence of SEQ ID No.3.

[0035] Furthermore, in the method, further, the primer composition is a primer composition consisting of the single-stranded DNA shown in SEQ ID No.1, the single-stranded DNA shown in SEQ ID No.2 and the single-stranded DNA shown in SEQ ID No.3.

[0036] Furthermore, in the method, the wheat stripe rust resistance of the wheat to be tested whose genotype at the AX-111915032 locus is GG genotype is higher or candidate higher than that of the wheat to be tested whose genotype at the AX-111915032 locus is AA genotype and / or AG genotype;

[0037] Among them, the GG genotype indicates that the nucleotide type of the AX-111915032 site in the wheat genome is the homozygous type of G; the AA genotype indicates that the nucleotide type of the AX-111915032 site in the wheat genome is the homozygous type of A; the AG genotype indicates that the nucleotide type of the AX-111915032 site in the wheat genome is the heterozygous type of G and A.

[0038] In the present application, the amplification product of the GG genotype at the AX-111915032 locus of the tested wheat carries FAM fluorescence, showing blue fluorescence; the amplification product of the AA genotype carries HEX fluorescence, showing red fluorescence, and the amplification product of the AG genotype shows green fluorescence. That is, the stripe rust resistance of the tested wheat whose amplification product shows blue fluorescence is higher or higher than that of the tested wheat whose amplification product shows red fluorescence or green fluorescence.

[0039] In the present application, the wheat to be tested may be a pure line or an inbred line. The inbred line may be a recombinant inbred line.

[0040] The present application also provides a method for wheat breeding, which comprises selecting wheat with a genotype of GG at the above-mentioned AX-111915032 locus as a parent for breeding, wherein the GG genotype indicates that the nucleotide type at the AX-111915032 locus in the wheat genome is a homozygous type of G.

[0041] In the present application, the product may be a reagent or a kit.

[0042] Compared with the prior art, the beneficial technical effects achieved by this application are as follows:

[0043] 1. This application discovered for the first time the wheat stripe rust resistance gene QTL and its linked molecular marker. The QTL was named QYR.gaas-2D, located on chromosome 2D, and tightly linked to AX-111915032 (12.7Mb).

[0044] 2. The results of detecting the genotype of the AX-111915032 locus of 111 natural wheat varieties showed that the primer combination provided in the present application had a good typing effect, and the primers could effectively identify whether the genotype of the test plant based on the AX-111915032 locus was GG genotype, AA genotype or AG genotype.

[0045] 3. The association analysis between the genotype and the stripe rust resistance phenotype of natural wheat varieties showed that there was a significant correlation between the genotype and the phenotype. This shows that the molecular marker (SNP site) AX-111915032 site and its corresponding primers provided in this application will help screen wheat stripe rust resistance germplasm resources, can be used for molecular marker-assisted selection breeding, and provide material reserves and technical support for the genetic improvement of wheat stripe rust resistance traits. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 The genotyping results of Kasp-2D-YR for 111 wheat varieties, among which the blue one is the Lantian 25 genotype GG, the red one is the Huixianhong genotype AA, and the green one is the heterozygous genotype GA. DETAILED DESCRIPTION

[0047] The present application is further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present application, not for limiting the scope of the present application. The examples provided below can be used as a guide for further improvements by ordinary technicians in the technical field, and do not constitute a limitation of the present application in any way.

[0048] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0049] The 111 wheat varieties described in the following examples are preserved by the applicant and disclosed in the document “Genome-WideAssociation Mapping of Adult-Plant Resistance to Stripe Rust in Common Wheat (Triticum aestivum). Plant Disease. 2020Aug;104(8):2174-2180.doi:10.1094 / PDIS-10-19-2116-RE.Epub 2020May 26.” The public can obtain the above-mentioned biological materials from the applicant. The obtained biological materials can only be used for verification of the content of this application and cannot be used for other purposes.

[0050] The dominant mixed stripe rust species described in the following embodiments are specifically pathogens mixed in equal proportions of CYR30, CYR31, CYR32, CYR33 and CYR34. It is disclosed in the document "Hu Chaoyue, Wang Fengtao, Lang Xiaowei, et al. Analysis of resistance of wheat stripe rust resistance genes to the main epidemic stripe rust species in China [J]. Chinese Agricultural Science, 2022, 55(03): 491-502." that the public can obtain the above-mentioned biological materials from the applicant, and the obtained biological materials can only be used for verification of the content of this application and cannot be used for other purposes.

[0051] The following examples used GraphPad Prism statistical software to process the data, and the experimental results were expressed as mean ± standard deviation and tested using t-test. * (P < 0.05) indicated a significant difference.

[0052] The quantitative tests in the following examples were repeated three times unless otherwise specified, and the results were averaged.

[0053] Example 1. Discovery of QTLs for Stripe Rust Resistance Genes in Wheat Material Lantian 25 and Acquisition of KASP Markers

[0054] 1. Acquisition of phenotype

[0055] The Lantian 25 / Huixianhong RIL population was planted in Chengdu, Sichuan and Tianshui, Gansu in 2019-2020 and 2020-2021. A completely randomized block design was used, with three replicates, single-row plots, 1m row length, 0.3m row width, and 30 seeds were evenly sown in each row. Field management was carried out according to local practices. The RIL population was identified for resistance in the adult stage using the current popular mixed strains of stripe rust in my country. The maximum disease severity (MDS) of the disease index showed continuous changes in the field, which was a typical quantitative trait inheritance. The modified CTAB method (Murray et al., 1980) was used to extract genomic DNA from young leaves of 235 families, and the DNA concentration was determined using a NanoDrop2000c spectrophotometer. The DNA sample was adjusted to a standard concentration of 50ng / ul, and then the DNA quality was detected using 0.8% agarose gel, and SNP typing was performed on the qualified DNA. SNP analysis was performed using a 50K SNP chip developed by the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences and Affymetrix Axiom.

[0056] 2. Construction of linkage map

[0057] After removing markers with heterozygous and missing rates greater than 10% between parents, Icimapping V4.1 was used to remove redundant markers, and then clusters were formed based on the genetic distance and chromosome position information between markers to construct a high-density genetic map, which contained a total of 5941 markers.

[0058] 3. QTL Analysis

[0059] The IciMapping 4.1ICIM-ADD method was used for QTL analysis, and the LOD value was selected as 2.5, and QYR.gaas-2D was located on chromosome 2D. It was tightly linked to AX-111915032 (12.7Mb), and could explain 4.9-8.5% of the phenotypic variation under different environmental conditions. Its flanking marker AX-111915032 was converted to Kasp-2D-YR. The nucleotide sequence of the SNP marker AX-111915032 was SEQ ID No. 4, position 37, which was a G / A polymorphism.

[0060] For the AX-111915032 locus, based on the principle of competitive allele-specific PCR, the primers were designed as follows:

[0061] Upstream primer AX-111915032-A (SEQ ID No. 1): 5'- GAAGGTGACCAAGTTCAT GCT CTACAAACATGGTTCACATTGAC-3' (the underline indicates the FAM fluorescent labeling sequence);

[0062] Upstream primer AX-111915032-B (SEQ ID No. 2): 5'- GAAGGTCGGAGTCAACGG ATT ACTACAAACATGGTTCACATTGAT-3' (the underline indicates the HEX fluorescent marker sequence);

[0063] Downstream primer AX-111915032-C (SEQ ID No. 3): 5'-TGCATCATCTCCACCGGA AC-3'.

[0064] The single-stranded DNA molecules shown in SEQ ID No. 1 and SEQ ID No. 3 above amplify the fragment in which the nucleotide at position 37 in SEQ ID No. 4 is G, and the fluorescence signal of the fluorescent group bound to the FAM fluorescent labeling sequence can be read by an instrument;

[0065] The single-stranded DNA molecules shown in SEQ ID No. 2 and SEQ ID No. 3 amplify the fragment in which the 37th nucleotide in SEQ ID No. 4 is A, and the fluorescence signal of the fluorescent group combined with the HEX fluorescent labeling sequence can be read by an instrument.

[0066] Table 1 KASP primer sequences for detecting stripe rust resistance QTL QYR.gaas-2D

[0067]

[0068]

[0069] Example 2: Genotypic and phenotypic identification of 111 wheat varieties

[0070] 1. 111 wheat varieties from different sources were planted in Pixian County, Sichuan and Tianshui, Gansu in 2014-2015 and 2015-2016. A completely randomized block design was used with three replicates, single-row plots, 1m long, 0.3m wide, and 30 seeds were evenly sown in each row. Field management was carried out according to local practices. The RIL population was identified for resistance in the adult stage using a mixed strain of stripe rust currently prevalent in my country, and the maximum severity MDS was recorded.

[0071] When 111 wheat samples were inoculated with a mixed species of dominant stripe rust at the adult stage and the control variety Huixian Red was seriously ill (the severity reached 80%), the severity of the disease was recorded for the 111 test materials, and the severity of the disease was recorded again every other week, for a total of three records, and the largest one was taken as the maximum severity of the test material.

[0072] The MDS value is the percentage of leaf area covered by pathogen spores to the total leaf area. For statistical methods, please refer to the literature: Lan Caixia. QTL mapping of adult plant resistance to stripe rust and powdery mildew in common wheat [D]. Chinese Academy of Agricultural Sciences, 2010.

[0073] 2. The genomic DNA of young leaves of 111 families was extracted using the improved CTAB method, and all genomic DNA was detected using the Kasp-2D-YR marker.

[0074] Competitive allele-specific PCR: KASP amplification requires three primers, two forward competitive primers (the 5' end of the primer has base sequences that are complementary to the fluorescent groups FAM and HEX, and the other sequences differ only at the SNP and InDel at the 3' end) and one reverse common primer. All reagents used are provided by LGC unless otherwise specified, and the reagent dosage, usage, and the entire experimental steps are carried out in accordance with the product instructions.

[0075] KASP labeled PCR amplification system (4 μL) is as follows: 0.048 μL Primer Mix, 2.0 μL 2×KASP Master Mix, 1.952 μL Template DNA (50 ng / μL), the ratio of Primer Mix is: 12% HEX primer (i.e., primer shown in SEQ ID No. 2), 12% FAM primer (i.e., primer shown in SEQ ID No. 1), 30% Common primer (i.e., primer shown in SEQ ID No. 3, the primer was commissioned to Shanghai Bioengineering to synthesize). 2×KASP master mix contains two universal fluorescent probes and two universal quenching probes synthesized for upstream primer tag sequences (FAM and HEX).

[0076] Amplification was performed using a 384-well PCR instrument (BIO-RAD, S1000TM Thermal Cycler) with the following program: 94°C for 15 min; 94°C for 20 s, 63-55°C for 1 min (1°C drop for each cycle), 10 cycles; 94°C for 20 s, 55°C for 60 s, 32 cycles. The PCR amplification product was placed in an autofocus fluorescence multifunctional microplate reader (PHERAstarplus SNP, BMG LABTECH) to read the final fluorescence data, and then the data was imported into Klustercaller v3.4 software (LGC, Hoddesdon, UK) for genotyping.

[0077] The results are shown in Tables 2, 3 and Figure 1 Among the 111 wheat varieties, 61 varieties showed the Lantian 25 genotype GG (blue), with an average stripe rust MDS of 37.8; 49 varieties showed the Huixianhong genotype AA (red), with an average stripe rust MDS of 44.2; 1 variety showed the heterozygous genotype AG (green), with an MDS of 39.3; statistical tests showed that the genetic effect of QPM.caas-2B reached a significant difference (P<0.05).

[0078] The GG genotype indicates that the nucleotide type at the AX-111915032 site in the wheat genome is the homozygous type of G; the AA genotype indicates that the nucleotide type at the AX-111915032 site in the wheat genome is the homozygous type of A; the AG genotype indicates that the nucleotide types at the AX-111915032 site in the wheat genome are the heterozygous type of G and A.

[0079] The amplification product of the GG genotype binds to the FAM fluorescent group and displays blue fluorescence; the amplification product of the AA genotype binds to the HEX fluorescent group and displays red fluorescence, and the amplification product of the AG genotype displays green fluorescence.

[0080] The results showed that the typing effect was good, and the primers could effectively identify whether the genotype of the test plant based on the AX-111915032 locus was GG genotype, AA genotype or AG genotype.

[0081] The stripe rust resistance of wheat with GG genotype at AX-111915032 in the wheat genome is higher or has a higher candidate than that of wheat with AA genotype and / or AG genotype at AX-111915032 in the wheat genome. That is, the stripe rust resistance of the tested wheat whose amplification product shows blue fluorescence is higher or has a higher candidate than that of the tested wheat whose amplification product shows red fluorescence or green fluorescence.

[0082] Table 2 Genotype detection results and stripe rust resistance of 111 wheat varieties

[0083]

[0084]

[0085]

[0086]

[0087]

[0088] GG is the Lantian 25 genotype; AA is the Huixianhong genotype.

[0089] Table 3 Effect of QYR.caas-2D 111 natural varieties on stripe rust resistance

[0090]

[0091] The present application has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present application, and without the need to carry out unnecessary experimental conditions, the present application can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present application provides specific embodiments, it should be understood that further improvements can be made to the present application. In a word, according to the principles of the present application, the present application is intended to include any changes, uses or improvements to the present application, including departure from the disclosed scope in the present application and changes made with conventional techniques known in the art.

Claims

1. Application, characterized in that, The application is the application of a substance for detecting the polymorphism or genotype of the AX-111915032 site in the following A1)-A6): A1) Identify or assist in identifying wheat stripe rust resistance; A2) preparing products for identifying or assisting in identifying wheat stripe rust resistance; A3) Screening or assisting in the screening of wheat varieties resistant to stripe rust; A4) preparing a product for screening or assisting in screening wheat varieties resistant to stripe rust; A5) Wheat breeding and / or assisted breeding; A6) preparing wheat breeding and / or breeding-assisted products; The AX-111915032 site is a SNP site in the wheat genome, which is the 37th nucleotide of SEQ ID No. 4, and its nucleotide type is G or A; The substance for detecting the polymorphism or genotype of the AX-111915032 site is a primer combination for amplifying a wheat genomic DNA fragment including the AX-111915032 site; The primer composition consists of a single-stranded DNA whose nucleotide sequence is the 22nd to 44th positions of SEQ ID No.1, a single-stranded DNA whose nucleotide sequence is the 22nd to 45th positions of SEQ ID No.2, and a single-stranded DNA whose nucleotide sequence is SEQ ID No.

3.

2. The use according to claim 1, characterized in that: The primer composition consists of the single-stranded DNA shown in SEQ ID No.1, the single-stranded DNA shown in SEQ ID No.2 and the single-stranded DNA shown in SEQ ID No.

3.

3. The primer composition according to claim 1 or 2.

4. A reagent comprising the composition according to claim 3.

5. A DNA molecule, wherein the nucleotide sequence of the DNA molecule is a DNA molecule shown in SEQ ID No.

4.

6. A method for identifying or assisting in identifying wheat stripe rust resistance, characterized in that: The method comprises using a substance for detecting the polymorphism or genotype of the AX-111915032 locus to detect the genotype of the AX-111915032 locus, and identifying or assisting in identifying the resistance to wheat stripe rust according to the genotype of the AX-111915032 locus of the wheat to be tested; The AX-111915032 site is a SNP site in the wheat genome, which is the 37th nucleotide of SEQ ID No. 4, and its nucleotide type is G or A; The substance for detecting the polymorphism or genotype of the AX-111915032 site is a primer combination for amplifying a wheat genomic DNA fragment including the AX-111915032 site; The primer composition consists of a single-stranded DNA whose nucleotide sequence is the 22nd to 44th positions of SEQ ID No.1, a single-stranded DNA whose nucleotide sequence is the 22nd to 45th positions of SEQ ID No.2, and a single-stranded DNA whose nucleotide sequence is SEQ ID No.

3.

7. The method according to claim 6, characterized in that The method for detecting the genotype of the AX-111915032 locus of the wheat to be tested comprises using the genomic DNA of the wheat to be identified as a template, performing PCR amplification using the primer combination described in claim 2, and obtaining a PCR product; The genotype of the AX-111915032 site is determined according to the sequencing result or the fluorescence signal of the PCR product.

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