Wheat taasadh-a1 gene tolerance methyldisulfonate snp site, kasp marker and application

Abstract

The application discloses a wheat TaASADH-A1 gene methyldisulfonate SNP site, a KASP marker and application, relates to the technical field of molecular marker assisted breeding, and use of the SNP site, the SNP site taking common wheat Chinese Spring RefSeq v1.1 as a reference genome, the SNP being a SNP site in the promoter region of a TraesCS5A02G398300 gene on the 5A chromosome of the wheat, corresponding to the 101th base from the 5' end of the sequence shown in SEQ ID NO:1, when the site is GG homozygous, the corresponding genotype is methyl; when the site is CC homozygous, the corresponding genotype is ethyl; the use is as follows: screening or assisting in screening methyl disulfonate resistant wheat, and the tolerance of the wheat to the methyl disulfonate is as follows: the wheat with genotype methyl homozygous is higher than or candidate is higher than the wheat with genotype ethyl homozygous.

Description

Technical Field

[0001] This invention relates to the field of molecular marker-assisted breeding technology, particularly to the SNP site of the wheat TaASADH-A1 gene resistant to mesosulfuron-methyl, the KASP marker, and its application. Background Technology

[0002] Weed management is a crucial aspect of ensuring sustainable wheat production. Weeds compete with wheat for nutrients, water, and light, inhibiting normal wheat growth and increasing the risk of lodging, causing yield losses of 9.8% to 23% globally. Grass weeds are the main pest group in wheat fields, with common species including jointed goatgrass, wild oat, wild oat, bluegrass, and Kentucky bluegrass. Mesosulfuron-methyl is one of the few herbicides that is highly effective in controlling grass weeds. Its mechanism of action involves inhibiting the activity of acetolactate synthase in susceptible plants, leading to the inhibition of branched-chain amino acid synthesis, thereby inhibiting cell division and ultimately causing the death of susceptible plants. This herbicide has both systemic and soil-adsorbing properties, thus possessing both foliar contact and soil residual activity. However, mesosulfuron-methyl can also cause phytotoxicity in wheat, leading to yield reduction, especially under improper application or specific environmental conditions. Therefore, developing molecular markers to assist in breeding wheat varieties tolerant to mesosulfuron-methyl is of great significance for cultivating herbicide-tolerant wheat varieties.

[0003] Competitive allele-specific PCR (KASP) technology, as a third-generation molecular marker detection platform, has become a core tool in crop molecular breeding due to its advantages of high throughput, low cost, and ease of operation. This technology enables precise genotyping of single nucleotide polymorphisms (SNPs) by designing two allele-specific primers (each linked to a FAM / HEX fluorescent group) and one universal primer. However, current research on KASP markers related to herbicide tolerance in wheat is still in its early stages. Therefore, developing efficient KASP markers with independent intellectual property rights that are closely linked to the mesosulfuron-methyl trait in wheat is of great significance for advancing the progress of wheat tolerance molecular breeding in my country.

[0004] Aspartate semialdehyde dehydrogenase (ASADH) plays a crucial role in the metabolism of microorganisms and certain plants, serving as a key enzyme in the biosynthesis of essential amino acids. Due to ASADH's critical role in essential amino acid biosynthesis, inhibiting its activity has become a promising target for developing novel antimicrobial agents and herbicides. Inhibiting ASADH activity in weeds can interfere with their essential amino acid synthesis, leading to inhibited weed growth or even death. Compared to traditional herbicides, ASADH-targeted herbicides exhibit higher selectivity and environmental friendliness. Since crops and weeds may differ in the structure or expression levels of ASADH, specific inhibition of weeds can be achieved through the rational design of inhibitors, reducing the risk of phytotoxicity to crops. Furthermore, these herbicides are readily degraded in soil, resulting in less environmental pollution and aligning with the requirements of sustainable development in modern agriculture. Therefore, in-depth exploration of functional sites in the wheat ASADH gene associated with metsulfuron-methyl tolerance and the development of efficient and specific molecular markers are of great theoretical and practical significance for enriching the molecular tool library for breeding wheat tolerant to metsulfuron-methyl and accelerating the process of breeding stress-resistant varieties. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide the SNP site for mesosulfuron-methyl resistance in the wheat TaASADH-A1 gene, the KASP marker, and its application.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows.

[0007] The purpose of the SNP site is as follows: Using the Chinese Spring RefSeqv1.1 genome of common wheat as a reference genome, the SNP is located in the promoter region of the TraesCS5A02G398300 gene on wheat chromosome 5A, corresponding to the 101st base from the 5' end of the sequence shown in SEQ ID NO: 1. When this site is homozygous for GG, the corresponding genotype is A; when this site is homozygous for CC, the corresponding genotype is B. The purpose is to screen or assist in the screening of wheat resistant to mesosulfuron-methyl. The tolerance of wheat to mesosulfuron-methyl is: wheat homozygous for genotype A is higher than or candidate wheat homozygous for genotype B.

[0008] A method for screening or assisting in screening wheat resistant to mesosulfuron-methyl includes the following steps: detecting whether the genotype of the wheat to be tested is GG or CC, wherein wheat with genotype GG has a higher tolerance to mesosulfuron-methyl than wheat with genotype CC.

[0009] The wheat with genotype GG is the wheat with a genotype of GG homozygote based on the G101C SNP site;

[0010] The wheat with genotype CC is the wheat with a genotype of CC homozygous based on the G101C SNP site;

[0011] The G101C SNP site is the SNP site in the promoter region of the TraesCS5A02G398300 gene on the wheat chromosome 5A, using the Chinese Spring RefSeq v1.1 genome as a reference genome. It corresponds to the 101st nucleotide from the 5' end of SEQ ID NO: 1.

[0012] More preferably, the step of detecting whether the genotype of the wheat to be tested is GG or CC is as follows:

[0013] (a1) Using the genomic DNA of the wheat to be tested as a template, PCR amplification was performed using a primer combination to obtain PCR amplification products; the primer combination consisted of upstream primer A shown in SEQ ID NO: 3, upstream primer B shown in SEQ ID NO: 4, and downstream universal primer C shown in SEQ ID NO: 5;

[0014] (a2) After completing step (a1), use a fluorescence detection instrument to read the fluorescence signal of the PCR amplification product. Based on the fluorescence signal, obtain the genotype of the wheat to be tested. If the fluorescence of the amplification product is consistent with the fluorescence of the FAM fluorescent group labeled with primer A, then the wheat sample to be tested is of the GG genotype; if the fluorescence of the amplification product is consistent with the fluorescence of the HEX fluorescent group labeled with primer B, then the wheat sample to be tested is of the CC genotype.

[0015] More preferably, the step of detecting whether the genotype of the wheat to be tested is GG or CC is as follows:

[0016] (b1) Using the genomic DNA of the wheat to be tested as a template, PCR amplification was performed using a primer combination to obtain PCR amplification products; the primer combination consisted of upstream primer A shown in SEQ ID NO: 3, upstream primer B shown in SEQ ID NO: 4, and downstream universal primer C shown in SEQ ID NO: 5;

[0017] (b2) Take the PCR amplification product obtained in step (b1) and perform sequencing;

[0018] (b3) Based on the sequencing results obtained in step (b2), obtain the genotype of the wheat to be tested.

[0019] A kit for identifying or assisting in the identification of wheat tolerance to mesosulfuron-methyl, comprising a primer combination for detecting whether the genotype of the wheat to be tested is GG or CC; the primer combination consists of upstream primer A shown in SEQ ID NO: 3, upstream primer B shown in SEQ ID NO: 4, and downstream universal primer C shown in SEQ ID NO: 5; the genotype GG is a homozygous GG genotype based on the G101C SNP site; the genotype CC is a homozygous CC genotype based on the G101C SNP site; the G101C SNP site is the SNP site in the promoter region of the TraesCS5A02G398300 gene on chromosome 5A of wheat, using the Chinese Spring RefSeqv1.1 as a reference genome, corresponding to the 101st nucleotide from the 5' end of SEQ ID NO: 1.

[0020] The molecular marker shown in SEQ ID NO: 1.

[0021] The application of the above-mentioned kits or molecular markers in the identification or auxiliary identification of wheat tolerance to mesosulfuron-methyl.

[0022] Application of the above-mentioned kits or molecular markers in screening or assisting screening of wheat resistant to mesosulfuron-methyl.

[0023] Application of the above-mentioned kits or molecular markers in wheat breeding for tolerance to mesosulfuron-methyl.

[0024] The application of primer combinations in the directional or assisted directional breeding of wheat lines resistant to mesosulfuron-methyl, wherein the primer combinations consist of upstream primer A shown in SEQ ID NO: 3, upstream primer B shown in SEQ ID NO: 4, and downstream universal primer C shown in SEQ ID NO: 5.

[0025] The beneficial effects of the above technical solution are as follows: Based on the SNP molecular site in the promoter region of the common wheat metsulfuron-methyl-related gene TaASADH-A1, the molecular marker site is located at the 101st base from the 5' end of the sequence shown in SEQ ID NO: 1, with a polymorphism of G / C. Based on this SNP site, a KASP marker, Kasp_TaASADH-A1, for detecting wheat metsulfuron-methyl resistance was developed. The type of wheat resistant to metsulfuron-methyl (TaASADH-A1) is characterized by FAM fluorescence distributed near the x-axis. G (i.e., GG homozygous), those carrying HEX fluorescence and distributed near the y-axis are the mesosulfuron-sensitive type (TaASADH-A1). C(i.e., CC homozygous). Identification of the seedling-stage metsulfuron-methyl phenotype in 532 common wheat varieties or germplasm resources showed that this marker can affect TaASADH-A1. G and TaASADH-A1 C Accurate typing of two allelic types is achieved. The molecular markers related to wheat tolerance to mesosulfuron-methyl provided by this invention enable efficient and accurate typing of mesosulfuron-methyl-related SNP sites, providing a standardized, high-throughput molecular detection tool for wheat breeding to tolerate the herbicide mesosulfuron-methyl. This tool can be used for screening and identifying the phenotypic traits of wheat varieties to tolerate mesosulfuron-methyl, providing important technical support for herbicide-tolerant wheat variety auxiliary breeding, and is expected to significantly improve the efficiency and accuracy of herbicide-tolerant variety breeding. Attached Figure Description

[0026] Figure 1 This is a schematic diagram showing the KASP primer positions at the 101st base from the 5' end of the sequence shown in SEQ ID NO: 1 for the two allelic types of the TaASADH-A1 gene related to metsulfuron-methyl resistance in common wheat. The boxes represent the 101st base from the 5' end of the sequence shown in SEQ ID NO: 1. The upstream primer position for the KASP marker is indicated by double underscores, and the downstream primer position by wavy lines. HAP1 indicates the genotype TaASADH-A1. G HAP2 indicates that the genotype is TaASADH-A1 C The sequence shown in the figure is the sequence of SEQ ID NO: 1.

[0027] Figure 2 This is a graph showing the detection results of the Kasp_TaASADH-A1 marker for wheat varieties / lines of this invention.

[0028] Figure 3 For different wheat varieties or germplasm under different environments, the TaASADH-A1 genotype is used. G and genotype TaASADH-A1 C A schematic diagram illustrating the association analysis results between wheat germplasm and herbicide resistance levels ("*" indicates P < 0.05, i.e., the difference reached a significant level; "**" and "****" indicate P < 0.01 and P < 0.0001, respectively, i.e., the difference reached a highly significant level; "GG" and "CC" indicate genotypes of TaASADH-A1). G and TaASADH-A1 C .

[0029] Figure 4This is a comparison of sequencing peaks of the promoter regions of the TaASADH-A1 gene from two allelic types. The sequences in the figure are the reverse complementary sequences of the promoter region of this gene. The arrows indicate the SNP site located 377 bp upstream of the start codon of this gene, and this site varies among different susceptible varieties.

[0030] Figure 5 This is a peak diagram for DNA concentration determination.

[0031] Figure 6 These are phenotypic diagrams of different wheat varieties after treatment with mesosulfuron-methyl. Detailed Implementation

[0032] The following embodiments illustrate the present invention in detail. All raw materials and equipment used in the present invention are commercially available products and can be directly obtained through market purchase. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods.

[0033] It should be understood that, when used in this application specification and the appended claims, the term "comprising" indicates the presence of the described features, integrals, steps, operations, elements and / or components, but does not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components and / or a collection thereof.

[0034] It should also be understood that the term “and / or” as used in this application specification and the appended claims means any combination of one or more of the associated listed items and all possible combinations, and includes such combinations.

[0035] References to "one embodiment" or "some embodiments" as described in this application specification mean that one or more embodiments of this application include the features described in connection with that embodiment.

[0036] Defined features, structure, or characteristics. Therefore, the phrases "in one embodiment," "in some embodiments," "in other embodiments," "in still other embodiments," etc., appearing in different places in this specification do not necessarily refer to the same embodiment, but rather mean "one or more, but not all, embodiments," unless otherwise specifically emphasized. The terms "comprising," "including," "having," and variations thereof mean "including, but not limited to," unless otherwise specifically emphasized.

[0037] Furthermore, in the description of this application and the appended claims, the terms "first," "second," "third," etc., are used only to distinguish descriptions and should not be construed as indicating or implying relative importance.

[0038] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] Example 1: Detection of different allelic types of the wheat metsulfuron-methyl-related gene TaASADH-A1 using the KASP marker Kasp_TaASADH-A1.

[0040] Based on the reference genome version of the Chinese Spring Genome Database (Chinese Spring RefSeq v1.1, database link: http: / / plants.ensembl.org / Triticum_aestivum / Info / Index), a pair of specific primers, named TraesCS5A02G398300-F / R, was designed targeting the promoter region of the TraesCS5A02G398300 gene (primer sequences are detailed in Table 2). Using genomic DNA from mesosulfuron-resistant varieties Chang 4640, Jinmai 85, and Jimai 32, and sensitive varieties Xinong 928, Hanyou 1, and Zhengnong 46 as templates, PCR amplification was performed using the aforementioned specific primers (herbicide damage levels are shown in Table 3). Partial sequencing results are shown in the peak diagram below. Figure 4 Sequencing and alignment analysis of the amplified sequences revealed 32 SNPs in the TraesCS5A02G398300 promoter region (sequencing range covering 2000 bp upstream of the start codon ATG) between susceptible and resistant varieties. The nucleotide sequences of these SNPs are shown in SEQ ID NO: 2. Based on this, we developed a KASP marker, Kasp_TaASADH-A1 (SEQ ID NO: 1), targeting the first SNP site upstream of the start codon (located 377 bp upstream of the start codon, a G / C mutation). Figure 1 As shown, the primer sequences are detailed in Table 1.

[0041] Table 1. Primer sequences for the KASP marker Kasp_TaASADH-A1 used to detect wheat herb resistance.

[0042]

[0043] Table 2 Primer sequences used for amplifying the promoter region of gene TraesCS5A02G398300

[0044]

[0045] The KASP marker was used to detect different allelic types of the wheat gene TaASADH-A1, which regulates tolerance to mesosulfuron-methyl. The main steps included PCR amplification and genotyping. Unless otherwise specified, all methods used in this embodiment were conventional. Most of the wheat varieties / lines used were well-known both domestically and internationally, with most information included in the following literature: “Jie Zhao, Lijing Sun, Huimin Gao, Mengyun Hu, Liming Mu, XiaohuCheng, Jianbing Wang, Yun Zhao, Qianying Li, Peinan Wang, Hui Li, YingjunZhang. Genome-wide association study of yield-related traits in common wheat (Triticum aestivum L.) under normal and drought treatment conditions. 2023,13:1098560.” All common wheat varieties / lines were preserved by the Wheat Research Center of the Institute of Grain and Oil Crops, Hebei Academy of Agricultural and Forestry Sciences.

[0046] (1) PCR amplification system and procedure. Genomic DNA was extracted from wheat leaves using the TPS method, and the precipitate was dissolved in 100 μL of ultrapure water. DNA quality was assessed by 1% agarose gel electrophoresis, requiring the extracted DNA to be free of obvious impurities, have clear bands, and show no degradation. DNA concentration was then determined (DNA concentration peaks are shown in the image). Figure 5 The solution was uniformly diluted to 28.3 ng / μL and used as a template for PCR amplification.

[0047] Preparation of KASP-labeled primer working solution: KASP primers were designed based on the SNP sequence of the wheat metsulfuron-methyl-related gene TaASADH-A1. This SNP site exhibits G / C polymorphism. The primer sequences are shown in Table 1. Take 12 μL each of the two upstream primers (100 μM) and 30 μL of the downstream primer (100 μM), add sterile ultrapure water to a final volume of 100 μL, mix well, and store at 4 ℃ for later use.

[0048] The PCR amplification system consisted of: 1.5 μl template DNA, 0.0417 μl primer working solution, 0.75 μl 2× KASP Master Mix (LGC, product number: KBS-1050-112), and sterile ultrapure water to a final volume of 3 μl.

[0049] The PCR reaction program was as follows: 94 °C pre-denaturation for 15 min; 94 °C denaturation for 20 s, annealing for 20 s (the first annealing temperature was 61 °C, and the temperature was reduced by 0.6 °C for each cycle) for a total of 10 cycles; 94 °C denaturation for 20 s, 55 °C annealing for 1 min for a total of 26 cycles; 72 °C extension for 3 min, and storage at 4 °C.

[0050] (2) After the PCR reaction, a fluorescence signal reader (Omega, BMG LABTECH, Germany) and a fluorescence detection system (Araya) were used to convert the fluorescence signal into analyzable values ​​for fluorescence data reading of the reaction products. The fluorescence scanning results were graphically displayed using the ggplot package in R software. G base types showed FAM fluorescence and were distributed near the x-axis; C base types showed HEX fluorescence and were distributed near the y-axis; samples with no detected signal were distributed near the origin (see...). Figure 2 ).

[0051] Application of the KASP marker Kasp_TaASADH-A1 to detect the herbicide damage level of common wheat varieties or germplasm

[0052] Of the 532 wheat varieties or germplasm accessions, 125 were of the TaASADH-A1G allelic type and 407 were of the TaASADH-A1C allelic type. These 532 varieties / lines were sown in Shijiazhuang City, Hebei Province, in 2022–2023, 2023–2024, and 2024–2025. Row lengths of 1.5 meters and 2 meters were set up for the control group and herbicide treatment group for each variety, respectively. A randomized complete block design was used. The control group was sown with 90 seeds per row, and the herbicide treatment group was sown with 120 seeds per row. The treatment groups were evenly divided into two parts: one half was treated with a low concentration of mesosulfuron-methyl, and the other half with a high concentration of mesosulfuron-methyl (the phenotypes of different wheat varieties after mesosulfuron-methyl treatment are shown in [reference needed]). Figure 6 During the experiment, standardized fertilization was carried out in the experimental field, and pests and diseases were controlled throughout the process. The optimal application time for mesosulfuron-methyl is the 3-6 leaf stage of wheat (before winter), when weeds are at the 2-5 leaf stage, and their absorption and translocation capabilities are strongest. This study used commercially available 30 g / L mesosulfuron-methyl dispersible oil suspension (Hebei Rongwei Biopharmaceutical Co., Ltd.). The dosage was set as follows: after dilution with deionized water, the pesticide was prepared at dosages of 15.75 g ai / ha (low concentration) and 31.50 g ai / ha (high concentration), and the treatment groups were treated accordingly. The severity of pesticide damage was classified into four levels as follows:

[0053] Mild damage (Level 1): The mortality rate of treated plants is less than 10%. Surviving samples showed only slight chlorosis at the leaf tips, and the plant height of treated plants was only slightly reduced (5%–10%) compared to the untreated control group.

[0054] Moderate damage (Level 2): ​​Mortality rate reaches approximately 40%. Affected plants exhibit extensive chlorosis, covering nearly 50% of the leaf area. Plant height is significantly inhibited in some wheat materials, with the average plant height of plants treated with susceptible genotypes being 50% lower than that of the control group.

[0055] Severe damage (Level 3): Mortality rate approaching 70%. Surviving plants exhibit severe chlorosis, affecting more than two-thirds of the leaf area. Plant height inhibition is extremely pronounced in some wheat materials, with treated plants reaching only 50% or less of the height of the control group.

[0056] Extremely harmful (Level 4): Nearly all treated plants die (≥95%), and the survival rate is negligible.

[0057] To improve data recording accuracy, we added "+" and "−" symbols to each level for further subdivision. For example, "2+" indicates a higher degree of herbicide damage than "2," while "2−" indicates a lower degree of herbicide damage than "2." In statistical analysis, the herbicide damage levels 1−, 1, 1+, 2−, 2, 2+, 3−, 3, 3+, 4−, and 4 were converted to numbers from 1 to 11, with higher values ​​indicating more severe herbicide damage to wheat, greater sensitivity to mesosulfuron-methyl, and weaker resistance. Table 3 shows the herbicide damage levels for wheat in different years.

[0058] Table 3. Detection results of the KASP marker Kasp_TaASADH-A1 for wheat herbicide resistance and data on herbicide damage levels.

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073] The results showed that the average herbicide damage severity of wheat varieties / lines carrying the TaASADH-A1 allele was lower than that of wheat varieties carrying the TaASADH-A1 allele in different years. C The wheat varieties / lines showed significant or highly significant differences between the two. Figure 3 (Table 4)

[0074] Table 4. Statistical analysis results of the relationship between the two allele genotypes of the TaASADH-A1 gene related to herbicide tolerance in common wheat and the severity of herbicide damage.

[0075]

[0076] Note: The statistical analysis used a two-tailed t-test.

[0077] This invention discloses a KASP marker for assisting in the breeding of wheat resistant to mesosulfuron-methyl. Developed based on the wheat aspartate semialdehyde dehydrogenase gene TaASADH-A1, it belongs to the field of marker-assisted breeding technology. The nucleotide sequence of this KASP marker is shown in SEQ ID NO: 1, and it contains a G / C single nucleotide polymorphism at base position 101. Using this marker, haplotypes of the TaASADH-A1 gene (gene number: TraesCS5A02G398300) can be distinguished and identified at any growth stage and in any tissue of wheat, thereby enabling rapid screening of wheat varieties resistant to the herbicide mesosulfuron-methyl. The KASP detection method provided by this invention is simple to operate, accurate, and unaffected by weather and environmental conditions. It can be efficiently applied to the screening and identification of mesosulfuron-methyl phenotypic traits in wheat, providing important technical support for the assisted breeding of mesosulfuron-methyl resistant wheat varieties.

[0078] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these examples without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0079] In the above embodiments, the descriptions of each embodiment have different focuses. For parts that are not described in detail or recorded in a certain embodiment, please refer to the relevant descriptions of other embodiments.

[0080] The above-described embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be included within the protection scope of the present invention.

Claims

1. The use of SNP sites in screening or assisting screening of wheat resistant to mesosulfuron-methyl, characterized in that, The SNP site is based on the Chinese Spring RefSeq v1.1 genome of common wheat as a reference genome. The SNP is the SNP site in the promoter region of the TraesCS5A02G398300 gene on wheat chromosome 5A, corresponding to the 101st nucleotide from the 5' end of the sequence shown in SEQ ID NO:

1. When this site is GG homozygous, the corresponding genotype is A; when this site is CC homozygous, the corresponding genotype is B. The purpose is to screen or assist in the screening of wheat resistant to mesosulfuron-methyl. The tolerance of wheat to mesosulfuron-methyl is higher in wheat of genotype A than in wheat of genotype B.

2. A method for screening or assisting in the screening of wheat resistant to mesosulfuron-methyl, characterized in that, The process includes the following steps: determining whether the wheat genotype is GG or CC. Wheat with genotype GG has a higher tolerance to mesosulfuron-methyl than wheat with genotype CC. The wheat with genotype GG is the wheat with a genotype of GG homozygote based on the G101C SNP site; The wheat with genotype CC is the wheat with a genotype of CC homozygous based on the G101C SNP site; The G101C SNP site is the SNP site in the promoter region of the TraesCS5A02G398300 gene on the wheat chromosome 5A, using the Chinese Spring RefSeq v1.1 genome as a reference genome. It corresponds to the 101st nucleotide from the 5' end of SEQ ID NO:

1.

3. The method according to claim 2, characterized in that, The steps for detecting whether the genotype of the wheat to be tested is GG or CC are as follows: (a1) Using the genomic DNA of the wheat to be tested as a template, PCR amplification was performed using a primer combination to obtain PCR amplification products; the primer combination consisted of upstream primer A shown in SEQ ID NO: 3, upstream primer B shown in SEQ ID NO: 4, and downstream universal primer C shown in SEQ ID NO: 5; (a2) After completing step (a1), use a fluorescence detection instrument to read the fluorescence signal of the PCR amplification product. Based on the fluorescence signal, obtain the genotype of the wheat to be tested. If the fluorescence of the amplification product is consistent with the fluorescence of the FAM fluorescent group labeled with primer A, then the wheat sample to be tested is of the GG genotype; if the fluorescence of the amplification product is consistent with the fluorescence of the HEX fluorescent group labeled with primer B, then the wheat sample to be tested is of the CC genotype.

4. The method according to claim 2, characterized in that, The steps for detecting whether the genotype of the wheat to be tested is GG or CC are as follows: (b1) Using the genomic DNA of the wheat to be tested as a template, PCR amplification was performed using a primer combination to obtain PCR amplification products; the primer combination consisted of upstream primer A shown in SEQ ID NO: 3, upstream primer B shown in SEQ ID NO: 4, and downstream universal primer C shown in SEQ ID NO: 5; (b2) Take the PCR amplification product obtained in step (b1) and perform sequencing; (b3) Based on the sequencing results obtained in step (b2), obtain the genotype of the wheat to be tested.

5. The application of the kit in identifying or assisting in the identification of wheat tolerance to mesosulfuron-methyl, characterized in that, The kit includes a primer combination for detecting whether the genotype of the wheat to be tested is GG or CC; the primer combination consists of upstream primer A shown in SEQ ID NO: 3, upstream primer B shown in SEQ ID NO: 4, and downstream universal primer C shown in SEQ ID NO: 5; the genotype GG is a homozygous genotype based on the G101C SNP site; the genotype CC is a homozygous genotype based on the G101C SNP site; the G101C SNP site is the SNP site in the promoter region of the TraesCS5A02G398300 gene on the wheat chromosome 5A, using the Chinese Spring RefSeq v1.1 genome as a reference genome, corresponding to the 101st nucleotide from the 5' end of SEQ ID NO:

1.

6. The application of the kit in screening or assisting screening for wheat resistant to mesosulfuron-methyl, characterized in that, The kit includes a primer combination for detecting whether the genotype of the wheat to be tested is GG or CC; the primer combination consists of upstream primer A shown in SEQ ID NO: 3, upstream primer B shown in SEQ ID NO: 4, and downstream universal primer C shown in SEQ ID NO: 5; the genotype GG is a homozygous genotype based on the G101C SNP site; the genotype CC is a homozygous genotype based on the G101C SNP site; the G101C SNP site is the SNP site in the promoter region of the TraesCS5A02G398300 gene on the wheat chromosome 5A, using the Chinese Spring RefSeq v1.1 genome as a reference genome, corresponding to the 101st nucleotide from the 5' end of SEQ ID NO:

1.

7. Application of the kit in wheat breeding for tolerance to mesosulfuron-methyl, characterized in that, The kit includes a primer combination for detecting whether the genotype of the wheat to be tested is GG or CC; the primer combination consists of upstream primer A shown in SEQ ID NO: 3, upstream primer B shown in SEQ ID NO: 4, and downstream universal primer C shown in SEQ ID NO: 5; the genotype GG is a homozygous genotype based on the G101C SNP site; the genotype CC is a homozygous genotype based on the G101C SNP site; the G101C SNP site is the SNP site in the promoter region of the TraesCS5A02G398300 gene on the wheat chromosome 5A, using the ChineseSpring RefSeq v1.1 genome as a reference genome, corresponding to the 101st nucleotide from the 5' end of SEQ ID NO:

1.

8. Application of primer combinations in the directional or assisted directional breeding of wheat lines resistant to mesosulfuron-methyl, wherein the primer combinations consist of upstream primer A shown in SEQ ID NO: 3, upstream primer B shown in SEQ ID NO: 4, and downstream universal primer C shown in SEQ ID NO: 5.

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