KASP Molecular Marker Linked to Wheat Stripe Rust Resistance Gene YrG363 and Its Application

Abstract

The present invention discloses KASP molecular markers linked to the wheat stripe rust resistance gene YrG363 and their applications, belonging to the field of agricultural biotechnology. The KASP molecular markers include Sicau-1BS.1 and Sicau-1BS.2, and their nucleotide sequences are as shown in SEQ ID NO.1-SEQ ID NO.2. The KASP molecular markers can be used for the fine mapping of YrG363 and molecular marker-assisted selection of the YrG363 gene, improving the efficiency of wheat disease-resistant breeding. The present invention overcomes the disadvantages in traditional disease-resistant breeding, such as the susceptibility of stripe rust resistance phenotype selection to environmental influence, poor stability and repeatability, and low efficiency, thus accelerating the breeding process of wheat varieties resistant to stripe rust. The KASP molecular markers do not rely on gel electrophoresis and can achieve high-throughput and large-scale detection of the YrG363 gene locus.

Description

Technical Field

[0001] The present invention relates to the field of agricultural biotechnology, and particularly to a KASP molecular marker linked to the wheat stripe rust resistance gene YrG363 and its application. Background Art

[0002] Wheat, as a widely cultivated food crop, is the main food for about 35%-40% of the global population. Wheat stripe rust is an airborne fungal disease caused by Puccinia striiformis West. f. sp. tritici. The occurrence of wheat stripe rust can lead to wheat yield reduction or even crop failure (Wellings, 2011, Euphytica, 179: 129-141). Utilizing disease-resistant genes and cultivating and promoting disease-resistant varieties are the most economical, environmentally friendly, and effective measures to control stripe rust.

[0003] So far, 86 officially named and dozens of tentatively named stripe rust resistance genes have been reported from wheat and its related species [Zhu et al. 2023, Theor. Appl. Genet. 136: 142; McIntosh, R. A. et al. 2021, Catalogue of gene symbols for wheat 2021 (supplement) Annu. Wheat Newsletter 67, 104-113], and only a few disease-resistant genes have been successfully applied to disease-resistant breeding. Due to the frequent variation of physiological races of stripe rust fungi, the emergence and prevalence of new virulent races result in the rapid loss of stripe rust resistance in resistant varieties after 3-5 years of large-scale promotion in production. The ancestral species of wheat carry rich genetic variations, including stripe rust resistance. Modern common wheat is an allohexaploid, and only a few ancestral species participated in the origin of wheat. Therefore, only a small part of the rich genetic variations of wheat ancestral species entered the background of common wheat, resulting in a narrow genetic basis of common wheat. Currently, there is a lack of effective resistance sources to wheat stripe rust in China, and it is urgent to explore new stripe rust resistance sources (Kang Zhensheng et al. 2015, Scientia Agricultura Sinica, 48: 3439-3453).

[0004] Wild emmer wheat is the ancestral species of modern common wheat and carries abundant stripe rust resistance genes, making it an important resource library for the genetic improvement of wheat stripe rust resistance (Huang et al. Annu. Rev. Phytopathol., 2016, 54: 279 - 301). SNP (Single Nucleotide Polymorphism) markers refer to the polymorphisms where a single nucleotide site A, T, G, or C at a specific position in the nucleotide sequence undergoes variation, resulting in changes in the DNA sequence. They can directly reflect the genetic polymorphisms at the DNA level. Their sources of variation are abundant and are distributed throughout the genome. The SNP-based Kompetitive Allele-Specific PCR (KASP) molecular marker technology performs marker genotyping and detection based on the specificity of SNPs, and has characteristics such as high throughput, accurate results, low cost, and simple operation. It has been widely used in gene mapping, cloning, and molecular marker selection (Semagn et al. 2014, Mol. Breeding, 33: 1 - 14).

[0005] The wild emmer wheat germplasm G363 preserved in the Wheat Research Institute of Sichuan Agricultural University shows resistance to the currently prevalent stripe rust races (CYR32, CYR33, CYR34, Zhong4, and HY46) in China throughout the growth period. In this invention, by constructing a genetic segregation population, a recessive stripe rust resistance gene was isolated from G363 and temporarily named YrG363. Developing high-throughput KASP molecular markers tightly linked to YrG363 has important value and significance for the rapid detection, automation, and platform-based molecular breeding of this gene, and for accelerating the breeding of stripe rust-resistant wheat varieties. Summary of the Invention

[0006] The objective of this invention is to provide KASP molecular markers linked to the wheat stripe rust resistance gene YrG363 and their applications to solve the problems existing in the above-mentioned prior art. The KASP molecular markers can be used for the fine mapping of YrG363 and for molecular marker-assisted selection of the YrG363 gene, thereby improving the efficiency of wheat disease-resistant breeding.

[0007] To achieve the above objective, this invention provides the following solutions:

[0008] This invention provides KASP molecular markers linked to the wheat stripe rust resistance gene YrG363, and the KASP molecular markers include Sicau-1BS.1 and Sicau-1BS.2;

[0009] The nucleotide sequence of Sicau-1BS.1 is shown in SEQ ID NO.1, with a T / C mutation at position 20, and the genotypes at this site include TT, CC, and TC genotypes;

[0010] The nucleotide sequence of Sicau-1BS.2 is shown in SEQ ID NO.2, with a C / A mutation at position 19, and the genotypes at this site include CC, AA, and CA genotypes.

[0011] The present invention also provides a primer set for amplifying the KASP molecular marker, and the primer set consists of the Sicau-1BS.1 primer set and the Sicau-1BS.2 primer set;

[0012] The Sicau-1BS.1 primer set consists of Sicau-1BS.1F1, Sicau-1BS.1F2, and Sicau-1BS.1R whose nucleotide sequences are shown in SEQ ID NO.3 - SEQ IN NO.5 in sequence;

[0013] The Sicau-1BS.2 primer set consists of Sicau-1BS.2F1, Sicau-1BS.2F2, and Sicau-1BS.2R whose nucleotide sequences are shown in SEQ ID NO.8 - SEQ IN NO.10 in sequence.

[0014] Furthermore, the 5' ends of Sicau-1BS.1F1 and Sicau-1BS.2F1 are modified with HEX fluorescent groups, and the 5' ends of Sicau-1BS.1F2 and Sicau-1BS.2F2 are modified with FAM fluorescent groups.

[0015] The present invention also provides a reagent or kit for detecting the wheat stripe rust resistance gene YrG363, including the primer set.

[0016] The present invention also provides a method for detecting the wheat stripe rust resistance gene YrG363, including the following steps:

[0017] Using the DNA of the wheat to be tested as a template, performing fluorescence quantitative PCR amplification with the primer set;

[0018] Collecting fluorescence signals, performing genotype typing on the amplification results, and judging whether the wheat to be tested contains YrG363 according to the color of the fluorescence signals or the genotype typing results.

[0019] Furthermore, if the fluorescence signal is blue or the base T corresponding to Sicau-1BS.1F1 and the base C corresponding to Sicau-1BS.2F1 are detected simultaneously, and the base C corresponding to Sicau-1BS.1F2 and the base A corresponding to Sicau-1BS.2F2 are not detected, it is determined that the wheat to be tested contains homozygous YrG363;

[0020] If the fluorescence signal is orange or the base C corresponding to Sicau-1BS.1F2 and the base A corresponding to Sicau-1BS.2F2 are detected simultaneously, and the base T corresponding to Sicau-1BS.1F1 and the base C corresponding to Sicau-1BS.2F1 are not detected, it is determined that the wheat to be tested does not contain YrG363;

[0021] If the fluorescence signal is green or the base T corresponding to Sicau-1BS.1F1 and the base C corresponding to Sicau-1BS.1F2 are detected simultaneously, and the base C corresponding to Sicau-1BS.2F1 and the base A corresponding to Sicau-1BS.2F2 are detected simultaneously, it is determined that the wheat to be tested contains heterozygous YrG363.

[0022] The present invention also provides a method for detecting the resistance of wheat to stripe rust, comprising the following steps:

[0023] Using the DNA of the wheat to be tested as a template, performing fluorescence quantitative PCR amplification with the primer set described above;

[0024] Collecting the fluorescence signal, performing genotype typing on the amplification result, and judging the resistance of the wheat to be tested to stripe rust according to the color of the fluorescence signal or the genotype typing result.

[0025] Furthermore, if the fluorescence signal is blue or the base T corresponding to Sicau-1BS.1F1 and the base C corresponding to Sicau-1BS.2F1 are detected simultaneously, and the base C corresponding to Sicau-1BS.1F2 and the base A corresponding to Sicau-1BS.2F2 are not detected, it is determined that the wheat to be tested has resistance to stripe rust;

[0026] If the fluorescence signal is orange or the base C corresponding to Sicau-1BS.1F2 and the base A corresponding to Sicau-1BS.2F2 are detected simultaneously, and the base T corresponding to Sicau-1BS.1F1 and the base C corresponding to Sicau-1BS.2F1 are not detected; or the fluorescence signal is green or the base T corresponding to Sicau-1BS.1F1 and the base C corresponding to Sicau-1BS.1F2 are detected simultaneously, and the base C corresponding to Sicau-1BS.2F1 and the base A corresponding to Sicau-1BS.2F2 are detected simultaneously, it is determined that the wheat to be tested does not have resistance to stripe rust.

[0027] The present invention also provides the application of the KASP molecular marker or the primer set as described above in any of the following:

[0028] Application in the detection and fine mapping of wheat stripe rust resistance gene YrG363;

[0029] Application in molecular marker-assisted selection breeding for wheat stripe rust resistance;

[0030] Application in the genetic transfer of wheat stripe rust resistance gene YrG363 to common wheat;

[0031] Application in the control of wheat stripe rust.

[0032] The present invention also provides a method for breeding stripe rust-resistant wheat, and the method includes any of the following:

[0033] Selecting wheat containing stripe rust resistance gene YrG363 according to the method for detecting wheat stripe rust resistance gene YrG363 as described above;

[0034] Or selecting wheat with stripe rust resistance according to the method for detecting wheat stripe rust resistance as described above.

[0035] The present invention discloses the following technical effects:

[0036] The present invention provides KASP markers linked to wheat stripe rust resistance gene YrG363, with genetic distances from YrG363 being 3.1 cM and 1.5 cM respectively. This molecular marker can be used for the fine mapping of YrG363 and molecular marker-assisted selection of the YrG363 gene, improving the efficiency of wheat disease-resistant breeding. The present invention overcomes the disadvantages in traditional disease-resistant breeding, such as the susceptibility of stripe rust resistance phenotype selection to environmental influence, poor stability and repeatability, and low efficiency, thereby accelerating the breeding process of wheat varieties resistant to stripe rust. The KASP molecular marker does not rely on gel electrophoresis and can achieve high-throughput and large-scale detection of the YrG363 gene locus. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0038] Figure 1Fluorescence reading results of the F2 generation plants of wheat LDN×LG363 - 60 detected by Sicau - 1BS.1; among them, the blue (HEX) fluorescence represents homozygous stripe rust resistant lines, the orange (FAM) fluorescence represents homozygous susceptible lines; the green fluorescence represents heterozygous lines; and the black fluorescence represents the blank control.

[0039] Figure 2 Fluorescence reading results of the F2 generation plants of wheat LDN×LG363 - 60 detected by Sicau - 1BS.2; among them, the blue (HEX) fluorescence represents homozygous stripe rust resistant lines, the orange (FAM) fluorescence represents homozygous susceptible lines; the green fluorescence represents heterozygous lines; and the black fluorescence represents the blank control. Detailed implementation manners

[0040] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0041] It should be understood that the terms used in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0042] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0043] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the specification of the present invention, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0044] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open - ended terms, meaning including but not limited to.

[0045] In this invention, the tetraploid durum wheat Langdon (LDN) and the wild emmer wheat G363 were provided by the Wheat Research Institute of Sichuan Agricultural University.

[0046] The mixed physiological races CYR32, CYR33, CYR34, Zhong4, and HY46 of Puccinia striiformis were provided by Researcher Jia Qiuzhen from the Institute of Plant Protection, Gansu Academy of Agricultural Sciences.

[0047] Example 1 Obtaining of Linked Molecular Markers of Wheat Stripe Rust Resistance Gene YrG363

[0048] 1. Test Materials

[0049] Using the stripe rust-susceptible wheat LDN as the female parent and the stripe rust-resistant wheat G363 as the male parent for hybridization to obtain the hybrid F1. The F1 generation was continuously self-crossed to obtain a disease-resistant F5 single plant LG363-60. LG363-60 was crossed with LDN to construct an F1 and an F2 resistance genetic segregation population consisting of 185 single plants.

[0050] In the autumn of 2023, 185 F2 single plants were planted in the experimental base of Gaoshan Village, Sichuan Agricultural University to identify the stripe rust resistance.

[0051] 2. Identification of Stripe Rust Resistance

[0052] For the field identification of stripe rust resistance, the currently prevalent mixed physiological races CYR32, CYR33, CYR34, Zhong4, and HY46 of Puccinia striiformis in China were used. The method for inoculating stripe rust in the field was as follows: Mix the mixed races of stripe rust and talcum powder at a mass ratio of 1:20, and inoculate the induced materials by the smearing method. The specific operation method was as follows: Wear gloves, smear the bacterial mixture on the thumb pad with the thumb and index finger, then gently pinch the newly grown young leaves of the induced materials, and inoculate the bacterial mixture at the wound of the young leaves. When the induction was completely diseased, stripe rust resistance was identified once a week for a total of three times, referring to the 0-9 level identification standard (Line and Qayoum, 1992, Virulence, aggressiveness, evolution, and distribution of races of Puccinia striiformis (the cause of stripe rust of wheat) in North America, 1968-87. Technical Bulletin-USDA), where levels 0-2 were highly resistant, levels 3-5 were moderately resistant, level 6 was moderately susceptible, and levels 7-9 were susceptible.

[0053] The results of the stripe rust resistance identification showed that in the F2 segregation population, 51 plants were disease-resistant (IT = 0-5) and 134 plants were susceptible (IT = 7-9), and the disease-resistant to susceptible ratio conforms to 1:3 (χ 2(=0.349, P = 0.555), indicating that the stripe rust resistance derived from G363 is controlled by a recessive single gene (temporarily named gene YrG363).

[0054] 3. Genomic DNA extraction

[0055] The genomic DNA of the leaves of the parental lines LDN, G363, and the F2 segregating population plants was extracted using the CTAB method (Rogers and Bendich 1985, Plant Mol. Biol., 1985, 5: 69 - 76).

[0056] 4. Bulked segregant transcriptome analysis

[0057] From the F2 population of LDN × LG363 - 60, 20 extremely resistant individuals and 20 extremely susceptible individuals were screened to construct resistant and susceptible bulks respectively, and BSR - seq sequencing was performed on a total of four samples, namely the parental lines LDN and G363. After removing the SNPs that were heterozygous in the two parents and the SNPs with a quality lower than 10% in the two parents and the resistant and susceptible bulks from the SNP data obtained by BSR - Seq, a total of 12,873 high - quality SNPs were obtained. By calculating the allelic differential variant frequency (ΔSNP - index) in the resistant and susceptible bulks, a total of 75 SNPs with ΔSNP - index ≥ 0.8 were obtained.

[0058] 5. Development of YrG363 - linked markers

[0059] Chromosome number distribution and sliding window mapping were performed on the 75 obtained SNPs, and it was found that 49 of them were located on chromosome 1B, and 17 of them were enriched in the 22 Mb interval at the end of 1BS. According to the associated region and the ΔSNP - index value, 2 highly reliable SNPs were identified in the resistant and susceptible parents and the resistant and susceptible bulks, located at positions 7,721,413 and 9,957,587 of chromosome 1B (TRIDC1BG001570 and TRIDC1BG002220) of the wild emmer wheat reference genome version V1. The 100 - base sequences before and after this SNP were extracted to design KASP markers, and the primer specificity was predicted using the wheat tribe multi - omics data website (http: / / 202.194.139.32 / ).

[0060] The genomic DNA of the resistant parental line G363, the resistant bulk, the susceptible parental line LDN, the susceptible bulk, and the offspring population was subjected to KASP amplification reaction using this KASP marker. The resistant and susceptible materials could be clearly genotyped. The two molecular markers were named Sicau - 1BS.1 (SEQ ID NO.1) and Sicau - 1BS.2 (SEQ ID NO.2) respectively. Genetic linkage analysis showed that the genetic distances between Sicau - 1BS.1 and Sicau - 1BS.2 and the stripe rust resistance gene YrG363 were 3.1 cM and 1.5 cM respectively.

[0061] SEQ ID NO.1:

[0062] 5'-AAGATGCAGGACTGGGGTCYGGTGCTCATCTCCTTCATGCTCTTCATCCTTCTGTCCCCAGGGCTTGTCATCCAGATC-3'(Note: There is a SNP site at the 20th base of the sequence shown in SEQ ID NO.1, and there is a T / C mutation at this site. Y represents T or C);

[0063] SEQ ID NO.2:

[0064] 5'-AGGTAGGGTCTTTCCAGCMAGATATTTCAATACACCATTTGAGCTTGGCAGACTAGACAACTCCAGATGT-3'(Note: There is a SNP site at the 19th base of the sequence shown in SEQ ID NO.2, and there is a C / A mutation at this site. M represents C or A).

[0065] The KASP molecular marker primer set for Sicau-1BS.1 includes 3 primers, namely: Sicau-1BS.1F1, Sicau-1BS.1F2 and Sicau-1BS.1R; The fluorescence primer sets for amplifying the Sicau-1BS.1 marker are: Sicau-1BS.1F1-HEX, Sicau-1BS.1F2-FAM and Sicau-1BS.1R (Table 1).

[0066] The KASP molecular marker primer set for Sicau-1BS.2 includes 3 primers, namely: Sicau-1BS.2F1, Sicau-1BS.2F2 and Sicau-1BS.2R; The fluorescence primer sets for amplifying the Sicau-1BS.2 marker are: Sicau-1BS.2F1-HEX, Sicau-1BS.2F2-FAM and Sicau-1BS.2R (Table 2).

[0067] Table 1 Primers related to Sicau-1BS.1

[0068]

[0069] Note: The underlined part is the HEX fluorescent group, and the bold part is the FAM fluorescent group.

[0070] Table 2 Primers related to Sicau-1BS.2

[0071]

[0072] Note: The underlined part is the HEX fluorescent group, and the bold part is the FAM fluorescent group.

[0073] The PCR amplification systems of the primers for Sicau-1BS.1 and Sicau-1BS.2 are the same. The reaction systems for PCR amplification are all: 5 μL HiGeno 2×Probe Mix B (Beijing Jiacheng Biotechnology Co., Ltd.), 0.5 μL genomic DNA (100 ng / μL), 1.4 μL primer mixture, and 3.1 μL ddH2O. Among them, the primer mixture contains: 0.168 μL FAM primer (10 mM / μL), 0.168 μL HEX primer (10 mM / μL), 0.42 μL common downstream primer (10 mM / μL), and 0.644 μL ddH2O.

[0074] PCR amplification program: Pre-denaturation at 95°C for 10 min; denaturation at 95°C for 20 s, annealing and extension at 61 - 55°C for 40 s, 10 cycles, with the annealing and extension temperature decreasing by 0.6°C for each cycle; denaturation at 95°C for 20 s, annealing and extension at 55°C for 60 s, 30 cycles.

[0075] According to the PCR amplification results, the genotyping is as follows:

[0076] (1) If the base T corresponding to Sicau-1BS.1F1-HEX and the base C corresponding to Sicau-1BS.2F1-HEX are detected simultaneously, and the base C corresponding to Sicau-1BS.1F2 and the base A corresponding to Sicau-1BS.2F2 are not detected, it is determined that the wheat to be tested contains the homozygous YrG363 gene;

[0077] (2) If the base C corresponding to Sicau-1BS.1F2-FAM and the base A corresponding to Sicau-1BS.2F2-FAM are detected simultaneously, and the base T corresponding to Sicau-1BS.1F1 and the base C corresponding to Sicau-1BS.2F1 are not detected, it is determined that the wheat to be tested does not contain the YrG363 gene;

[0078] (3) If T and C are detected simultaneously for Sicau-1BS.1F1-HEX and Sicau-1BS.1F2-FAM, and C and A are detected simultaneously for Sicau-1BS.2F1-HEX and Sicau-1BS.2F2-FAM, it is determined that the wheat to be tested contains the heterozygous YrG363 gene.

[0079] Example 2 Application of the KASP molecular marker linked to the wheat stripe rust resistance gene YrG363

[0080] Using the tetraploid wheat line LDN susceptible to stripe rust as the female parent and the tetraploid wheat LG363-60 resistant to stripe rust as the male parent for hybridization, an F2 segregation population consisting of 185 plants was obtained. The 185 individual plants obtained were detected by KASP markers. The specific method was as follows: The DNA of 185 individual plants was extracted by the CTAB method; using this as a template, a fluorescence primer set for amplifying Sicau-1BS.1 and Sicau-1BS.2 markers was used as primers for fluorescence quantitative PCR amplification to complete the fluorescence reading.

[0081] Figure 1 - Figure 2 Regarding the fluorescence reading results, the genotype of the plants detected with HEX (blue) fluorescence consistent with LG363-60 was designated as A, which was a stripe rust resistant line, and the genotype of the plants showing FAM (orange) fluorescence like LDN was designated as B, which was a stripe rust susceptible line. The plants detected with green fluorescence signals were heterozygous lines. The genotypes of 82 randomly selected wheat plants from the LDN×LG363-60 F2 segregation population and the field stripe rust identification results are shown in Table 3. The detection results were consistent with the field identification results, indicating that the stripe rust resistant gene YrG363 of the present invention indeed had a significant effect on increasing stripe rust resistance, and the molecular markers provided by the present invention could accurately track the stripe rust resistant gene YrG363 of wheat, predict the stripe rust resistance characteristics of wheat, and enable high-throughput and large-scale detection of the YrG363 gene locus.

[0082] Table 3 Genotyping results of Sicau-1BS.1 and Sicau-1BS.2 markers for LDN×LG363-60 hybrid F2 plants

[0083]

[0084]

[0085] Note: S indicates susceptible to stripe rust, and R indicates resistant to stripe rust.

[0086] In summary, using the molecular markers Sicau-1BS.1 and Sicau-1BS.2 linked to the gene YrG363 developed by the present invention, high-throughput detection of YrG363 can be achieved, which is of great significance for fine mapping of this gene, molecular marker-assisted selection breeding, creation of wheat materials resistant to stripe rust, and improvement of wheat disease resistance breeding efficiency.

[0087] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should fall within the protection scope determined by the claims of the present invention.

Claims

1. KASP molecular marker linked to wheat stripe rust resistance gene YrG363, characterized in that: The KASP molecular markers include Sicau-1BS.1 and Sicau-1BS.2; The nucleotide sequence of Sicau-1BS.1 is shown in SEQ ID NO.1, and there is a T / C mutation at position 20, and the genotypes of this site include TT, CC and TC genotypes; The nucleotide sequence of Sicau-1BS.2 is shown in SEQ ID NO.2, and there is a C / A mutation at position 19. The genotypes of this site include CC, AA and CA genotypes.

2. A primer set for amplifying the KASP molecular marker according to claim 1, characterized in that: The primer set consists of Sicau-1BS.1 primer set and Sicau-1BS.2 primer set; The Sicau-1BS.1 primer set consists of Sicau-1BS.1F1, Sicau-1BS.1F2, and Sicau-1BS.1R, the nucleotide sequences of which are shown in SEQ ID NO.3 to SEQ ID NO.5 in sequence; The Sicau-1BS.2 primer set consists of Sicau-1BS.2F1, Sicau-1BS.2F2 and Sicau-1BS.2R, whose nucleotide sequences are shown in SEQ ID NO.8-SEQ ID NO.10 in sequence.

3. The primer set according to claim 2, characterized in that The 5' ends of the Sicau-1BS.1F1 and Sicau-1BS.2F1 are modified with a HEX fluorescent group, and the 5' ends of the Sicau-1BS.1F2 and Sicau-1BS.2F2 are modified with a FAM fluorescent group.

4. A reagent or kit for detecting wheat stripe rust resistance gene YrG363, characterized in that: Comprising the primer set of claim 2 or 3.

5. A method for detecting wheat stripe rust resistance gene YrG363, characterized in that: The following steps are involved: Using the wheat DNA to be tested as a template, the primer set of claim 3 is used to perform fluorescent quantitative PCR amplification; The fluorescence signal was collected, the amplification results were genotyped, and whether the tested wheat contained YrG363 was determined based on the color of the fluorescence signal.

6. The method according to claim 5, characterized in that If the fluorescence signal is blue, it means that the base T corresponding to Sicau-1BS.1F1 and the base C corresponding to Sicau-1BS.2F1 are detected at the same time, and the base C corresponding to Sicau-1BS.1F2 and the base A corresponding to Sicau-1BS.2F2 are not detected, then it is judged that the tested wheat contains homozygous YrG363; If the fluorescent signal is orange, it means that the base C corresponding to Sicau-1BS.1F2 and the base A corresponding to Sicau-1BS.2F2 are detected at the same time, and the base T corresponding to Sicau-1BS.1F1 and the base C corresponding to Sicau-1BS.2F1 are not detected, then it is judged that the tested wheat does not contain YrG363; If the fluorescence signal is green, that is, the base T corresponding to Sicau-1BS.1F1 and the base C corresponding to Sicau-1BS.1F2 are detected at the same time, and the base C corresponding to Sicau-1BS.2F1 and the base A corresponding to Sicau-1BS.2F2 are detected at the same time, then it is judged that the tested wheat contains heterozygous YrG363.

7. A method for detecting wheat stripe rust resistance, characterized in that: The following steps are involved: Using the wheat DNA to be tested as a template, the primer set of claim 3 is used to perform fluorescent quantitative PCR amplification; The fluorescence signal was collected, the amplification results were genotyped, and the stripe rust resistance of the tested wheat was determined according to the color of the fluorescence signal.

8. The method according to claim 7, characterized in that If the fluorescent signal is blue, it means that the base T corresponding to Sicau-1BS.1F1 and the base C corresponding to Sicau-1BS.2F1 are detected at the same time, and the base C corresponding to Sicau-1BS.1F2 and the base A corresponding to Sicau-1BS.2F2 are not detected; it is judged that the tested wheat has stripe rust resistance; If the fluorescence signal is orange, it means that the base C corresponding to Sicau-1BS.1F2 and the base A corresponding to Sicau-1BS.2F2 are detected at the same time, and the base T corresponding to Sicau-1BS.1F1 and the base C corresponding to Sicau-1BS.2F1 are not detected; or the fluorescence signal is green, it means that the base T corresponding to Sicau-1BS.1F1 and the base C corresponding to Sicau-1BS.1F2 are detected at the same time, and the base C corresponding to Sicau-1BS.2F1 and the base A corresponding to Sicau-1BS.2F2 are detected at the same time, then it is judged that the tested wheat is not resistant to stripe rust.

9. Use of the KASP molecular marker according to claim 1 or the primer set according to any one of claims 2 to 3 in any of the following: Application in the detection and fine positioning of wheat stripe rust resistance gene YrG363; Application in molecular marker-assisted selection breeding for wheat resistance to stripe rust; Application of wheat stripe rust resistance gene YrG363 in genetic transfer to common wheat; Application in the prevention and control of wheat stripe rust.

10. A method for breeding wheat resistant to stripe rust, characterized in that: The method includes any of the following: Breeding wheat containing the stripe rust resistance gene YrG363 according to the method of any one of claims 5 to 6; Or, according to the method described in any one of claims 7-8, wheat with stripe rust resistance is bred.

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