KASP molecular marker linked with wheat stripe rust resistance gene YrG363 and application of KASP molecular marker

Abstract

The invention discloses a KASP molecular marker linked with a wheat stripe rust resistance gene YrG363 and application of the KASP molecular marker, and belongs to the technical field of agricultural biology. The KASP molecular marker comprises Sicau-1BS. 1 and Sicau-1BS. 2, and the nucleotide sequence of the KASP molecular marker is as shown in SEQ ID NO. 1 to SEQ ID NO. 2. The KASP molecular marker can be used for fine positioning of YrG363 and molecular marker-assisted selection of the YrG363 gene, so that the wheat disease-resistant breeding efficiency is improved. According to the method, the defects that stripe rust resistance phenotype selection in traditional disease resistance breeding is easily influenced by the environment, the stability and repeatability are poor, and the efficiency is low are overcome, and then the breeding process of the stripe rust resistant wheat variety is accelerated. The KASP molecular marker does not depend on gel electrophoresis, and high-throughput and large-scale detection of the YrG363 gene locus can be realized.

Description

Technical Field

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

[0002] Wheat is a widely planted food crop and the main food for about 35%-40% of the world's 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 a reduction in wheat yield or even a total crop failure (Wellings, 2011, Euphytica, 179:129-141). Using disease-resistant genes and cultivating and promoting disease-resistant varieties are the most economical, environmentally friendly and effective measures to control stripe rust.

[0003] To date, 86 officially named and dozens of tentatively named stripe rust resistance genes have been reported from wheat and its closely related species [Zhu et al. 2023, Theor. Appl. Genet. 136: 142; McIntosh, RA et al. 2021, Catalogue of gene symbols for wheat 2021 (supplement) Annu. Wheat Newsletter 67, 104-113], and only a few disease resistance genes have been successfully applied to disease resistance breeding. Due to the frequent variation of physiological subspecies of stripe rust, the emergence and prevalence of new toxic subspecies have led to the rapid loss of stripe rust resistance after resistant varieties were widely promoted in production for 3-5 years. The ancestral species of wheat carry rich genetic variation, 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 few of the rich genetic variation of the ancestral species of wheat entered the common wheat background, resulting in a narrow genetic base of common wheat. Currently, there is a lack of effective sources of resistance to wheat stripe rust in my country, and there is an urgent need to discover new sources of resistance (Kang Zhensheng et al. 2015, Chinese Agricultural Science, 48: 3439-3453).

[0004] Wild emmer wheat is the ancestral species of modern common wheat. It carries abundant stripe rust resistance genes and is an important resource for genetic improvement of wheat stripe rust resistance (Huang et al. Annu. Rev. Phytopathol., 2016, 54: 279-301). SNP (Single Nucleotide Polymorphism) marker refers to a single nucleotide site A, T, G or C at a specific position in the nucleotide sequence, which changes the DNA sequence and causes a polymorphic marker, which can directly reflect the polymorphism of DNA level inheritance. Its sources of variation are rich and distributed throughout the genome. The competitive allele-specific PCR (Kompetitive Allele-Specific PCR, KASP) molecular marker technology based on SNP uses the specificity of SNP to perform marker typing and detection. It has the characteristics of high throughput, accurate results, low cost, and simple operation. It is widely used in gene positioning, cloning and molecular marker selection (Semagn et al. 2014, Mol. Breeding, 33: 1-14).

[0005] Wild emmer wheat germplasm G363 preserved in the Wheat Research Institute of Sichuan Agricultural University shows full growth period resistance to the currently popular stripe rust fungus species (CYR32, CYR33, CYR34, Zhong4 and HY46) in my country. The present invention isolates a recessive stripe rust resistance gene from G363 by constructing a genetic isolation population, which is temporarily named YrG363. The development of a high-throughput KASP molecular marker closely linked to YrG363 is of great value and significance for the rapid detection, automation, and platform-based molecular breeding of the gene, and for accelerating the selection of wheat varieties resistant to stripe rust. Summary of the invention

[0006] The purpose of the present invention is to provide a KASP molecular marker linked to the wheat stripe rust resistance gene YrG363 and its application to solve the problems existing in the above-mentioned prior art. The KASP molecular marker can be used for the fine positioning of YrG363 and molecular marker-assisted selection of the YrG363 gene, thereby improving the efficiency of wheat disease resistance breeding.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a KASP molecular marker linked to the wheat stripe rust resistance gene YrG363, wherein the KASP molecular marker includes Sicau-1BS.1 and Sicau-1BS.2;

[0009] 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;

[0010] 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.

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

[0012] 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;

[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 ID NO.10 in sequence.

[0014] Furthermore, 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.

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

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

[0017] Using the wheat DNA to be tested as a template, the primer set is used to perform fluorescent quantitative PCR amplification;

[0018] The fluorescence signal is collected, and the amplification result is genotyped. It is determined whether the wheat to be tested contains YrG363 based on the color of the fluorescence signal or the genotyping result.

[0019] Further, 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 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 determined that the tested wheat contains homozygous YrG363;

[0020] If the fluorescent signal is orange or 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, 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 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, it is determined that the wheat to be tested contains heterozygous YrG363.

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

[0023] Using the wheat DNA to be tested as a template, the primer set is used to perform fluorescent quantitative PCR amplification;

[0024] The fluorescence signal is collected, the amplification result is genotyped, and the stripe rust resistance of the tested wheat is determined according to the color of the fluorescence signal or the genotyping result.

[0025] Further, 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 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;

[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 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 or 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, it is judged that the tested wheat is not resistant to stripe rust.

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

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

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

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

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

[0032] The present invention also provides a method for breeding wheat resistant to stripe rust, the method comprising any one of the following:

[0033] Breeding wheat containing the stripe rust resistance gene YrG363 according to the method for detecting the stripe rust resistance gene YrG363;

[0034] Or, wheat with stripe rust resistance can be bred according to the method for detecting wheat stripe rust resistance.

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

[0036] The present invention provides a KASP marker linked to the wheat stripe rust resistance gene YrG363, with a genetic distance of 3.1 cM and 1.5 cM from YrG363, respectively. The molecular marker can be used for the fine positioning of YrG363 and molecular marker-assisted selection of the YrG363 gene, thereby improving the efficiency of wheat disease resistance breeding. The present invention overcomes the shortcomings of stripe rust resistance phenotype selection in traditional disease resistance breeding, such as being easily affected by the environment, poor stability and repeatability, and low efficiency, thereby accelerating the breeding process of wheat stripe rust resistance varieties. The KASP molecular marker does not rely on gel electrophoresis, and can achieve high-throughput and large-scale detection of the YrG363 gene site. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0038] Figure 1The fluorescence readings of Sicau-1BS.1 on the F2 generation of wheat LDN×LG363-60 are shown in Figure 1. Among them, blue (HEX) fluorescence is the homozygous stripe rust-resistant strain, orange (FAM) fluorescence is the homozygous susceptible strain; green fluorescence is the heterozygous strain; black fluorescence is the blank control.

[0039] Figure 2 The fluorescence readings of Sicau-1BS.2 on the F2 generation of wheat LDN×LG363-60 are shown in Figure 2. Among them, blue (HEX) fluorescence represents the homozygous stripe rust-resistant strain, orange (FAM) fluorescence represents the homozygous susceptible strain, green fluorescence represents the heterozygous strain, and black fluorescence represents the blank control. DETAILED DESCRIPTION

[0040] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0041] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0042] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may 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 associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0043] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0044] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

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

[0046] The mixed physiological races of stripe rust CYR32, CYR33, CYR34, Zhong4 and HY46 were provided by Jia Qiuzhen, a researcher at the Institute of Plant Protection, Gansu Academy of Agricultural Sciences.

[0047] Example 1 Obtaining a molecular marker linked to the wheat stripe rust resistance gene YrG363

[0048] 1. Test materials

[0049] The stripe rust-susceptible wheat LDN was used as the female parent and the stripe rust-resistant wheat G363 was used as the male parent to obtain the hybrid F1. The F1 generation was self-pollinated continuously to obtain an F5 disease-resistant plant LG363-60. LG363-60 was hybridized with LDN to construct the F1 and F2 resistance genetic segregation populations including 185 plants.

[0050] In the autumn of 2023, 185 F2 plants will be planted at the Gaoshan Village Experimental Base of Sichuan Agricultural University to identify their resistance to stripe rust.

[0051] 2. Stripe rust resistance identification

[0052] The stripe rust resistance identification in the field uses the current popular stripe rust mixed physiological subspecies CYR32, CYR33, CYR34, Zhong4 and HY46 in my country. The field stripe rust inoculation method is: mix the stripe rust mixed subspecies with talcum powder at a mass ratio of 1:20, and inoculate the inducing material by smearing method. The specific operation method is as follows: wear gloves, use the thumb and index finger to smear the fungus mixture on the thumb, then gently pinch the young leaves that have just grown from the inducing material, and inoculate the fungus mixture on the wound of the young leaves. When the disease is fully induced, stripe rust resistance identification is carried out once a week for a total of three surveys, 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), among which 0-2 is highly resistant, 3-5 is moderately resistant, 6 is moderately susceptible, and 7-9 is susceptible.

[0053] The results of stripe rust resistance identification showed that among the F2 segregating population, 51 strains were resistant (IT = 0-5) and 134 strains were susceptible (IT = 7-9), and the resistance-susceptibility ratio was 1:3 (χ 2=0.349, P=0.555), indicating that the stripe rust resistance derived from G363 was controlled by a recessive single gene (tentatively named gene YrG363).

[0054] 3. Genomic DNA Extraction

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

[0056] 4. Pooled transcriptome analysis

[0057] From the F2 population of LDN×LG363-60, 20 extremely resistant plants and 20 extremely susceptible plants were selected to construct resistance and sensitivity pools, and four samples were sequenced with the parents LDN and G363 for BSR-seq. After removing the SNPs that showed heterozygous in the parents and the SNPs with a quality of less than 10% in the parents and resistance and sensitivity pools, a total of 12,873 high-quality SNPs were obtained from the SNP data obtained by BSR-Seq. By calculating the allele difference variation frequency (ΔSNP-index) in the resistance and sensitivity pool, a total of 75 SNPs with ΔSNP-index ≥ 0.8 were obtained.

[0058] 5. Development of YrG363 linkage marker

[0059] The chromosome number distribution and sliding window plotting of the 75 SNPs obtained revealed that 49 of them were located on chromosome 1B, of which 17 were enriched in the 22Mb interval at the end of 1BS. Based on the association region and ΔSNP-index value, two high-confidence SNPs were identified in the resistant and susceptible parents and the resistant and susceptible pool, located at bases 7721413 and 9957587 of chromosome 1B (TRIDC1BG001570 and TRIDC1BG002220) of the wild emmer wheat reference genome V1. The 100 base sequences before and after the SNP were extracted to design KASP markers, and the primer specificity was predicted using the Triticum multi-omics data website (http: / / 202.194.139.32 / ).

[0060] The KASP marker was used to perform KASP amplification reaction on the genomic DNA of the disease-resistant parent G363, the disease-resistant pool and the susceptible parent LDN, the disease-susceptible pool and the offspring population. The resistant and susceptible materials could be clearly typed. 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 of Sicau-1BS.1 and Sicau-1BS.2 to 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: the 20th base of the sequence shown in SEQ ID NO.1 is a SNP site, which has a T / C mutation, and Y represents T or C);

[0063] SEQ ID NO.2:

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

[0065] The KASP molecular marker primer set of Sicau-1BS.1 includes three primers, namely: Sicau-1BS.1F1, Sicau-1BS.1F2 and Sicau-1BS.1R; the fluorescent primer sets used to amplify 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 of Sicau-1BS.2 includes three primers: Sicau-1BS.2F1, Sicau-1BS.2F2 and Sicau-1BS.2R; the fluorescent primer sets used to amplify the Sicau-1BS.2 marker are: Sicau-1BS.2F1-HEX, Sicau-1BS.2F2-FAM and Sicau-1BS.2R (Table 2).

[0067] Table 1 Sicau-1BS.1 related primers

[0068]

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

[0070] Table 2 Sicau-1BS.2 related primers

[0071]

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

[0073] The PCR amplification system of the primers for Sicau-1BS.1 and Sicau-1BS.2 was the same. The reaction system for PCR amplification was: 5μL HiGeno 2×Probe Mix B (Beijing Jiacheng Biotechnology Co., Ltd.), 0.5μL genomic DNA (100ng / μL), 1.4μL primer mixture and 3.1μL ddH2O. Among them, the primer mixture contained: 0.168μL FAM primer (10mM / μL), 0.168μL HEX primer (10mM / μL), 0.42μL common downstream primer (10mM / μL) and 0.644μL 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 in 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 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 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 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, it is determined that the wheat to be tested does not contain the YrG363 gene;

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

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

[0080] The tetraploid wheat line LDN susceptible to stripe rust was used as the female parent and the tetraploid wheat line LG363-60 resistant to stripe rust was used as the male parent to obtain an F2 segregation population containing 185 plants. The 185 plants obtained were tested for KASP markers by using the CTAB method to extract DNA from the 185 plants; using it as a template, using the fluorescent primer set for amplifying Sicau-1BS.1 and Sicau-1BS.2 markers as primers for fluorescence quantitative PCR amplification, and completing the fluorescence reading.

[0081] Figure 1-Figure 2 The fluorescence reading results are as follows. The plant genotype that detects HEX (blue) fluorescence consistent with LG363-60 is recorded as A, which is a stripe rust resistant strain. The plant genotype that shows FAM (orange) fluorescence like LDN is recorded as B, which is a stripe rust susceptible strain. The green fluorescence signal is detected as a heterozygous strain. The genotypes of 82 wheat plants randomly selected from the LDN×LG363-60 F2 segregation population and the field stripe rust identification results are shown in Table 3. The detection results are consistent with the field identification results, indicating that the stripe rust resistance gene YrG363 of the present invention does have a significant effect of increasing stripe rust resistance, and the molecular marker provided by the present invention can accurately track the wheat stripe rust resistance gene YrG363, predict the stripe rust resistance characteristics of wheat, and realize high-throughput and large-scale detection of the YrG363 gene site.

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

[0083]

[0084]

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

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

[0087] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all 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 described in 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 described in claim 2 or 3 is used to perform fluorescent quantitative PCR amplification; The fluorescence signal is collected, and the amplification result is genotyped. It is determined whether the wheat to be tested contains YrG363 based on the color of the fluorescence signal or the genotyping result.

6. The method according to claim 5, characterized in that If the fluorescent signal is blue or 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 determined that the tested wheat contains homozygous YrG363; If the fluorescent signal is orange or 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, it is determined that the wheat to be tested does not contain YrG363; 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 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, it is determined that the wheat to be tested 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 described in claim 2 or 3 is used to perform fluorescent quantitative PCR amplification; The fluorescence signal is collected, the amplification result is genotyped, and the stripe rust resistance of the tested wheat is determined according to the color of the fluorescence signal or the genotyping result.

8. The method according to claim 7, characterized in that If the fluorescent signal is blue or 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 or 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 or 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, 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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