Linked molecular marker and primer for detecting wheat Puccinia stelleriana resistance gene and application of linked molecular marker and primer

Abstract

The invention discloses a linked molecular marker and primer for detecting a wheat Puccinia stelleriana gene and application. The method is not influenced by external environmental factors and is simple and convenient to operate. Primers of molecular markers K-71551 and K-71810 in close linkage with the wheat Puccinia graminis resistance gene are utilized, PCR amplification electrophoresis detection is carried out, germplasm materials carrying the wheat Puccinia graminis resistance gene can be screened out according to the size of a target band, the molecular marker can accelerate research and utilization of the excellent resistance gene of the wheat Puccinia graminis, and the application prospect is wide. A new material is provided for wheat disease-resistant genetic improvement.

Description

Technical Field

[0001] The invention relates to the field of wheat molecular breeding, and in particular to a linkage molecular marker, a primer and an application thereof for detecting a wheat stem rust resistance gene. Background Art

[0002] Wheat stem rust is a disease caused by Puccinia graminis f.sp.tritici (Pgt), which seriously affects the world's wheat production and poses a serious threat to wheat leaves, stems, leaf sheaths and husks. In my country, it mainly occurs in the coastal areas of East China, the Yangtze River Basin, the southern winter wheat areas, Northeast China, Inner Mongolia in North China, and the Northwest spring wheat areas. In severe epidemic years, it can cause a 74% to 84% reduction in wheat production, or even a total loss of production.

[0003] By designing tightly linked molecular markers for specific DNA sequences, the relationship between genotype and phenotype can be identified, individuals with specific genotypes can be efficiently screened out, and the efficiency of molecular breeding can be greatly improved. In most cases, the phenotype is not only affected by a single gene. Through co-dominant molecular markers, phenotypic changes caused by different alleles can be accurately identified and distinguished. Using co-dominant molecular markers, heterozygous and homozygous individuals can be distinguished. This is particularly important for variety improvement, especially in selective breeding to ensure the accurate transmission of excellent genes.

[0004] Therefore, biotechnology can be used to introduce stem rust resistance genes into excellent wheat varieties. Traditional breeding methods have long cycles, are labor-intensive, difficult to identify phenotypes, and have low breeding efficiency. Molecular marker-assisted selection breeding technology can effectively improve breeding efficiency and solve this problem. Summary of the invention

[0005] In order to solve at least some of the technical problems in the prior art, the present invention phenotypic identification of wheat strains was conducted, and it was found that the sdauP29 wheat strain showed an immune phenotype to most physiological races of stem rust fungi, exhibiting good resistance and having great application value. By constructing a RIL population, the resistance locus of its immunity was located on 7BL, and the recombinants of the F2:3 strains were further screened, and the interval was further narrowed, and the tightly linked marker K-71551 was identified to be co-segregated with the phenotype. This marker can well distinguish between immune and susceptible strains, and no fragments of the same size can be amplified in Chinese Spring and Fielder wheat materials, which further proves that this marker can well identify the stem rust resistance gene of 7BL and can improve the efficiency of molecular marker-assisted selection. Specifically, the present invention includes the following contents.

[0006] The first aspect of the present invention provides a primer for detecting a wheat stem rust resistance gene-linked molecular marker, wherein the primer comprises:

[0007] (1) K-71551F: the sequence of which is shown in SEQ ID NO. 1, K-71551R: the sequence of which is shown in SEQ ID NO. 2; and / or

[0008] (2) K-71810F: its sequence is shown in SEQ ID NO.3, K-71810R: its sequence is shown in SEQ ID NO.4.

[0009] In certain embodiments, according to the primers for detecting molecular markers linked to wheat stem rust resistance genes of the present invention, the molecular markers include 1058 bp and / or 560 bp nucleic acid fragments amplified by the primers.

[0010] In certain embodiments, according to the primers for detecting molecular markers linked to wheat stem rust resistance genes of the present invention, the molecular markers have sequences shown in SEQ ID NO.5 and / or SEQ ID NO.6.

[0011] The second aspect of the present invention provides a wheat stem rust resistance gene-linked molecular marker, which is obtained by amplification using the primers of the present invention.

[0012] The third aspect of the present invention provides a kit for detecting wheat stem rust resistance gene-linked molecular markers, comprising the primers of the present invention.

[0013] The fourth aspect of the present invention provides the use of the primers, molecular markers or kit in wheat genetic analysis.

[0014] In certain embodiments, according to the use of the present invention, the genetic analysis includes genetic diversity analysis, germplasm identification or assisted breeding.

[0015] The fifth aspect of the present invention provides a method for identifying whether a plant contains a wheat stem rust resistance gene, which includes the step of detecting whether there is a wheat stem rust resistance gene-linked molecular marker in the DNA of the plant to be identified, wherein the molecular marker includes a 1058 bp and / or 560 bp nucleic acid fragment amplified by the primers described in the first aspect.

[0016] In certain embodiments, the method for identifying whether a plant comprises a wheat stem rust resistance gene according to the present invention comprises the following steps:

[0017] (1) extracting sample DNA from the plant sample to be identified;

[0018] (2) a step of amplifying the nucleic acid containing the molecular marker from the sample DNA by PCR using primers; and

[0019] (3) Identifying the plant by detecting the length of the sample PCR product by electrophoresis.

[0020] In certain embodiments, according to the method for identifying whether a plant contains a wheat stem rust resistance gene according to the present invention, the reaction system for amplification comprises: 1 μL of DNA template, 5 μL of 2×Taq Master Mix, 0.5 μL of primer K-71551F at a concentration of 10 μmol / L, 0.4 μL of primer K-71551R at a concentration of 10 μmol / L, double distilled water is added to 10 μL, and the reaction procedure is: 95°C for 5 min; 95°C for 15 s, 55°C for 15 s, 72°C for 30 s, 35 cycles; 72°C for 5 min.

[0021] In certain embodiments, according to the method for identifying whether a plant contains a wheat stem rust resistance gene according to the present invention, the reaction system for amplification comprises: 1 μL of DNA template, 5 μL of 2×Taq Master Mix, 0.5 μL of primer K-71810F at a concentration of 10 μmol / L, 0.5 μL of primer K-71810R at a concentration of 10 μmol / L, double distilled water is added to 10 μL, and the reaction procedure is: 95°C for 5 min; 94°C for 15 s, 57°C for 15 s, 72°C for 1 min, 35 cycles; 72°C for 5 min.

[0022] A sixth aspect of the present invention provides a translocation wheat breeding method, comprising:

[0023] (1) using wheat sdauP29 carrying a stem rust resistance gene as the male parent and wheat variety Chinese spring susceptible to stem rust as the female parent for hybridization, using single seed propagation to perform phenotypic identification on each strain of the RIL population, further performing phenotypic analysis on the phenotypic segregation secondary segregation population, and performing genotypic identification using the primers, molecular markers, kits or methods described in the present invention to isolate homozygous offspring strains;

[0024] (2) identifying the homozygous strains of the progeny, selecting the strains whose phenotypes do not segregate, and identifying the genotypes of the strains using the primers, molecular markers, kits or methods of the present invention, to determine the resistant strains whose molecular markers and phenotypes co-segregate; or

[0025] (1') using sdauP29 wheat material as the donor parent and Chinese Spring, a wheat variety susceptible to stem rust, as the recurrent parent, backcrossing to obtain a BC1F1 generation population, and retaining individual plants carrying the stem rust resistance gene using the primers, molecular markers, kits or methods described in the present invention;

[0026] (2') Self-pollinating individual plants of the BC1F1 generation carrying the stem rust resistance gene to obtain a BC1F2 generation population and planting it, identifying the stem rust resistance of the individual plants by spraying spores, and detecting it using the primers, molecular markers, kits or methods described in the present invention.

[0027] The invention is not affected by external environmental factors and is simple and convenient to operate. The primers of molecular markers K-71551 and K-71810 closely linked to the wheat stem rust resistance gene are used, and the germplasm material carrying the wheat stem rust resistance gene is screened out according to the size of the target band through PCR amplification and electrophoresis detection. The molecular marker can accelerate the research and utilization of excellent wheat stem rust resistance genes and provide new materials for wheat disease resistance genetic improvement. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The sequence amplified by molecular marker K-71551 on wheat chromosome sdauP29 (the yellow content is the primer sequence).

[0029] Figure 2 Detection results of molecular marker K-71551 in secondary segregation population samples of wheat sdaup29RIL ​​population (M, DL2000 Marker bands are 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp respectively; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, F7 generation individual plants; 16, sdauP29; 17, Chinese Spring).

[0030] Figure 3 Phenotypic diagram of segregating populations in the RIL population.

[0031] Figure 4 The sequence amplified by molecular marker K-71810 on wheat chromosome sdauP29.

[0032] Figure 5 Identification phenotype of stem rust resistance of some individuals with different molecular markers K-71810 genotype in wheat sdauP29 BC1F2 population samples.

[0033] Figure 6 The detection results of molecular marker K-71810 in wheat sdauP29 BC1F2 generation population samples (M, DL2000Marker bands are 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, P29, Chinese Spring). DETAILED DESCRIPTION

[0034] 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.

[0035] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that the upper and lower limits of the scope and each intermediate value therebetween are specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and 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.

[0036] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the invention belongs. 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 related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0037] Molecular markers

[0038] In a first aspect of the present invention, a specific molecular marker is provided, which has a characteristic sequence for distinguishing whether a plant (e.g., wheat) carries a stem rust resistance gene. In the present invention, stem rust refers to a rust-like phenotype caused by the wheat-specific subspecies Pgt of Puccinia graminis, in which the infected part presents bright yellow, reddish brown or brown spore piles, epidermal rupture, and spore scattering.

[0039] The molecular marker ("characteristic sequence") in the present invention refers to a gene fragment that only appears in stem rust-resistant wheat and is linked to the stem rust-resistant gene and is closely related to stem rust resistance, wherein the stem rust-resistant gene refers to a resistance gene located on 7BL in the hybrid offspring of wheat sdauP29 and Chinese spring wheat.

[0040] Primer combinations

[0041] In one aspect of the present invention, a specific primer combination is provided, which is used to amplify the molecular marker described in the present invention, comprising a forward primer and a reverse primer. In a specific embodiment, the forward primer used to amplify K-71551 has a sequence shown in SEQ ID NO.1, and the reverse primer has a sequence shown in SEQ ID NO.2. In another specific embodiment, the forward primer used to amplify K-71810 has a sequence shown in SEQ ID NO.3, and the reverse primer has a sequence shown in SEQ ID NO.4.

[0042] In a specific embodiment, the molecular marker of the present invention has a sequence shown in SEQ ID NO. 5. In a specific embodiment, the molecular marker of the present invention has a sequence shown in SEQ ID NO. 6.

[0043] Reagent test kit

[0044] One aspect of the present invention provides a test kit for detecting wheat stem rust resistance gene linked molecular markers, which comprises a primer combination of the present invention and an optional reagent for polymerase chain reaction. The reagent for polymerase chain reaction comprises any reagent used by conventional PCR, such as polymerase, buffer, etc.

[0045] In addition to the above components, the kits of the invention may also be provided with detailed instructions for use, storage and troubleshooting.The kits may also optionally be provided in a suitable apparatus, preferably for robotic manipulation in a high throughput setting.

[0046] In certain embodiments, the components of the kit of the present invention can be provided as dry powders. When reagents and / or components are provided as dry powders, the powder can be restored to its original state by adding a suitable solvent. It is expected that the solvent can also be arranged in another container. The container will usually include at least one vial, test tube, flask, bottle, syringe and / or other container means, wherein the solvent is optionally placed in equal parts. The kit can also include a means for a second container containing a sterile, pharmaceutically acceptable buffer and / or other solvent.

[0047] In certain embodiments, the components of the kit of the present invention may be provided in the form of a solution, such as an aqueous solution. In the case of being present in an aqueous solution state, the concentration or content of these components can be easily determined by those skilled in the art according to different needs. For example, for storage purposes, the concentration of the components may be present in a higher form, and when in working state or use, the concentration may be reduced to the working concentration by, for example, diluting the above-mentioned higher concentration solution.

[0048] In the case where there is more than one component in the kit, the kit will also typically include a second, third or other additional container in which the additional components can be placed separately. In addition, combinations of various components can be included in the container. Any combination or reagent described herein can be a component in the kit.

[0049] application

[0050] In one aspect of the present invention, the use of the primers, molecular markers or kits of the present invention in wheat genetic analysis is provided, and the genetic analysis includes but is not limited to genetic diversity analysis, germplasm identification, assisted breeding, etc. In a specific embodiment, the application includes the identification of wheat translocation lines with 7B stem rust resistance genotype.

[0051] Identification methods

[0052] One aspect of the present invention provides a method for identifying whether a plant contains the wheat 7B stem rust resistance gene, which includes the step of detecting whether there is a wheat stem rust resistance gene-linked molecular marker in the DNA of the plant to be identified, wherein the molecular marker has a sequence shown in SEQ ID NO.5 and / or SEQ ID NO.6.

[0053] In the present invention, the method for detecting the presence of wheat stem rust resistance gene-linked molecular markers can adopt any method known in the art, examples of which include methods of amplifying nucleic acids containing characteristic sequences and methods of directly sequencing plant genomes.

[0054] In certain embodiments, the method of identifying whether a plant contains a wheat stem rust resistance gene of the present invention comprises the step of amplifying a gene sequence containing a characteristic sequence from DNA of a plant sample to be identified by PCR using forward and reverse primers.

[0055] In an exemplary embodiment, a method for identifying whether a plant comprises a wheat stem rust resistance gene comprises the following steps:

[0056] (1) extracting sample DNA from the plant sample to be identified;

[0057] (2) a step of amplifying the nucleic acid containing the molecular marker from the sample DNA by PCR using a forward primer and a reverse primer;

[0058] (3) Identify whether the plant contains the wheat stem rust resistance gene by detecting the length of the sample PCR product by electrophoresis.

[0059] In step (1), the "plant sample to be identified" refers to the whole plant or a part of the plant, for example, a plant root, a leaf or a plant stem, etc. In step (3), the length of the sample PCR product is detected by electrophoresis, and the sample contains a stem rust resistance gene amplification fragment of 1058 bp and / or 560 bp.

[0060] Breeding methods

[0061] The present invention also provides a breeding method for translocation wheat, particularly a breeding method for translocation wheat containing the wheat 7B stem rust resistance genotype, comprising the steps of using the primers, molecular markers, kits or identification methods of the present invention, wherein the phenotypic identification can be carried out by a spore spraying method, which is known in the art, for example, by spraying spores of the wheat-specific race Pgt of Puccinia graminearum to determine the stem rust resistance phenotype.

[0062] Example 1

[0063] 1. Development of molecular marker K-71551

[0064] First, the third-generation sequencing of the laboratory wheat variety sdaup29 was performed and its sequence was compared with the Chinese Spring v2.1 reference genome to identify the INDEL loci. The primers were designed based on the Chinese Spring reference genome and the sdaup29 chromosome sequence using the NCBI primer designing tools website and TBtools-II software ( Figure 1 ), genomic DNA of wheat sdauP29 and RIL populations were extracted respectively, and specific INDEL molecular markers were screened for molecular marker positioning after polymerase chain reaction amplification and electrophoresis detection. sdauP29 is a hybrid of Elytrium longispinum and common wheat.

[0065] 2. Linkage analysis using molecular marker K-71551 combined with phenotype

[0066] At Shandong Agricultural University, an artificial climate chamber was used to carry out generation. The wheat variety carrying the sdauP29 resistance gene was used as the male parent and the wheat variety susceptible to stem rust, Chinese spring ph1b, was used as the female parent for hybridization. The single-grain method was used to carry out generation to the F7 generation. The phenotypic identification of each strain of the RIL population was carried out, and it was found that the single plant 142 still showed phenotypic separation. Further phenotypic analysis and genotypic identification of its secondary segregation population found that the K-71551 marker was completely consistent with the phenotype, proving that it is a tightly linked marker of the sdauP29 resistance gene and can be used for molecular marker selection.

[0067] The phenotype of the homozygous lines of the offspring was identified, with 10 seeds per plant. Lines with non-segregating phenotypes were selected for molecular marker identification of genotypes. K-7155F / R was able to amplify a 1058bp band in the resistant lines with consistent phenotypes, while only a 1555bp band was amplified in the susceptible lines with consistent phenotypes, indicating that the marker was co-segregated with the phenotype and could be used as a tightly linked molecular marker.

[0068] Specifically, the method for identifying wheat stem rust resistance genotype based on molecular marker K-71551 comprises the following steps:

[0069] (1) Extraction of sample genomic DNA using the CTAB method

[0070] Cut 2-3cm wheat leaves and put them into a 2mL centrifuge tube (put 2 small steel balls in advance). After freezing the leaves with liquid nitrogen, quickly grind them into powder (the parameters of the plant cell tissue disruptor are set to a frequency of 40Hz and a time of 40sec), add 800μL of CTAB extraction buffer preheated to 65℃ in a water bath, and water bath at 65℃ for 30min. Then, add an equal volume of three mixed solutions (phenol: chloroform: isoamyl alcohol = 25:24:1), mix well and let stand for 3min, centrifuge at 12000rpm for 10min, absorb 700μL of supernatant, add 700μL of isopropanol and 70μL of sodium acetate, mix well, and let stand for 15min. Subsequently, centrifuge at 12000rpm for 10min, pour off the supernatant, and the white precipitate is genomic DNA. After washing twice with 600μL 70% ethanol, dissolve the precipitate with ddH2O and store at low temperature for later use.

[0071] (2) PCR amplification using extracted DNA as template

[0072] The PCR reaction system includes: 1 μL of DNA template, 5 μL of 2×Taq Master Mix, 0.5 μL of 10 μmol / L primers K-71551F and K-71551R, and double distilled water to 10 μL.

[0073] PCR reaction program: 95°C for 5 min; 94°C for 15 s, 57°C for 15 s, 72°C for 30 s, 35 cycles; 72°C for 5 min.

[0074] (3) Detection of PCR products by 1% agarose gel.

[0075] The electrophoresis results showed that primers K-71551F and K-71551R amplified a 1058 bp band, indicating that individual plants 1, 2, 3, 10, and 12 of the F7 generation secondary segregation population of sdaup29 carried the stem rust resistance gene of chromosome 7BL of sdauP29; no band could be amplified or the band size was inconsistent, indicating that individual plants 4, 5, 6, 7, 8, 9, 11, 13, 14, and 15 of the F7 generation secondary segregation population of sdaup29 did not contain the resistance gene ( Figure 2 Primers K-71551F and K-71551R can amplify a 1058 bp band in the resistant families with consistent offspring phenotypes, indicating that all offspring plants carry the resistance gene ( Figure 3 ).

[0076] Example 2

[0077] 1. Development of molecular marker K-71810

[0078] Primers were designed based on the conservative sequences of Chinese Spring 2.1 and sdauP29 wheat material reference sequences using the NCBI primer designing tools website ( Figure 4 ), extracted the genomic DNA of Chinese Spring and sdauP29 respectively, and compared the differences after PCR amplification and electrophoresis detection. Based on the differences in the bands between the two, specific primers for identifying the stem rust resistance gene were designed.

[0079] 2. Identification of the wheat stem rust resistance gene using molecular marker K-71810 assisted selection

[0080] At Shandong Agricultural University, an artificial climate chamber was used for additional generation. The sdauP29 wheat material was used as the donor parent and the stem rust-susceptible wheat variety Chinese Spring was used as the recurrent parent. Combined with molecular marker-assisted selection, one backcross was performed to obtain the BC1F1 generation population. The molecular marker K-71810 was used for auxiliary selection to retain the individual plants carrying the stem rust resistance gene. The BC1F1 generation individual plants carrying the stem rust resistance gene were self-pollinated once to obtain the BC1F2 generation population and planted in the artificial climate chamber. The stem rust resistance of the individual plants was identified by spraying spores (key strains such as Figure 5 ) and molecular marker-assisted detection was performed. All strains that could amplify the 560 bp fragment showed resistance to stem rust MTGSM, and the identification results of molecular markers were consistent with the phenotype.

[0081] The method for identifying wheat stem rust resistance genotype based on molecular marker K-71810 comprises the following steps:

[0082] (1) Extraction of sample genomic DNA using the CTAB method

[0083] Cut 2-3cm wheat leaves and put them into a 2mL centrifuge tube (put in 2 4mm steel balls in advance). After freezing the leaves with liquid nitrogen, quickly grind them into powder, add 800μL of CTAB extraction buffer preheated to 65℃, and water bath at 65℃ for 45min. Then, add an equal volume of chloroform, mix well and let stand for 3min, centrifuge at 12000rpm for 10min, extract 700μL of supernatant, add 700μL of isopropanol and 70μL of sodium acetate, mix well, and let stand for 15min. Subsequently, centrifuge at 12000rpm for 10min, pour off the supernatant, and precipitate as genomic DNA. After washing twice with 600μL 70% ethanol, dissolve the precipitate with double distilled water and store at low temperature for later use.

[0084] (2) PCR amplification using extracted DNA as template

[0085] The PCR reaction system includes: 1 μL of DNA template, 5 μL of 2×Taq Master Mix, 0.5 μL of 10 μmol / L primers K-71810F and K-71810R, and double distilled water to 10 μL.

[0086] The PCR reaction program was 95°C for 5 min; 95°C for 15 s, 57°C for 15 s, 72°C for 60 s, 35 cycles; 72°C for 5 min.

[0087] (3) Detection of PCR products by 8% polyacrylamide gel.

[0088] The electrophoresis results showed that primers K-71810F and K-71810R amplified a 560 bp band, indicating that wheat sdauP29BC1F2 generation plants 6, 8, 9, 11, 13, 17, 22, and 27 carried the stem rust resistance gene; and amplified a 526 bp DNA band, indicating that wheat sdauP29 BC1F2 generation plants 1, 2, 3, 4, 5, 7, 10, 12, 14, 15, 16, 18, 19, 20, 21, 23, 24, 25, and 26 did not contain the stem rust resistance gene ( Figure 6 ).

[0089] The molecular markers in the present invention are closely linked to the stem rust resistance gene in wheat. The molecular markers K-71551 and K-71810 can improve the efficiency of molecular marker-assisted selection and can be directly applied to wheat stem rust resistance production practice, providing technical support for the creation and breeding of wheat improved lines carrying stem rust resistance genes through molecular marker-assisted selection.

[0090] 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 application description and examples are exemplary only.

Claims

1. A primer for detecting a wheat stem rust resistance gene-linked molecular marker, characterized in that: The primers include: (1) K-71551F: the sequence of which is shown in SEQ ID NO. 1, K-71551R: the sequence of which is shown in SEQ ID NO. 2; and / or (2) K-71810F: its sequence is shown in SEQ ID NO.3, K-71810R: its sequence is shown in SEQ ID NO.

4.

2. The primer for detecting wheat stem rust resistance gene linked molecular marker according to claim 1, characterized in that: The molecular marker has the sequence shown in SEQ ID NO.5 and / or SEQ ID NO.

6.

3. A molecular marker linked to a wheat stem rust resistance gene, characterized in that: Amplified by the primers described in claim 1 or 2.

4. A kit for detecting molecular markers linked to wheat stem rust resistance genes, characterized in that: Comprising the primers described in claim 1 or 2.

5. Use of the primer according to claim 1 or 2, the molecular marker according to claim 3 or the kit according to claim 4 in wheat genetic analysis.

6. The use according to claim 5, characterized in that: The genetic analysis includes genetic diversity analysis, germplasm identification or assisted breeding.

7. A method for identifying whether a plant contains a wheat stem rust resistance gene, characterized in that: The method comprises the step of detecting whether there is a molecular marker linked to a wheat stem rust resistance gene in the DNA of the plant to be identified, wherein the molecular marker is amplified using the primers described in claim 1 or 2.

8. The method for identifying whether a plant contains a wheat stem rust resistance gene according to claim 7, characterized in that: The following steps are involved: (1) extracting sample DNA from the plant sample to be identified; (2) a step of amplifying the nucleic acid containing the molecular marker from the sample DNA by PCR using primers; and (3) Identifying the plant by detecting the length of the sample PCR product by electrophoresis.

9. The method for identifying whether a plant contains a wheat stem rust resistance gene according to claim 7, characterized in that: The reaction system used for amplification included: 1 μL of DNA template, 5 μL of 2× Taq Master Mix, 0.5 μL of primer K-71551F at a concentration of 10 μmol / L, 0.4 μL of primer K-71551R at a concentration of 10 μmol / L, double distilled water was added to 10 μL, and the reaction program was: 95°C for 5 min; 95°C for 15 s, 55°C for 15 s, 72°C for 30 s, 35 cycles; 72°C for 5 min; Preferably, the reaction system for amplification includes: 1 μL of DNA template, 5 μL of 2×Taq Master Mix, 0.5 μL of primer K-71810F at a concentration of 10 μmol / L, 0.5 μL of primer K-71810R at a concentration of 10 μmol / L, double distilled water is added to 10 μL, and the reaction program is: 95°C for 5 min; 94°C for 15 s, 57°C for 15 s, 72°C for 1 min, 35 cycles; 72°C for 5 min; Preferably, amplification of DNA bands of 1058 bp and / or 560 bp indicates that the sample contains a stem rust resistance gene, and failure to amplify a band of the same size indicates that the sample does not contain a stem rust resistance gene.

10. A method for breeding translocation wheat, characterized in that: include: (1) using wheat sdauP29 carrying a stem rust resistance gene as the male parent and wheat variety Chinese spring susceptible to stem rust as the female parent for hybridization, using single seed propagation to perform phenotypic identification on each strain of the RIL population, further performing phenotypic analysis on the phenotypic segregation secondary segregation population, and performing genotypic identification using the primers described in claim 1 or 2, the molecular markers described in claim 3, the kit described in claim 4, or the method described in claim 8 or 9, to isolate homozygous offspring strains; (2) performing progeny homozygous strain identification on the progeny homozygous strains, selecting strains with non-segregating phenotypes and performing genotyping identification using the primers described in claim 1 or 2, the molecular markers described in claim 3, the kit described in claim 4, or the method described in any one of claims 7 to 9, and determining resistant strains with co-segregation of the molecular markers and phenotypes; or (1') using sdauP29 wheat material as the donor parent and Chinese spring wheat variety susceptible to stem rust as the recurrent parent, backcrossing to obtain a BC1F1 generation population, and retaining individual plants carrying the stem rust resistance gene using the primers described in claim 1 or 2, the molecular markers described in claim 3, the kit described in claim 4, or the method described in any one of claims 7 to 9; (2') Self-pollinating individual plants of the BC1F1 generation carrying the stem rust resistance gene to obtain a BC1F2 generation population and planting it, identifying the stem rust resistance of the individual plants by spraying spores, and detecting using the primers described in claim 1 or 2, the molecular markers described in claim 3, the kit described in claim 4, or the method described in any one of claims 7 to 9.

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