Molecular marker and primer for detecting leaf rust resistance gene of wheat and application of molecular marker and primer

Abstract

The invention discloses a molecular marker and a primer for detecting a leaf rust resistant gene of wheat and application of the molecular marker and the primer. The method is not influenced by external environmental factors and is simple and convenient to operate. Primers of molecular markers Lsdau-33 and Xsdau3829 in close linkage with the wheat leaf rust resistance gene are utilized, PCR amplification electrophoresis detection is carried out, germplasm materials carrying the wheat leaf rust 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 wheat leaf rust resistance gene, 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 molecular marker, a primer and an application thereof for detecting a wheat leaf rust resistance gene. Background Art

[0002] Wheat is one of the most important food crops in the world. However, the current growth in wheat production is unlikely to meet the needs of global population growth in the coming decades. A key measure to address this challenge is to improve wheat's resistance to various diseases, which can recover the 15-20% yield loss caused by diseases each year. Leaf rust is a fungal disease of wheat. Frequent mutations in leaf rust toxicity genes and large-scale monoculture of disease-resistant varieties often lead to the "loss" of disease-resistant genes. Therefore, in-depth research on the genetics of wheat leaf rust resistance and continuous research to discover and mark new genes for wheat leaf rust resistance is a long and arduous task.

[0003] At present, nearly 100 leaf rust resistance genes have been found in wheat. Except for Lr4, Lr5, Lr6, Lr7 and Lr8, which have not yet been isolated as single gene lines, most of the others have been located on specific wheat chromosomes. These genes often have great differences in resistance under different genetic backgrounds, regions and temperature conditions, and many genes have lost their resistance. Therefore, while scientists are discovering resistance genes in wheat, they are also gradually paying attention to discovering new excellent resistance sources from closely related species of wheat.

[0004] Studies have shown that cultivating and planting disease-resistant varieties is the most economical and effective measure to solve the problem of wheat leaf rust. However, there are currently few effective resistance sources available for production. The 7E chromosome of Elytium longipedum carries multiple disease-resistant genes, which is currently known to be the main disease-resistant gene. Using biotechnology methods, disease-resistant genes can be transferred to promoted wheat varieties susceptible to leaf rust. However, traditional methods are time-consuming and labor-intensive, phenotypic identification is difficult, and breeding efficiency is low. Molecular marker-assisted selection breeding can effectively 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 provides a wheat leaf rust resistance gene linked molecular marker, detection primer and application, thereby providing a theoretical basis for wheat leaf rust resistance molecular breeding. Specifically, the present invention includes the following contents.

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

[0007] (1) Xsdau3829F: whose sequence is shown in SEQ ID NO.1; Xsdau3829R: whose sequence is shown in SEQ ID NO.2; and / or

[0008] (2) Lsdau-33F: its sequence is shown in SEQ ID NO.3; Lsdau-33R: its sequence is shown in SEQ ID NO.4.

[0009] In certain embodiments, according to the primers for detecting molecular markers linked to wheat leaf rust resistance genes of the present invention, the molecular markers include 401 bp and / or 746 bp nucleic acid fragments amplified by the primers.

[0010] In certain embodiments, according to the primers for detecting molecular markers linked to wheat leaf 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 molecular marker linked to a wheat leaf rust resistance gene, 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 molecular markers linked to wheat leaf rust resistance genes, comprising the primers described in 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 leaf rust resistance gene, which includes the step of detecting whether there is a wheat leaf rust resistance gene-linked molecular marker in the DNA of the plant to be identified, wherein the molecular marker includes a 401bp and / or 746bp 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 leaf 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 leaf rust resistance gene according to the present invention, the reaction system for amplification comprises: 1 μL of DNA template, 10 μL of 2×Taq Master Mix, 1 μL of primer Lsdau-33F at a concentration of 10 μmol / L, 1 μL of primer Lsdau-33R at a concentration of 10 μmol / L, and double distilled water is added to 20 μL, and the reaction program of the PCR is: 95°C for 5 min; 95°C for 30 s, 62°C increment -0.7°C for 30 s, 72°C for 30 s, 10 cycles; 95°C for 5 min; 95°C for 30 s, 55°C for 30 s, 72°C for 30 s, 22 cycles, 72°C for 10 min.

[0021] In certain embodiments, according to the method for identifying whether a plant contains a wheat leaf rust resistance gene according to the present invention, the reaction system for amplification includes: 1 μL of DNA template, 7.5 μL of 2×Taq Master Mix, 1 μL of primer Xsdau3829F with a concentration of 10 μmol / L, 1 μL of primer Xsdau3829R with a concentration of 10 μmol / L, and double distilled water is added to 15 μL, and the reaction program of the PCR is: 95°C for 5 min; 95°C for 30 s, 60°C for 30 s, 72°C for 30 s, 35 cycles; 72°C for 10 min.

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

[0023] (1) hybridizing KS11695 and CS Ph1b mutant to obtain an F1 generation population, hybridizing the F1 generation population with the CS Ph1b mutant to obtain a BC1F1 generation population, determining the genotype of the hybrid offspring using the primers, molecular markers, kits or methods described in the present invention, and performing leaf rust phenotype identification to obtain wheat-Elygropyrum elongatum short segment translocation line hybrids carrying a leaf rust resistance gene;

[0024] (2) self-pollinating the hybrid once to obtain individual seeds carrying a leaf rust resistance gene;

[0025] (3) Harvesting seeds from individual plants, and determining the genotype of the seeds of the individual plants using the primers, molecular markers, kits or methods described in the present invention. If all seeds carry the leaf rust resistance gene, the individual plant is a homozygous translocation line wheat.

[0026] The invention is not affected by external environmental factors and is simple and convenient to operate. The primers of the molecular markers closely linked to the wheat leaf rust resistance gene are used to perform PCR amplification and electrophoresis detection, and the germplasm material carrying the wheat leaf rust resistance gene is screened out according to the size of the target band. The molecular markers of the invention can accelerate the research and utilization of excellent wheat leaf rust resistance genes, and provide new materials for the genetic improvement of wheat disease resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The sequence amplified by the molecular marker Xsdau3829 on chromosome 7E of Elymus elongatus (the yellow content is the primer sequence).

[0028] Figure 2 Detection results of molecular marker Xsdau3829 in the BC1F2 population samples of the wheat-Elymus elongatus short segment translocation line (M, DL2000 Marker, the bands are 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp respectively; 1, 2, 3, 4, BC1F2 generation individual plants; 5, DL2000 Marker; 6, donor parent KS11695 (7DS.7el1L); 7, Chinese Spring Ph1b mutant).

[0029] Figure 3 The DNA fragments amplified by molecular markers were blasted in the genomes of diploid Elytrigia elongata and Triticum aestivum.

[0030] Figure 4 Identification of leaf rust resistance phenotypes of individuals with different molecular marker Xsdau3829 genotypes in the wheat-Elygropyrum elongatum short segment translocation line.

[0031] Figure 5 The sequence amplified by the molecular marker Lsdau-33 on chromosome 7E of Elymus elongatus (the yellow content is the primer sequence).

[0032] Figure 6 Detection results of molecular marker Lsdau-33 in BC1F2 population samples of wheat-Elymus elongatus short segment translocation line (M, DL2000 Marker bands are 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp respectively; 1, 2, BC1F2 generation single plants; 3, donor parent KS11695 (7DS.7el1L); 4, Chinese Spring Ph1b mutant).

[0033] Figure 7 The DNA fragments amplified by molecular markers were blasted in the genomes of common wheat and thinleaf elytris.

[0034] Figure 8Identification of leaf rust resistance phenotypes of individuals with different molecular marker Lsdau-33 genotypes in the BC1F2 population of the wheat-Elygropyrum elongatum short segment translocation line. DETAILED DESCRIPTION

[0035] 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.

[0036] 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.

[0037] 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.

[0038] Molecular markers

[0039] In a first aspect of the present invention, a specific molecular marker is provided, which has a characteristic sequence for distinguishing whether a plant (such as wheat) carries a leaf rust resistance gene. In the present invention, leaf rust refers to a rust-like phenotype of the infected part caused by wheat leaf rust.

[0040] The molecular marker ("characteristic sequence") in the present invention refers to a gene fragment that only appears in leaf rust-resistant wheat, is linked to the leaf rust-resistant gene, and is closely related to the leaf rust-resistant phenotype.

[0041] Primer combinations

[0042] 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, including a forward primer and a reverse primer. In a specific embodiment, the forward primer used to amplify Xsdau3829 has the sequence shown in SEQ ID NO.1, and the reverse primer has the sequence shown in SEQ ID NO.2. In another specific embodiment, the forward primer used to amplify Lsdau-33 has the sequence shown in SEQ ID NO.3, and the reverse primer has the sequence shown in SEQ ID NO.4.

[0043] In a specific embodiment, the molecular marker amplified from SEQ ID NO.1-2 has the sequence shown in SEQ ID NO.5. In another specific embodiment, the molecular marker amplified from SEQ ID NO.3-4 has the sequence shown in SEQ ID NO.6.

[0044] Reagent test kit

[0045] One aspect of the present invention provides a kit for detecting wheat leaf rust resistance gene linked molecular markers, which comprises the primer combination of the present invention and an optional reagent for polymerase chain reaction. The reagent for polymerase chain reaction includes any reagent used in conventional PCR, such as polymerase, buffer, etc.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] application

[0051] 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 the leaf rust resistance genotype of the wheat-Elygropyrum elongatum short segment translocation line.

[0052] Identification methods

[0053] One aspect of the present invention provides a method for identifying whether a plant contains a wheat leaf rust resistance gene, which includes the step of detecting whether there is a wheat leaf rust resistance gene-linked molecular marker in the DNA of the plant to be identified.

[0054] In the present invention, the method for detecting the presence of a wheat leaf rust resistance gene-linked molecular marker can be any method known in the art, examples of which include methods for amplifying nucleic acids containing characteristic sequences and methods for directly sequencing plant genomes.

[0055] In certain embodiments, the method of identifying whether a plant contains a wheat leaf 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.

[0056] In an exemplary embodiment, a method for identifying whether a plant comprises a wheat leaf rust resistance gene comprises the following steps:

[0057] (1) extracting sample DNA from the plant sample to be identified;

[0058] (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;

[0059] (3) Determine whether the plant contains the wheat leaf rust resistance gene by detecting the length of the sample PCR product by electrophoresis.

[0060] 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 leaf rust resistance gene amplification fragment of 401 bp and / or 746 bp.

[0061] Breeding methods

[0062] The present invention also provides a translocation wheat breeding method, in particular a homozygous wheat-Elygropyrum longissima short segment translocation wheat breeding method of leaf rust resistance genotype, comprising the steps of using the primers, molecular markers, kits or identification methods of the present invention.

[0063] In a preferred embodiment, the method comprises:

[0064] (1) hybridizing KS11695 and CS Ph1b mutant to obtain an F1 generation population, hybridizing the F1 generation population with the CS Ph1b mutant to obtain a BC1F1 generation population, determining the genotype of the hybrid offspring using the primers, molecular markers, kits or methods described in the present invention, and performing leaf rust phenotype identification to obtain wheat-Elygropyrum elongatum short segment translocation line hybrids carrying a leaf rust resistance gene;

[0065] (2) self-pollinating the hybrid to obtain individual seeds carrying a leaf rust resistance gene;

[0066] (3) Harvesting seeds from individual plants, and determining the genotype of the seeds of the individual plants using the primers, molecular markers, kits or methods described in the present invention. If all seeds carry the leaf rust resistance gene, the individual plant is a homozygous translocation line wheat.

[0067] Wherein, in step (1), when a molecular marker linked to a leaf rust resistance gene is detected and the hybrid has a leaf rust resistance phenotype, the hybrid offspring is determined to be a wheat-Elygropyrum elongatum short segment translocation line hybrid carrying a leaf rust resistance gene. In step (2), methods known in the art can be used, such as infection with wheat leaf rust spores to determine the leaf rust resistance phenotype.

[0068] Example 1

[0069] 1. Development of molecular marker Xsdau3829

[0070] The invention uses an induced (Chinese Spring ph1b mutant) wheat-Thinopyrum elongatum leaf rust resistance short segment translocation line, according to the published Chinese Spring D genome sequence (Chinese Spring v4.0) and Thinopyrum elongatum (Thinopyrum elongatum v1.0 D-3458) genome database, and through Primer Premier 6.0 software, develops a pair of primers for Thinopyrum elongatum 7E specific molecular markers, wherein the specific molecular marker has a specific amplification band in the wheat-Thinopyrum elongatum leaf rust resistance short segment translocation line but has no amplification band in common wheat varieties, respectively extracts genomic DNA of wheat and Thinopyrum elongatum, and after PCR amplification and sequencing analysis, the obtained amplified sequence is from Thinopyrum elongatum, thereby designing specific primers for identifying the Thinopyrum elongatum leaf rust resistance short segment.

[0071] 2. Transfer and utilization of wheat leaf rust resistance genes by assisted selection with molecular marker Xsdau3829

[0072] In the greenhouse of Shandong Agricultural University, a hybrid combination (KS11695×CS Ph1b mutant) was configured to obtain F1, which was then hybridized with CS Ph1b mutant to obtain BC1F1. The obtained plants were identified by molecular markers. Based on the molecular marker identification, BC1F1 carrying the target gene and exogenous fragments was self-pollinated to obtain BC1F2. With the help of molecular markers, a wheat-Elygropyrum longipedula short fragment translocation line was obtained, and leaf rust resistance was identified. Figure 2 It can be seen that there is no amplified band of Xsdau3829 in wheat, but there is a 746 bp band in Elymus ovata and wheat-Elymus ovata short fragment translocation line that is closely linked to the leaf rust resistance gene and can be used for molecular marker-assisted selection of leaf rust resistance genes.

[0073] The corresponding DNA fragment ( Figure 1 ) is a specific sequence on the 7E genome of Elytrium longipedum, which is closely linked and co-segregated with the leaf rust resistance gene. The relevant sequence information can be used to distinguish between disease-resistant and disease-susceptible plants of wheat leaf rust and used for molecular breeding assisted selection. This molecular marker can accurately mark the leaf rust resistance gene in Elytrium longipedum with an accuracy rate of more than 93%.

[0074] The method for identifying a wheat broad-spectrum leaf rust resistance genotype based on the molecular marker Xsdau3829 comprises the following steps:

[0075] (1) Extraction of sample genomic DNA using the CTAB method

[0076] Cut 2-3cm wheat leaves and put them into a 2mL centrifuge tube. After freezing the leaves with liquid nitrogen, quickly grind them into powder, add 400μL 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 350μL of supernatant, add 350μL of isopropanol and 35μL 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 500μL 70% ethanol, dissolve the precipitate in water and store at low temperature for later use.

[0077] (2) PCR amplification using extracted DNA as template

[0078] The PCR reaction system includes: 1 μL of DNA template, 7.5 μL of 2×Taq Master Mix, 1 μL of primers Xsdau3829F and Xsdau3829R at a concentration of 10 μmol / L, and water is added to 15 μL.

[0079] The PCR reaction program was 95°C for 5 min; 95°C for 30 s, 60°C for 30 s, 72°C for 30 s, 35 cycles; 72°C for 10 min.

[0080] (3) Detection of PCR products by 1% agarose gel

[0081] The electrophoresis results showed that primer Xsdau3829 amplified a 746 bp band, indicating that wheat-Elymus elongatus short segment translocation line BC1F2 generation single plants 1 and 4 carried the leaf rust resistance gene; no band could be amplified, indicating that wheat-Elymus elongatus short segment translocation line BC1F2 generation single plants 2 and 3 did not carry the leaf rust resistance gene ( Figure 2 Primer Xsdau3829 could amplify a 746 bp band in both plants 1 and 4 of the wheat-Elygropyrum short segment translocation line BC1F2, and the plants showed resistance to the disease. However, it could not amplify a band in plants 2 and 3 of the wheat-Elygropyrum short segment translocation line BC1F2, and the plants showed susceptible to the disease ( Figure 2 ).Depend on Figure 2 , Figure 4 It can be seen that Xsdau3829 has no amplified band in wheat, but a 746 bp band in Thinopyrum and wheat-Thinopyrum short fragment translocation lines is closely linked to the leaf rust resistance gene and the leaf rust resistance phenotype ( Figure 4 ), which can be used for molecular marker-assisted selection of leaf rust resistance genes.

[0082] Example 2

[0083] 1. Development of the molecular marker Lsdau-33

[0084] According to the published Chinese Spring D genome sequence (Chinese Spring Triticum4.0chromosomes) and Thinopyrum elongatum chromosome 7 genome database, primers were designed according to the conservative segment of the reference sequence in the Thinopyrum elongatum genome database using PrimerPremier 6.0 software ( Figure 5 ), genomic DNA of wheat and Elytrimus elongatus was extracted respectively, and after PCR amplification and sequencing analysis, the amplified sequence obtained was from Elytrimus elongatus, thus specific primers for identifying molecular markers linked to leaf rust resistance genes were designed.

[0085] The corresponding DNA fragment ( Figure 5 ) is a specific sequence on the 7E genome of Elymus elongatus, which is closely linked to the leaf rust resistance gene and co-segregates. The relevant sequence information can be used to distinguish between disease-resistant and disease-susceptible plants of wheat leaf rust and used for molecular breeding assisted selection. This molecular marker can accurately mark the leaf rust resistance gene in Elymus elongatus with an accuracy rate of more than 97%.

[0086] 2. Transfer and utilization of wheat leaf rust resistance genes by assisted selection with molecular marker Lsdau-33

[0087] In the greenhouse of Shandong Agricultural University, a hybrid combination (KS11695×CS Ph1b mutant) was configured to obtain F1; the obtained F1 was hybridized with the CS-Ph1b mutant to obtain BC1F1, and the obtained plants were identified by molecular markers. On the basis of molecular marker identification, BC1F1 carrying the target gene and exogenous fragments was self-pollinated to obtain BC1F2. With the help of molecular markers, a wheat-Elygropyrum elongatum short fragment translocation line was obtained, and leaf rust resistance was identified.

[0088] The method for identifying wheat leaf rust resistance genotype based on molecular marker Lsdau-33 comprises the following steps:

[0089] (1) Extraction of sample genomic DNA using the CTAB method

[0090] Cut 2-3cm wheat leaves and put them into a 2mL centrifuge tube. After freezing the leaves with liquid nitrogen, quickly grind them into powder, add 400μL 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 350μL of supernatant, add 350μL of isopropanol and 35μL 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 500μL 70% ethanol, dissolve the precipitate in water and store at low temperature for later use.

[0091] (2) PCR amplification using extracted DNA as template

[0092] The PCR reaction system includes: 1 μL of DNA template, 10 μL of 2×Taq Master Mix, 1 μL of primers Lsdau-33F and Lsdau-33R at a concentration of 10 μmol / L, and water is added to 20 μL.

[0093] PCR reaction program: 95°C for 5 min; 95°C for 30 s, 62°C increasing by -0.7°C for 30 s, 72°C for 30 s, 10 cycles; 95°C for 5 min; 95°C for 30 s, 55°C for 30 s, 72°C for 30 s, 22 cycles; 72°C for 10 min.

[0094] (3) Detection of PCR products by 1% agarose gel

[0095] The electrophoresis results showed that the primer Lsdau-33 amplified a 401 bp band, indicating that the wheat-Elymus elongatus short segment translocation line BC1F2 generation single plants 1 and 3 carried the leaf rust resistance gene; no band could be amplified, indicating that the wheat-Elymus elongatus short segment translocation line BC1F2 generation single plants 2 and 4 did not carry the leaf rust resistance gene ( Figure 6 Primer Lsdau-33 could amplify a 401 bp band in both plants 1 and 3 of the wheat-Elygropyrum short segment translocation line BC1F2, and the plants showed resistance to the disease. However, it could not amplify a band in plants 2 and 4 of the wheat-Elygropyrum short segment translocation line BC1F2, and the plants showed susceptible to the disease ( Figure 8 ).

[0096] Depend on Figure 6 , Figure 8 It can be seen that there is no amplified band of Lsdau-33 in wheat, but there is a 401 bp band in Elymus ovata and wheat-Elymus ovata short fragment translocation line, which is closely linked to the leaf rust resistance gene and the leaf rust resistance phenotype, and can be used for molecular marker-assisted selection of leaf rust resistance genes.

[0097] 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 molecular marker linked to a wheat leaf rust resistance gene, characterized in that: The primers include: (1) Xsdau3829F: whose sequence is shown in SEQ ID NO.1; Xsdau3829R: whose sequence is shown in SEQ ID NO.2; and / or (2) Lsdau-33F: its sequence is shown in SEQ ID NO.3; Lsdau-33R: its sequence is shown in SEQ ID NO.

4.

2. The primer for detecting a wheat leaf 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 leaf 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 leaf 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 leaf rust resistance gene, characterized in that: The method comprises the steps of detecting whether there is a molecular marker linked to a wheat leaf rust resistance gene in the DNA of the plant to be identified, wherein the primers used to amplify the molecular marker include: (1) Xsdau3829F: whose sequence is shown in SEQ ID NO.1; Xsdau3829R: whose sequence is shown in SEQ ID NO.2; and / or (2) Lsdau-33F: its sequence is shown in SEQ ID NO.3; Lsdau-33R: its sequence is shown in SEQ ID NO.

4.

8. The method for identifying whether a plant contains a wheat leaf 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 PCR product of the sample by electrophoresis; Preferably, the reaction system for amplification includes: 1 μL of DNA template, 10 μL of 2×Taq Master Mix, 1 μL of primer Lsdau-33F with a concentration of 10 μmol / L, 1 μL of primer Lsdau-33R with a concentration of 10 μmol / L, and double distilled water to 20 μL. The reaction program of the PCR is: 95°C for 5 min; 95°C for 30 s, 62°C with an increment of -0.7°C for 30 s, 72°C for 30 s, 10 cycles; 95°C for 5 min; 95°C for 30 s, 55°C for 30 s, 72°C for 30 s, 22 cycles, 72°C for 10 min; Preferably, the reaction system for amplification includes: 1 μL of DNA template, 7.5 μL of 2×Taq Master Mix, 1 μL of primer Xsdau3829F with a concentration of 10 μmol / L, 1 μL of primer Xsdau3829R with a concentration of 10 μmol / L, and double distilled water to 15 μL. The reaction program of the PCR is: 95°C for 5 min; 95°C for 30 s, 60°C for 30 s, 72°C for 30 s, 35 cycles; 72°C for 10 min.

9. The method for identifying whether a plant contains a wheat leaf rust resistance gene according to claim 7, characterized in that: Amplification of DNA bands of 401 bp and / or 746 bp indicates that the sample contains a leaf rust resistance gene, and failure to amplify bands of the same size indicates that the sample does not contain a leaf rust resistance gene.

10. A method for breeding translocation wheat, characterized in that: include: (1) hybridizing KS11695 and CS Ph1b mutant to obtain an F1 population, hybridizing the F1 population with the CS Ph1b mutant to obtain a BC1F1 population, determining the genotype of the hybrid offspring 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 performing leaf rust phenotype identification to obtain a wheat-Elygropyrum elongatum short segment translocation line hybrid carrying a leaf rust resistance gene; (2) self-pollinating the hybrid to obtain individual seeds carrying a leaf rust resistance gene; (3) Harvesting seeds from individual plants, and determining the genotype of the seeds of the individual plants 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; if all the seeds carry the leaf rust resistance gene, the individual plant is a homozygous translocation line wheat.

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