Wheat powdery mildew resistance gene linked molecular marker, primer combination and application

Abstract

The invention discloses a wheat powdery mildew resistance gene linked molecular marker, a primer combination and application. The molecular marker is closely linked with the powdery mildew resistant gene, can be directly applied to wheat powdery mildew resistant production practice, provides technical support for breeding a wheat improved line carrying the powdery mildew resistant gene, and can improve the efficiency of molecular marker-assisted selection, thereby accelerating the research and utilization of the good powdery mildew resistant gene of wheat. And a new material is provided for wheat disease-resistant genetic improvement. In addition, the method has the advantages of being not affected by external environmental factors, easy and convenient to operate and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of wheat molecular breeding, and particularly relates to a wheat powdery mildew resistance gene linkage molecular marker, a primer combination and an application. Background Art

[0002] Wheat powdery mildew is an important disease that seriously affects the world's wheat production and is caused by Blumeria graminis, a wheat-specific fungus of the Poaceae family. Wheat powdery mildew is widely distributed around the world, especially in temperate regions such as Europe, North America, Asia, and Australia. It has many physiological subspecies and rapid mutations. It is distributed in all major wheat-growing areas, posing a serious threat to the safe production of wheat. In China, wheat powdery mildew may occur in almost all wheat-growing areas, especially in the main wheat-producing areas such as the North China Plain, the Huanghuaihai River Basin, and the Northwest. The impact on wheat yield depends on the early and late occurrence of the disease and the severity of the disease, which can cause a maximum reduction in wheat yield of more than 50%.

[0003] Wild emmer wheat (Triticum dicoccoides, 2n = 4x = 28, AABB) is the wild tetraploid ancestor of common wheat (Triticum aestivum, 2n = 6x = 42, AABBDD), and its genome consists of AA and BB, which has a high homology with the A and B chromosome groups of common wheat. Wild emmer wheat originated in the Fertile Crescent in the Middle East and is widely distributed in the current Israel, Turkey, Iran, Syria and other countries.

[0004] There is a resistance gene at the end of the long arm of chromosome 4A of wild emmer wheat on the northern slope of the Evolution Valley in Israel, which can effectively resist powdery mildew. Through the method of distant hybridization, this gene can be transferred to the promoted wheat varieties susceptible to powdery mildew. However, traditional methods are time-consuming and labor-intensive, phenotypic identification is difficult, and breeding efficiency is low. Therefore, there is an urgent need to develop molecular markers linked to powdery mildew resistance genes for genetic analysis such as assisted selection breeding. Summary of the invention

[0005] In order to solve at least some of the problems in the above-mentioned prior art, the present invention uses a molecular marker closely linked to the wheat powdery mildew resistance gene MlNFS10 to perform PCR amplification and electrophoresis detection, and can screen out germplasm materials carrying the wheat powdery mildew resistance gene MlNFS10 according to the size of the target band. Specifically, the present invention includes the following contents.

[0006] The first aspect of the present invention provides a primer combination for detecting a wheat powdery mildew resistance gene-linked molecular marker, wherein the primer combination comprises a first primer pair and / or a second primer pair, wherein the first primer pair comprises a forward primer SDAU-8259F having a sequence as shown in SEQ ID NO.1 and a reverse primer SDAU-8259R having a sequence as shown in SEQ ID NO.2, and the second primer pair comprises a forward primer SDAU-6050F having a sequence as shown in SEQ ID NO.3 and a reverse primer SDAU-6050R having a sequence as shown in SEQ ID NO.4.

[0007] In certain embodiments, according to the primer combination for detecting molecular markers linked to wheat powdery mildew resistance genes of the present invention, the molecular markers have sequences shown in SEQ ID NO.5 and / or SEQ ID NO.6.

[0008] The second aspect of the present invention provides a wheat powdery mildew resistance gene-linked molecular marker, which is obtained by amplifying the primer combination of the present invention.

[0009] The third aspect of the present invention provides a kit for detecting molecular markers linked to wheat powdery mildew resistance genes, comprising the primer combination described in the present invention.

[0010] The fourth aspect of the present invention provides the use of the primer combination, the molecular marker, or the kit of the present invention in wheat genetic analysis.

[0011] In certain embodiments, according to the use of the present invention, the genetic analysis includes genetic diversity analysis, germplasm identification or assisted breeding.

[0012] The fifth aspect of the present invention provides a method for identifying whether a plant contains a wheat powdery mildew resistance gene, which comprises the step of using the primer combination of the present invention to detect whether there is a wheat powdery mildew resistance gene-linked molecular marker in the DNA of the plant to be identified.

[0013] In certain embodiments, the method for identifying whether a plant comprises a wheat powdery mildew resistance gene according to the present invention comprises the following steps:

[0014] (1) extracting sample DNA from the plant sample to be identified;

[0015] (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; and

[0016] (3) Identifying the plant by detecting the length of the sample PCR product by electrophoresis.

[0017] In certain embodiments, according to the method for identifying whether a plant contains a wheat powdery mildew resistance gene according to the present invention, amplification of 241 bp and / or 377 bp DNA bands indicates that the sample contains the powdery mildew resistance gene, and failure to amplify a band of the same size indicates that the sample does not contain the powdery mildew resistance gene.

[0018] A sixth aspect of the present invention provides a translocation wheat breeding method, comprising:

[0019] (1) using wild emmer wheat NFS10 as a donor parent carrying a powdery mildew resistance gene and a powdery mildew susceptible wheat variety Shannong 47 as a recurrent parent for hybridization, and then hybridizing and backcrossing with a powdery mildew susceptible wheat variety Zhongyu 1428 to obtain a BC2F1 generation population, using the primer combination, the molecular marker, the kit, or the method of the present invention to determine the genotype of the backcross offspring, and perform powdery mildew phenotype identification to obtain a Shannong-wild emmer wheat short segment translocation line hybrid carrying a powdery mildew resistance gene;

[0020] (2) self-pollinating the hybrid to obtain a BC2F2 population, identifying the powdery mildew resistance of individual plants by spraying spores, and obtaining individual seeds carrying a powdery mildew resistance gene;

[0021] (3) Harvesting individual plants to obtain BC2F3 generation seeds, and determining the genotype of the individual plant seeds using the primer combination, the molecular marker, the kit, or the method described in the present invention. If all the seeds carry the powdery mildew resistance gene, the individual plant is a homozygous translocation line wheat.

[0022] The molecular marker of the present invention is closely linked to the powdery mildew resistance gene, can be directly applied to the production practice of wheat powdery mildew resistance, provides technical support for breeding wheat improved lines carrying powdery mildew resistance genes, can improve the efficiency of molecular marker-assisted selection, thereby accelerating the research and utilization of excellent wheat powdery mildew resistance genes, and provides new materials for wheat disease resistance genetic improvement. In addition, the present invention has the advantages of not being affected by external environmental factors, being simple and convenient to operate, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the sequence amplified by the molecular marker SDAU-8259 on chromosome 4A of wild emmer wheat (the yellow content is the primer sequence).

[0024] Figure 2These are the detection results of the molecular marker SDAU-8259 in the BC2F1 generation population samples of the transferred and utilized materials (the bands represent M, 1-10 from left to right, and the M band represents 2000bp, 1000bp, 750bp, 500bp, 250bp, and 100bp from top to bottom; in the amplified bands, 1 and 2 represent NFS10; 3 and 4 represent SFS2-12; 5 and 6 represent BC2F1 generation plants; 7 and 8 represent Zhongyu 1428; 9 and 10 represent Shannong 47).

[0025] Figure 3 These are the detection results of the molecular marker SDAU-8259 in the BC2F3 generation population samples of the transferred and utilized materials (the bands represent M, 1-8 from left to right, the M band is 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp from top to bottom; 1, 2, 3, 4, 5, 6 and 7 represent BC2F3 generation individual plants; 8 represents SFS2-12).

[0026] Figure 4 The powdery mildew resistance phenotype of individual plants with SDAU-8259 genotypes and some different molecular markers in the BC2F2 generation population of the transfer material was identified.

[0027] Figure 5 This is the sequence amplified by the molecular marker SDAU-6050 on chromosome 4A of wild emmer wheat (the yellow content is the primer sequence).

[0028] Figure 6 These are the detection results of the molecular marker SDAU-6050 in the BC2F1 generation population samples of the transferred and utilized materials (the bands represent M, 1-10 from left to right, and the M band represents 2000bp, 1000bp, 750bp, 500bp, 250bp, and 100bp from top to bottom; 1 and 2 represent NFS10; 3 and 4 represent SFS2-12; 5 and 6 represent BC2F1 generation individual plants; 7 and 8 represent Zhongyu 1428; 9 and 10 represent Shannong 47).

[0029] Figure 7 It is the detection result of molecular marker SDAU-6050 in the BC2F3 generation population sample of the transferred and utilized material (the bands represent M, 1-16 from left to right, and the M band represents 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp from top to bottom; 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 represent BC2F3 generation single plants; 11 and 12 represent NFS10; 13 and 14 represent SFS2-12; 15 represents the susceptible background material Zhongyu 1428; 16 represents the susceptible background material Shannong 47).

[0030] Figure 8 The powdery mildew resistance phenotype of individual plants of SDAU-6050 genotype with some different molecular markers in the BC2F2 generation population of the transfer material was identified. DETAILED DESCRIPTION

[0031] 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.

[0032] 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.

[0033] 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.

[0034] Molecular markers

[0035] In one 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 powdery mildew resistance gene. SDAU-8259 and SDAU-6050 are located within 5Mb of the MlNFS10 localization interval, and powdery mildew refers to powdery mildew caused by the wheat-specific fungus Blumeria graminacea of ​​the Poaceae family.

[0036] The molecular marker ("characteristic sequence") in the present invention refers to a gene fragment that only appears in powdery mildew-resistant wheat and is linked to the powdery mildew resistance gene and closely related to powdery mildew resistance, wherein the powdery mildew resistance gene refers to a resistance gene MlNFS10 located at the end of the long arm of chromosome 4A of wild two-grain wheat originating from the northern slope of the Evolution Canyon in Israel.

[0037] In the present invention, the molecular marker specifically refers to a sequence linked to a resistance gene against the wheat-specific fungus Blumeria graminearum of the Poaceae family.

[0038] In a preferred embodiment, the molecular marker has a sequence shown in SEQ ID NO. 5. In another preferred embodiment, the molecular marker has a sequence shown in SEQ ID NO. 6.

[0039] Primer combinations

[0040] In one aspect of the present invention, a primer combination for detecting a molecular marker linked to a wheat powdery mildew resistance gene is provided, which is used to amplify the molecular marker described in the present invention, and includes a forward primer and a reverse primer. In a preferred embodiment, the forward primer is SDAU-8259F having a sequence shown in SEQ ID NO.1, and the reverse primer is SDAU-8259R having a sequence shown in SEQ ID NO.2. In another preferred embodiment, the forward primer is SDAU-6050F having a sequence shown in SEQ ID NO.3, and the reverse primer is SDAU-6050R having a sequence shown in SEQ ID NO.4.

[0041] Reagent test kit

[0042] One aspect of the present invention provides a kit for detecting wheat powdery mildew 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] application

[0048] In one aspect of the present invention, the primer combination, molecular marker or kit of the present invention is used in wheat genetic analysis, 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 germplasm identification of powdery mildew resistant wheat. In another specific embodiment, the application includes assisted breeding of powdery mildew resistant wheat.

[0049] Identification methods

[0050] One aspect of the present invention provides a method for identifying whether a plant contains a wheat powdery mildew resistance gene, which comprises the step of using the primer combination of the present invention to detect whether there is a wheat powdery mildew resistance gene-linked molecular marker in the DNA of the plant to be identified.

[0051] In the present invention, the method for detecting the presence of a molecular marker linked to a wheat powdery mildew resistance gene may adopt any method known in the art, examples of which include methods of amplifying a nucleic acid containing a characteristic sequence, and methods of directly sequencing a plant genome.

[0052] In certain embodiments, the method of identifying whether a plant contains a wheat powdery mildew 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.

[0053] In an exemplary embodiment, a method for identifying whether a plant comprises a wheat powdery mildew resistance gene comprises the following steps:

[0054] (1) extracting sample DNA from the plant sample to be identified;

[0055] (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;

[0056] (3) Identify whether the plant contains the wheat powdery mildew resistance gene by detecting the length of the sample PCR product by electrophoresis.

[0057] In step (1), the "plant sample to be identified" refers to the whole plant or a part of the plant, such as plant roots, leaves or plant stems.

[0058] In a preferred embodiment, in step (2), the ratio of forward primer to reverse primer is 1:1. PCR reaction program: 95℃5min; 95℃30s, 55℃30s, 72℃30s, 35 cycles; 72℃5min. The reaction system for amplification includes: 1μL DNA template, 5μL 2×Taq Master Mix, 0.4μL primer SDAU-8259F with a concentration of 10μmol / L, 0.4μL primer SDAU-8259R with a concentration of 10μmol / L, and double distilled water to 10μL. In step (3), the length of the sample PCR product is detected by electrophoresis. A 241bp DNA band is amplified, indicating that the sample contains the powdery mildew resistance gene. Failure to amplify a band of the same size indicates that the sample does not contain the powdery mildew resistance gene.

[0059] In another preferred embodiment, in step (2), the ratio of forward primer to reverse primer is 1:1. PCR reaction program: 95℃ pre-denaturation for 5min; 95℃ denaturation for 30s, 61℃ annealing for 30s, 72℃ extension for 30s, then each cycle decreases by 1℃ until 55℃, for a total of 6 cycles; 95℃ denaturation for 30s, 55℃ annealing for 30s, 72℃ extension for 30s, 29 cycles; 72℃ extension for 5min. The reaction system for amplification includes: 1μL DNA template, 5μL 2×Taq Master Mix, 0.4μL primer SDAU-6050F with a concentration of 10μmol / L, 0.4μL primer SDAU-6050R with a concentration of 10μmol / L, and double distilled water to 10μL. In step (3), the length of the sample PCR product is detected by electrophoresis. A 377 bp DNA band is amplified, indicating that the sample contains the powdery mildew resistance gene. Failure to amplify a band of the same size indicates that the sample does not contain the powdery mildew resistance gene.

[0060] Breeding methods

[0061] The present invention also provides a translocation wheat breeding method, in particular a homozygous Shannong-wild emmer wheat short segment translocation wheat breeding method of powdery mildew resistance genotype, comprising the steps of using the primer combination, molecular marker, kit or identification method of the present invention, wherein when the powdery mildew resistance gene is detected and has a powdery mildew resistance phenotype, the hybrid offspring is determined to be a Shannong-wild emmer wheat short segment translocation hybrid carrying the powdery mildew resistance gene. In step (2), the spore spraying method is not particularly limited and can be carried out by methods known in the art.

[0062] Example 1

[0063] The development of the molecular marker SDAU-8259 is shown below.

[0064] Using the primer design website, primers were designed in the conservative region of the reference sequence around the MlNFS10 gene at the end of the long arm of chromosome 4A based on the wild emmer wheat reference gene (Zavitan V1.0) and the scaffold-level genome assembled by resequencing of the disease-resistant parent NFS10 ( Figure 1 ), the genomic DNA of the resistant parent NFS10 and the susceptible parent SFS2-12 were extracted respectively, and the differences were compared after PCR amplification and electrophoresis detection. The amplified sequence obtained was from NFS10, and the specific primers for identifying the MlNFS10 gene were designed.

[0065] Example 2

[0066] The following shows the transfer and utilization of the wheat powdery mildew resistance gene MlNFS10 by assisted selection using the molecular marker SDAU-8259.

[0067] The wild emmer wheat NFS10 was used as the donor parent carrying MlNFS10 in an artificial climate chamber, and the powdery mildew-susceptible wheat variety Shannong 47 was used as the susceptible parent not carrying MlNFS10. Then, it was hybridized and backcrossed with the powdery mildew-susceptible wheat variety Zhongyu 1428. During each backcrossing process, the molecular marker SDAU-8259 was used for auxiliary selection to retain the hybrids carrying MlNFS10. The BC2F1 generation of individual plants carrying MlNFS10 was self-pollinated once to obtain the BC2F2 generation population and planted in an artificial climate chamber. The powdery mildew resistance of the individual plants was identified by spraying spores ( Figure 4 ), retain the individual plants with good agronomic traits and good powdery mildew resistance, harvest them according to the individual plants to obtain BC2F3 generation seeds, take 7 seeds from each individual plant for molecular marker-assisted detection, if all of them carry the MlNFS10 gene, it proves that the individual plant is homozygous material ( Figure 3 ), which was then sown in the field and purified according to conventional methods.

[0068] Example 3

[0069] The following is a method for identifying wheat powdery mildew resistance genotypes based on the molecular marker SDAU-8259.

[0070] 1. Extract sample genomic DNA using the CTAB method

[0071] 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.

[0072] 2. PCR amplification using extracted DNA as template

[0073] The PCR reaction system includes: 1 μL of DNA template, 5 μL of 2×Taq Master Mix, 0.4 μL of 10 μmol / L primers SDAU-8259F and SDAU-8259R, and double distilled water to 10 μL.

[0074] The PCR reaction program was 95°C for 5 min; 95°C for 30 s, 55°C for 30 s, 72°C for 30 s, 35 cycles; 72°C for 5 min.

[0075] 3.1% agarose gel detection of PCR products

[0076] The electrophoresis results showed that primers SDAU-8259F and SDAU-8259R amplified a 241 bp band, indicating that the MlNFS10 gene was carried; failure to amplify a band or the band size was inconsistent indicated that the MlNFS10 gene was not contained ( Figure 2 ). Primers SDAU-8259F and SDAU-8259R can amplify a 241bp band in the BC2F3 generation plants used for transfer, indicating that all BC2F3 generation plants of the short fragment translocation line carry the MlNFS10 gene, proving that the parent BC2F2 generation plant that produced the BC2F3 generation population is homozygous ( Figure 3 ).

[0077] Example 4

[0078] The development of the molecular marker SDAU-6050 is shown below.

[0079] Using the primer design website, primers were designed in the conservative region of the reference sequence around the MlNFS10 gene at the end of the long arm of chromosome 4A based on the wild emmer wheat reference gene (Zavitan V1.0) and the scaffold-level genome assembled by resequencing of the disease-resistant parent NFS10 ( Figure 5 ), the genomic DNA of the resistant parent NFS10 and the susceptible parent SFS2-12 were extracted respectively, and the differences were compared after PCR amplification and electrophoresis detection. The amplified sequence obtained was from NFS10, and the specific primers for identifying the MlNFS10 gene were designed.

[0080] Example 5

[0081] The following shows the transfer and utilization of the wheat powdery mildew resistance gene MlNFS10 using molecular marker SDAU-6050 assisted selection.

[0082] The artificial climate chamber was used for additional generation. Wild emmer wheat NFS10 was used as the donor parent carrying MlNFS10, and the powdery mildew-susceptible wheat variety Shannong 47 was used as the susceptible parent not carrying MlNFS10. Then, it was hybridized and backcrossed with the powdery mildew-susceptible wheat variety Zhongyu 1428. During each backcrossing process, the molecular marker SDAU-6050 was used for auxiliary selection to retain the hybrids carrying MlNFS10. The BC2F1 generation of individual plants carrying MlNFS10 was self-pollinated once to obtain the BC2F2 generation population and planted in the artificial climate chamber. The powdery mildew resistance of the individual plants was identified by spraying spores ( Figure 8 ), retain the individual plants with good agronomic traits and good powdery mildew resistance, harvest them according to the individual plants to obtain BC2F3 generation seeds, take 10 seeds from each individual plant for molecular marker-assisted detection, if all of them carry the MlNFS10 gene, it proves that the individual plant is homozygous material ( Figure 7 ), which was then sown in the field and purified according to conventional methods.

[0083] Example 6

[0084] The following is a method for identifying wheat powdery mildew resistance genotypes based on the molecular marker SDAU-6050.

[0085] 1. Extract sample genomic DNA using the CTAB method

[0086] The CTAB method was used to extract DNA from wheat leaves. The specific steps are as follows:

[0087] 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.

[0088] 2. PCR amplification using extracted DNA as template

[0089] The PCR reaction system includes: 1 μL of DNA template, 5 μL of 2×Taq Master Mix, 0.4 μL of 10 μmol / L primers SDAU-6050F and SDAU-6050R, and double distilled water to 10 μL.

[0090] The PCR reaction program was pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 61°C for 30 s, and extension at 72°C for 30 s, with the temperature decreasing by 1°C in each subsequent cycle until it reached 55°C, for a total of 6 cycles; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s, for 29 cycles; and extension at 72°C for 5 min.

[0091] 3.1% agarose gel detection of PCR products

[0092] The electrophoresis results showed that primers SDAU-6050F and SDAU-6050R amplified a 377 bp band, indicating that the MlNFS10 gene was carried; failure to amplify a band or the band size was inconsistent indicated that the MlNFS10 gene was not contained ( Figure 6 Primers SDAU-6050F and SDAU-6050R can amplify a 377bp band in the transferred BC2F3 generation plants, indicating that all BC2F3 generation plants carry the MlNFS10 gene, proving that the parental BC2F2 generation plant that produced the BC2F3 generation population is homozygous ( Figure 7 ).

[0093] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A primer combination for detecting molecular markers linked to wheat powdery mildew resistance genes, characterized in that: The primer combination includes a first primer pair and / or a second primer pair, wherein the first primer pair includes a forward primer SDAU-8259F with a sequence as shown in SEQ ID NO.1 and a reverse primer SDAU-8259R with a sequence as shown in SEQ ID NO.2, and the second primer pair includes a forward primer SDAU-6050F with a sequence as shown in SEQ ID NO.3 and a reverse primer SDAU-6050R with a sequence as shown in SEQ ID NO.

4.

2. The primer combination for detecting wheat powdery mildew resistance gene-linked molecular markers 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 powdery mildew resistance gene, characterized in that: Amplified by the primer combination of claim 1 or 2.

4. A kit for detecting molecular markers linked to wheat powdery mildew resistance genes, characterized in that: Comprising the primer combination of claim 1 or 2.

5. Use of the primer combination 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 powdery mildew resistance gene, characterized in that: The method comprises the step of using the primer combination described in claim 1 or 2 to detect whether there is a wheat powdery mildew resistance gene linked molecular marker in the DNA of the plant to be identified.

8. The method for identifying whether a plant contains a wheat powdery mildew 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 a forward primer and a reverse primer; 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 powdery mildew resistance gene according to claim 8, characterized in that: Amplification of DNA bands of 241 bp and / or 377 bp indicates that the sample contains a powdery mildew resistance gene, and failure to amplify bands of the same size indicates that the sample does not contain a powdery mildew resistance gene.

10. A method for breeding translocation wheat, characterized in that: include: (1) Using wild emmer wheat NFS10 as a donor parent carrying a powdery mildew resistance gene and using a powdery mildew susceptible wheat variety Shannong 47 as a recurrent parent for hybridization, and then hybridizing and backcrossing with a powdery mildew susceptible wheat variety Zhongyu 1428 to obtain a BC2F1 generation population, determining the genotype of the backcross progeny using the primer combination described in claim 1 or 2, the molecular marker described in claim 3, the kit described in claim 4, or the method described in any one of claims 7 to 9, and performing powdery mildew phenotype identification to obtain a Shannong-wild emmer wheat short segment translocation line hybrid carrying a powdery mildew resistance gene; (2) self-pollinating the hybrid to obtain a BC2F2 generation population, identifying the powdery mildew resistance of individual plants by spraying spores, and obtaining individual seeds carrying a powdery mildew resistance gene; (3) Harvesting individual plants to obtain BC2F3 generation seeds, and determining the genotype of the individual seeds using the primer combination described in claim 1 or 2, the molecular marker 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 powdery mildew resistance gene, the individual plant is a homozygous translocation line wheat.

Images