Wheat gibberellic disease-resistant gene linked molecular marker, detection primer and application

Abstract

The invention discloses a wheat gibberellic disease-resistant gene linked molecular marker, a detection primer and application. The molecular marker Z-538 is linked with a gibberellic disease-resistant gene, can be directly applied to wheat gibberellic disease-resistant production practice, provides a technical support for breeding a wheat improved line carrying the gibberellic disease-resistant gene, and can improve the efficiency of molecular marker-assisted selection, thereby accelerating the research and utilization of the excellent wheat gibberellic disease-resistant gene, and having a wide application prospect. 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 scab resistance gene linkage molecular marker, a detection primer and an application thereof. 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 meet this challenge is to improve wheat's resistance to various diseases, which can recover the 15-20% yield loss caused by diseases each year. Fusarium graminearum, a fungal disease of wheat, causes wheat ear rot, which in turn threatens wheat yield and quality. In recent years, wheat fusarium rust has become an important disease that seriously threatens wheat production.

[0003] Studies have shown that breeding and planting disease-resistant varieties is the most economical and effective measure to solve the problem of wheat fusarium head blight. At present, the fusarium head blight resistance gene can be transferred to the promoted wheat varieties susceptible to fusarium head blight in production. However, the traditional method is time-consuming and labor-intensive, phenotypic identification is difficult, and the breeding efficiency is low. Therefore, it is urgent to develop molecular markers linked to fusarium head blight resistance genes for genetic analysis such as assisted selection breeding. Summary of the invention

[0004] In order to solve the technical problems in the prior art, the present invention provides a wheat scab resistance gene linked molecular marker, detection primers and applications. Specifically, the present invention includes the following contents.

[0005] The first aspect of the present invention provides a detection primer for detecting a wheat fusarium resistance gene linked molecular marker, wherein the detection primer comprises a forward primer Z-538F having a sequence as shown in SEQ ID NO.1 and a reverse primer Z-538R having a sequence as shown in SEQ ID NO.2.

[0006] In certain embodiments, according to the detection primers for detecting molecular markers linked to wheat ergot resistance genes of the present invention, the molecular marker has a sequence shown in SEQ ID NO.3.

[0007] The second aspect of the present invention provides a wheat scab resistance gene-linked molecular marker, which is amplified by the detection primers of the present invention.

[0008] The third aspect of the present invention provides a kit for detecting molecular markers linked to wheat scab resistance genes, comprising the detection primers described in the present invention.

[0009] The fourth aspect of the present invention provides use of the detection primers, the molecular markers or the kit according to the present invention in wheat genetic analysis.

[0010] In certain embodiments, according to the use of the present invention, the genetic analysis includes genetic diversity analysis, germplasm identification or assisted breeding.

[0011] The fifth aspect of the present invention provides a method for identifying whether a plant contains a wheat ergot resistance gene, which comprises the step of using the detection primers described in the present invention to detect whether there is a wheat ergot resistance gene-linked molecular marker in the DNA of the plant to be identified.

[0012] In certain embodiments, the method for identifying whether a plant comprises a wheat scab resistance gene according to the present invention comprises the following steps:

[0013] (1) extracting sample DNA from the plant sample to be identified;

[0014] (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;

[0015] (3) Identifying the plant by detecting the length of the sample PCR product by electrophoresis.

[0016] In certain embodiments, according to the method for identifying whether a plant contains a wheat ergot resistance gene according to the present invention, a 157 bp DNA band is amplified, indicating that the sample contains the ergot resistance gene, and a 169 bp DNA band is amplified, indicating that the sample does not contain the ergot resistance gene.

[0017] The sixth aspect of the present invention provides a breeding method for wheat with a gene for resistance to ergot disease, which comprises: hybridizing a susceptible parent of wheat with a resistant parent of wheat carrying a gene for resistance to ergot disease, and then selfing, and during each generation of selfing, using the detection primers, molecular markers, kits or identification methods described in the present invention for auxiliary selection, thereby retaining wheat strains carrying QFhb.2D and performing phenotypic identification on them.

[0018] The molecular marker of the present invention is linked to the scab resistance gene, can be directly applied to the scab resistance production practice of wheat, provides technical support for the selection of improved wheat lines carrying scab resistance genes, can improve the efficiency of molecular marker-assisted selection, thereby accelerating the research and utilization of excellent scab resistance genes for wheat, and provides new materials for genetic improvement of wheat disease resistance. In addition, the present invention has the advantages of not being affected by external environmental factors, being simple and convenient to operate, accelerating the breeding process, and having broad application prospects in wheat molecular breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1A is the specific detection result of molecular marker Z-538 in the disease-resistant parent, and B is the specific detection result of molecular marker Z-538 in the disease-susceptible parent.

[0020] Figure 2 These are the detection results of the molecular marker Z-538 in the F4 population samples of the hybrid offspring of the resistant parent and the susceptible parent (the 5th lane from left to right is the Marker, and the DL2000 Marker bands are 2000bp, 1000bp, 750bp, 500bp, 250bp, and 100bp, respectively; the remaining lanes are F4 generation plants).

[0021] Figure 3 The figure shows the identification phenotype of Fusarium fusarium resistance of individual plants with different molecular marker Z-538 genotypes in the F4 generation population, which is the hybrid of the resistant parent and the susceptible parent. DETAILED DESCRIPTION

[0022] 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.

[0023] 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.

[0024] 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.

[0025] Molecular markers

[0026] One aspect of the present invention provides a specific molecular marker having a characteristic sequence for distinguishing whether a plant (such as wheat) carries a gene for resistance to Fusarium head blight.

[0027] The molecular marker ("characteristic sequence") in the present invention refers to a gene fragment that only appears in wheat resistant to fusarium sphaeroides and is linked to the fusarium sphaeroides resistance gene and is closely related to fusarium sphaeroides resistance, wherein the fusarium sphaeroides resistance gene refers to the fusarium sphaeroides resistance gene QFhb.2D located on wheat chromosome 2D. In a preferred embodiment, the molecular marker has a sequence shown in SEQ ID NO.3.

[0028] Detection primers

[0029] In one aspect of the present invention, a detection primer for detecting a molecular marker linked to a wheat scab resistance gene is provided, which is used to amplify the molecular marker described in the present invention, and comprises a forward primer and a reverse primer. In another preferred embodiment, the forward primer is Z-538F having a sequence shown in SEQ ID NO.1, and the reverse primer is Z-538R having a sequence shown in SEQ ID NO.2.

[0030] Reagent test kit

[0031] One aspect of the present invention provides a kit for detecting wheat scab resistance gene linked molecular markers, which comprises the detection primer 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] application

[0037] In one aspect of the present invention, the use of the detection 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 germplasm identification of wheat resistant to scab. In another specific embodiment, the application includes assisted breeding of wheat resistant to scab.

[0038] Identification methods

[0039] One aspect of the present invention provides a method for identifying whether a plant contains a wheat scab resistance gene, which comprises the step of using the detection primers of the present invention to detect whether there is a wheat scab resistance gene-linked molecular marker in the DNA of the plant to be identified.

[0040] In the present invention, the method for detecting the presence of a wheat ergot 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.

[0041] In certain embodiments, the method of the present invention for identifying whether a plant contains a wheat scab resistance gene 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.

[0042] In an exemplary embodiment, a method for identifying whether a plant comprises a wheat scab resistance gene comprises the following steps:

[0043] (1) extracting sample DNA from the plant sample to be identified;

[0044] (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;

[0045] (3) Identify whether the plant contains the wheat ergot resistance gene by detecting the length of the sample PCR product by electrophoresis.

[0046] 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.

[0047] 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℃10min. The reaction system for amplification includes: 1μL DNA template, 5μL 2×Taq Master Mix, 0.7μL primer Z-538F with a concentration of 10μmol / L, 0.7μL primer Z-538R with a concentration of 10μmol / L, and water is added to 10μL. In step (3), a 157bp DNA band is amplified, indicating that the sample contains the ergot resistance gene, and a 169bp DNA band is amplified, indicating that the sample does not contain the ergot resistance gene.

[0048] Breeding methods

[0049] The present invention also provides a method for breeding wheat with a fusarium resistant gene, in particular a method for breeding wheat with a homozygous fusarium resistant genotype, comprising the steps of using the detection primers, molecular markers, kits or identification methods of the present invention, wherein when the fusarium resistant gene is detected and has a fusarium resistant phenotype, the hybrid offspring is determined as a hybrid carrying the fusarium resistant gene. In the breeding method of the present invention, the above-mentioned primers are used to amplify a 157bp DNA band, indicating that the offspring contains the fusarium resistant gene, and amplify a 169bp DNA band, indicating that the offspring does not contain the fusarium resistant gene.

[0050] Example 1

[0051] The development of molecular marker Z-538 is shown below.

[0052] Based on the comparison of the resequencing data of the disease-resistant parent ZNY029 and the disease-susceptible parent ZNY045, the Indel site near QFhb.2D on chromosome 2D was extracted, and primers were designed based on the differential sites between the parents. The reference genome was ChineseSpring RefSeq v2.1. Figure 1 , this Indel site has a difference of 12bp. Wheat genomic DNA was extracted from the disease-resistant parent and the susceptible parent, respectively, and the specificity of the primers was detected by polyacrylamide electrophoresis after PCR amplification, so as to design specific primers for identifying the QFhb.2D gene. The disease-resistant parent ZNY029 and the susceptible parent ZNY045 are the offspring of Yangmai 158.

[0053] Example 2

[0054] The following shows the screening of strains containing QFhb.2D using molecular marker Z-538.

[0055] The disease-resistant parent ZNY029 and the susceptible parent ZNY045 were hybridized and then self-pollinated. During each generation of self-pollination, molecular marker Z-538 was used for auxiliary selection to retain wheat lines carrying QFhb.2D and to conduct phenotypic identification.

[0056] Example 3

[0057] The following is a method for identifying the wheat ergot resistance gene QFhb.2D based on the molecular marker Z-538.

[0058] 1. Extract sample genomic DNA using the CTAB method

[0059] The CTAB method is used to extract DNA from wheat samples, and the specific steps are as follows:

[0060] 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.

[0061] 2. PCR amplification using extracted DNA as template

[0062] The PCR reaction system includes: 1 μL of DNA template, 5 μL of 2×Taq Master Mix, 0.7 μL of primers Z-538F and Z-538R with a concentration of 10 μmol / L, and water is added to 10 μL.

[0063] The PCR reaction program is 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 10 min.

[0064] Detection of PCR products by 3.8% polyacrylamide gel electrophoresis

[0065] Figure 2 In the figure, A is a 157bp band type, indicating that this individual plant contains QFhb.2D, B is a 169bp band type, indicating that this individual plant does not contain QFhb.2D, and H is a heterozygous band type.

[0066] 4. Use the single flower drip method to inject Fusarium graminearum No. 4 around the wheat ovary, bag and moisturize for 48 hours, then remove the bag. Count the incidence of different genotypes of plants on the 16th day, such as Figure 3 , keep the environment moist during this period to ensure good disease control.

[0067] 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 detection primer for detecting a wheat scab resistance gene linked molecular marker, characterized in that: The detection primers include a forward primer Z-538F whose sequence is shown in SEQ ID NO.1 and a reverse primer Z-538R whose sequence is shown in SEQ ID NO.

2.

2. The detection primer for detecting wheat scab resistance gene linked molecular marker according to claim 1, characterized in that: The molecular marker has the sequence shown in SEQ ID NO.

3.

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

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

5. Use of the detection 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 scab resistance gene, characterized in that: The method comprises the step of using the detection primers described in claim 1 or 2 to detect whether there is a wheat scab 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 scab 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 scab resistance gene according to claim 8, characterized in that: Amplification of a 157 bp DNA band indicates that the sample contains a gene resistant to ergot disease, and amplification of a 169 bp DNA band indicates that the sample does not contain a gene resistant to ergot disease.

10. A method for breeding wheat with a gene resistant to scab, characterized in that: include: A susceptible wheat parent and a resistant wheat parent carrying a fusarium head blight resistance gene are hybridized and then self-pollinated. During each generation of self-pollination, auxiliary selection is performed using the detection 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, thereby retaining the wheat strain carrying the fusarium head blight resistance gene and conducting phenotypic identification thereof.

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