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

Abstract

The invention discloses a wheat gibberellic disease-resistant gene linked molecular marker, a primer combination and application. The molecular marker W17 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 primer combination and an application. 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, primer combination and application. Specifically, the present invention includes the following contents.

[0005] In a first aspect of the present invention, a primer combination for detecting a wheat ergot resistance gene-linked molecular marker is provided, wherein the primer combination comprises a forward primer W17-F having a sequence as shown in SEQ ID NO.1 and a reverse primer W17-R having a sequence as shown in SEQ ID NO.2.

[0006] In certain embodiments, according to the primer combination for detecting molecular markers linked to wheat ergot resistance genes of the present invention, the molecular markers have 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 primer combination 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 primer combination of the present invention.

[0009] The fourth aspect of the present invention provides use of the primer combination, the molecular marker 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 primer combination of 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; and

[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 551 bp DNA band is amplified, indicating that the sample contains the ergot resistance gene, and a 598 bp DNA band is amplified, indicating that the sample does not contain the ergot resistance gene.

[0017] A sixth aspect of the present invention provides a method for breeding wheat having a scab-resistant gene, comprising:

[0018] (1) Using a disease-resistant material carrying a scab-resistant gene as a donor parent and a wheat variety susceptible to scab as a recurrent parent, performing continuous backcrossing to obtain a BC4F1 generation population, determining the genotype of the backcross offspring using the primer combination, the molecular marker, the kit or the method of the present invention, and performing scab-resistant phenotype identification to obtain a hybrid carrying a scab-resistant gene;

[0019] (2) self-pollinating the hybrid to obtain a BC4F2 generation population, identifying the fusarium resistance of individual plants by single seed transmission, and obtaining individual seeds carrying the fusarium resistance gene;

[0020] (3) Harvesting individual plants to obtain BC4F3 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 ergot resistance gene, then the individual plant is a homozygous wheat carrying the ergot resistance gene.

[0021] 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

[0022] Figure 1 A is the sequence amplified by the molecular marker W17 in the material that does not carry the allele variation of resistance to ergot; B is the sequence amplified by the molecular marker W17 in the material that carries the allele variation of resistance to ergot (the sequence amplified by the primer is in the arrow, and the middle marker position is the difference series).

[0023] Figure 2 The detection results of molecular marker W17 in wheat BC4F2 generation population samples (M, DL2000 Marker; 1 is the resistant parent, 2 is the susceptible parent, 3, 4, 5, 6, 7, 8, 9 and 10 are BC4F2 generation individual plants).

[0024] Figure 3 To identify the phenotype of ergot resistance of individual plants with different molecular marker W17 genotypes in the wheat BC4F2 population. DETAILED DESCRIPTION

[0025] 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.

[0026] 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.

[0027] 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.

[0028] Molecular markers

[0029] 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.

[0030] The molecular marker ("characteristic sequence") in the present invention refers to a gene fragment that only appears in wheat resistant to fusarium spp. and is linked to a fusarium spp. resistance gene and is closely related to fusarium spp. resistance, wherein the fusarium spp. resistance gene refers to the fusarium spp. resistance gene QFhb-5 located on wheat chromosome 5A. In a preferred embodiment, the molecular marker has a sequence shown in SEQ ID NO.3.

[0031] Primer combinations

[0032] In one aspect of the present invention, a primer combination for detecting a wheat scab resistance gene-linked molecular marker 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 a preferred embodiment, the forward primer is W17-F having a sequence shown in SEQ ID NO.1, and the reverse primer is W17-R having a sequence shown in SEQ ID NO.2.

[0033] Reagent test kit

[0034] One aspect of the present invention provides a kit for detecting wheat scab 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] application

[0040] 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 wheat resistant to scab. In another specific embodiment, the application includes assisted breeding of wheat resistant to scab.

[0041] Identification methods

[0042] 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 primer combination 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.

[0043] 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.

[0044] 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.

[0045] In an exemplary embodiment, a method for identifying whether a plant comprises a wheat scab resistance gene comprises the following steps:

[0046] (1) extracting sample DNA from the plant sample to be identified;

[0047] (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;

[0048] (3) Identify whether the plant contains the wheat ergot resistance gene by detecting the length of the sample PCR product by electrophoresis.

[0049] 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.

[0050] 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 W17-F with a concentration of 10μmol / L, 0.7μL primer W17-R with a concentration of 10μmol / L, and water is added to 10μL. In step (3), a 551bp DNA band is amplified, indicating that the sample contains the ergot resistance gene, and a 598bp DNA band is amplified, indicating that the sample does not contain the ergot resistance gene.

[0051] Breeding methods

[0052] The present invention also provides a breeding method for carrying a scab-resistant gene, in particular a breeding method for a homozygous scab-resistant wheat genotype, comprising the steps of using the primer combination, molecular marker, kit or identification method of the present invention, wherein when the scab-resistant gene is detected and has a scab-resistant phenotype, the hybrid offspring is determined to be a hybrid carrying the scab-resistant gene. In step (2), the single seed propagation method is not particularly limited and can be carried out by methods known in the art.

[0053] Example 1

[0054] The development of molecular marker W17 is shown below.

[0055] The software was used to obtain sequence information by resequencing the whole genome of the resistant parent SNDH-28. The obtained sequencing data were aligned to the reference genome (Fielder). Primers were designed based on the reference sequences around the differential sites between the susceptible parent Fielder genome database and the resistant parent SNDH-28 ( Figure 1 ), extracting genomic DNA from the two parental wheats, and after PCR amplification and sequencing analysis, the obtained amplified sequences were different, so specific primers were designed to identify the fusarium resistance gene on chromosome 5A of wheat. SNDH-28 is a hybrid of the tetraploid wheat London and Aegilops tauschii.

[0056] Example 2

[0057] The following shows the transfer and utilization of the wheat ergot resistance gene QFhb-5 using the molecular marker W17-assisted selection.

[0058] The artificial climate chamber was used for generation, with the disease-resistant material SNDH-28 as the donor parent and the susceptible material Fielder as the recurrent parent. Combined with molecular marker-assisted selection, four backcrosses were performed to obtain the BC4F1 generation population. Then, the BC4F1 generation individual plants were self-pollinated once to obtain the BC4F2 generation population and planted in the artificial climate chamber. The single seed transmission method was used to identify the fusarium resistance of the individual plants ( Figure 3 ), retaining individual plants with good resistance to ergot.

[0059] Example 3

[0060] The following is a method for identifying wheat ergot resistance genotypes based on the molecular marker W17.

[0061] 1. Extract sample genomic DNA using the CTAB method

[0062] The CTAB method is used to extract DNA from wheat samples, and the specific steps are as follows:

[0063] 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 700μL 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 500μL of supernatant, add 500μL of isopropanol and 50μL 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.

[0064] 2. PCR amplification using extracted DNA as template

[0065] The PCR reaction system includes: 1 μL of DNA template, 5 μL of 2×Taq Master Mix, 0.7 μL of primers W17-F and W17-R with a concentration of 10 μmol / L, and water is added to 10 μL.

[0066] 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.

[0067] 3. Detection of PCR products by polyacrylamide gel

[0068] The results of electrophoresis showed that primers W17-F and W17-R amplified a 551 bp band, indicating that wheat BC4F2 generation plants 7, 8, 9, and 10 carried the allele variant gene for resistance to fusarium scurf; primers W17-F and W17-R amplified a 598 bp band, indicating that wheat BC4F2 generation plants 3, 4, 5, and 6 did not carry the allele variant gene for resistance to fusarium scurf. Figure 2 ).

[0069] 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 a molecular marker linked to a wheat scab resistance gene, characterized in that: The primer combination includes a forward primer W17-F whose sequence is shown in SEQ ID NO.1 and a reverse primer W17-R whose sequence is shown in SEQ ID NO.

2.

2. The primer combination for detecting wheat scab resistance gene linked molecular markers 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 primer combination of claim 1 or 2.

4. A kit for detecting molecular markers linked to wheat scab resistance genes, characterized in that: Comprising the primer combination described in 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 scab 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 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: Amplifying a DNA band of 551 bp indicates that the sample contains a gene resistant to ergot disease, and amplifying a DNA band of 598 bp 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: (1) Using a disease-resistant material carrying a fusarium head blight resistance gene as a donor parent and a wheat variety susceptible to fusarium head blight as a recurrent parent, performing continuous backcrossing to obtain a BC4F1 generation population, determining the genotype of the backcross offspring 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 fusarium head blight phenotype identification to obtain a hybrid carrying a fusarium head blight resistance gene; (2) self-pollinating the hybrid to obtain a BC4F2 generation population, identifying the fusarium resistance of individual plants by single seed transmission, and obtaining individual seeds carrying the fusarium resistance gene; (3) Harvesting individual plants to obtain BC4F3 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 ergot resistance gene, then the individual plant is homozygous wheat carrying the ergot resistance gene.

Images