Linked molecular marker for detecting stripe rust resistance gene of wheat, primer combination and application

Abstract

The invention discloses a linked molecular marker and primer combination for detecting a stripe rust resistant gene of wheat and application of the linked molecular marker and primer combination. The method is not influenced by external environmental factors and is simple and convenient to operate. A primer of the molecular marker S-61635 in close linkage with the wheat stripe rust resistance gene is utilized, PCR amplification electrophoresis detection is carried out, germplasm materials carrying the wheat stripe rust resistance gene can be screened out according to the size of a target stripe, the molecular marker can accelerate research and utilization of the excellent wheat stripe 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 linkage molecular marker, a primer combination and an application thereof for detecting a wheat stripe rust resistance gene. Background Art

[0002] Wheat stripe rust is an airborne leaf disease caused by the specialized pathogenic fungus Puccinia striiformis f.sp.tritici. In severe cases, it can even harm the stem, ear, and leaf sheath. China is the world's largest and relatively independent wheat stripe rust epidemic area. In severe cases, the disease can reduce wheat production by 40%-60%, posing a major threat to my country's food safety production. Designing tightly linked molecular markers for specific DNA sequences can identify the relationship between genotype and phenotype, efficiently screen out individuals with specific genotypes, and greatly improve the efficiency of molecular breeding.

[0003] Therefore, research shows that cultivating and planting disease-resistant varieties is the most economical and effective measure to solve the problem of wheat stripe rust. Traditional breeding methods have a long cycle, consume manpower, are difficult to identify phenotypes, and have low breeding efficiency. With the help of molecular marker-assisted selection breeding technology, breeding efficiency can be effectively improved and this problem can be solved. Summary of the invention

[0004] In order to solve at least some of the technical problems in the prior art, the present invention phenotypically identified wheat strains and found that the SN40 strain showed an immune phenotype to most physiological subspecies of stripe rust, exhibiting good resistance and having great application value. By constructing a RIL population, the immune resistance site was located on 2BL, and the recombinants of the F2:3 strains were further screened to further narrow the interval, and the tightly linked marker S-61635 was identified to be co-separated with the phenotype. This marker can well distinguish between immune and susceptible strains. After amplification with the marker primer in wheat materials such as Fielder, Huixianhong, Mingxian 169, and CB037, enzyme cutting could not obtain a fragment type consistent with Shannong 40, which further proved that this marker can well identify the stripe rust resistance gene of 2BL and can improve the efficiency of molecular marker-assisted selection. Specifically, the present invention includes the following contents.

[0005] The first aspect of the present invention provides a primer combination for detecting a wheat stripe rust resistance gene-linked molecular marker, wherein the primer combination comprises:

[0006] (1) S-61635F: a forward primer whose sequence is shown in SEQ ID NO. 1;

[0007] (2) S-61635R: a reverse primer whose sequence is shown in SEQ ID NO.2.

[0008] In certain embodiments, according to the primer combination for detecting molecular markers linked to wheat stripe rust resistance genes of the present invention, the molecular markers have a sequence shown in SEQ ID NO.3.

[0009] The second aspect of the present invention provides a wheat stripe rust resistance gene-linked molecular marker, which is amplified by the primer combination of the present invention.

[0010] The third aspect of the present invention provides a kit for detecting molecular markers linked to wheat stripe rust resistance genes, comprising the primer combination of the present invention.

[0011] The fourth aspect of the present invention provides the use of the primer combination, molecular marker or kit of the present invention in wheat genetic analysis.

[0012] In certain embodiments, according to the use of the present invention, the genetic analysis includes genetic diversity analysis, germplasm identification or assisted breeding.

[0013] The fifth aspect of the present invention provides a method for identifying whether a plant contains a wheat stripe rust resistance gene, which includes the step of detecting whether there is a wheat stripe rust resistance gene-linked molecular marker in the DNA of the plant to be identified, wherein the molecular marker is amplified using the primer combination described in the present invention.

[0014] In certain embodiments, the method for identifying whether a plant comprises a wheat stripe rust resistance gene according to the present invention comprises the following steps:

[0015] (1) extracting sample DNA from the plant sample to be identified;

[0016] (2) a step of amplifying the nucleic acid containing the molecular marker from the sample DNA by PCR using primers; and

[0017] (3) Identifying the plant by detecting the length of the sample PCR product by electrophoresis.

[0018] In certain embodiments, according to the method for identifying whether a plant contains a wheat stripe rust resistance gene according to the present invention, the reaction system for amplification comprises: 1 μL of DNA template, 5 μL of 2×Taq Master Mix, 0.5 μL of primer S-61635F at a concentration of 10 μmol / L, 0.5 μL of primer S-61635R at a concentration of 10 μmol / L, double distilled water is added to 10 μL, and the reaction procedure is: 95°C for 5 min; 95°C for 30 s, 56°C for 30 s, 72°C for 15 s, 33 cycles; 72°C for 5 min; enzyme digestion using BmgbI endonuclease at 37°C for 3 h, 65°C for 20 min.

[0019] In certain embodiments, according to the method for identifying whether a plant contains a wheat stripe rust resistance gene according to the present invention, a 581 bp DNA band is amplified, and two bands of 256 bp and 325 bp in length appear after restriction endonuclease BmgBI digestion, indicating that the sample contains the stripe rust resistance gene, and the sample does not contain two bands of 256 bp and 325 bp in length after digestion, indicating that the sample does not contain the stripe rust resistance gene.

[0020] A sixth aspect of the present invention provides a method for breeding wheat having a stripe rust resistance gene, comprising:

[0021] A susceptible wheat parent and a resistant wheat parent carrying a stripe rust resistance gene are hybridized and then self-pollinated. During each generation of self-pollination, the primer combination, the molecular marker, the kit, or the method described in the present invention are used for auxiliary selection, thereby retaining the wheat strain carrying the stripe rust resistance gene and conducting phenotypic identification thereof.

[0022] The invention is not affected by external environmental factors and is simple and convenient to operate. The primers of the molecular marker S-61635 closely linked to the wheat stripe rust resistance gene are used, and the germplasm material carrying the wheat stripe rust resistance gene is screened out according to the size of the target band through PCR amplification and electrophoresis detection. The molecular marker of the invention can accelerate the research and utilization of excellent wheat stripe rust resistance genes and provide new materials for the genetic improvement of wheat disease resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the sequence information of the molecular marker S-61635 amplified in resistant / susceptible wheat (the yellow content indicates the forward primer sequence, the green content indicates the reverse primer sequence, and the red content indicates the enzyme cutting region).

[0024] Figure 2 These are the detection results of molecular marker S-61635 in wheat materials such as SN40, Fielder, Huixianhong, Mingxian169, and CB037 (M, DL2000 Marker bands from top to bottom are 2000bp, 1500bp, 1000bp, 750bp, 500bp, and 250bp; the bands from left to right are SN40, SN40, Fielder, Fielder, Huixianhong, Mingxian169, and CB037).

[0025] Figure 3These are the detection results of molecular marker S-61635 in wheat SN40, Fielder, Huixianhong, Mingxian169, CB037 and other materials after enzyme digestion reaction (M, DL2000 Marker bands from top to bottom are 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp; the bands from left to right are SN40, SN40, Fielder, Fielder, Huixianhong, Mingxian169, CB037).

[0026] Figure 4 To identify the stripe rust resistance phenotype of some individuals with different molecular marker S-61635 genotypes in wheat SN40 and susceptible material Fielder population samples. DETAILED DESCRIPTION

[0027] 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.

[0028] 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.

[0029] 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.

[0030] Molecular markers

[0031] In a first aspect of the present invention, a specific molecular marker is provided, which has a characteristic sequence for distinguishing whether a plant (e.g., wheat) carries a stripe rust resistance gene. In the present invention, stripe rust refers to an airborne leaf disease caused by the specialized pathogenic fungus Puccinia striata CYR32, and the infected part shows a large number of spore piles and a stripe rust-like phenotype.

[0032] The molecular marker ("characteristic sequence") in the present invention refers to a gene fragment that only appears in stripe rust-resistant wheat and is linked to the stripe rust-resistant gene and is closely related to stripe rust resistance, wherein the stripe rust-resistant gene refers to a resistance gene located on the 2BL of wheat SN40.

[0033] In a preferred embodiment, the molecular marker has the sequence shown in SEQ ID NO.3.

[0034] Primer combinations

[0035] In one aspect of the present invention, a primer combination for detecting a wheat stripe rust 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 S-61635F having a sequence shown in SEQ ID NO.1, and the reverse primer is S-61635R having a sequence shown in SEQ ID NO.2.

[0036] Reagent test kit

[0037] One aspect of the present invention provides a kit for detecting wheat stripe 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] application

[0043] In one aspect of the present invention, there is provided an application of the primer combination, molecular marker or kit of the present invention in wheat genetic analysis, wherein the genetic analysis includes but is not limited to genetic diversity analysis, germplasm identification, assisted breeding, etc. In a specific embodiment, the application includes identification of wheat germplasm with a stripe rust resistance genotype.

[0044] Identification methods

[0045] One aspect of the present invention provides a method for identifying whether a plant contains a wheat stripe rust resistance gene, which includes the step of detecting whether there is a wheat stripe rust resistance gene-linked molecular marker in the DNA of the plant to be identified.

[0046] In the present invention, the method for detecting the presence of wheat stripe rust resistance gene-linked molecular markers can adopt any method known in the art, examples of which include methods of amplifying nucleic acids containing characteristic sequences and methods of directly sequencing plant genomes.

[0047] In certain embodiments, the method of identifying whether a plant contains a wheat stripe 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.

[0048] In an exemplary embodiment, a method for identifying whether a plant comprises a wheat stripe rust resistance gene comprises the following steps:

[0049] (1) extracting sample DNA from the plant sample to be identified;

[0050] (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;

[0051] (3) Determine whether the plant contains the wheat stripe rust resistance gene by detecting the length of the sample PCR product by electrophoresis.

[0052] 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. In step (3), the length of the sample PCR product is detected by electrophoresis, and a 581 bp DNA band is amplified. After enzyme digestion, two bands of 256 bp and 325 bp appear, indicating that the sample contains a stripe rust resistance gene.

[0053] Breeding methods

[0054] The present invention also provides a method for breeding wheat with a stripe rust resistance gene, comprising the steps of using the primers, molecular markers, kits or identification methods of the present invention, wherein the phenotypic identification can be carried out using a spore smearing method, which is known in the art, for example, by smearing spores of the wheat-specific subspecies CYR32 of the stripe rust fungus to determine the stripe rust resistance phenotype.

[0055] In certain embodiments, the disease-resistant material Shannong 40 is used as the female parent, and the susceptible material Huixianhong and / or Fielder etc. is used as the male parent to obtain the hybrid F1 generation, and the F1 is self-pollinated to obtain the F2 generation segregation population and further self-pollinated to obtain the F2:3 family. Through phenotypic identification of the parents and the segregation population, the stripe rust resistance gene contained in Shannong 40 is genetically analyzed, and the stripe rust CYR32 race is inoculated to identify the disease resistance at the seedling stage, and the primers, molecular markers, kits or methods of the present invention are used for genotyping to separate the homozygous strains of the offspring;

[0056] Then, the homozygous offspring strains are identified, and the strains with non-segregating phenotypes are selected for genotyping using the primers, molecular markers, kits or methods of the present invention to determine the resistant strains with co-segregation of molecular markers and phenotypes.

[0057] Example 1

[0058] The development of the molecular marker S-61635 is shown below.

[0059] By sequencing the third generation of wheat variety SN40 in the laboratory, its sequence was compared with the genome of the susceptible variety Fielder, and the INDEL loci were identified. Primers were designed based on the chromosome sequence of SN40 using a primer design website and software ( Figure 1 ), the primer sequence S61635F is as shown in SEQ ID NO.1, the primer sequence S61635R is as shown in SEQ ID NO.2, the genomic DNA of wheat SN40 and susceptible population are extracted respectively, and the specific INDEL molecular markers are screened out for molecular marker positioning through polymerase chain reaction amplification and electrophoresis detection.

[0060] Example 2

[0061] The following shows linkage analysis using molecular marker S-61635 combined with phenotype.

[0062] At Shandong Agricultural University, an artificial climate chamber was used to carry out generation. The wheat SN40 variety carrying the resistance gene was used as the female parent, and the wheat varieties susceptible to stripe rust, Fielder and Huixianhong, were used as the male parents to obtain the hybrid F1 generation. The F1 was self-pollinated to obtain the F2 generation segregation population and further self-pollinated to obtain the F2:3 family. At the same time, Shannong 40 was used as the female parent and Mingxian 169 as the male parent to create a reserve population. Through the phenotypic identification of the parents and the segregation population, the stripe rust resistance gene contained in Shannong 40 was genetically analyzed.

[0063] The offspring strains were phenotypically identified and the strains were selected for genotype identification using molecular markers. S-61635F / R was able to amplify bands of 256bp and 325bp after restriction digestion in phenotypically consistent resistant strains, while only a 576bp band was amplified after restriction digestion in phenotypically consistent susceptible strains, indicating that the marker was co-segregated with the phenotype and could be used as a tightly linked molecular marker.

[0064] Example 3

[0065] The following is a method for identifying wheat stripe rust resistance genotypes based on the molecular marker S-61635.

[0066] 1. Extract sample genomic DNA using the CTAB method

[0067] Cut 2-3cm wheat leaves and put them into a 2mL centrifuge tube (put 2 small steel balls in advance). After freezing the leaves with liquid nitrogen, quickly grind them into powder (the parameters of the plant cell tissue disruptor are set to a frequency of 40Hz and a time of 40sec), add 800μL of CTAB extraction buffer preheated to 65℃ in a water bath, and water bath at 65℃ for 30min. Then, add an equal volume of three mixed solutions (phenol: chloroform: isoamyl alcohol = 25:24:1), mix well and let stand for 3min, centrifuge at 12000rpm for 10min, absorb 700μL of supernatant, add 700μL of isopropanol and 70μL of sodium acetate, mix well, and let stand for 15min. Subsequently, centrifuge at 12000rpm for 10min, pour off the supernatant, and the white precipitate is genomic DNA. After washing twice with 600μL 70% ethanol, dissolve the precipitate with ddH2O and store at low temperature for later use.

[0068] 2. PCR amplification using extracted DNA as template

[0069] The PCR reaction system includes: 1 μL of DNA template, 5 μL of 2×Taq Master Mix, 0.5 μL of 10 μmol / L primers S-61635F and S-61635R, and double distilled water to 10 μL.

[0070] PCR reaction program: 95℃5min; 95℃30s, 56℃30s, 72℃15s, 33 cycles; 72℃5min; 37℃3h, 65℃20min enzyme digestion with BmgbI endonuclease;

[0071] PCR products were detected by 3.1% agarose gel.

[0072] The electrophoresis results showed that primers S-61635F and S-61635R amplified 256 bp and 325 bp bands after restriction digestion, indicating that SN40 strains 1 and 2 carried the stripe rust resistance gene of chromosome 2BL; no bands could be amplified or the band sizes did not match, indicating that susceptible strains 3, 4, 5, 6, and 7 did not contain the resistance gene ( Figure 3 ).

[0073] The molecular marker in the present invention is closely linked to the stripe rust resistance gene in wheat. The use of the molecular marker S-61635 can improve the efficiency of molecular marker-assisted selection and can be directly applied to the production practice of wheat stripe rust resistance, providing technical support for the creation and breeding of wheat improved lines carrying stripe rust resistance genes through molecular marker-assisted selection.

[0074] 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 combination for detecting molecular markers linked to wheat stripe rust resistance genes, characterized in that: The primer combination comprises: (1) S-61635F: a forward primer whose sequence is shown in SEQ ID NO. 1; (2) S-61635R: a reverse primer whose sequence is shown in SEQ ID NO.

2.

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

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

9. The method for identifying whether a plant contains a wheat stripe rust resistance gene according to claim 8, characterized in that: The reaction system used for amplification includes: 1 μL DNA template, 5 μL 2×Taq Master Mix, 0.5 μL primer S-61635F at a concentration of 10 μmol / L, 0.5 μL primer S-61635R at a concentration of 10 μmol / L, double distilled water to 10 μL, and the reaction program is: 95°C for 5 min; 95°C for 30 s, 56°C for 30 s, 72°C for 15 s, 33 cycles; 72°C for 5 min; enzyme digestion with endonuclease at 37°C for 3 h, 65°C for 20 min; Preferably, a 581 bp DNA band is amplified, and two bands of 256 bp and 325 bp appear after enzyme digestion, indicating that the sample contains the stripe rust resistance gene. After enzyme digestion, no bands of 256 bp and 325 bp appear, indicating that the sample does not contain the stripe rust resistance gene.

10. A method for breeding wheat with a stripe rust resistance gene, characterized in that: include: A susceptible wheat parent and a resistant wheat parent carrying a stripe rust resistance gene are hybridized and then self-pollinated. During each generation of self-pollination, auxiliary selection is performed 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, so as to retain the wheat strain carrying the stripe rust resistance gene and conduct phenotypic identification on it.

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