KASP molecular marker linked with wheat stalk rust resistance gene Sr26 and application of KASP molecular marker

Abstract

The invention discloses a KASP molecular marker linked with a wheat stalk rust resistance gene Sr26 and application of the KASP molecular marker, and relates to the technical field of biomolecular detection. The nucleotide sequence of the KASP molecular marker is as shown in SEQ ID NO. 1. According to the present invention, the SNP site closely linked with the wheat Puccinia graminis L. rust resistance gene Sr26 is obtained through the genome comparative analysis, and the KASP molecular marker closely linked with the Sr26 is developed based on the SNP site, and has the co-separation characteristic with the resistance. The KASP molecular marker can be used for detecting whether wheat contains the Puccinia graminicola Sr26 or not, the detection process is simple, convenient and rapid, the flux is high, and a wheat Puccinia graminicola plant containing the Sr26 gene can be accurately identified. The method has important significance on wheat germplasm resource Puccinia graminicola-resistant gene screening, wheat Puccinia graminicola-resistant material creation or wheat Puccinia graminicola-resistant molecule auxiliary breeding.

Description

Technical Field

[0001] The present invention relates to the technical field of biological molecule detection, in particular to a KASP molecular marker linked to a wheat stem rust resistance gene Sr26 and an application thereof. Background Art

[0002] Wheat (Triticum aestivum L., AABBDD, 2n=6x=42) is a widely cultivated staple crop. Wheat growth is often plagued by various diseases, resulting in yield and quality losses. Wheat stem rust, caused by the wheat-specific form of the fungus Puccinia graminis (Puccinia graminis Pers. f. sp. tritici Eriks. & Henn., Pgt), is a major disease in wheat production. Breeding and utilizing genetically resistant varieties is considered the most effective and sustainable strategy for controlling this disease. First discovered in 1999, the highly pathogenic wheat stem rust race Ug99 mutates rapidly and exhibits strong virulence, purportedly activating one or more of the widely used resistance genes Sr21, Sr24, Sr31, and Sr36. Currently, over 80 stem rust (Sr) resistance genes have been discovered, 70 of which have been formally named or mapped to different chromosomes. Most of these resistance genes have lost resistance to Ug99. The stem rust resistance gene Sr26 confers resistance to all currently known field races of stem rust, including Ug99 and its related variants. Therefore, in addition to vigorously exploring and utilizing this resistance gene, further broadening the genetic basis of disease-resistant breeding materials is an important issue that needs to be addressed.

[0003] The present invention intends to develop a KASP molecular marker linked to the wheat stem rust resistance gene Sr26, in order to provide technical support for the screening of wheat germplasm resources for the stem rust resistance gene Sr26, the creation of wheat stem rust resistance materials or molecular assisted breeding of wheat stem rust resistance. Summary of the Invention

[0004] The present invention aims to provide a KASP molecular marker linked to the wheat stem rust resistance gene Sr26 and its application to address the above-mentioned problems in the prior art. The KASP molecular marker can be used to detect whether wheat contains the stem rust resistance gene Sr26. The detection process is simple, rapid, and high-throughput, and can accurately identify wheat plants that are stem rust-resistant and contain the Sr26 gene.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a KASP molecular marker linked to the wheat stem rust resistance gene Sr26. The nucleotide sequence of the KASP molecular marker is shown in SEQ ID NO.1.

[0007] The present invention also provides a KASP primer set for identifying the wheat stem rust resistance gene Sr26, comprising a forward primer 1, a forward primer 2, and a universal reverse primer; the nucleotide sequences of the forward primer 1 and the forward primer 2 are shown as SEQ ID NO. 2 and SEQ ID NO. 3, respectively, and the nucleotide sequence of the universal reverse primer is shown as SEQ ID NO. 4;

[0008] The forward primer 1 and the forward primer 2 are labeled with different fluorescent groups.

[0009] The present invention also provides the use of the KASP primer set in preparing a product for identifying the wheat stem rust resistance gene Sr26.

[0010] Furthermore, the product is a kit.

[0011] The present invention also provides a product for identifying the wheat stem rust resistance gene Sr26, comprising the above-mentioned KASP primer set.

[0012] Furthermore, the product is a kit.

[0013] The present invention also provides the use of the above-mentioned KASP molecular marker, KASP primer set or product in identifying the wheat stem rust resistance gene Sr26.

[0014] The present invention also provides a method for identifying the wheat stem rust resistance gene Sr26, comprising the following steps:

[0015] Extracting the genomic DNA of the wheat material to be tested;

[0016] The genomic DNA is used as a template, and the above-mentioned KASP primer set is used to perform fluorescent quantitative PCR amplification to obtain an amplified product;

[0017] Analyzing the amplified product to obtain genotype data, and obtaining the genotype of the wheat material to be tested;

[0018] The genotype showing only the fluorescent label of the forward primer 1 is denoted as X:X;

[0019] The genotype showing only the fluorescent label of the forward primer 2 was recorded as Y:Y;

[0020] The genotypes of the fluorescent markers of the forward primer 1 and the forward primer 2 are simultaneously displayed and recorded as X:Y or Y:X;

[0021] Determine whether the wheat material to be tested contains the stem rust resistance gene Sr26 according to the obtained genotype of the wheat material to be tested:

[0022] When the genotype is X:X, X:Y or Y:X, it is determined that the tested wheat material contains the stem rust resistance gene Sr26;

[0023] When the genotype is Y:Y, it is determined that the wheat material to be tested does not contain the stem rust resistance gene Sr26.

[0024] Furthermore, the reaction system of the fluorescent quantitative PCR amplification is: 0.1 μg DNA template, 5 μL FLU-ARMS V5F, 0.5 μL KASP primer set and 3.5 μL ddH2O.

[0025] Furthermore, the reaction procedure of the fluorescent quantitative PCR amplification is: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 5 seconds, annealing at 58°C for 20 seconds, and 45 cycles.

[0026] The present invention discloses the following technical effects:

[0027] The present invention identified a single-nucleotide polymorphism (SNP) site tightly linked to the wheat stem rust resistance gene Sr26 through genomic alignment analysis. Based on the SNP site, a KASP molecular marker tightly linked to Sr26 was developed, which co-segregates with resistance. This KASP molecular marker can be used to detect the presence of the Sr26 stem rust resistance gene in wheat. The detection process is simple, rapid, and high-throughput, and can accurately identify wheat stem rust-resistant plants containing the Sr26 gene. This method is of great significance for screening wheat germplasm resources for stem rust resistance genes, developing wheat stem rust-resistant materials, or molecularly assisted breeding for wheat stem rust resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0029] Figure 1 This is the gene structure diagram of Sr26;

[0030] Figure 2 Genotyping test results for the ZK-SR26E3 marker; the test materials involved are Avocet, Chinese Spring, Zhou 8425B, and Xiaoyan 81;

[0031] Figure 3 The genotyping results of 14 Chinese wheat varieties in Example 3;

[0032] Figure 4 The genotyping results of 8 Australian wheat varieties in Example 3;

[0033] Figure 5 The genotyping results of some Huanghuai wheat region promoted varieties and strain population 1 collected in Example 4;

[0034] Figure 6 The genotyping results of some Huanghuai wheat region promoted varieties and strain group 2 collected in Example 4;

[0035] Figure 7 The genotyping results of some Huanghuai wheat region promoted varieties and strain group 3 collected in Example 4;

[0036] Figure 8 The genotyping results of some Huanghuai wheat region promoted varieties and strain group 4 collected in Example 4;

[0037] Figure 9 The genotyping results of some Huanghuai wheat region promoted varieties and strain population 5 collected in Example 4;

[0038] Figure 10 These are the genotyping results of some Huanghuai wheat region promoted varieties and strain population 6 collected in Example 4. DETAILED DESCRIPTION

[0039] 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 rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0040] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0041] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice 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 associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0042] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0043] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0044] Example 1 Acquisition of KASP Molecular Markers Tightly Linked to the Sr26 Gene

[0045] Using sequence alignment website platforms and software (such as NCBI, SnapGene), the Sr26 gene (structure diagram see Figure 1 The full-length sequence was aligned with the published genome sequences of Chinese Spring, Zhou 8425B, and wheat 10+ Genomes. SNPs within highly similar sequence fragments were identified and converted into KASP markers for genotyping. KASP primers were designed using the Wheatomics website (http: / / wheatomics.sdau.edu.cn / ) and synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0046] The present invention designs and obtains a KASP molecular marker ZK-SR26E3 that is tightly linked to the Sr26 gene, and its nucleotide sequence is shown in SEQ ID NO.1.

[0047] SEQ ID NO.1:

[0048] 5′-GGAATCCAACTTCRCCASAGACTATGACCCTCGGAACTTCCCTTTCAACAATTCG CAGAACCAGATAACGCAGAACAGGTAAGCCTCCAAGGA-3′; wherein, R is G or A; and S is G or C.

[0049] The KASP primer set designed for genotyping detection of the KASP molecular marker ZK-SR26E3 is shown in Table 1.

[0050] The genotyping of ZK-SR26E3 marker was tested using the test materials Avocet, Chinese Spring, Zhou 8425B and Xiaoyan 81. The results are shown in Figure 2 , the results showed that the genotyping was obvious.

[0051] Table 1 KASP primer sets

[0052]

[0053] Note: 5′-GAAGGTGACCAAGTTCATGCT-3′ is a FAM fluorescent universal primer. The FAM fluorescent group is observed and read at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. 5′-GAAGGTCGGAGTCAACGGATT-3′ is a HEX fluorescent universal primer. The HEX fluorescent group is observed and read at an excitation wavelength of 535 nm and an emission wavelength of 556 nm.

[0054] Example 2 Genotyping and Identification Method for Containing Stem Rust Resistance Gene Sr26

[0055] Method for identifying whether wheat materials contain the stem rust resistance gene Sr26:

[0056] (1) Extracting the genomic DNA of the wheat material to be tested using the CTAB method;

[0057] (2) Using the genomic DNA of the wheat material to be tested as a template, the KASP primer set for genotyping detection of the KASP molecular marker ZK-SR26E3 obtained in Example 1 was used to perform fluorescent quantitative PCR amplification to obtain an amplified product;

[0058] The reaction system for fluorescence quantitative PCR amplification was as follows: 0.1 μg DNA template, 5 μL FLU-ARMS V5F, 0.5 μL KASP primer set, and 3.5 μL ddH2O; the concentration of each primer was 10 μM.

[0059] The reaction program of fluorescence quantitative PCR amplification was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 5 s, annealing at 58°C for 20 s, and 45 cycles.

[0060] (3) Analyze the amplified products using Bio-Rad CFX Maestro software to obtain genotype data and the genotype of the wheat material to be tested;

[0061] The genotype of the homozygous allele with FAM fluorescence is X:X, the genotype of the homozygous allele with HEX fluorescence is Y:Y, and the genotype of the heterozygote is X:Y or Y:X;

[0062] (4) Determine whether the plant to be tested contains the stem rust resistance gene Sr26 based on the genotype:

[0063] When the genotype is X:X, X:Y or Y:X, it is determined that the tested wheat material contains the stem rust resistance gene Sr26;

[0064] When the genotype is Y:Y, it is determined that the wheat material to be tested does not contain the stem rust resistance gene Sr26.

[0065] Example 3 Application of KASP molecular markers in identifying wheat varieties containing the stem rust resistance gene Sr26

[0066] The identification method of Example 2 was used to perform genotyping on 14 Chinese wheat varieties, 8 Australian wheat varieties, 4 disease-resistant varieties Avocet, Eagle, Kite, Avocet+Lr34, and 1 susceptible variety Chinese Spring. At the same time, the gold standard (PCR detection method) was used to detect the wheat stem rust resistance Sr26 gene. The results are shown in Tables 2 and Figure 3-Figure 4 .

[0067] The results showed that the disease-resistant varieties Avocet, Eagle, Kite, Avocet+Lr34 and the artificial hybrid contained the Sr26 gene, while the remaining 22 wheat materials did not contain the Sr26 gene and were clearly typed. These results indicate that ZK-SR26E3 can be used for the molecular detection of the wheat stem rust resistance gene Sr26 and the screening of disease-resistant wheat.

[0068] Table 2 Genotyping results of 14 Chinese wheat varieties, 8 Australian wheat varieties, 4 disease-resistant varieties and 1 susceptible variety

[0069]

[0070]

[0071] Example 4 Application of KASP molecular markers in genetically diverse populations

[0072] The identification method of Example 2 was used to perform genotyping on some of the Huanghuai wheat region promoted varieties and lines (as shown in Tables 3-8), the positive control disease-resistant variety Avocet, the negative control variety Chinese Spring, and the artificial hybrid Avocet + Chinese Spring. The results are shown in Tables 3-8 and Figure 5-10 The results showed that, except for the positive control and artificial hybrids, the other wheat varieties did not contain the Sr26 gene.

[0073] The above results indicate that ZK-SR26E3 can identify the presence or absence of the Sr26 gene and is suitable for screening germplasm resources for the wheat stem rust resistance gene Sr26.

[0074] Table 3 Genotyping results of some promoted varieties and line population 1 collected in the Huanghuai wheat region

[0075]

[0076]

[0077] Table 4 Genotyping results of some promoted varieties and line population 2 collected in the Huanghuai wheat region

[0078]

[0079]

[0080] Table 5 Genotyping results of some promoted varieties and line population 3 in the Huanghuai wheat region

[0081]

[0082]

[0083] Table 6 Genotyping results of some promoted varieties and line population 4 collected in the Huanghuai wheat region

[0084]

[0085]

[0086] Table 7 Genotyping results of some promoted varieties and line population 5 collected in the Huanghuai wheat region

[0087]

[0088]

[0089] Table 8 Genotyping results of some promoted varieties and line population 6 collected in the Huanghuai wheat region

[0090]

[0091] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A KASP molecular marker linked to the wheat stem rust resistance gene Sr26, characterized in that: The nucleotide sequence of the KASP molecular marker is shown in SEQ ID NO.

1.

2. A KASP primer set for identifying the wheat stem rust resistance gene Sr26, characterized in that: It comprises a forward primer 1, a forward primer 2 and a universal reverse primer; the nucleotide sequences of the forward primer 1 and the forward primer 2 are shown in SEQ ID NO.2 and SEQ ID NO.3 respectively, and the nucleotide sequence of the universal reverse primer is shown in SEQ ID NO.4; The forward primer 1 and the forward primer 2 are labeled with different fluorescent groups.

3. Use of the KASP primer set according to claim 2 in preparing a product for identifying the wheat stem rust resistance gene Sr26.

4. The use according to claim 3, characterized in that The product is a test kit.

5. A product for identifying wheat stem rust resistance gene Sr26, characterized in that: Comprising the KASP primer set according to claim 2.

6. The product according to claim 5, characterized in that The product is a test kit.

7. Use of the KASP molecular marker according to claim 1, the KASP primer set according to claim 2, or the product according to claim 5 or 6 in identifying the wheat stem rust resistance gene Sr26.

8. A method for identifying the wheat stem rust resistance gene Sr26, characterized in that: The following steps are involved: Extracting the genomic DNA of the wheat material to be tested; The genomic DNA is used as a template, and the KASP primer set according to claim 2 is used to perform fluorescent quantitative PCR amplification to obtain an amplified product; Analyzing the amplified product to obtain genotype data, and obtaining the genotype of the wheat material to be tested; The genotype showing only the fluorescent label of the forward primer 1 is denoted as X:X; The genotype showing only the fluorescent label of the forward primer 2 was recorded as Y:Y; The genotypes of the fluorescent markers of the forward primer 1 and the forward primer 2 are simultaneously displayed and recorded as X:Y or Y:X; Determine whether the wheat material to be tested contains the stem rust resistance gene Sr26 according to the obtained genotype of the wheat material to be tested: When the genotype is X:X, X:Y or Y:X, it is determined that the tested wheat material contains the stem rust resistance gene Sr26; When the genotype is Y:Y, it is determined that the wheat material to be tested does not contain the stem rust resistance gene Sr26.

9. The method according to claim 8, characterized in that The reaction system for the fluorescent quantitative PCR amplification is: 0.1 μg DNA template, 5 μL FLU-ARMS V5F, 0.5 μL KASP primer set and 3.5 μL ddH2O.

10. The method according to claim 8, characterized in that The reaction procedure of the fluorescent quantitative PCR amplification was as follows: pre-denaturation at 95° C. for 5 min; denaturation at 95° C. for 5 s, annealing at 58° C. for 20 s, and 45 cycles.

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