Co-dominant function marker for detecting wheat powdery mildew resistance related genes, multiple PCR amplification system and application

Abstract

The invention provides a co-dominant functional marker for detecting wheat powdery mildew resistance related genes, a multiplex PCR amplification system and application, and belongs to the field of molecular biology and crop genetic breeding. Sequence information of the Pm26 gene and the Pm68 gene is utilized to develop co-dominant functional markers XPm26 and XPm68-2 of the Pm26 and the Pm68, the XPm26 and the XPm68-2 are combined to form a multiple PCR amplification system for detecting the two wheat powdery mildew resistance genes, the wheat powdery mildew resistance genes Pm26 and Pm68 are identified at the same time through one-time PCR reaction, the gene Pm26 and Pm68 can be used for detecting genetic segregation populations, and the gene Pm26 and Pm68 can be used for detecting the powdery mildew resistance of the wheat. And identifying an exchange event of the Pm26 and the Pm68 to obtain a novel powdery mildew-resistant wheat germplasm with double-gene homozygous polymerization of the Pm26 and the Pm68, so that a novel, practical and efficient functional marker-assisted selection technology is provided for breeding new powdery mildew-resistant wheat varieties.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and crop genetics and breeding, and specifically relates to codominant functional markers, multiplex PCR amplification systems, and applications for detecting wheat powdery mildew resistance-related genes. Background Technology

[0002] Powdery mildew is one of the most important fungal diseases threatening global wheat production, causing significant yield losses and quality declines. Breeding resistant varieties using disease-resistant genes is considered the most economical, environmentally friendly, and sustainable strategy for controlling this disease. The powdery mildew resistance genes Pm26 and Pm68, located on the wheat 2BS chromosome, originate from the wild emmer wheat line TTD140 (Zhu et al. An atypical NLR pair TdCNL1 / TdCNL5 from wild emmerconfers powdery mildew resistance in wheat. Nature Genetics. 2025, 57:1553-1562) and the durum wheat line TRI 1796 (He et al. An NLR pair in the Pm68 locus conferspowdery mildew resistance in durum and common wheat. Nature Communications. 2025, 16:9039), respectively. The homologous sequence of the Pm26 gene is located at 21.77 Mb on chromosome 2BS of the Chinese *C. cylindrica* reference genome, while the homologous sequence of Pm68 is located at 25.62 Mb on chromosome 2BS. Since Pm26 and Pm68 originate from different chromosomes 2BS, these two genes are clearly mutually exclusive, and their physical locations on the Chinese *C. cylindrica* genome are quite close, making linkage to form a homozygous Pm26-Pm68 linkage pattern extremely difficult. Because Pm26 and Pm68 are 3.85 Mb apart, exchange may occur at some frequency in their hybrid populations, but this frequency is likely low. Therefore, there is an urgent need to develop an efficient screening system to identify exchange events in their hybrid populations, thereby achieving linkage between Pm26 and Pm68.

[0003] Molecular marker-assisted selection has become a key tool for overcoming the aforementioned bottlenecks in modern crop genetic improvement. In particular, codominant functional markers can clearly distinguish between homozygous and heterozygous genotypes of target genes, ensuring accurate tracking of these genes; while multiplex PCR technology can simultaneously detect two or more gene loci in a single reaction, significantly improving genotype identification efficiency and saving costs. Therefore, it is necessary to develop a rapid screening method using molecular markers to identify low-frequency exchange events, enabling efficient and precise aggregation of Pm26 and Pm68 and the formation of linkage relationships. This has positive practical significance for accelerating the breeding process of broad-spectrum, durable powdery mildew-resistant wheat varieties. Summary of the Invention

[0004] To address some shortcomings in existing technologies, this invention provides co-dominant functional markers, multiplex PCR amplification systems, and applications for detecting wheat powdery mildew resistance-related genes. Utilizing the sequence information of the Pm26 gene (Genbank accession number: PP781952) and the Pm68 gene (Genbank accession number: PQ655412), this invention develops co-dominant functional markers XPm26 and XPm68-2 for Pm26 and Pm68. This invention combines XPm26 and XPm68-2 into a multiplex PCR amplification system for detecting multiple wheat powdery mildew resistance genes, enabling the simultaneous identification of wheat powdery mildew resistance genes Pm26 and Pm68 in a single PCR reaction. This system can be used to detect genetically segregating populations, identify exchange events between Pm26 and Pm68, and obtain new powdery mildew-resistant wheat germplasm with homozygous aggregation of both Pm26 and Pm68 genes. This provides a novel, practical, and efficient functional marker-assisted selection technology for breeding new wheat varieties resistant to powdery mildew.

[0005] To achieve the above-mentioned technical objectives, the present invention employs the following technical means:

[0006] This invention first provides codominant functional markers for wheat powdery mildew resistance-related genes. The codominant functional markers include the functional marker XPm26 for the wheat powdery mildew resistance gene Pm26 and the functional marker XPm68-2 for the wheat powdery mildew resistance gene Pm68. The primers for amplifying the functional marker XPm26 are shown in SEQ ID No:1 and SEQ ID No:2, and the primers for amplifying the functional marker XPm68-2 are shown in SEQ ID No:3 and SEQ ID No:4.

[0007] The nucleotide sequences of the primers used to amplify XPm26 are as follows:

[0008] XPm26-F: 5'-GGATTAGTTCTGGATATACAGT-3' (SEQ ID No: 1);

[0009] XPm26-R: 5'-ACATCATCAACCAAGACCAAGT-3' (SEQ ID No: 2);

[0010] The nucleotide sequences of the primers used to amplify XPm28 are as follows:

[0011] XPm68-2-F: 5'-TCGGCCGTTCCTATTCGCCATTG-3' (SEQ ID No: 3),

[0012] XPm68-2-R: 5'-CCACACCACATAGGTTGGTGCAT-3' (SEQ ID No: 4).

[0013] The present invention also provides primers for detecting wheat powdery mildew resistance-related genes or the above-mentioned codominant functional markers, wherein the genes include wheat powdery mildew resistance-related genes Pm26 and Pm68; the primers include primer pairs shown in SEQ ID No:1 and SEQ ID No:2 and primer pairs shown in SEQ ID No:3 and SEQ ID No:4.

[0014] The present invention also provides a reagent or kit for detecting wheat powdery mildew resistance-related genes Pm26 and Pm68, or for detecting the above-mentioned co-dominant functional markers, wherein the reagent or kit contains primers for amplifying the above-mentioned functional markers.

[0015] The present invention also provides a multiplex PCR amplification system for detecting wheat powdery mildew resistance-related genes, wherein the genes include wheat powdery mildew resistance-related genes Pm26 and Pm68, and the amplification system includes amplification primers of the above-mentioned codominant functional markers or the above-mentioned primers.

[0016] This invention also provides the application of any of the above-mentioned codominant functional markers, primers, reagents or kits, or multiplex PCR amplification systems in detecting polymerized wheat powdery mildew resistance genes Pm26 and Pm68 in materials.

[0017] Preferably, the application includes any one of the following:

[0018] (1) To identify or assist in identifying whether the sample to be tested carries the wheat powdery mildew resistance gene Pm26 and / or Pm68;

[0019] (2) Identify the exchange events between Pm26 and Pm68, and identify the wheat that has exchanged between Pm26 and Pm68;

[0020] (3) Screening for materials carrying Pm26-Pm68 linked chromosomes;

[0021] (4) Breed new wheat varieties resistant to powdery mildew.

[0022] This invention also provides a method for detecting polymerized materials containing powdery mildew resistance genes Pm26 and Pm68, the method comprising:

[0023] The genomic DNA of the test material was amplified by PCR using the above primers, and the amplification products were detected. If the specific target products XPm26 and XPm68-2 could be obtained at the same time, then the material with the aggregated anti-powdery mildew genes Pm26 and Pm68 was obtained.

[0024] Preferably, the PCR reaction system used in the PCR amplification procedure comprises: 50 ng template DNA, 1× PCR buffer, and 1.5 mmol / L... -1 MgCl2, 200 mmol L -1 The final concentrations of the four primers (dNTP, XPm26-F, XPm26-R, XPm68-2-F, and XPm68-2-R) were each 2 μmol / L. -1 Add 1 U Taq DNA polymerase and replenish the reaction system to 25 μL with sterile distilled water.

[0025] Preferably, the PCR amplification reaction program includes: pre-denaturation at 94°C for 3 minutes; denaturation at 94°C for 20 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 60 seconds, 34 cycles; extension at 72°C for 5 minutes; and storage at 4°C.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] This invention develops highly specific and stable co-dominant functional markers for Pm26 and Pm68. A multiplex PCR amplification system is constructed by combining the two functional markers XPm26 and XPm68-2, enabling simultaneous identification of the Pm26 and Pm68 genes in a single PCR reaction. This system can be used to detect genetically segregating populations, rapidly screen for low-frequency exchange events, and achieve efficient and precise aggregation of Pm26 and Pm68, establishing linkage relationships. This has significant practical implications for accelerating the breeding process of broad-spectrum, long-lasting powdery mildew resistant wheat varieties.

[0028] Using the detection system of this invention, genetically recombinant plants of Pm26 and Pm68 were successfully and rapidly identified from the segregating population of the F2 offspring of the wheat variety Yangmai 158-Pm26 (containing Pm26) and the wheat variety Yangmai 158-Pm68 (containing Pm68), thus obtaining new wheat germplasm with close linkage between Pm26 and Pm68. This invention provides an efficient and precise functional marker-assisted selection technology for the aggregation breeding of wheat powdery mildew resistance genes Pm26 and Pm68. Attached Figure Description

[0029] Figure 1 The image shows the amplification results of multiplex PCR detection of functional markers XPm26 and XPm68-2 in parental materials. In the image, M: DNA molecular weight marker DL2000; 1-3: common wheat variety Chinese Spring susceptible to powdery mildew; 4-6: common wheat variety Yangmai 158 susceptible to powdery mildew; 7-9: powdery mildew resistant wheat variety Yangmai 158-Pm26; 10-12: powdery mildew resistant wheat variety Yangmai 158-Pm68; the band at 377 bp indicated by the arrow is the specific band containing Pm26, and the band at 164 bp is the specific band containing Pm68.

[0030] Figure 2 To detect the genotype of the F2 generation of the cross between wheat lines Yangmai 158-Pm26 and Yangmai 158-Pm68 using the functional markers XPm26 and XPm68-2; in the figure, M: DNA molecular weight marker DL2000. 1-21: Different individual plants from the F2 generation of the cross between wheat lines Yangmai 158-Pm26 and Yangmai 158-Pm68, lanes 1, 2, 3, 5, 6, 7, 8, 1 The genotypes of individual plants 1, 12, 16, 17, and 18 are both heterozygous for Pm26 and Pm68; the genotypes of individual plants in lanes 4, 13, 14, and 20 are homozygous for Pm26 with only the Pm26 gene; the genotypes of individual plants in lanes 9, 10, 15, and 19 are homozygous for Pm68 with only the Pm68 gene; lane 21 is an exchanged individual plant containing both Pm26 and Pm68 genes, and its genotype is homozygous for Pm26 and heterozygous for Pm68.

[0031] Figure 3 Genotyping of families containing tightly linked chromosome segments of Pm26-Pm68; In the figure, M: DNA molecular weight marker DL2000; 1-12: Genotypes of different individuals in families containing tightly linked chromosome segments of Pm26-Pm68, homozygous for both Pm26 and Pm68; 13: Powdery mildew-susceptible common wheat Yangmai 158; 14: Powdery mildew-resistant wheat line Yangmai 158-Pm26 (containing Pm26); 15: Powdery mildew-resistant parental material wheat line Yangmai 158-Pm68 (containing Pm68). Detailed Implementation

[0032] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited thereto. In the embodiments of the present invention, unless otherwise described, conventional experimental methods are used. The processes involved in the embodiments, unless otherwise described, are those that can be understood and easily implemented by those skilled in the art based on the product manual or basic knowledge in the field, and therefore will not be described in detail.

[0033] The wheat materials and sources used in the following examples are as follows:

[0034] The common wheat variety "Chinese Spring" susceptible to powdery mildew is for public use and was provided by the Jiangsu Lixiahe Regional Agricultural Science Research Institute.

[0035] The common wheat variety Yangmai 158, susceptible to powdery mildew, is publicly available and was provided by the Jiangsu Lixiahe Regional Agricultural Science Research Institute.

[0036] Yangmai 158-Pm26 was created by the inventor. It is a pure line containing the Pm26 gene formed by crossing Yangmai 158 as a recurrent parent with the wheat line Pm26-40 (which is publicly known and provided by the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences) and self-pollinating (this material was identified by the molecular marker XPm26 provided by this invention).

[0037] Yangmai 158-Pm68 is a pure line carrying Pm68, created by the inventors. See He et al. An NLR pairin the Pm68 locus confers powdery mildew resistance in durum and commonwheat. Nature Communications. 2025,16:9039, with the original number BC3F5Line1 in the article.

[0038] Example 1: Development of codominant functional marker combinations and establishment of multiplex PCR amplification system

[0039] Based on the sequence differences between the Pm26 gene (Genbank accession number: PP781952) and the Pm68 gene (Genbank accession number: PQ655412), codominant functional markers were designed.

[0040] The Pm26 and Pm68 gene sequences were compared and analyzed with the genomes of the powdery mildew-susceptible common wheat varieties Chinese Spring (http: / / 202.194.139.32) and Yangmai 158 (http: / / 202.194.139.32), respectively. Specific functional markers XPm26 and XPm68-2 were developed, and the primer sequences are as follows:

[0041] XPm26-F: 5'-GGATTAGTTCTGGATATACAGT-3' (SEQ ID No: 1);

[0042] XPm26-R: 5'-ACATCATCAACCAAGACCAAGT-3' (SEQ ID No: 2);

[0043] XPm68-2-F: 5'-TCGGCCGTTCCTATTCGCCATTG-3' (SEQ ID No: 3),

[0044] XPm68-2-R: 5'-CCACACCACATAGGTTGGTGCAT-3' (SEQ ID No: 4).

[0045] To achieve simultaneous detection of two powdery mildew resistance genes, Pm26 and Pm68, in the same reaction system, this study established a multiplex PCR system containing two primer pairs, XPm26 and XPm68-2. This significantly improves detection efficiency, saving time and reagent costs; furthermore, it allows for simultaneous analysis of multiple target genes in the same reaction, making it suitable for large-scale population detection and marker-assisted selection.

[0046] Using wheat varieties such as Chinese Spring, Yangmai 158, Yangmai 158-Pm26, and Yangmai 158-Pm68 as templates, PCR verification was performed simultaneously using the specific functional markers XPm26 and XPm68-2.

[0047] PCR reaction system: A 25 μL reaction system contains approximately 50 ng template DNA, 1× PCR buffer, 1.5 mmol L⁻¹ MgCl₂, and 200 mmol L⁻¹ PCR buffer. -1 dNTPs, with a final concentration of 2 μmol / L for each of the four primers. -1 Add 1 U Taq DNA polymerase and replenish the reaction system to 25 μL with sterile distilled water.

[0048] PCR reaction procedure: 94℃ pre-denaturation for 3 minutes; 94℃ denaturation for 20 seconds, 55℃ annealing for 30 seconds, 72℃ extension for 60 seconds, 34 cycles; 72℃ final extension for 5 minutes; store at 4℃.

[0049] PCR products were detected by 8% polyacrylamide gel electrophoresis (PAGE): PCR products were mixed with 5 μL of loading buffer and separated by electrophoresis on an 8% non-denaturing polyacrylamide gel. Electrophoresis was performed at a constant voltage of 120 V for 2 hours and then stained with silver.

[0050] The results are as follows Figure 1As shown, lanes 1-3 represent amplification bands of the common wheat variety *Chinese Spring* susceptible to powdery mildew, where the larger band is the background band formed by XPm26 amplification (322 bp) and the smaller band is the background band formed by XPm68-2 amplification (150 bp). Lanes 4-6 represent amplification bands of the common wheat variety *Yangmai 158* susceptible to powdery mildew, where the larger band is the background band formed by XPm26 amplification (322 bp) and the smaller band is the background band formed by XPm68-2 amplification (150 bp). Lanes 7-9 represent amplification bands of the wheat variety *Yangmai 158-Pm26*, which is the target band formed by XPm26 amplification of the Pm26 gene (377 bp). Lanes 10-12 represent amplification bands of the wheat variety *Yangmai 158-Pm68*, where the larger band is the background band formed by XPm26 amplification (322 bp). The smaller band is the target band formed by XPm68-2 amplification of the Pm68 gene, with a size of 164 bp. It can be seen that the functional marker XPm26 can amplify a specific band of 377 bp in materials containing the Pm26 gene, while this band is not present in materials without the Pm26 gene. Similarly, the functional marker XPm68-2 can amplify a specific band of 164 bp in materials containing the Pm68 gene, but this band is not present in materials without the Pm68 gene.

[0051] In summary, multiplex PCR can distinguish whether the material to be identified contains the aggregation of the Pm26, Pm68, or both Pm26 and Pm68 genes, and whether the gene carrier is in a heterozygous or homozygous state. Both XPm26 and XPm68-2 can stably amplify specific bands of the expected size with clear band patterns.

[0052] Example 2: Screening for Pm26-Pm68 linked chromosomes in the F2 population

[0053] In this embodiment, the wheat cultivar Yangmai 158-Pm26 carrying Pm26 was crossed with the wheat cultivar Yangmai 158-Pm68 carrying Pm68, and self-crossed to form F2. After DNA extraction using the CTAB method, the F2 plants were validated using multiplex PCR with the developed co-dominant functional markers XPm26 and XPm68-2. The validation steps were as shown in Example 1, and the validation results were as follows. Figure 2 As shown.

[0054] from Figure 2It can be seen that lanes 1, 2, 3, 5, 6, 7, 8, 11, 12, 16, 17, and 18 all have specific bands of 377 bp and 164 bp, respectively, indicating that these monoclonal plants all contain Pm26 and Pm68, but these two genes are in a heterozygous state. Lanes 4, 13, 14, and 20 only have a specific band of 377 bp, indicating that these four monoclonal plants are homozygous for Pm26. Lanes 9, 10, 15, and 19 only have a specific band of 164 bp, indicating that these four monoclonal plants are homozygous for Pm68. According to the amplified bands, the monoclonal plant involved in lane 21 is homozygous for Pm26 and heterozygous for Pm68, indicating that this monoclonal plant contains a heterozygous Pm26-Pm68 linked chromosome. This monoclonal plant was retained for subsequent experiments.

[0055] Example 3: Creation and identification of stable lines homozygous for the Pm26-Pm68 double gene.

[0056] In this embodiment, the exchanged single plants containing heterozygous Pm26-Pm68 linkage selected in Example 2 were self-crossed. The multiplex PCR system of Example 1 was used to detect and screen the self-crossed progeny for homozygosity. The process was followed and selected for multiple generations. In each generation, single plants that showed homozygous banding at both Pm26 and Pm68 sites were preferentially retained, while heterozygous single plants or those containing only a single disease resistance gene were gradually eliminated.

[0057] After three generations of targeted screening and verification, the test results are as follows: Figure 3 As shown in the diagram, lanes 1-12 represent the genotype bands of self-crossed progeny of heterozygous Pm26-Pm68 linked exchange single plants, with both genes in a homozygous state; lane 13 represents the negative control, Yangmai 158, a common wheat variety susceptible to powdery mildew; lane 14 represents the positive control, Yangmai 158-Pm26, containing Pm26; and lane 15 represents the positive control, Yangmai 158-Pm68, containing Pm68. These results indicate that stable aggregated lines with homozygous Pm26 and Pm68 were successfully obtained. These lines simultaneously possess the characteristic bands of 377 bp and 164 bp at the molecular level, with stable and consistent band patterns and no segregation. This demonstrates that Pm26 and Pm68 have been stably linked together and are both in a homozygous state.

[0058] In summary, this invention provides a combination of codominant functional markers for detecting multiple wheat powdery mildew resistance genes, a multiplex PCR amplification system, and applications. Utilizing the sequence information of the Pm26 gene (Genbank accession number: PP781952) and the Pm68 gene (Genbank accession number: PQ655412), this invention develops codominant functional markers XPm26 and XPm68-2 for Pm26 and Pm68. This invention combines XPm26 and XPm68-2 into a system for detecting multiple wheat powdery mildew resistance genes. A multiplex PCR amplification system for detecting multiple wheat powdery mildew resistance genes was developed, enabling the simultaneous identification of wheat powdery mildew resistance genes Pm26 and Pm68 in a single PCR reaction. This system can be used to detect genetically segregating populations, identify exchange events between Pm26 and Pm68, and obtain new powdery mildew-resistant wheat germplasm with homozygous aggregation of both Pm26 and Pm68 genes. This improves the efficiency and reliability of cumulative breeding of disease resistance genes and provides a novel, practical, and efficient functional marker-assisted selection technology for breeding new wheat varieties resistant to powdery mildew.

[0059] The embodiments described above are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A codominant functional marker for wheat powdery mildew resistance-related genes, characterized in that, The codominant functional markers include the functional marker XPm26 for the wheat powdery mildew resistance gene Pm26 and the functional marker XPm68-2 for the wheat powdery mildew resistance gene Pm68. The primers for amplifying the functional marker XPm26 are shown in SEQ ID No:1 and SEQ ID No:2, and the primers for amplifying the functional marker XPm68-2 are shown in SEQ ID No:3 and SEQ ID No:

4.

2. Primers for detecting wheat powdery mildew resistance-related genes or the co-dominant functional marker of claim 1, characterized in that, The genes include wheat powdery mildew resistance-related genes Pm26 and Pm68; the primers include the primer pairs shown in SEQ ID No:1 and SEQ ID No:2 and the primer pairs shown in SEQ ID No:3 and SEQ ID No:

4.

3. A reagent or kit, characterized in that, The reagent or kit is used to detect wheat powdery mildew resistance-related genes Pm26 and Pm68, or to detect the codominant functional marker of claim 1, wherein the reagent or kit contains primers for amplifying the functional marker of claim 1.

4. A multiplex PCR amplification system for detecting wheat powdery mildew resistance-related genes, characterized in that, The genes include wheat powdery mildew resistance-related genes Pm26 and Pm68, and the amplification system includes amplification primers of the codominant functional markers as described in claim 1 or primers as described in claim 2.

5. The use of any one of the codominant functional markers of claim 1, the primers of claim 2, the reagents or kits of claim 3, and the multiplex PCR amplification system of claim 4 in detecting polymerized wheat powdery mildew resistance genes Pm26 and Pm68 materials.

6. The application according to claim 5, characterized in that, The application includes any of the following: (1) To identify or assist in identifying whether the sample to be tested carries the wheat powdery mildew resistance gene Pm26 and / or Pm68; (2) Identify the exchange events between Pm26 and Pm68, and identify the wheat that has exchanged between Pm26 and Pm68; (3) Screening for materials carrying Pm26-Pm68 linked chromosomes; (4) Breed new wheat varieties resistant to powdery mildew.

7. A method for detecting polymeric materials containing powdery mildew resistance genes Pm26 and Pm68, characterized in that, The method includes: Using the primers described in claim 2, PCR amplification of the genomic DNA of the test material was performed, and the amplification products were detected. If the specific target products XPm26 and XPm68-2 could be obtained simultaneously, then the material with aggregated anti-powdery mildew genes Pm26 and Pm68 was obtained.

8. The method according to claim 8, characterized in that, The PCR reaction system used in the PCR amplification procedure includes: 50 ng template DNA, 1× PCR buffer, and 1.5 mmol / L. -1 MgCl2, 200 mmol L -1 The final concentrations of the four primers (dNTP, XPm26-F, XPm26-R, XPm68-2-F, and XPm68-2-R) were each 2 μmol / L. -1 Add 1 U Taq DNA polymerase and replenish the reaction system to 25 μL with sterile distilled water.

9. The method according to claim 8, characterized in that, The PCR amplification reaction procedure includes: pre-denaturation at 94℃ for 3 minutes; denaturation at 94℃ for 20 seconds, annealing at 55℃ for 30 seconds, extension at 72℃ for 60 seconds, 34 cycles; extension at 72℃ for 5 minutes; storage at 4℃.

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