Molecular marker for efficiently tracking wheat powdery mildew resistance gene and application of molecular marker

Abstract

The invention provides a molecular marker for efficiently tracking a wheat powdery mildew resistance gene and application of the molecular marker, and belongs to the technical fields of molecular biology, agricultural biology and genetic breeding science. The invention discloses four diagnostic molecular markers Pm26-M, Pm36-M, TdPm60-M and Pm68-M capable of accurately tracking powdery mildew resistance genes Pm26, Pm36, TdPm60 and Pm68 of wheat respectively, and an application of the four diagnostic molecular markers Pm26-M, Pm36-M, TdPm60-M and Pm68-M. The molecular marker can be independently used, and can also form a mixed detection system to carry out multiple PCR amplification, so that efficient detection of a plurality of powdery mildew resistance genes is realized; the molecular marker has the characteristics of good stability, high resolution and good repeatability in PCR amplification, and an efficient and reliable tool is provided for molecular marker-assisted selection and precise transformation of the wheat powdery mildew resistance gene.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology, agricultural biology, and genetic breeding science and technology, specifically involving molecular markers for efficiently tracking wheat powdery mildew resistance genes and their applications. Background Technology

[0002] Wheat is one of the world's most important food crops. Powdery mildew, caused by wheat powdery mildew fungus, is a widespread and devastating disease that leads to severe yield reduction and quality deterioration, posing a serious threat to the sustainability of wheat production. Discovering and utilizing highly effective disease-resistant genes is the most economical and environmentally friendly strategy for controlling this disease. Therefore, developing molecular markers that can accurately track superior disease-resistant genes is of great significance for breeding wheat varieties resistant to powdery mildew.

[0003] Pm26 originates from the wild emmer wheat line TTD140 and is located on the short arm of chromosome 2B. It is a recessive gene regulated by a pair of NLR genes, TdCNL1 and TdCNL5 (Zhu et al. An atypical NLR pair TdCNL1 / TdCNL5 from wild emmer confers powdery mildew resistance in wheat. Nature Genetics, 2025, 57: 864-873). Pm36 originates from the wild emmer wheat line MG29896 and its genetic behavior follows the dominant single-gene inheritance pattern. This gene has been finely mapped to the Bin-0.29-0.76 region on chromosome 5BL, and its function has been confirmed as encoding a transmembrane domain tandem kinase (Li et al. A membrane associated tandem kinase from wildemmer wheat confers broad-spectrum resistance to powdery mildew. Nature Communications, 2024, 15: 2763). Pm60 originates from the Urartu wheat variety PI428309 (Zou et al. TheNB-LRR gene Pm60 confers powdery mildew resistance in wheat. New Phytologist, 2018, 218: 298-309). Studies have shown that the homolog of Pm60 in wild emmer wheat, MlIW172 (i.e., TdPm60), also possesses powdery mildew resistance (Wu et al. Functional characterization of powdery mildew resistance gene MlIW172, a new Pm60 allele and its allelic variation in wildemmer wheat. Journal of Genetics and Genomics, 2022, 49: 787-795).Pm68 is a powdery mildew resistance gene discovered in durum wheat TRI 1796. Genetic and transgenic analysis has confirmed that Pm68 is controlled by a pair of NLR genes (He et al. An NLR pair in the Pm68 locus confers powdery mildew resistance in durum and common wheat. Nature Communications, 2025, 16: 6427).

[0004] In summary, Pm26, Pm36, TdPm60, and Pm68 are superior powdery mildew resistance genes originating from different closely related wheat species. They possess different inheritance patterns, molecular mechanisms, and resistance spectra, providing rich and valuable genetic resources for wheat disease resistance breeding. However, in traditional breeding, accurately tracking these genes through phenotypic identification suffers from technical bottlenecks such as low efficiency, poor accuracy, and lengthy processes. Therefore, to achieve efficient aggregation of multiple genes or to study the complex resistance effects among genes, developing specific molecular markers that are closely linked to these disease resistance genes and enable precise genotyping is crucial for overcoming the aforementioned breeding bottlenecks. Summary of the Invention

[0005] To address some shortcomings in existing technologies, this invention provides molecular markers for efficiently tracking wheat powdery mildew resistance genes and their applications. This invention discloses four diagnostic molecular markers, Pm26-M, Pm36-M, TdPm60-M, and Pm68-M, capable of accurately tracking wheat powdery mildew resistance genes Pm26, Pm36, TdPm60, and Pm68, respectively, and their applications. These molecular markers can be PCR-amplified in materials containing wheat powdery mildew resistance genes Pm26, Pm36, TdPm60, and Pm68 to produce specific products of 638 bp, 453 bp, 230 bp, and 341 bp, respectively. The molecular markers can be used alone or incorporated into a mixed detection system for multiplex PCR amplification, achieving efficient detection of multiple powdery mildew resistance genes. The molecular markers of this invention exhibit good stability, high resolution, and excellent reproducibility during PCR amplification, providing an efficient and reliable tool for marker-assisted selection and precise breeding of wheat powdery mildew resistance genes.

[0006] To achieve the above-mentioned technical objectives, the present invention employs the following technical means:

[0007] This invention first provides molecular markers for efficiently tracking wheat powdery mildew resistance genes. These molecular markers include any or any combination of the molecular markers Pm26-M, TdPm60-M, Pm68-M, and Pm36-M for the wheat powdery mildew resistance genes Pm26, TdPm60, Pm36, and Pm68. The primers for these molecular markers include:

[0008] The primers for the molecular marker Pm26-M are shown in SEQ ID No:1 and SEQ ID No:2:

[0009] Pm26-MF: 5'-TGGAAGAGTTCAACAGCACATA-3' (SEQ ID No: 1);

[0010] Pm26-MR: 5'-GTTCTCCTTACACCAGTTCTCC-3' (SEQ ID No: 2);

[0011] The primers for the molecular marker TdPm60-M are shown in SEQ ID No:3 and SEQ ID No:4:

[0012] TdPm60-MF: 5'-TATTAATGGGTATAATAGTGGTG-3' (SEQ ID No: 3);

[0013] TdPm60-MF: 5'-CGGTAGGCCATGGAAAGAAGAC-3' (SEQ ID No: 4);

[0014] The primers for the molecular marker Pm68-M are shown in SEQ ID No:5 and SEQ ID No:6:

[0015] Pm68-MF: 5'-CTACCCACGATCTGTATGATGGT-3' (SEQ ID No: 5);

[0016] Pm68-MR: 5'-GTATGCTCCAATGCGCGTAGATC-3' (SEQ ID No: 6);

[0017] The primers for the molecular marker Pm36-M are shown in SEQ ID No:7 and SEQ ID No:8:

[0018] Pm36-MF: 5'-TCATAGTTGCCTTCTCACTCTTG-3' (SEQ ID No: 7);

[0019] Pm36-MR: 5'-ACCGCATTACCAGGACATAAAG-3' (SEQ ID No: 8).

[0020] The present invention also provides primers for detecting the above-mentioned molecular markers, said primers comprising any one or any combination of the following:

[0021] The amplification primers for the molecular marker Pm26-M are shown in SEQ ID No:1 and SEQ ID No:2:

[0022] Pm26-MF: 5'-TGGAAGAGTTCAACAGCACATA-3' (SEQ ID No: 1);

[0023] Pm26-MR: 5'-GTTCTCCTTACACCAGTTCTCC-3' (SEQ ID No: 2);

[0024] The amplification primers for the molecular marker TdPm60-M are shown in SEQ ID No:3 and SEQ ID No:4:

[0025] TdPm60-MF: 5'-TATTAATGGGTATAATAGTGGTG-3' (SEQ ID No: 3);

[0026] TdPm60-MF: 5'-CGGTAGGCCATGGAAAGAAGAC-3' (SEQ ID No: 4);

[0027] The amplification primers for the molecular marker Pm68-M are shown in SEQ ID No:5 and SEQ ID No:6:

[0028] Pm68-MF: 5'-CTACCCACGATCTGTATGATGGT-3' (SEQ ID No: 5);

[0029] Pm68-MR: 5'-GTATGCTCCAATGCGCGTAGATC-3' (SEQ ID No: 6);

[0030] The amplification primers for the molecular marker Pm36-M are shown in SEQ ID No:7 and SEQ ID No:8:

[0031] Pm36-MF: 5'-TCATAGTTGCCTTCTCACTCTTG-3' (SEQ ID No: 7);

[0032] Pm36-MR: 5'-ACCGCATTACCAGGACATAAAG-3' (SEQ ID No: 8).

[0033] The present invention also provides reagents or kits for detecting multiple wheat powdery mildew resistance genes, wherein the reagents or kits contain the primers described above.

[0034] Preferably, the reagent or kit comprises any of the following:

[0035] (1) Any one of the amplification primers Pm26-M, TdPm60-M, Pm68-M and Pm36-M;

[0036] (2) Amplification primer combinations for Pm26-M and TdPm60-M;

[0037] (3) Amplification primer combinations for Pm26-M, TdPm60-M and Pm68-M;

[0038] (4) Amplification primer combinations of Pm26-M, TdPm60-M, Pm68-M and Pm36-M.

[0039] This invention also provides the application of the above-mentioned molecular markers, primers, reagents or kits in the efficient and simultaneous tracking of multiple wheat powdery mildew resistance genes.

[0040] Preferably, the application includes any one of the following:

[0041] (1) To identify or assist in identifying whether the sample carries one or more of the wheat powdery mildew resistance genes Pm26, Pm36, TdPm60 and Pm68;

[0042] (2) Simultaneous tracking of Pm26, Pm36, TdPm60 and Pm68 in the context of hexaploid wheat;

[0043] (3) Transgenic genes Pm26, Pm36, TdPm60 and Pm68 that resist powdery mildew;

[0044] (4) Research on multi-gene aggregation.

[0045] This invention also provides a method for efficiently and simultaneously tracking multiple wheat powdery mildew resistance genes, the method comprising:

[0046] The genomic DNA of the test material was amplified by PCR using the primers described above, and the amplification products were detected. If a specific target product was obtained, the material was considered to carry the corresponding powdery mildew resistance gene; otherwise, the test material was considered to not carry the corresponding powdery mildew resistance gene.

[0047] Preferably, in the PCR amplification, the 25 μL PCR reaction system comprises: 50 ng genomic DNA of the test material, 1× PCR buffer, and 1.5 mmol / L. -1 MgCl2, 200 mmol L -1 dNTPs, 1 U Taq DNA polymerase, and sterile deionized water to bring the volume to 25 μL.

[0048] Preferably, the PCR reaction system further includes amplification primers;

[0049] When the amplification primer is any one of Pm26-M, TdPm60-M, Pm68-M and Pm36-M, the final concentration of the amplification primer is 0.4 μM.

[0050] When the amplification primers are a combination of Pm26-M and TdPm60-M, the final concentration of the Pm26-M amplification primer is 0.25 μM, and the final concentration of the TdPm60-M amplification primer is 0.35 μM.

[0051] When the amplification primers are a combination of Pm26-M, TdPm60-M and Pm68-M, the final concentration of the amplification primers Pm26-M and Pm68-M is 0.2 μM, and the final concentration of the amplification primers TdPm60-M is 0.4 μM.

[0052] When the amplification primers are a combination of Pm26-M, TdPm60-M, Pm68-M and Pm36-M, the final concentration of the amplification primers Pm26-M, Pm36-M and Pm68-M is 0.2 μM, and the final concentration of the amplification primer TdPm60-M is 0.4 μM.

[0053] 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, 32 cycles; extension at 72°C for 5 minutes; and storage at 4°C.

[0054] Preferably, the specific target products are the molecular markers Pm26-M, TdPm60-M, Pm68-M, and Pm36-M corresponding to the wheat powdery mildew resistance genes Pm26, TdPm60, Pm68, and Pm36, respectively.

[0055] The molecular markers Pm26-M, TdPm60-M, Pm68-M, and Pm36-M have sizes of 638 bp, 230 bp, 341 bp, and 453 bp, respectively.

[0056] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0057] The multiplex PCR system provided by this invention, composed of the molecular marker combination Pm26-M, Pm36-M, TdPm60-M, and Pm68-M, can effectively track Pm26, Pm36, TdPm60, and Pm68 simultaneously in the background of hexaploid wheat. Moreover, the above molecular markers all have the advantages of stable amplification of polymorphic bands, high resolution, and good reproducibility. Furthermore, there is no non-specific amplification in wheat materials that do not contain the above four genes, which can greatly improve the accuracy and efficiency of breeding. The four molecular markers can be used individually or constructed into a mixed detection system for multiplex PCR amplification to achieve efficient detection of four powdery mildew resistance genes.

[0058] The molecular markers described in this invention exhibit good stability, high resolution, and excellent reproducibility in PCR amplification. This invention has developed reaction systems for duplex, triplet, and quadruplex PCR. The optimized asymmetric concentration ratios of these reaction systems enable accurate, stable, and direct rapid and synchronous genotyping of powdery mildew resistance genes Pm26, Pm36, TdPm60, and Pm68 in wheat breeding. The multiplex PCR system described in this invention provides an efficient and reliable tool for marker-assisted selection and precise transgenic development of wheat powdery mildew resistance genes. The molecular markers described in this invention have significant application value in transgenic and multi-gene aggregation studies of powdery mildew resistance genes Pm26, Pm36, TdPm60, and Pm68. Attached Figure Description

[0059] Figure 1 Molecular markers Pm26-M and TdPm60-M and their constructed duplex PCR system were used to detect Yangmai 158 and wild emmer wheat No.487; in the figure, 1 represents Yangmai 158 and 2 represents wild emmer wheat No.487. The experiment was set up with 3 replicates, and the arrows indicate the target bands.

[0060] Figure 2: Molecular marker Pm68-M and its triple PCR system constructed together with Pm26-M and TdPm60-M were used to detect Yangmai 158 and wild emmer wheat No.487; in the figure, 1 represents Yangmai 158 and 2 represents wild emmer wheat No.487. The experiment was set up with 3 replicates, and the arrows indicate the target bands.

[0061] Figure 3 The molecular marker Pm36-M and its quadruple PCR system constructed with Pm26-M, TdPm60-M, and Pm68 were used to detect Yangmai 158 and wild emmer wheat No.487. In the figure, 1 represents Yangmai 158 and 2 represents wild emmer wheat No.487. The experiment was set up with 3 replicates. The arrows indicate the target bands.

[0062] Figure 4Multiplex PCR system was used to detect the hybrid offspring of Yangmai 158 and wild emmer wheat No. 487; Figure 1-16 are single plants of the hybrid offspring of the two, 17 is the negative control Yangmai 158, 18 is the positive control wild emmer wheat No. 487, and the arrows indicate the target bands. Detailed Implementation

[0063] 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.

[0064] Example 1:

[0065] Primers were designed based on the DNA sequences of Pm26 (GenBank accession number: PP781951), TdPm60 (GenBank accession number: MW375699), Pm68 (GenBank accession number: PQ655406), and Pm36 (GenBank accession number: OQ361691). Primers were designed using Primer Premier 5.0 software, with the following design principles: amplification product length between 200-650 bp and easily distinguishable; annealing temperature (Tm) of each primer close to 55℃; and high specificity for the target gene confirmed by Primer-BLAST alignment on NCBI.

[0066] Specific molecular markers were developed based on the wheat powdery mildew resistance genes Pm26, TdPm60, Pm68 and Pm36, namely Pm26-M, TdPm60-M, Pm68-M and Pm36-M, respectively.

[0067] The amplification primers for the molecular marker Pm26-M include:

[0068] Pm26-MF: 5'-TGGAAGAGTTCAACAGCACATA-3' (SEQ ID No: 1);

[0069] Pm26-MR: 5'-GTTCTCCTTACACCAGTTCTCC-3' (SEQ ID No: 2);

[0070] The amplification primers for the molecular marker TdPm60-M include:

[0071] TdPm60-MF: 5'-TATTAATGGGTATAATAGTGGTG-3' (SEQ ID No: 3);

[0072] TdPm60-MF: 5'-CGGTAGGCCATGGAAAGAAGAC-3' (SEQ ID No: 4);

[0073] The amplification primers for the molecular marker Pm68-M include:

[0074] Pm68-MF: 5'-CTACCCACGATCTGTATGATGGT-3' (SEQ ID No: 5);

[0075] Pm68-MR: 5'-GTATGCTCCAATGCGCGTAGATC-3' (SEQ ID No: 6);

[0076] The amplification primers for the molecular marker Pm36-M include:

[0077] Pm36-MF: 5'-TCATAGTTGCCTTCTCACTCTTG-3' (SEQ ID No: 7);

[0078] Pm36-MR: 5'-ACCGCATTACCAGGACATAAAG-3' (SEQ ID No: 8).

[0079] Using DNA from Yangmai 158 (publicly known and used, provided by Jiangsu Lixiahe Agricultural Science Research Institute) and wild emmer wheat No. 487 (from the US Germplasm Resources Information Network, website: https: / / npgsweb.ars-grin.gov / gringlobal / search) as templates, conventional PCR was performed.

[0080] The PCR reaction system consisted of approximately 50 ng template DNA, 1× PCR buffer, and 1.5 mmol / L of other reagents in a 25 μL reaction volume. -1 MgCl2, 200 mmol L -1 dNTPs, with a final concentration of 0.4 μmol / L for each amplification primer. -1 Add 1 U Taq DNA polymerase and replenish the reaction system to 25 μL with sterile distilled water.

[0081] PCR reaction procedure: 94℃ pre-denaturation for 3 minutes; 94℃ denaturation for 20 seconds, 55℃ annealing for 30 seconds, 72℃ extension for 60 seconds, 32 cycles; 72℃ extension for 5 minutes; store at 4℃.

[0082] PCR products were electrophoresed on a 1% agarose gel. The results showed that each primer pair amplified a single, bright, specific band at the expected location, with no non-specific amplification. Figure 1 , Figure 2 and Figure 3 The results show the detection performance of single primer pairs, with specific products for the Pm26, TdPm60, Pm68, and Pm36 genes being 638 bp, 230 bp, 341 bp, and 453 bp, respectively. The standard working concentration for each primer pair in the above results is 0.4 μM.

[0083] Example 2:

[0084] Based on the amplification primers obtained in Example 1, this embodiment combines them to obtain a double PCR reaction system, a triple PCR reaction system, and a quadruple PCR reaction system. The concentration ratio of the amplification primers in the PCR reaction system is adjusted to obtain a stable and balanced detection system.

[0085] The specific steps are as follows:

[0086] (1) Establishment of the double PCR reaction system:

[0087] Amplification was performed using the PCR system described in Example 1. Agarose gel electrophoresis results showed that the Pm26-M band was bright, while the TdPm60-M band was significantly weaker, indicating an uneven signal distribution in the doublet PCR reaction system. To address this, the concentrations of the primers in the PCR reaction system were appropriately adjusted: the final concentration of each primer for Pm26-M was adjusted to 0.25 μM, and the final concentration of each primer for TdPm60-M was adjusted to 0.35 μM, before performing doublet PCR detection.

[0088] The duplex PCR reaction system consisted of approximately 50 ng template DNA, 1×PCR buffer, and 1.5 mmol / L of other reagents in a 25 μL reaction volume. -1 MgCl2, 200 mmol L -1 The final concentrations of dNTP and Pm26-M primers were each 0.25 μmol / L. -1 The final concentrations of the two primers, TdPm60-M, were each 0.35 μmol / L. -1 Add 1 U Taq DNA polymerase and replenish the reaction system to 25 μL with sterile distilled water; the PCR reaction procedure is the same as in Example 1.

[0089] The agarose gel electrophoresis results of the duplex PCR products are as follows: Figure 1The display showed two clear bands of corresponding size (Pm26-M (638 bp) and TdPm60-M (230 bp)), with the brightness of the Pm26-M specific band decreasing and the brightness of the TdPm60-M specific band increasing.

[0090] (2) Establishment of the triple PCR reaction system:

[0091] This step further introduces Pm68-M amplification primers into the duplex PCR reaction system obtained in step (1). Considering the agarose gel electrophoresis of the duplex PCR products (…), Figure 1 When performing triplet PCR of Pm26-M, the brightness of the Pm26-M specific band decreased, while the brightness of the TdPm60-M specific band increased, but the effect was not significant. Therefore, when performing triplet PCR of Pm26-M, TdPm60-M, and Pm68-M, the primer concentrations in the reaction system were further adjusted. Through a series of tests, it was found that the concentration of TdPm60-M primer had to be further increased to 0.4 μM, while maintaining Pm26-M and Pm68-M at 0.2 μM, in order to make all three bands (Pm26-M: 638 bp, TdPm60-M: 230 bp, Pm68-M: 341 bp) clearly visible. At this point, the concentration of TdPm60-M was twice that of Pm26-M and Pm68-M. The triplet system was initially stable.

[0092] Therefore, the concentrations of each primer in Pm26-M were adjusted to 0.2 μM, and the concentrations of each primer in TdPm60-M were adjusted to 0.4 μM. Given that single-primer pair amplification resulted in a stronger amplification band from Pm68-M (primer concentration at 0.4 μM),... Figure 2) Therefore, when performing triple PCR, the concentration of each primer of Pm68-M was reduced by half and adjusted to 0.2 μM.

[0093] Therefore, the specific PCR reaction system for triple PCR is as follows: 25 μL reaction system containing approximately 50 ng template DNA, 1× PCR buffer, and 1.5 mmol / L... -1 MgCl2, 200 mmol L -1 The final concentrations of the two primer pairs, Pm26-M and Pm68-M, were each 0.2 μmol / L. -1 The final concentrations of the two primers, TdPm60-M, were each 0.4 μmol / L. -1 Add 1 U Taq DNA polymerase and replenish the reaction system to 25 μL with sterile distilled water; the PCR reaction procedure is the same as in Example 1.

[0094] Agarose gel electrophoresis showed that the specific amplification products of the three markers in wild emmer wheat No. 487 had uniform brightness and high resolution. Figure 2) .

[0095] (3) Establishment and optimization of the quadruple PCR reaction system:

[0096] In this step, a fourth pair of amplification primers, Pm36-M (product size 453 bp), is introduced into the triplet PCR reaction system obtained in step (2) to construct a quadruple PCR reaction system. Based on the experience that TdPm60-M always requires a higher concentration in duplex and triplet constructions, the core hypothesis is proposed—in a quadruple competitive environment, TdPm60-M needs to be "concentration compensated," while the concentrations of the other three primers need to be reduced to decrease competitive inhibition. Given that during single primer pair amplification, the amplification band of Pm36-M (each primer concentration at 0.4 μM) is strong ( Figure 3 Therefore, when performing quadruple PCR, the concentration of each primer of Pm36-M was reduced by half and adjusted to 0.2 μM.

[0097] In this embodiment, a key technical obstacle was identified: in this specific four-gene combination, the TdPm60-M gene was at a competitive disadvantage in multiplex amplification, and conventional isoconcentration strategies resulted in missed detection. By reducing the primer concentrations of Pm26-M, Pm36-M, and Pm68-M to half the working concentration of their individual primer pairs (0.2 μM), while increasing the TdPm60-M primer concentration to twice that of the former (0.4 μM), balanced and stable amplification of all four targets was successfully achieved. This specific "2-fold concentration ratio" constitutes the core of the stable, efficient, and reliable quadruple PCR detection method of this invention.

[0098] Therefore, the specific PCR reaction system for quadruple PCR is as follows: 25 μL of reaction system contains approximately 50 ng template DNA, 1× PCR buffer, and 1.5 mmol / L of... -1 MgCl2, 200 mmol L -1 The final concentrations of the three primer pairs (Pm26-M, Pm36-M, and Pm68-M) were each 0.2 μmol / L. -1 The final concentrations of the two primers, TdPm60-M, were each 0.4 μmol / L. -1 Add 1 U Taq DNA polymerase and replenish the reaction system to 25 μL with sterile distilled water; the PCR reaction procedure is the same as in Example 1.

[0099] Agarose gel electrophoresis showed that the brightness of the specific amplification products of the four markers in wild emmer wheat No. 487 decreased, but the resolution of each specific band was high and did not affect the interpretation of the results. Figure 3).

[0100] In summary, the molecular markers of this invention can be used to detect wheat materials carrying four powdery mildew resistance genes: Pm26, Pm36, TdPm60, and Pm68. This invention discovered that wild emmer wheat No. 487 carries these four powdery mildew resistance genes. (No. 487 is the experimental number provided by the inventors; the original germplasm resource bank number is PI352323. This material originates from the Germplasm Resources Information Network (GRI), website: https: / / npgsweb.ars-grin.gov / gringlobal / search).

[0101] Example 3:

[0102] The disease-susceptible wheat variety Yangmai 158 (publicly known and provided by the Jiangsu Lixiahe Agricultural Science Research Institute) was crossed with wild emmer wheat No. 487. Then, Yangmai 158 was used as the recurrent parent and backcrossed twice with the hybrid. After self-pollination, a new wheat germplasm was formed.

[0103] The hybrid offspring of Yangmai 158 and wild emmer wheat No. 487 were detected using the quadruple PCR system established in Example 2. The results of agarose gel electrophoresis are as follows: Figure 4 As shown. Lanes 1-16 are the hybrid offspring of the two, lane 17 is the negative control Yangmai 158, and lane 19 is the positive control wild emmer wheat No. 487. Among them, the negative control Yangmai 158 in lane 17 did not have any amplification products, while the positive control wild emmer wheat No. 487 in lane 19 had specific bands corresponding to the four genes: Pm26-M product of 638 bp, Pm36-M product of 453 bp, Pm68-M product of 341 bp, and TdPm60-M product of 230 bp. In addition, four specific bands appeared in lanes 1 and 3-13, consistent with the bands in wild emmer wheat No. 487, indicating that these plants carry the four genes Pm26, Pm36, TdPm60, and Pm68; three bands of 638 bp, 453 bp, and 341 bp appeared in lanes 14 and 15, indicating that these two plants contain the three genes Pm26, Pm36, and Pm68; only one specific band of 453 bp appeared in lane 2, indicating that this plant only contains the Pm36 gene.

[0104] In summary, this invention discloses four diagnostic molecular markers, Pm26-M, Pm36-M, TdPm60-M, and Pm68-M, capable of accurately tracking wheat powdery mildew resistance genes Pm26, Pm36, TdPm60, and Pm68, respectively, and their applications. These molecular markers can be PCR-amplified into specific products of 638 bp, 453 bp, 230 bp, and 341 bp, respectively, in materials containing wheat powdery mildew resistance genes Pm26, Pm36, TdPm60, and Pm68. The molecular markers can be used alone or incorporated into a mixed detection system for multiplex PCR amplification, achieving efficient detection of multiple powdery mildew resistance genes. The molecular markers of this invention exhibit good stability, high resolution, and excellent reproducibility during PCR amplification, providing an efficient and reliable tool for marker-assisted selection and precise transfer of wheat powdery mildew resistance genes.

[0105] 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 molecular marker for efficiently tracking wheat powdery mildew resistance genes, characterized in that, The molecular markers include any or any combination of the molecular markers Pm26-M, TdPm60-M, Pm68-M, and Pm36-M for the wheat powdery mildew resistance genes Pm26, TdPm60, Pm36, and Pm68, and the primers for the molecular markers include: The primers for the molecular marker Pm26-M are shown in SEQ ID No:1 and SEQ ID No:2: Pm26-MF: 5'-TGGAAGAGTTCAACAGCACATA-3' (SEQ ID No: 1); Pm26-MR: 5'-GTTCTCCTTACACCAGTTCTCC-3' (SEQ ID No: 2); The primers for the molecular marker TdPm60-M are shown in SEQ ID No:3 and SEQ ID No:4: TdPm60-MF: 5'-TATTAATGGGTATAATAGTGGTG-3' (SEQ ID No: 3); TdPm60-MF: 5'-CGGTAGGCCATGGAAAGAAGAC-3' (SEQ ID No: 4); The primers for the molecular marker Pm68-M are shown in SEQ ID No:5 and SEQ ID No:6: Pm68-MF: 5'-CTACCCACGATCTGTATGATGGT-3' (SEQ ID No: 5); Pm68-MR: 5'-GTATGCTCCAATGCGCGTAGATC-3' (SEQ ID No: 6); The primers for the molecular marker Pm36-M are shown in SEQ ID No:7 and SEQ ID No:8: Pm36-MF: 5'-TCATAGTTGCCTTCTCACTCTTG-3' (SEQ ID No: 7); Pm36-MR: 5'-ACCGCATTACCAGGACATAAAG-3' (SEQ ID No: 8).

2. Primers for detecting the molecular marker of claim 1, characterized in that, The primers include any one or any combination of the following: The amplification primers for the molecular marker Pm26-M are shown in SEQ ID No:1 and SEQ ID No:2: Pm26-MF: 5'-TGGAAGAGTTCAACAGCACATA-3' (SEQ ID No: 1); Pm26-MR: 5'-GTTCTCCTTACACCAGTTCTCC-3' (SEQ ID No: 2); The amplification primers for the molecular marker TdPm60-M are shown in SEQ ID No:3 and SEQ ID No:4: TdPm60-MF: 5'-TATTAATGGGTATAATAGTGGTG-3' (SEQ ID No: 3); TdPm60-MF: 5'-CGGTAGGCCATGGAAAGAAGAC-3' (SEQ ID No: 4); The amplification primers for the molecular marker Pm68-M are shown in SEQ ID No:5 and SEQ ID No:6: Pm68-MF: 5'-CTACCCACGATCTGTATGATGGT-3' (SEQ ID No: 5); Pm68-MR: 5'-GTATGCTCCAATGCGCGTAGATC-3' (SEQ ID No: 6); The amplification primers for the molecular marker Pm36-M are shown in SEQ ID No:7 and SEQ ID No:8: Pm36-MF: 5'-TCATAGTTGCCTTCTCACTCTTG-3' (SEQ ID No: 7); Pm36-MR: 5'-ACCGCATTACCAGGACATAAAG-3' (SEQ ID No: 8).

3. A reagent or kit for detecting multiple wheat powdery mildew resistance genes, characterized in that, The reagent or kit contains the primers as described in claim 2.

4. The reagent or kit according to claim 3, characterized in that, The reagent or kit contains any of the following: (1) Any one of the amplification primers Pm26-M, TdPm60-M, Pm68-M and Pm36-M; (2) Amplification primer combinations for Pm26-M and TdPm60-M; (3) Amplification primer combinations for Pm26-M, TdPm60-M and Pm68-M; (4) Amplification primer combinations of Pm26-M, TdPm60-M, Pm68-M and Pm36-M.

5. The application of the molecular marker of claim 1, the primer of claim 2, and the reagent or kit of claim 3 in the efficient and simultaneous tracking of multiple wheat powdery mildew resistance genes.

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 carries one or more of the wheat powdery mildew resistance genes Pm26, Pm36, TdPm60 and Pm68; (2) Simultaneous tracking of Pm26, Pm36, TdPm60 and Pm68 in the context of hexaploid wheat; (3) Transgenic genes Pm26, Pm36, TdPm60 and Pm68 that resist powdery mildew; (4) Research on multi-gene aggregation.

7. A method for efficiently and simultaneously tracking multiple wheat powdery mildew resistance genes, characterized in that, The method includes: The primers described in claim 2 are used to perform PCR amplification on the genomic DNA of the test material, and the amplification products are detected. If a specific target product can be obtained, the material is a material carrying the corresponding powdery mildew resistance gene; otherwise, the test material is a material that does not carry the corresponding powdery mildew resistance gene.

8. The method according to claim 7, characterized in that, In the PCR amplification, the 25 μL PCR reaction system included: 50 ng of genomic DNA to be tested, 1× PCR buffer, and 1.5 mmol / L. -1 MgCl2, 200 mmol L -1 dNTPs, 1U Taq DNA polymerase, and sterile deionized water to bring the volume to 25μL.

9. The method according to claim 8, characterized in that, The PCR reaction system also includes amplification primers; When the amplification primer is any one of Pm26-M, TdPm60-M, Pm68-M and Pm36-M, the final concentration of the amplification primer is 0.4 μM. When the amplification primers are a combination of Pm26-M and TdPm60-M, the final concentration of the Pm26-M amplification primer is 0.25 μM, and the final concentration of the TdPm60-M amplification primer is 0.35 μM. When the amplification primers are a combination of Pm26-M, TdPm60-M and Pm68-M, the final concentration of the amplification primers Pm26-M and Pm68-M is 0.2 μM, and the final concentration of the amplification primers TdPm60-M is 0.4 μM. When the amplification primers are a combination of Pm26-M, TdPm60-M, Pm68-M and Pm36-M, the final concentration of the amplification primers Pm26-M, Pm36-M and Pm68-M is 0.2 μM, and the final concentration of the amplification primer TdPm60-M is 0.4 μM.

10. The method according to claim 7, characterized in that, The PCR amplification reaction procedure includes: 94℃ pre-denaturation for 3 minutes; 94℃ denaturation for 20 seconds, 55℃ annealing for 30 seconds, 72℃ extension for 60 seconds, 32 cycles; 72℃ extension for 5 minutes; storage at 4℃.

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