Wheat avrlr15 gene and primer set and kit for detecting and / or identifying wheat leaf rust pathogenicity

Abstract

The present application relates to the application of wheat AvrLr15 gene, primer set and kit in detecting and / or identifying the virulence of wheat leaf rust fungus, and belongs to the technical field of pathogenic microorganism detection.The present application provides the application of wheat AvrLr15 gene in detecting and / or identifying the virulence of wheat leaf rust fungus, wherein the CDS region nucleotide sequence of the AvrLr15 gene is shown as SEQ ID NO.1.The primer set of the present application can directly detect and / or identify the virulence of wheat leaf rust fungus to the wheat leaf rust resistant material TcLr15, and has good specificity, reliability and stability, and can be used as a primer set of specific molecular marker for detecting and / or identifying the virulence of wheat leaf rust fungus to TcLr15.

Description

Technical Field

[0001] This invention relates to the field of pathogen detection technology, and in particular to the application of the wheat AvrLr15 gene, primer set, and kit in the detection and / or identification of wheat leaf rust virulence. Background Technology

[0002] Wheat leaf rust is a fungal disease caused by *Puccinia tirticina*, and it is one of the most widespread and serious wheat diseases, causing yield reductions and significant economic losses in major wheat-producing areas. The rational use and distribution of wheat varieties containing disease-resistant genes is an economical, safe, and effective method for controlling the occurrence and spread of wheat leaf rust. However, large-scale planting of a single disease-resistant variety can exert selective pressure on the virulence genes of the pathogen population due to the resistance genes, leading to the loss of rust resistance in the host wheat and subsequently triggering disease outbreaks, causing severe losses to wheat production. Therefore, virulence monitoring of wheat leaf rust populations in the field is crucial for guiding the breeding and proper use of rust-resistant wheat varieties in production practices.

[0003] With advancements in molecular-level technologies, methods such as molecular fingerprinting, molecular markers, and gene amplification have begun to be applied to the identification of wheat leaf rust fungi. These methods mainly include restriction fragment length polymorphism (RFLP), random amplified fragment length polymorphism (RAPD), simple repeat sequence analysis (SSR), sequence-associated amplified polymorphism (AFLP), and single nucleotide polymorphism (SNP) analysis. Although numerous molecular markers, such as AFLP, RAPD, SSR, and EST-SSR, have been developed based on conventional PCR detection methods, specific molecular markers for the virulence / avirulence genes of specific wheat leaf rust fungi are rarely reported.

[0004] The wheat leaf rust resistance gene Lr15 is a seedling-stage resistance gene located on wheat chromosome 2D. When co-existing with adult-stage resistance genes, this gene significantly enhances the rust resistance of wheat plants. Currently, the Lr15 gene still provides good resistance to most physiological races of wheat leaf rust fungi. Therefore, to prevent the loss of resistance to the Lr15 gene, it is urgent to develop reasonable and effective control measures. However, existing technologies lack direct and effective detection or identification methods to determine the toxicity of wheat leaf rust fungi to wheat plants containing the Lr15 gene. Summary of the Invention

[0005] The purpose of this invention is to provide the application of the wheat AvrLr15 gene, primer set, and kit in the detection and / or identification of wheat leaf rust virulence, so as to solve the problem that the existing technology cannot directly and effectively detect and / or identify the virulence of wheat leaf rust to wheat plants containing the Lr15 gene.

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

[0007] This invention provides the application of the wheat AvrLr15 gene in detecting and / or identifying the virulence of wheat leaf rust fungi, the nucleotide sequence of the CDS region of the AvrLr15 gene is shown in SEQ ID NO.1.

[0008] The present invention also provides a primer set for detecting the wheat AvrLr15 gene, the primer set comprising: AvrLr15 M1-F primer, AvrLr15 M1-R primer and AvrLr15 M2 primer;

[0009] The nucleotide sequence of the AvrLr15 M1-F primer is shown in SEQ ID NO.5, the nucleotide sequence of the AvrLr15 M1-R primer is shown in SEQ ID NO.6, and the nucleotide sequence of the AvrLr15 M2 primer is shown in SEQ ID NO.7.

[0010] The present invention also provides the application of the primer set described herein in the detection and / or identification of wheat leaf rust virulence.

[0011] The present invention also provides the application of the primer set described herein in the preparation of products for detecting and / or identifying the virulence of wheat leaf rust.

[0012] The present invention also provides a kit for detecting and / or identifying the virulence of wheat leaf rust fungus, comprising the following components in parts by volume: 0.5-2 parts DNA template, 10.5-14.5 parts DNA polymerase, 1.6-2.4 parts of the AvrLr15 M1-F primer, 0.8-1.2 parts of the AvrLr15 M1-R primer, 0.8-1.2 parts of the AvrLr15 M2 primer, and 2.7-10.8 parts water.

[0013] Preferably, the concentration of the AvrLr15 M1-F primer is 1–10 mmol / L, the concentration of the AvrLr15M1-R primer is 1–10 mmol / L, and the concentration of the AvrLr15 M2 primer is 1–10 mmol / L.

[0014] The present invention also provides the application of the kit in detecting and / or identifying the virulence of wheat leaf rust fungi.

[0015] The present invention has the following technical effects and advantages: the primer set of the present invention can stably detect specific bands in wheat leaf rust fungi that are non-toxic to the wheat leaf rust resistant material TcLr15, while no corresponding bands are amplified in wheat leaf rust fungi that are toxic to TcLr15. This indicates that the primer set of the present invention can directly detect and identify the toxicity of wheat leaf rust fungi to TcLr15, and has good specificity, reliability and stability. It is applicable to all tested wheat leaf rust fungi. Therefore, it can be used as a primer set for specific molecular markers to detect and / or identify the toxicity of wheat leaf rust fungi to TcLr15. It is of great significance for the localization and cloning of toxic / non-toxic genes of wheat leaf rust fungi, the identification and structural analysis of wheat leaf rust fungi races, and the revelation of the variation patterns of wheat leaf rust fungi races. Attached Figure Description

[0016] Figure 1 The results of β-Actin internal reference gene detection for 21 physiological races of wheat leaf rust in Example 2 are shown in the figure. Lane M is the marker, and lanes 1 to 22 are ddH2O, wheat leaf rust physiological races V1, V2, V3, V4, V4, V5, V6, V7, A8, A9, A10, A11, A12, A13, A14, A15, A16, A17, A18, A19, A20, and A21, respectively.

[0017] Figure 2 This is the result of β-Actin internal reference gene detection for 120 wheat leaf rust samples in Example 2. In the figure, lane M is the marker, and lanes 1 to 120 are the wheat leaf rust samples numbered 1 to 120 in Table 2, respectively.

[0018] Figure 3 The figure shows the virulence test results of 21 physiological races of wheat leaf rust in Example 3. Lane M is the marker, and lanes 1 to 22 are ddH2O, wheat leaf rust physiological races V1, V2, V3, V4, V4, V5, V6, V7, A8, A9, A10, A11, A12, A13, A14, A15, A16, A17, A18, A19, A20, and A21, respectively.

[0019] Figure 4 The results show the toxicity test results of 120 wheat leaf rust samples in Example 3. In the figure, lane M is the marker, lane H2O is ddH2O, and lanes 1 to 120 are the wheat leaf rust samples numbered 1 to 120 in Table 2.

[0020] Figure 5The figure shows the pathogenicity phenotypes of 21 wheat leaf rust physiological races against the wheat leaf rust resistant material TcLr15 and the wheat susceptible material Thatcher in Example 3. In the figure, V15 indicates that the wheat leaf rust physiological race is toxic to TcLr15, and A15 indicates that the wheat leaf rust physiological race is non-toxic to TcLr15. Detailed Implementation

[0021] This invention provides the application of the wheat AvrLr15 gene in detecting and / or identifying the virulence of wheat leaf rust fungi, the nucleotide sequence of the CDS region of the AvrLr15 gene is shown in SEQ ID NO.1.

[0022] The present invention also provides a primer set for detecting the wheat AvrLr15 gene, the primer set comprising: AvrLr15 M1-F primer, AvrLr15 M1-R primer and AvrLr15 M2 primer;

[0023] The nucleotide sequence of the AvrLr15 M1-F primer is shown in SEQ ID NO.5, the nucleotide sequence of the AvrLr15 M1-R primer is shown in SEQ ID NO.6, and the nucleotide sequence of the AvrLr15 M2 primer is shown in SEQ ID NO.7.

[0024] The present invention also provides the application of the primer set described herein in the detection and / or identification of wheat leaf rust virulence.

[0025] The present invention also provides the application of the primer set described herein in the preparation of products for detecting and / or identifying the virulence of wheat leaf rust.

[0026] The present invention also provides a kit for detecting and / or identifying the virulence of wheat leaf rust fungus, comprising the following components in parts by volume: 0.5 to 2 parts of DNA template, preferably 1 part; 10.5 to 14.5 parts of DNA polymerase, preferably 12.5 parts; 1.6 to 2.4 parts of the AvrLr15 M1-F primer, preferably 2 parts; 0.8 to 1.2 parts of the AvrLr15 M1-R primer, preferably 1 part; 0.8 to 1.2 parts of the AvrLr15 M2 primer, preferably 1 part; and 2.7 to 10.8 parts of water, preferably 7.5 parts.

[0027] In this invention, the DNA polymerase is 2×Es Taq MasterMix, purchased from Jiangsu Kangwei Reagent Biotechnology Co., Ltd.

[0028] In this invention, the concentration of the AvrLr15 M1-F primer is 1–10 mmol / L, preferably 5 mmol / L; the concentration of the AvrLr15 M1-R primer is 1–10 mmol / L, preferably 5 mmol / L; the concentration of the AvrLr15 M2 primer is 1–10 mmol / L, preferably 5 mmol / L; and the concentration of the DNA template is 10–100 ng / μL, preferably 30 ng / μL.

[0029] In this invention, the PCR reaction conditions of the kit are: 94℃ pre-denaturation for 1 min, 94℃ denaturation for 30 s, 55-62℃ annealing for 1 min, 72℃ extension for 2 min, 30-40 cycles; preferably 94℃ pre-denaturation for 1 min, 94℃ denaturation for 30 s, 59℃ annealing for 1 min, 72℃ extension for 2 min, 35 cycles.

[0030] The present invention also provides the application of the kit in detecting and / or identifying the virulence of wheat leaf rust fungi.

[0031] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0032] In this invention, the leaf rust-susceptible wheat material 'Thatcher' and the leaf rust-resistant near-isogenic wheat material 'TcLr15' (the leaf rust-resistant near-isogenic wheat material 'TcLr15' is a wheat material with Thatcher as its genetic background and containing the leaf rust-resistant gene Lr15, variety number RL6052 from Agricultural Research Service (ARS, https: / / www.ars.usda.gov / midwest-area / stpaul / cereal-disease-lab / docs / cereal-rusts / wheat-leaf-rust / ) both came from the Wheat Leaf Rust Research Center of Hebei Agricultural University;

[0033] In this invention, 21 physiological races of wheat leaf rust were collected from 2020 to 2023. Among them, V1 to V7 are physiological races of wheat leaf rust that are toxic to TcLr15, and A8 to A21 are physiological races of wheat leaf rust that are not toxic to TcLr15. They were collected from the Wheat Leaf Rust Research Center of Hebei Agricultural University. Detailed information is shown in Table 1.

[0034] Table 1. Physiological races of wheat leaf rust

[0035] serial number Physiological race names of leaf rust fungi Year of collection Collection Area V1 THTT 2020 Hebei V2 THTS 2021 Henan V3 SHJF 2020 Hebei V4 DHSJ 2022 Shandong V5 TKSQ 2023 Hebei V6 260 2023 Shandong V7 S21 2023 Hebei A8 PHNT 2021 Hebei A9 SHST 2020 Shandong A10 FHJJ 2021 Hebei A11 FHTT 2023 Hebei A12 FKKT 2021 Henan A13 DHSJ 2023 Hebei A14 S13 2023 Hebei A15 S10 2023 Shandong A16 S11 2023 Shandong A17 S12 2023 Hebei A18 S14 2023 Hebei A19 S15 2023 Henan A20 671 2023 Hebei A21 509 2023 Hebei

[0036] In this invention, 120 wheat leaf rust samples were collected from different regions of Hebei, Henan and Shandong provinces. The toxicity level indicated that the wheat leaf rust sample was toxic to TcLr15 (V15), and the toxicity level indicated that the wheat leaf rust sample was non-toxic to TcLr15 (A15). The samples were collected from the Wheat Leaf Rust Research Center of Hebei Agricultural University. Detailed information is shown in Table 2.

[0037] Table 2 Information on wheat leaf rust samples

[0038]

[0039]

[0040]

[0041] In the reagents of this invention, 2×Es Taq MasterMix was purchased from Jiangsu Kangwei Reagent Biotechnology Co., Ltd.

[0042] Example 1: Primer design and acquisition of wheat leaf rust genome

[0043] (1) Primer design: Point mutations were performed at four sites in the nucleotide sequence of the CDS region of the wheat leaf rust avirulence gene AvrLr15 (as shown in SEQ ID NO.1, NCBI ID OQ458736.1). Specifically, based on SEQ ID NO.1, the C at 238 bp was mutated to G, the GCC at 273-275 bp was deleted, the T at 279 bp was mutated to G, and the A at 286 bp was mutated to T, thus obtaining the nucleotide sequence of the CDS region of the wheat leaf rust virulence gene avrLr15 (as shown in SEQ ID NO.3). SNP sites in the nucleotide sequences of the CDS regions of the AvrLr15 and avrLr15 genes were analyzed. Primers AvrLr15M1-F, AvrLr15M1-R, and AvrLr15M1-R were designed based on the SNP sites in the ORF regions of the AvrLr15 and avrLr15 genes, respectively. M2 was synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the nucleotide sequences of each primer are shown in Table 3.

[0044] Table 3. Nucleotide sequences of each primer

[0045] Primer name Sequence (5'~3') SEQ ID NO. AvrLr15 M1-F ATGCACTGCCTCTTCTACGTC 5 AvrLr15M1-R GGCCCGCCGTAGCCGTAGAG 6 AvrLr15M2 TTTCATCAACAGACCCGGC 7

[0046] The nucleotide sequence of the CDS region of the wheat leaf rust avirulence gene AvrLr15 is shown in SEQ ID NO.1.

[0047] SEQ ID NO.1:

[0048] ATGCACTGCCTCTTCTACGTCGCCTGCTTCTTGGCCGTTCTCCAGTCAGCACTCGCTGTCCCAACACTCGCGCCACGAGCAGACACGACCGCCACCAAAGGCCAATCAGACCAGAAGTGCTGGGGCTTAGGCTTGGGTTACTGCGGGGGATTATATGCCGGCCTC GGCTCGTTTGGATTTGACCCGTTCGGCTTGTACACCCTCGGCTTGAACAGCTTATATGGCTACGGCGGACTGCCCTTCGGCCTGGCTCTCTCTACGGCTACGGCGGGCCGGGTCTGTTGATGAAAGATGCTCCGGGGGCTGGTATAACTCCCAGCACATCGGCT

[0049] The amino acid sequence of the AvrLr15 protein it encodes is shown in SEQ ID NO.2.

[0050] SEQ ID NO.2:

[0051] MHCLFYVACFLAVLQSALAVPTLAPRADTTATKGQSDQKCWGLGLGYCGGLYAGLGSFGFDPFGLYTLGLNSLYGYGGLPFGAGSLYGYGGPGLLMKDAPGAGITPSTSA

[0052] The nucleotide sequence of the CDS region of the wheat leaf rust virulence gene avrLr15 is shown in SEQ ID NO.3.

[0053] SEQ ID NO.3:

[0054] ATGCACTGCCTCTTCTACGTCGCCTGCTTCTTGGCCGTTCTCCAGTCAGCACTCGCTGTCCCAACACTCGCGCCACGAGCAGACACGACCGCCACCAAAGGCCAATCAGACCAGAAGTGCTGGGGCTTAGGCTTGGGTTACTGCGGGGGATTATATGCCGGCC TCGGCTCGTTTGGATTTGACCCGTTCGGCTTGTACACCCTTGGCCTGAACAGCTTGTATGGCTACGGCGGACTGGCCTTCGGCCTGGCTCTCTACGGCTACGGCGGTGGGCTGTTGTTGAAAGATGCTCCAGGGGCTGGTATAACTTCCAGCACATCGGCG

[0055] The amino acid sequence of the avrLr15 protein it encodes is shown in SEQ ID NO.4.

[0056] SEQ ID NO.4:

[0057] MHCLFYVACFLAVLQSALAVPTLAPRADTTATKGQSDQKCWGLGLGYCGGLYAGLGSFGFDPFGLYTLGLNSLYGYGGLAFGAGSLYGYGGGLLLKDAPGAGITSSTSA

[0058] (2) Obtaining the genome of wheat leaf rust fungi: The genomic DNA of urediniospores of 21 physiological races of wheat leaf rust fungi in Table 1 and 120 wheat leaf rust fungi standard samples in Table 2 were extracted in large quantities using the CTAB method disclosed by Huang et al. in the literature (Huang X, Zeller FJ, HsamSL, et al. Chromosomal location of AFLP markers in common wheat utilizing nulli-tetrasomic stocks[J]. Genome, 2000, 43(2):298-305). The genomic DNA of each wheat leaf rust fungi was obtained and its concentration was adjusted to 30 ng / μL to obtain the DNA template of each wheat leaf rust fungi.

[0059] Example 2: Detection of β-Actin internal reference gene in wheat leaf rust fungus

[0060] Using the genomic DNA of urediniospores from the 21 physiological races of wheat leaf rust fungi in Table 1 and the 120 wheat leaf rust fungi standards in Table 2 as DNA templates for each wheat leaf rust fungus, and using β-Actin internal reference gene primers as detection primers (as shown in Table 4), the β-Actin internal reference gene of each wheat leaf rust fungus was detected, with ddH2O as a control. Specifically:

[0061] The detection system was constructed as follows: The detection system consisted of 12.5 μL of 2×Es Taq MasterMix, 1 μL of β-Actin-F primer at a concentration of 5 mmol / L, 1 μL of β-Actin-R primer at a concentration of 5 mmol / L, 1 μL of DNA templates of various wheat leaf rust fungi at a concentration of 30 ng / μL, and the remainder ddH2O.

[0062] The detection program was set as follows: PCR amplification was performed at 94℃ for 1 min, followed by denaturation at 94℃ for 30 s, annealing at 59℃ for 1 min, and extension at 72℃ for 2 min, for a total of 35 cycles. A final extension at 72℃ for 10 min was then performed to obtain the PCR amplification products. Each PCR amplification product was subjected to 1% agarose gel electrophoresis. The detection results of the β-Actin internal reference gene in each wheat leaf rust fungus are as follows: Figures 1-2 As shown.

[0063] Table 4. Nucleotide sequences of primers for the β-Actin internal reference gene.

[0064] Primer name Sequence (5'~3') SEQ ID NO. β-Actin-F GTTCTACAACGAGCTCCGTGTC 8 β-Actin-R GACATACATTGCTGGGCAAC 9

[0065] The results showed that the β-Actin internal reference gene band was successfully amplified from the DNA templates of the 21 physiological races of wheat leaf rust in Table 1 and the 120 wheat leaf rust standards in Table 2, indicating that the DNA templates of each wheat leaf rust can be used for virulence detection.

[0066] Example 3: Virulence detection of wheat leaf rust

[0067] Using the genomic DNA of urediniospores from the 21 physiological races of wheat leaf rust in Table 1 and the 120 wheat leaf rust standards in Table 2 as DNA templates for each wheat leaf rust, and the primer set described in Example 1 as detection primers, the virulence of each wheat leaf rust was tested, with ddH2O as a control. Specifically:

[0068] (1) Construction of detection system: The detection system is in 25 μL, including 12.5 μL of 2×Es Taq MasterMix, 2 μL of AvrLr15 M1-F primer at a concentration of 5 mmol / L, 1 μL of AvrLr15 M1-R primer at a concentration of 5 mmol / L, 1 μL of AvrLr15 M2 primer at a concentration of 5 mmol / L, 1 μL of DNA template of each wheat leaf rust fungus at a concentration of 30 ng / μL, and the remainder ddH2O;

[0069] (2) Setting up the detection program: The PCR amplification program was set as follows: 94℃ pre-denaturation for 1 min, followed by 94℃ denaturation for 30 s → 59℃ annealing for 1 min → 72℃ extension for 2 min, for a total of 35 cycles, and finally 72℃ extension for 10 min to obtain the PCR amplification products; each PCR amplification product was subjected to 1% agarose gel electrophoresis, and the virulence detection results of each wheat leaf rust fungus were as follows. Figures 3-4 As shown;

[0070] The 21 physiological races of wheat leaf rust fungi listed in Table 1 were inoculated onto wheat leaf rust resistant material TcLr15 and wheat susceptible material Thatcher, respectively. The pathogenic phenotypes of each wheat leaf rust physiological race on wheat plants were observed, and the results are as follows: Figure 5 As shown.

[0071] The results showed that, using the primer set of the present invention for virulence detection, no bands were amplified in 7 physiological races of wheat leaf rust that are virulent to TcLr15 and 21 wheat leaf rust standards that are virulent to TcLr15, but bands of approximately 291 bp were amplified in 14 physiological races of wheat leaf rust that are not virulent to TcLr15 and 99 wheat leaf rust standards that are not virulent to TcLr15. Furthermore, the virulence detection results were completely consistent with the pathogenic phenotype of each physiological race of wheat leaf rust on wheat plants, indicating that the primer set of the present invention can be used to detect and identify the virulence of wheat leaf rust to TcLr15.

[0072] Example 4: Virulence detection of wheat leaf rust

[0073] The virulence of wheat leaf rust fungus was tested using the method described in Example 3, except that in this example:

[0074] The detection system, in 25 μL volumes, included 14.5 μL of 2×Es Taq MasterMix, 2.4 μL of AvrLr15 M1-F primer at a concentration of 5 mmol / L, 1.2 μL of AvrLr15 M1-R primer at a concentration of 5 mmol / L, 1.2 μL of AvrLr15 M2 primer at a concentration of 5 mmol / L, 0.5 μL of DNA templates from various wheat leaf rust fungi at a concentration of 30 ng / μL, and the remainder ddH2O.

[0075] The PCR amplification program was set as follows: 94℃ pre-denaturation for 1 min, followed by 94℃ denaturation for 30 s → 62℃ annealing for 1 min → 72℃ extension for 2 min, for a total of 30 cycles, and finally 72℃ extension for 10 min to obtain the PCR amplification product.

[0076] Example 5: Virulence detection of wheat leaf rust

[0077] The virulence of wheat leaf rust fungus was tested using the method described in Example 3, except that in this example:

[0078] The detection system, in 25 μL volumes, includes 10.5 μL of 2×Es Taq MasterMix, 1 μL of AvrLr15 M1-F primer at a concentration of 5 mmol / L, 0.5 μL of AvrLr15 M1-R primer at a concentration of 5 mmol / L, 0.5 μL of AvrLr15 M2 primer at a concentration of 5 mmol / L, 2 μL of DNA templates of various wheat leaf rust fungi at a concentration of 30 ng / μL, and the remainder ddH2O.

[0079] The PCR amplification program was set as follows: 94℃ pre-denaturation for 1 min, followed by 94℃ denaturation for 30 s → 58℃ annealing for 1 min → 72℃ extension for 2 min, for a total of 30 cycles, and finally 72℃ extension for 10 min to obtain the PCR amplification product.

[0080] The results showed that, after repeated virulence tests, specific bands were consistently detected in wheat leaf rust fungi that were non-toxic to TcLr15, while no corresponding bands were amplified in wheat leaf rust fungi that were toxic to TcLr15. This indicates that the primer set of the present invention has good specificity, reliability, and stability, and is applicable to all tested wheat leaf rust fungi. Therefore, it can be used as a primer set for detecting and / or identifying the virulence of wheat leaf rust fungi to TcLr15.

[0081] As can be seen from the above embodiments, the present invention provides the application of the wheat AvrLr15 gene, primer set, and kit in the detection and / or identification of wheat leaf rust virulence. The primer set of the present invention can be used to detect the virulence of all wheat leaf rusts to TcLr15, and has the advantages of good specificity, reliability, and stability.

[0082] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting wheat leaf rust. AvrLr15 The application of primer sets for the CDS region of a gene in the detection and / or identification of wheat leaf rust virulence, characterized in that... The primer set includes: AvrLr15 M1-F primers, AvrLr15 M1-R primers and AvrLr15 M2 primers; The AvrLr15 The nucleotide sequence of the M1-F primer is shown in SEQ ID NO.

5. AvrLr15 The nucleotide sequence of the M1-R primer is shown in SEQ ID NO.

6. AvrLr15 The nucleotide sequence of the M2 primer is shown in SEQ ID NO.

7. The primer set amplified a specific band in wheat leaf rust fungi that are non-toxic to TcLr15, but did not amplify a band in wheat leaf rust fungi that are toxic to TcLr15.

2. A method for detecting wheat leaf rust. AvrLr15 The application of primer sets for the CDS region of a gene in the preparation of products for detecting and / or identifying the virulence of wheat leaf rust fungi, characterized in that... The primer set includes: AvrLr15 M1-F primers, AvrLr15 M1-R primers and AvrLr15 M2 primers; The AvrLr15 The nucleotide sequence of the M1-F primer is shown in SEQ ID NO.

5. AvrLr15 The nucleotide sequence of the M1-R primer is shown in SEQ ID NO.

6. AvrLr15 The nucleotide sequence of the M2 primer is shown in SEQ ID NO.

7. The primer set amplified a specific band in wheat leaf rust fungi that are non-toxic to TcLr15, but did not amplify a band in wheat leaf rust fungi that are toxic to TcLr15.

3. The application as described in claim 2, characterized in that... The product is a kit for detecting and / or identifying the virulence of wheat leaf rust fungi.

4. The application according to claim 3, characterized in that, In the kit, the AvrLr15 The concentration of the M1-F primer is 1–10 mmol / L. AvrLr15 The concentration of the M1-R primer is 1–10 mmol / L. AvrLr15 The concentration of the M2 primer is 1–10 mmol / L.

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