Application of wheat AvrLr15 gene, primer group and kit in detection and / or identification of wheat puccinia puccinia virulence

Abstract

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

Description

Technical Field

[0001] The present invention relates to the technical field of pathogenic microorganism detection, and particularly relates to the application of wheat AvrLr15 gene, primer sets and kits in detecting and / or identifying the virulence of wheat leaf rust fungus. Background Art

[0002] Wheat leaf rust is a fungal disease caused by the infection of Puccinia triticina, and is one of the wheat diseases with wide prevalence and serious harm, which can cause yield reduction and huge economic losses to the main wheat producing areas. Reasonable use and layout of wheat resistant varieties containing disease-resistant genes are economic, safe and effective methods for preventing and controlling the occurrence and spread of wheat leaf rust. However, large-scale planting of a single resistant variety will exert selective pressure on the virulence genes of the pathogen population by the disease-resistant genes, resulting in the loss of rust resistance of the host wheat, and then triggering the epidemic of the disease, bringing serious losses to wheat production. Therefore, it is very important for production practice to monitor the virulence of the wheat leaf rust fungus population in the field, and then guide the breeding and correct use of wheat varieties resistant to leaf rust.

[0003] With the progress of molecular-level technologies, methods such as molecular fingerprint analysis, molecular markers and gene amplification have begun to be applied to the identification of leaf rust fungus, mainly including restriction fragment length polymorphism analysis (RFLP), random amplified polymorphic DNA analysis (RAPD), simple sequence repeat analysis (SSR), amplified fragment length polymorphism analysis (AFLP) and single nucleotide polymorphism analysis (SNP), etc. Although a large number of molecular markers such as AFLP, RAPD, SSR and EST-SSR have been developed according to conventional PCR detection methods, there are few reports on specific molecular markers for specific virulence / avirulence genes of wheat leaf rust fungus.

[0004] The wheat leaf rust resistance gene Lr15 is a wheat seedling resistance gene located on wheat chromosome 2D. When this gene coexists with adult plant resistance genes, it can significantly improve the rust resistance of wheat plants. At present, the Lr15 gene still has good resistance to most physiological races of wheat leaf rust fungus. Therefore, in order to avoid the loss of resistance of the Lr15 gene, it is urgent to formulate reasonable and effective prevention and control measures. However, there is no relevant detection or identification method in the prior art that can directly and effectively judge the virulence of wheat leaf rust fungus to wheat plants containing the Lr15 gene. Summary of the Invention

[0005] The purpose of the present invention is to provide the application of wheat AvrLr15 gene, primer sets and kits in detecting and / or identifying the virulence of wheat leaf rust fungus, so as to solve the problem that the virulence of wheat leaf rust fungus to wheat plants containing the Lr15 gene cannot be directly and effectively detected and / or identified in the prior art.

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

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

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

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

[0010] The present invention also provides the application of the primer set as described above in detecting and / or identifying the virulence of wheat leaf rust fungus.

[0011] The present invention also provides the application of the primer set as described above in preparing a product for detecting and / or identifying the virulence of wheat leaf rust fungus.

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

[0013] Preferably, the concentration of the AvrLr15 M1-F primer is 1 - 10 mmol / L, the concentration of the AvrLr15 M1-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 as described above in detecting and / or identifying the virulence of wheat leaf rust fungus.

[0015] The present invention has the following technical effects and advantages: The primer set of the present invention can stably detect specific bands in the wheat leaf rust fungus that is avirulent to the wheat leaf rust-resistant material TcLr15, while no corresponding bands are amplified in the wheat leaf rust fungus that is virulent to TcLr15. This indicates that the primer set of the present invention can directly detect and identify the virulence of the wheat leaf rust fungus to TcLr15, 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 specific molecular markers to detect and / or identify the virulence of the wheat leaf rust fungus to TcLr15, and is of great significance for research such as the mapping and cloning of the virulence / avirulence genes of the wheat leaf rust fungus, the identification and structural analysis of wheat leaf rust fungus races, and the revelation of the variation law of wheat leaf rust fungus races. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0018] Figure 3 It is the virulence detection result of 21 physiological races of wheat leaf rust fungus in Example 3. In the figure, lane M is Marker, and lanes 1 to 22 are ddH2O, physiological races V1, V2, V3, V4, V4, V5, V6, V7, A8, A9, A10, A11, A12, A13, A14, A15, A16, A17, A18, A19, A20, A21 of wheat leaf rust fungus respectively;

[0019] Figure 4 It is the virulence detection result of 120 wheat leaf rust fungus specimens in Example 3. In the figure, lane M is Marker, lane H2O is ddH2O, and lanes 1 to 120 are the wheat leaf rust fungus specimens numbered 1 to 120 in Table 2 respectively;

[0020] Figure 5For the pathogenic phenotypes of 21 physiological races of wheat leaf rust fungus on the wheat leaf rust-resistant material TcLr15 and the wheat susceptible material Thatcher in Example 3, V15 in the figure indicates that the physiological race of the wheat leaf rust fungus is virulent to TcLr15, and A15 indicates that the physiological race of the wheat leaf rust fungus is avirulent to TcLr15. Detailed implementation mode

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

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

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

[0024] The present invention also provides the application of the primer set in detecting and / or identifying the virulence of wheat leaf rust fungus.

[0025] The present invention also provides the application of the primer set in preparing a product for detecting and / or identifying the virulence of wheat leaf rust fungus.

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

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

[0028] In the present 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; the concentration of the DNA template is 10 - 100 ng / μL, preferably 30 ng / μL.

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

[0030] The present invention also provides the application of the described 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 in conjunction with the examples, but they should not be construed as limiting the protection scope of the present invention.

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

[0033] In the present invention, 21 physiological races of wheat leaf rust fungi were collected respectively from 2020 to 2023, among which V1 - V7 are physiological races of wheat leaf rust fungi toxic to TcLr15, and A8 - A21 are physiological races of wheat leaf rust fungi non-toxic to TcLr15, from the Wheat Leaf Rust Research Center of Hebei Agricultural University, and the detailed information is shown in Table 1;

[0034] Table 1 Information table of physiological races of wheat leaf rust fungi

[0035] Number Name of Puccinia triticina Physiological Race Collection Year Collection Region 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 the present invention, 120 wheat leaf rust fungus specimens were collected from different regions of Hebei, Henan, and Shandong provinces. Among them, a virulent specimen means that the wheat leaf rust fungus specimen is virulent to TcLr15 (V15), and an avirulent specimen means that the wheat leaf rust fungus specimen is avirulent to TcLr15 (A15). They are from the Wheat Leaf Rust Research Center of Hebei Agricultural University, and the detailed information is shown in Table 2.

[0037] Table 2 Information Table of Wheat Leaf Rust Fungus Specimens

[0038]

[0039]

[0040]

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

[0042] Example 1: Primer Set Design and Acquisition of Wheat Leaf Rust Fungus Genome

[0043] (1) Primer set design: Four sites in the CDS region nucleotide sequence of the wheat leaf rust fungus avirulence gene AvrLr15 (shown as SEQ ID NO.1, NCBI ID is OQ458736.1) were subjected to point mutations, that is, based on SEQ ID NO.1, the C at the 238th bp was mutated to G, the GCC at the 273 - 275th bp was deleted, the T at the 279th bp was mutated to G, and the A at the 286th bp was mutated to T, obtaining the CDS region nucleotide sequence of the wheat leaf rust fungus virulence gene avrLr15 (shown as SEQ ID NO.3); SNPs in the CDS region nucleotide sequences of the AvrLr15 gene and the avrLr15 gene were analyzed, and primers AvrLr15M1-F, AvrLr15 M1-R, and AvrLr15 M2 were designed based on the SNPs in the ORF regions of the AvrLr15 gene and the avrLr15 gene respectively, and were commissioned to be synthesized by Sangon Biotech (Shanghai) Co., Ltd. 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 CDS region nucleotide sequence of the wheat leaf rust fungus avirulence gene AvrLr15 is shown as SEQ ID NO.1.

[0047] SEQ ID NO.1:

[0048] ATGCACTGCCTCTTCTACGTCGCCTGCTTCTTGGCCGTTCTCCAGTCAGCACTCGCTGTCCCAACACTCGCGCCACGAGCAGACACGACCGCCACCAAAGGCCAATCAGACCAGAAGTGCTGGGGCTTAGGCTTGGGTTACTGCGGGGGATTATATGCCGGCCTCGGCTCGTTTGGATTTGACCCGTTCGGCTTGTACACCCTCGGCTTGAACAGCTTATATGGCTACGGCGGACTGCCCTTCGGCGCTGGCTCTCTCTACGGCTACGGCGGGCCGGGTCTGTTGATGAAAGATGCTCCGGGGGCTGGTATAACTCCCAGCACATCGGCT

[0049] The amino acid sequence of the encoded AvrLr15 protein 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 fungus virulence gene avrLr15 is shown in SEQ ID NO.3.

[0053] SEQ ID NO.3:

[0054] ATGCACTGCCTCTTCTACGTCGCCTGCTTCTTGGCCGTTCTCCAGTCAGCACTCGCTGTCCCAACACTCGCGCCACGAGCAGACACGACCGCCACCAAAGGCCAATCAGACCAGAAGTGCTGGGGCTTAGGCTTGGGTTACTGCGGGGGATTATATGCCGGCCTCGGCTCGTTTGGATTTGACCCGTTCGGCTTGTACACCCTTGGCCTGAACAGCTTGTATGGCTACGGCGGACTGGCCTTCGGCGCTGGCTCTCTCTACGGCTACGGCGGTGGGCTGTTGTTGAAAGATGCTCCAGGGGCTGGTATAACTTCCAGCACATCGGCG

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

[0056] SEQ ID NO.4:

[0057] MHCLFYVACFLAVLQSALAVPTLAPRADTTATKGQSDQKCWGLGLGYCGGLYAGLGSFGFDPFGLYTLGLNSLYGYGGLAFGAGSLYGYGGGLLLKDAPGAGITSSTSA

[0058] (2) Acquisition of the Puccinia triticina genome: Referring to the CTAB method disclosed by Huang et al. in the literature (Huang X, Zeller FJ, Hsam SL, 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 uredospores of 21 Puccinia triticina physiological races in Table 1 and 120 Puccinia triticina specimens in Table 2 was extracted in large quantities to obtain the genomic DNA of each Puccinia triticina, and its concentration was adjusted to 30 ng / μL to obtain the DNA template of each Puccinia triticina.

[0059] Example 2: Detection of the β-Actin internal reference gene of Puccinia triticina

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

[0061] Construct a detection system: The detection system is calculated as 25 μL, including 12.5 μL of 2×Es Taq MasterMix, 1 μL of β-Actin-F primer with a concentration of 5 mmol / L, 1 μL of β-Actin-R primer with a concentration of 5 mmol / L, 1 μL of the DNA template of each wheat leaf rust fungus with a concentration of 30 ng / μL, and the remaining ddH2O;

[0062] Set the detection program: Set the PCR amplification program as: pre-denaturation at 94°C for 1 min, followed by denaturation at 94°C for 30 s → annealing at 59°C for 1 min → extension at 72°C for 2 min, for a total of 35 cycles, and finally extension at 72°C for 10 min to obtain the PCR amplification product; Perform 1% agarose gel electrophoresis on each PCR amplification product, and the detection results of the β-Actin reference gene of each wheat leaf rust fungus are as Figure 1-2 shown.

[0063] Table 4 Nucleotide sequences of primers for the β-Actin 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 reference gene bands were successfully amplified from the DNA templates of 21 physiological races of wheat leaf rust fungus in Table 1 and 120 urediniospore specimens of wheat leaf rust fungus in Table 2, indicating that the DNA templates of each wheat leaf rust fungus can be used for virulence detection.

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

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

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

[0069] (2) Setting of the detection procedure: Set the PCR amplification procedure as follows: pre-denaturation at 94°C for 1 min, followed by denaturation at 94°C for 30 s → annealing at 59°C for 1 min → extension at 72°C for 2 min, for a total of 35 cycles, and finally extension at 72°C for 10 min to obtain the PCR amplification product; perform 1% agarose gel electrophoresis on each PCR amplification product, and the virulence detection results of each wheat leaf rust fungus are as Figure 3-4 shown;

[0070] Inoculate 21 physiological races of wheat leaf rust fungus in Table 1 on wheat leaf rust-resistant material TcLr15 and wheat susceptible material Thatcher respectively, and observe the pathogenic phenotypes of each physiological race of wheat leaf rust fungus on wheat plants. The results are as Figure 5 shown.

[0071] The results show that when using the primer set of the present invention for virulence detection, no bands were amplified in 7 physiological races of wheat leaf rust fungus that are virulent to TcLr15 and 21 samples of wheat leaf rust fungus that are virulent to TcLr15, while bands of about 291 bp in size were amplified in 14 physiological races of wheat leaf rust fungus that are avirulent to TcLr15 and 99 samples of wheat leaf rust fungus that are avirulent to TcLr15; and the virulence detection results are completely consistent with the pathogenic phenotypes of each physiological race of wheat leaf rust fungus on wheat plants, indicating that the primer set of the present invention can be used for detecting and identifying the virulence of wheat leaf rust fungus to TcLr15.

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

[0073] The virulence detection of wheat leaf rust fungus was carried out by the method described in Example 3, except that in this example:

[0074] The detection system is 25 μL in total, including 14.5 μL of 2×Es Taq MasterMix, 2.4 μL of AvrLr15 M1-F primer with a concentration of 5 mmol / L, 1.2 μL of AvrLr15 M1-R primer with a concentration of 5 mmol / L, 1.2 μL of AvrLr15 M2 primer with a concentration of 5 mmol / L, 0.5 μL of DNA template of each wheat leaf rust fungus with a concentration of 30 ng / μL, and the remaining ddH2O;

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

[0076] Example 5: Virulence detection of Puccinia triticina

[0077] The method described in Example 3 was used for the virulence detection of Puccinia triticina. The difference is that in this example:

[0078] The detection system was 25 μL in volume, including 10.5 μL of 2×Es Taq MasterMix, 1 μL of AvrLr15 M1-F primer with a concentration of 5 mmol / L, 0.5 μL of AvrLr15 M1-R primer with a concentration of 5 mmol / L, 0.5 μL of AvrLr15 M2 primer with a concentration of 5 mmol / L, 2 μL of DNA template of each Puccinia triticina with a concentration of 30 ng / μL, and the remaining ddH2O;

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

[0080] The results showed that after multiple repeated virulence detections and verifications, specific bands could be stably detected in Puccinia triticina that was avirulent to TcLr15, while the corresponding bands were not amplified in Puccinia triticina that was virulent to TcLr15, indicating that the primer set of the present invention has good specificity, reliability, and stability, and is applicable to all tested Puccinia triticina. Therefore, it can be used as a primer set for specific molecular markers to detect and / or identify the virulence of Puccinia triticina to TcLr15.

[0081] As can be seen from the above examples, the present invention provides the application of wheat AvrLr15 gene, primer set, and kit in detecting and / or identifying the virulence of Puccinia triticina. The primer set of the present invention can be used to detect the virulence of all Puccinia triticina 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 of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. Application of wheat AvrLr15 gene in detecting and / or identifying the virulence of wheat leaf rust, characterized in that: The nucleotide sequence of the CDS region of the AvrLr15 gene is shown in SEQ ID NO.

1.

2. A primer set for detecting the wheat AvrLr15 gene according to claim 1, characterized in that: The primer set includes: AvrLr15 M1-F primer, AvrLr15 M1-R primer and AvrLr15 M2 primer; 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.

3. Use of the primer set according to claim 2 in detecting and / or identifying the virulence of wheat leaf rust.

4. Use of the primer set according to claim 2 in preparing a product for detecting and / or identifying the virulence of wheat leaf rust.

5. A kit for detecting and / or identifying the virulence of wheat leaf rust, characterized in that: The invention comprises the following components in parts by volume: 0.5 to 2 parts of DNA template, 10.5 to 14.5 parts of DNA polymerase, 1.6 to 2.4 parts of the AvrLr15 M1-F primer according to claim 2, 0.8 to 1.2 parts of the AvrLr15 M1-R primer according to claim 2, 0.8 to 1.2 parts of the AvrLr15 M2 primer according to claim 2, and 2.7 to 10.8 parts of water.

6. The kit according to claim 5, characterized in that The concentration of the AvrLr15 M1-F primer is 1 to 10 mmol / L, the concentration of the AvrLr15 M1-R primer is 1 to 10 mmol / L, and the concentration of the AvrLr15 M2 primer is 1 to 10 mmol / L.

7. Use of the kit according to claim 5 or 6 in detecting and / or identifying the virulence of wheat leaf rust.

Images