A specific primer pair for identifying wheat special strains of rice blast and application thereof

Abstract

This invention belongs to the field of biotechnology and discloses a specific primer pair for identifying wheat-specific strains of *Blastomyces oryzae* and its application. Based on the analysis of the genomes of *Blastomyces oryzae* strains specializing in different hosts, this invention identified a conserved fragment in the gene of the wheat-specific strain. Through design, PCR amplification, agarose gel electrophoresis, and cloning sequencing, three pairs of specific primers were ultimately screened to identify the wheat-specific strain of *Blastomyces oryzae*. Using the screened specific primer pairs, the wheat-specific strain of *Blastomyces oryzae* was qualitatively identified by PCR amplification of the test samples. The identification method is simple, efficient, and accurate, and has practical application value. This invention provides molecular markers for screening wheat-specific strains of *Blastomyces oryzae* and offers a convenient and accurate method for observing and studying these strains.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and discloses a specific primer pair for identifying a wheat special strain of Magnaporthe oryzae and application thereof. BACKGROUND

[0002] Magnaporthe oryzae (Berk.) Barron et Jol. Magnaporthe oryzae has a wide host range and can infect more than 50 different hosts such as rice, wheat, and barley. The wheat pathogenic type of Magnaporthe oryzae is also known as the wheat special strain of Magnaporthe oryzae. In recent years, wheat special strains have caused wheat blast in the Americas, Africa, and Bangladesh in Asia, causing serious losses to wheat production. The wheat special strain can not only infect the leaves of wheat plants but also the ears of wheat plants, and ear infection can cause greater economic impact. Infection of the leaf axis or flower stalk can hinder the transport of photosynthetic products, killing the upper part of the ear inflorescence. The pathogen is easily transmitted over long distances through seeds and air currents, and is difficult to control due to its rapid development of drug resistance.

[0003] Due to the similarity of different host special strains, morphological methods cannot effectively distinguish them. Identification by inoculating different hosts takes a long time and is not accurate. For example, under laboratory conditions, rice special strains can also infect some other crops. Genome sequencing methods can effectively distinguish different host special strains of Magnaporthe oryzae, but genome sequencing is time-consuming and can affect the diagnosis time of the pathogen, causing the field disease control to be not timely. In addition, the genetic difference between the wheat special strain of Magnaporthe oryzae and other host special strains of Magnaporthe oryzae is very small, and the special strain of Magnaporthe oryzae on weeds such as crabgrass and finger millet in the rice field has a great influence on the accurate identification of the wheat special strain, so it is very important to develop effective, accurate, and rapid identification molecular markers.

[0004] Patent No. CN119193916B discloses a primer probe combination for identifying wheat blast, a product and application thereof. The specific marker primer probe combination is designed based on the wheat blast gene, and the primer probe combination can accurately distinguish wheat blast, Gibberella and Magnaporthe oryzae. At the same time, based on the designed primer probe combination, RPA amplification technology and nucleic acid colloidal gold detection test strips are used to detect wheat blast, Gibberella and Magnaporthe oryzae, and the detection method can be specifically detected. However, the primer is only limited to distinguishing between the rice special strain and the wheat special strain of Magnaporthe oryzae, and cannot distinguish between the wheat special strain and the weed special strain of Magnaporthe oryzae.

[0005] After searching, no relevant research on distinguishing the wheat special strain of Magnaporthe oryzae from the weed special strains of Magnaporthe oryzae on crabgrass and finger millet has been found. SUMMARY

[0006] In view of the deficiencies of the prior art, the application provides a specific primer pair for identifying a wheat special strain of Magnaporthe oryzae and application thereof.

[0007] To solve the above technical problems, the technical solutions of the present application are as follows:

[0008] A specific primer pair for identifying Magnaporthe oryzae f. sp. tritici strains, the specific primer pair is a first primer pair, a second primer pair or a third primer pair; wherein the nucleotide sequence of the first primer pair is shown as SEQ ID NO. 1-2; the nucleotide sequence of the second primer pair is shown as SEQ ID NO. 3-4; and the nucleotide sequence of the third primer pair is shown as SEQ ID NO. 5-6.

[0009] The present application also protects a detection reagent for identifying Magnaporthe oryzae f. sp. tritici strains, the detection reagent comprising any of the above-mentioned primer pairs.

[0010] Further, the detection reagent further comprises at least one of the following: dNTPs, DNA polymerase, reaction solvent or buffer.

[0011] The present application also protects the use of the primer pair and the detection reagent in identifying Magnaporthe oryzae f. sp. tritici strains.

[0012] The present application also protects a method for identifying Magnaporthe oryzae f. sp. tritici strains, comprising the following steps:

[0013] 1) using the primer pair of claim 1 or the detection reagent of any one of claims 2-3 to perform PCR amplification on different sample DNAs to obtain amplification products; 2) performing electrophoresis detection on the amplification products to interpret the results; observing the band size of the amplification products, using the first primer pair, if a band appears at 1189bp, then the sample to be tested is Magnaporthe oryzae f. sp. tritici, or the sample to be tested contains Magnaporthe oryzae f. sp. tritici; if there is no band at 1189bp after amplification, then the sample to be tested does not contain Magnaporthe oryzae f. sp. tritici, or the sample to be tested does not belong to Magnaporthe oryzae f. sp. tritici; using the second primer pair, if a band appears at 710bp, then the sample to be tested is Magnaporthe oryzae f. sp. tritici, or the sample to be tested contains Magnaporthe oryzae f. sp. tritici; if there is no band at 710bp after amplification, then the sample to be tested does not contain Magnaporthe oryzae f. sp. tritici, or the sample to be tested does not belong to Magnaporthe oryzae f. sp. tritici; using the third primer pair, if a band appears at 1297bp, then the sample to be tested is Magnaporthe oryzae f. sp. tritici, or the sample to be tested contains Magnaporthe oryzae f. sp. tritici; if there is no band at 1297bp after amplification, then the sample to be tested does not contain Magnaporthe oryzae f. sp. tritici, or the sample to be tested does not belong to Magnaporthe oryzae f. sp. tritici.

[0014] Further, the PCR amplification reaction conditions are: (1) 95℃, 5 min, (2) 94℃, 30 s, 60℃, 30 s, 72℃, 30 s, repeat 35 cycles, (3) 72℃, 7 min.

[0015] Compared with the prior art, the present application has the following beneficial effects:

[0016] Compared with the traditional inoculation method for identifying different hosts, the method of the present application can obtain results in a shorter time and is not affected by the growth cycle and environmental conditions of the pathogenic bacteria, and can more accurately identify the wheat-specialized strain of Magnaporthe oryzae, and the Magnaporthe oryzae specialized to the field weeds Digitaria sanguinalis and Eleusine indica, thereby avoiding the problem of inaccurate identification caused by the fact that the rice-specialized strain can infect other crops. The primer pair of the present application has high sensitivity and strong specificity, and even if the genetic difference between the wheat-specialized strain of Magnaporthe oryzae and other host-specialized strains of Magnaporthe oryzae is extremely small, the inventors of the present application can accurately identify the wheat-specialized strain of Magnaporthe oryzae by detecting the subtle differences in DNA sequences, thereby reducing the interference of the host-specialized strains on the field weeds. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The DNA amplification results of the different host-specialized strains of Magnaporthe oryzae using different primer pairs are shown in Table 1.

[0018] Figure 2 The alignment results of the amplification and sequencing results using the first primer pair (WBS1 primer pair) and the target gene fragment WB18 are shown in Table 2.

[0019] Figure 3 The alignment results of the amplification and sequencing results using the second primer pair (WBS2 primer pair) and the target gene fragment WB18 are shown in Table 3.

[0020] Figure 4 The alignment results of the amplification and sequencing results using the third primer pair (WBS3 primer pair) and the target gene fragment WB18 are shown in Table 4. DETAILED DESCRIPTION

[0021] The technical solutions of the present application will be further described in detail below in combination with the drawings and specific embodiments, but the present application is not limited to the following technical solutions.

[0022] Example 1: Screening of specific gene fragments for identifying wheat-specialized strains of Magnaporthe oryzae

[0023] The genomes of 11 Magnaporthe oryzae wheat special strain B71 and 29 genomes of other different host special strains of Magnaporthe oryzae were analyzed, and samtools was used to analyze the conserved fragments (similarity greater than 99%) in the wheat special strain of Magnaporthe oryzae, and the fragments not contained in other special strains, and 46 fragments were preliminarily screened. Further analysis of the 46 fragments by blast in different strain genomes found that one fragment WB18 was aligned in the wheat special strain of Magnaporthe oryzae, and was not aligned in other special strains of Magnaporthe oryzae.

[0024] The sequence of WB18 is shown as SEQ ID NO: 17.

[0025]

[0026] The information of 11 U. tritici strains and 29 U. other host strains for genome analysis is shown in Table 1.

[0027] Table 1. Information of strains for genome analysis

[0028]

[0029] Example 2. Design of primers for identifying U. tritici strains

[0030] According to the fragment WB18 obtained in Example 1, 8 pairs of specific primers were finally determined by preliminary design with Primer5 software, PCR amplification test, agarose gel electrophoresis and cloning sequencing according to the primer length, Tm value and the size of amplified fragment. The primer sequences are shown in Table 2.

[0031] Table 2. Primer sequences and related information table for amplifying target sequence WB18

[0032]

[0033] Example 3. Identification of U. tritici strains and verification by using specific primers

[0034] The DNA of U. tritici strains, U. oryzae strains, U. allii strains and U. striatum strains were used as templates for PCR amplification detection of the above primers, and the amplification conditions were (1) 95℃, 5min, (2) 94℃, 30s, 60℃, 30s, 72℃, 30s, repeated for 35 cycles, (3) 72℃, 7min.

[0035] The results are shown in Table 3. Figure 1As shown, it is indicated that WBS1, WBS2 and WBS3 can only amplify specific bands for the DNA of the wheat pathogen. By observing the band size of the amplification product, if a band appears at 1189bp using the first primer pair, the sample to be tested is a wheat special strain of Magnaporthe oryzae, or the sample to be tested contains a wheat special strain of Magnaporthe oryzae; if there is no band at 1189bp after amplification, the sample to be tested does not contain a wheat special strain of Magnaporthe oryzae, or the sample to be tested is not a wheat special strain of Magnaporthe oryzae; using the second primer pair, if a band appears at 710bp, the sample to be tested is a wheat special strain of Magnaporthe oryzae, or the sample to be tested contains a wheat special strain of Magnaporthe oryzae; if there is no band at 710bp after amplification, the sample to be tested does not contain a wheat special strain of Magnaporthe oryzae, or the sample to be tested is not a wheat special strain of Magnaporthe oryzae; using the third primer pair, if a band appears at 1297bp, the sample to be tested is a wheat special strain of Magnaporthe oryzae, or the sample to be tested contains a wheat special strain of Magnaporthe oryzae; if there is no band at 1297bp after amplification, the sample to be tested does not contain a wheat special strain of Magnaporthe oryzae, or the sample to be tested is not a wheat special strain of Magnaporthe oryzae. It is indicated that the three primer pairs can be used as specific molecular markers for wheat special strains, and can be used for rapid identification, field detection or evolution analysis of wheat special strains of Magnaporthe oryzae, and provide a reliable molecular tool for distinguishing different host special strains.

[0036] The fragments amplified by WBS1, WBS2 and WBS3 primer pairs were connected with pClone007 Versatile Simple Vector, and were transferred into E. coli competent cells, and were cultured in LB medium containing ampicillin at 37°C in the dark for 16h. The colonies grown were sent to a sequencing company for sequencing, and the sequencing results of the target fragments were compared with the genome sequence of the wheat special strain of Magnaporthe oryzae.

[0037] As shown in Table 2, the results show that the fragments amplified by WBS1, WBS2 and WBS3 primers are successfully compared with the specific fragments of the wheat special strain of Magnaporthe oryzae, which further indicates that the three primer pairs WBS1, WBS2 and WBS3 can be used to detect the specific fragments WB18 of the wheat special strain of Magnaporthe oryzae, and can be used for rapid identification, field detection or evolution analysis of the wheat special strain of Magnaporthe oryzae. Figures 2-4 As shown in Table 2, the results show that the fragments amplified by WBS1, WBS2 and WBS3 primers are successfully compared with the specific fragments of the wheat special strain of Magnaporthe oryzae, which further indicates that the three primer pairs WBS1, WBS2 and WBS3 can be used to detect the specific fragments WB18 of the wheat special strain of Magnaporthe oryzae, and can be used for rapid identification, field detection or evolution analysis of the wheat special strain of Magnaporthe oryzae.

Claims

1. A specific primer pair for identifying Magnaporthe grisea f. sp. tritici strains, characterized in that, The specific primer pair is a first primer pair, a second primer pair or a third primer pair; wherein the nucleotide sequences of the first primer pair are shown as SEQ ID NO. 1-2; the nucleotide sequences of the second primer pair are shown as SEQ ID NO. 3-4; and the nucleotide sequences of the third primer pair are shown as SEQ ID NO. 5-6.

2. A detection reagent for identifying Magnaporthe grisea f. sp. tritici strains, characterized by, The detection reagent comprises any primer pair of claim 1.

3. The detection reagent according to claim 2, characterized in that, The detection reagent further comprises at least one of dNTPs, DNA polymerase, reaction solvent or buffer.

4. Use of the primer pair of claim 1 or the detection reagent of any one of claims 2-3 in identifying Magnaporthe oryzae wheat special strain.

5. A method for identifying a strain of Magnaporthe grisea specialis tritici, characterized in that, The method comprises: 1) using the primer pair of claim 1 or the detection reagent of any one of claims 2-3 to perform PCR amplification on the DNA of the sample to be tested to obtain an amplification product; 2) performing electrophoresis detection on the amplification product to interpret the results; observing the band size of the amplification product, using the first primer pair, if a band appears at 1189bp, then the sample to be tested is Magnaporthe oryzae wheat special strain, or the sample to be tested contains Magnaporthe oryzae wheat special strain; if no band appears at 1189bp after amplification, then the sample to be tested does not contain Magnaporthe oryzae wheat special strain, or the sample to be tested is not Magnaporthe oryzae wheat special strain; using the second primer pair, if a band appears at 710bp, then the sample to be tested is Magnaporthe oryzae wheat special strain, or the sample to be tested contains Magnaporthe oryzae wheat special strain; if no band appears at 710bp after amplification, then the sample to be tested does not contain Magnaporthe oryzae wheat special strain, or the sample to be tested is not Magnaporthe oryzae wheat special strain; using the third primer pair, if a band appears at 1297bp, then the sample to be tested is Magnaporthe oryzae wheat special strain, or the sample to be tested contains Magnaporthe oryzae wheat special strain; if no band appears at 1297bp after amplification, then the sample to be tested does not contain Magnaporthe oryzae wheat special strain, or the sample to be tested is not Magnaporthe oryzae wheat special strain.

6. The method of claim 5, wherein: The reaction conditions of the PCR amplification are: (1) 95℃, 5min, (2) 94℃, 30s, 60℃, 30s, 72℃, 30s, repeat for 35 cycles, (3) 72℃, 7min.

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