A SNP molecular marker for detecting the mating type gene MAT1 of rice blast fungus and its application

Abstract

This invention relates to the fields of plant protection and molecular biology, specifically to an SNP molecular marker for detecting the mating type gene MAT1 of *Oryza sativa* and its application. The polymorphic site of the SNP molecular marker of this invention is located at Chr7.1156809, and the polymorphism of the SNP molecular marker is either T or C. More specifically, when the polymorphic base is T, the mating type of the *Oryza sativa* strain is determined to be MAT1-1; when the polymorphic base is C, the mating type of the *Oryza sativa* strain is determined to be MAT1-2. Detection of the SNP molecular marker of this invention enables high-throughput detection in 96,384, and 1536-well plates, suitable for large-scale, high-throughput detection of the *Oryza sativa* mating type gene MAT1. This invention eliminates the need for cumbersome procedures such as enzyme digestion, electrophoresis, and sequencing during the detection process, reducing PCR product aerosol contamination and the use of toxic substances such as EB.

Description

Technical Field

[0001] This invention relates to the fields of plant protection and molecular biology, specifically to an SNP molecular marker for detecting the mating type gene MAT1 of rice blast fungus and its application. Background Technology

[0002] Sexual reproduction is a common mode of reproduction in fungi. It refers to the process by which a pair of compatible male and female gametes combine to produce sexual spores, thus forming a new individual. Fungal sexual reproduction includes two types: homothallic mating and heterothallic mating. Homothallic mating is self-fertile, while heterothallic mating is infertile and requires the fusion of individuals with different mating types to complete sexual reproduction. The sexual reproduction and mating compatibility of fungi are mainly controlled by mating type genes. Homothallic fungi typically possess only one mating type gene locus; while most heterothallic fungi possess a pair of highly heterologous mating type gene loci. In heterothallic ascomycetes, the highly heterologous mating type gene loci are MAT1-1 and MAT1-2. Therefore, the mating type of heterothallic mating can be determined by detecting mating type genes.

[0003] The asexual form of the rice blast fungus is *Pyricularia grisea*, belonging to the Deuteromycetes; the sexual form is *Magnaporthe grisea*, also belonging to the Ascomycetes. Rice blast, caused by *Pyricularia grisea*, is a global disease affecting rice crops, resulting in yield losses of 11%–30% annually worldwide. Besides rice, *Pyricularia grisea* can also infect important crops such as barley, wheat, and millet, as well as weeds such as *Eriocaulon buergerianum*, *Digitaria sanguinalis*, and *Barnyardgrass*. It is well known that sexual reproduction is the primary factor in pathogen genetic variation in many fungi. Although the importance of sexual reproduction in *Pyricularia grisea* is often masked by the high frequency of asexual reproduction, and sexual reproduction has not yet been observed in nature, increasing indirect evidence suggests that sexual reproduction may have played a significant role in the population evolution of *Pyricularia grisea*. Therefore, mating types are an important aspect of population genetics research on *Pyricularia grisea*. Understanding the population characteristics formed by the mating types of rice blast fungus during geographical differentiation and phylogenetic evolution will help to grasp the inherent laws of the population's phylogenetic evolution, genetic variation, and epidemic occurrence more comprehensively and accurately, so as to lay a theoretical foundation for rice disease resistance breeding and variety layout.

[0004] Currently, the conventional molecular markers for detecting the MAT1 mating type gene of *Blastomyces oryzae*, as published in existing literature, require agarose gel electrophoresis after PCR amplification. The entire detection process is cumbersome and costly. The nucleic acid dyes and other reagents used are harmful to the environment and human health, and the automation level is low, resulting in low throughput. Furthermore, non-specific amplification sometimes occurs, leading to inaccurate results and significantly limiting detection efficiency. Therefore, there is an urgent need to establish a rapid, simple, efficient, and environmentally friendly method for detecting the mating type gene of *Blastomyces oryzae*. Summary of the Invention

[0005] The purpose of this invention is to provide a method for detecting the MAT1 mating type gene of rice blast fungus, suitable for large-scale, high-throughput testing.

[0006] In a first aspect, the present invention provides an SNP molecular marker, wherein the SNP molecular marker is K_MAT1, and the polymorphism of K_MAT1 is T or C. The polymorphic site of the molecular marker K_MAT1 is located at Chr7.1156809 (reference genome: Pyricularia oryzae strain LpKY97; Sequence ID: CP050926.1).

[0007] More specifically, if the polymorphic site base is T, the mating type of the rice blast strain is determined to be MAT1-1; if the polymorphic site base is C, the mating type of the rice blast strain is determined to be MAT1-2.

[0008] Secondly, the present invention provides a primer combination for detecting the molecular marker K_MAT1, wherein the primer sequences contain specific primers:

[0009] MAT1-1P: 5'-TGGTTGGCCGCGTTGATT-3' (as shown in SEQ ID NO.1);

[0010] MAT1-2P: 5'-CTGGTTGGCCGCGTTGATC-3' (as shown in SEQ ID NO.2);

[0011] Universal primers:

[0012] MAT1P: 5'-ATGACTGTGAGGACGCTGTG-3' (as shown in SEQ ID NO.3).

[0013] Preferably, the specific primers MAT1-1P and MAT1-2P are respectively linked to different fluorescent adapter sequences. According to some embodiments of the present invention, the fluorescent adapter sequences are FAM and HEX adapter sequences. Further, the FAM and HEX adapter sequences are respectively linked to the 5' ends of the two specific primers.

[0014] The specific MAT1-1P, after adding the adapter sequence, is as shown in SEQ ID NO.4: gaaggtgaccaagttcatgctTGGTTGGCCGCGTTGATT;

[0015] The specific MAT1-2P, after adding the adapter sequence, is as shown in SEQ ID NO.5: gaaggtcggagtcaacggattCTGGTTGGCCGCGTTGATC.

[0016] Based on the understanding of those skilled in the art, this invention seeks protection for the application of the above-described primer combination in the preparation of a mating type detection kit for rice blast fungus.

[0017] Thirdly, the present invention provides a kit for detecting the mating type of rice blast fungus, the kit containing primer combinations with sequences as shown in SEQ ID NO.1-3 or SEQ ID NO.3-5.

[0018] The present invention also claims protection for the use of the above-described SNP molecular markers, primer combinations, or kits in the detection of the mating type gene MAT1 of rice blast fungus.

[0019] Fourthly, the present invention provides a method for detecting the mating type gene MAT1 of rice blast fungus, comprising:

[0020] S1. Extract genomic DNA from the mycelium of the rice blast fungus to be identified;

[0021] S2. Using the genomic DNA extracted in step S1 as a template, PCR amplification was performed using primer combinations with sequences as shown in SEQ ID NO.3-5;

[0022] S3. If a fluorescent signal corresponding to the fluorescent adapter sequence in SEQ ID NO.4 is detected, the polymorphic site base of Chr7.1156809 is T, and the mating type of the rice blast strain to be identified is determined to be MAT1-1; if a fluorescent signal corresponding to the fluorescent adapter sequence in SEQ ID NO.5 is detected, the polymorphic site base of Chr7.1156809 is C, and the mating type of the strain is determined to be MAT1-2.

[0023] The method for detecting the mating type gene MAT1 of rice blast fungus provided by the present invention includes step S2, which involves adding genomic DNA of rice blast fungus mycelia to be identified to a microplate, drying it, adding KASP reaction mixture, and performing PCR amplification using primer combinations with sequences as shown in SEQ ID NO. 3-5.

[0024] The beneficial effects of this invention are as follows:

[0025] This invention is the first to discover a SNP molecular marker associated with the detection of the MAT1 mating type gene of rice blast fungus. This invention also provides a rapid method for detecting the MAT1 mating type gene of rice blast fungus based on KASP technology.

[0026] The method provided by this invention determines the mating type of the rice blast fungus gene MAT1 by detecting the polymorphism of SNP molecular markers. It can achieve high-throughput detection in 96,384, and 1536-well plates, and is suitable for large-scale, high-throughput detection of the rice blast fungus mating type gene MAT1. This invention eliminates the need for cumbersome procedures such as enzyme digestion, electrophoresis, and sequencing during the detection process, reducing PCR product aerosol contamination and the use of toxic substances such as EB. Attached Figure Description

[0027] Figure 1 This is a genotyping diagram of the MAT1 gene detected using conventional molecular markers and gel electrophoresis in Example 2 of the present invention.

[0028] Figure 2 This is the typing diagram of the SNP molecular marker K_MAT1 detected using KASP technology in Example 2 of the present invention.

[0029] Figure 3 The genotyping diagram of the SNP molecular marker K2_MAT1 was detected using KASP technology in Comparative Example 1 of this invention. Detailed Implementation

[0030] To illustrate the technical content, objectives, and effects of this invention in detail, the following description is provided in conjunction with embodiments and accompanying drawings. The embodiments described in the following exemplary embodiments are not intended to limit the scope of this invention. Those skilled in the art can make various modifications and substitutions to this invention without departing from its spirit and intent. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0031] Example 1 Primer Design

[0032] Based on literature information, SNP mining was performed on the MAT1 gene locus sequence on chromosome 7 of the *Pyricularia oryzae* genome. DNAMAN was used to compare and analyze the sequences of six samples with known MAT1 genotypes. Numerous differentially expressed sites were found within the MAT1 gene. Based on the conservation of upstream and downstream loci, and whether the locus variations matched the MAT1 gene, four candidate SNPs or Indels for marker design were selected. For each of the four candidate sites, 1-2 sets of KASP primers were designed using Primer5 software for amplification testing, resulting in seven sets of KASP candidate primers. Genomic DNA from 22 *Pyricularia oryzae* strains was amplified, fluorescence detected, and analyzed using these seven sets of KASP primers. Based on the genotyping effect of the markers, the optimal marker set K_MAT1 was selected (Table 1). This marker is located at sequence 1156809 (reference genome: *Pyricularia oryzae* strain LpKY97; Sequence). The SNP site with a T / C base in ID:CP050926.1 is named K_MAT1.

[0033] Primers were synthesized by Invitrogen. Each set of primers contained three primers, with two specific primers having FAM and HEX fluorescent linker sequences attached to their 5' ends, respectively.

[0034] A marker designed based on the KASP reaction principle and single-base difference of materials can be used for high-throughput detection of the MAT1 gene of *Blastoma oryzae*. The marker consists of three primers: two specific primers with fluorescent adapter sequences specific to the KASP reaction reagent from LGC attached to their 5' ends, and one universal primer. Specific information is shown in Table 1. If a fluorescent signal is detected corresponding to the fluorescent adapter sequence attached to primer MAT1-1P, and the SNP site detected by K_MAT1 is T, the strain is determined to be of mating type MAT1-1. If a fluorescent signal is detected corresponding to the fluorescent adapter sequence attached to primer MAT1-2P, and the SNP site detected by K_MAT1 is C, the strain is determined to be of mating type MAT1-2.

[0035] Table 1 K_MAT1 tag information

[0036]

[0037]

[0038] Example 2: Detection of KASP reaction with SNP molecular markers

[0039] 1. Primers designed based on the molecular markers described in this invention are used to detect the mating type gene MAT1 of rice blast fungus in high throughput using the KASP reaction, and the above primer combination is adopted.

[0040] 2. Genomic DNA was extracted from the mycelia of rice blast fungus using the lysis-precipitation method.

[0041] 3. KASP Reaction Test

[0042] KASP Reaction Assay: The KASP reaction assay was performed on the LGC SNPline genotyping platform. 20 ng of DNA sample was added to a microplate, dried, and then KASP reaction mixture was added; the reaction system is shown in Table 2. PCR amplification was performed in a water bath thermal cycler; the Touchdown PCR reaction conditions are shown in Table 3. After the reaction, the fluorescence data of the KASP reaction products were read using a Pherastar scanner; the fluorescence scan results were automatically converted into images. The LGC SNPline genotyping platform and its accompanying reagents and consumables used in this invention were purchased from LGC Ltd., UK.

[0043] Table 2 Reaction system for KASP detection

[0044] Components 2μl system 10μl system DNA template 5ng-50ng 25ng-250ng 2×Flu AS-PCR Mix 1 μl 5μl Specific allele primer - Fam (10 μM) 0.03μl 0.15μl Specific allele primer - Hex (10 μM) 0.03μl 0.15μl Universal primers (Com) 0.06μl 0.30μl water Add water to 2μl Add water to 10μl

[0045] Table 3 PCR reaction conditions

[0046]

[0047]

[0048] The KASP reaction was used to detect the MAT1 gene in 22 single-spore strains of *Bacillus oryzae* collected and isolated from Daweishan, Liuyang, using the marker K_MAT1. Simultaneously, whole-genome resequencing of these strains was performed, and detection and verification were conducted using conventional molecular markers (Tredway et al., 2003) and agarose gel electrophoresis. The results are shown in [Figure number missing]. Figure 1 , Figure 2 See Table 4. K_MAT1 marker detection results showed that two strains had a base T at the K_MAT1 test site, indicating a mating type of MAT1-1; 20 strains had a base C at the K_MAT1 test site, indicating a mating type of MAT1-2. The KASP detection results were completely consistent with the genome resequencing and conventional molecular marker detection results.

[0049] Table 4. K_MAT1 markers, conventional molecular markers, and resequencing results

[0050]

[0051]

[0052] Detection of KASP reaction with different primers in Comparative Example 1

[0053] In this comparative study, KASP primers were designed using the SNP sites listed in Table 5 (primer information is shown in Table 5), and the collected rice blast single-spore strains were tested for KASP reaction.

[0054] Table 5 shows the primer information tested in the comparative examples.

[0055]

[0056] The results showed that this set of KASP primers could not achieve correct genotyping of strain MAT1, and the genotyping effect was as follows: Figure 3 .

[0057] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention. sequence list <110> Yuan Longping High-Tech Agriculture Co., Ltd., Hunan Longping High-Tech Seed Industry Research Institute Co., Ltd., and Hunan Yahua Seed Industry Research Institute <120> A SNP molecular marker for detecting the mating type gene MAT1 of rice blast fungus and its application <130> KHP221115031.6 <160> 7 <170> SIPOSequenceListing 1.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <400> 1 tggttggccg cgttgatt 18 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <400> 2 ctggttggcc gcgttgatc 19 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 atgactgtga ggacgctgtg 20 <210> 4 <211> 39 <212> DNA <213> Artificial Sequence <400> 4 gaaggtgacc aagttcatgc ttggttggcc gcgttgatt 39 <210> 5 <211> 40 <212> DNA <213> Artificial Sequence <400> 5 gaaggtcgga gtcaacggat tctggttggc cgcgttgatc 40 <210> 6 <211> twenty three <212> DNA <213> Artificial Sequence <400> 6 actttcttca ccttttcctt ccg 23 <210> 7 <211> twenty three <212> DNA <213> Artificial Sequence <400> 7 actttcttca ccttttcctt ccc 23

Claims

1. A primer combination for detecting SNP molecular markers, characterized in that, The sequences of the primer combination are shown in SEQ ID NO.1-3; The polymorphic site of the SNP molecular marker is located at Chr7.1156809, reference genome: Pyricularia oryzaestrain LpKY97, Sequence ID: CP050926.1; the polymorphism of the SNP molecular marker is T or C.

2. The primer combination according to claim 1, characterized in that, The polymorphic site base is T, indicating that the mating type of the rice blast strain is MAT1-1; the polymorphic site base is C, indicating that the mating type of the rice blast strain is MAT1-2.

3. The primer combination according to claim 1 or 2, characterized in that, The primers shown in SEQ ID NO.1 and SEQ ID NO.2 are connected to different fluorescent adapter sequences.

4. The use of the primer combination according to any one of claims 1-3 in the preparation of a detection kit for the mating type gene MAT1 of rice blast fungus.

5. A kit for detecting the mating type gene MAT1 of rice blast fungus, characterized in that, The kit contains primer combinations with sequences as shown in SEQ ID NO.1-3 or SEQ ID NO.3-5.

6. The use of SNP molecular markers or primer combinations according to any one of claims 1-3 or the kit according to claim 5 in detecting the mating type gene MAT1 of rice blast fungus; The polymorphic site of the SNP molecular marker is located at Chr7.1156809, reference genome: Pyricularia oryzaestrain LpKY97, Sequence ID: CP050926.1; the polymorphism of the SNP molecular marker is T or C.

7. The application according to claim 6, characterized in that, The polymorphic site base is T, indicating that the mating type of the rice blast strain is MAT1-1; the polymorphic site base is C, indicating that the mating type of the rice blast strain is MAT1-2.

8. A method for detecting the mating type gene MAT1 of rice blast fungus, characterized in that, include: S1. Extract genomic DNA from the mycelium of the rice blast fungus to be identified; S2. Using the genomic DNA extracted in step S1 as a template, PCR amplification was performed using primer combinations with sequences as shown in SEQ ID NO.3-5; S3. If a fluorescent signal corresponding to the fluorescent adapter sequence in SEQ ID NO.4 is detected, the polymorphic site base of Chr7.1156809 is T, and the mating type of the rice blast strain to be identified is determined to be MAT1-1; if a fluorescent signal corresponding to the fluorescent adapter sequence in SEQ ID NO.5 is detected, the polymorphic site base of Chr7.1156809 is C, and the mating type of the strain is determined to be MAT1-2.

9. The method according to claim 8, characterized in that, Step S2 involves adding genomic DNA of the mycelium of the rice blast fungus to be identified into a microplate, drying it, adding KASP reaction mixture, and performing PCR amplification using primer combinations with sequences as shown in SEQ ID NO. 3-5.

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