A molecular marker for identifying the mating type of Agrocybe poplarii and its application

Abstract

The present invention belongs to the technical field of molecular breeding of edible fungi, and specifically discloses a molecular marker for identifying the mating type of Agrocybe poplar and its application. The mating type is A1, A2, B1 or B2, the molecular marker of mating type A1 is hda1; the molecular marker of mating type A2 is hda2; the molecular marker of mating type B1 is prb1; and the molecular marker of mating type B2 is prb2. The amino acid sequence of hda1 protein is shown in SEQ ID No.1; the amino acid sequence of hda2 protein is shown in SEQ ID No.5; the amino acid sequence of prb1 protein is shown in SEQ ID No.9; and the amino acid sequence of prb2 protein is shown in SEQ ID No.13. By using the molecular marker provided by the present invention, the mating type typing can be identified by molecular detection means in the breeding of Agrocybe poplar and its application is shortened, the breeding process is shortened, and the selection of excellent germplasm is accelerated.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular breeding of edible fungi, and particularly relates to a molecular marker for identifying the mating type of Agrocybe poplar and its application. Background Art

[0002] In the life history of heterothallic basidiomycetes, the formation of heterologous dikaryons is generally considered to be a prerequisite for fruiting body formation. The unlinked A and B mating sites in tetrapolar heterothallic basidiomycetes control the sexual reproduction process, including the formation of lock-like structures, nuclear migration, septum fusion, and nuclear fusion. Through the biological functions of mating type genes, the self-pollination behavior of basidiomycetes is inhibited, thereby maintaining the diversity of the intraspecific population, which is crucial for genetic breeding.

[0003] Willow Agrocybe Agrocybe salicacola Agaricus yangliutiantou (Agrocybe yangliutiantou) is an edible fungus with high nutritional and medicinal value. It has a typical quadripolar mating system (mating type A1A2B1B2) of basidiomycetes. Its monokaryotic strains contain two unlinked mating type loci, commonly referred to as the A and B mating type loci. Therefore, the sexual monokaryotic basidiospores formed during meiosis can produce four mating types: A1B1, A2B2, A1B2, and A2B1. Monokaryotic strains of different karyotypes must hybridize with monokaryotic strains of complementary karyotypes to produce fruiting bodies. For example, hybridization between A1B1 and A2B2, or A1B2 and A2B1, can both produce fruiting bodies; however, hybridization between A1B1 and A1B2 or A2B1 will not produce fruiting bodies.

[0004] Traditional mating type identification is done by hybridization, which relies on the presence or absence of a lock-like union after hybridization between two mononuclear bodies to determine whether the mating types are the same or different. This method is time-consuming, inefficient, and its accuracy is greatly affected by the subjective influence of the experimenter. With the development of genome sequencing technology, genome sequencing analysis has revealed that the mating type of Agrocybe salsa ( Agrocybe salicacola )'s mating type gene sequence and the use of molecular detection methods to identify mating type typing can shorten the breeding process and accelerate industrial development. Summary of the Invention

[0005] The main purpose of the present invention is to provide a mating type gene of a Pleurotus ostreatus strain and its application to solve the problem of mating type identification in the breeding process of Pleurotus ostreatus in the background art. Specifically, the present invention provides the following technical solutions:

[0006] The present invention provides a molecular marker for the mating type of Agrocybe poplarii, wherein the mating type is A1, A2, B1 or B2.

[0007] The molecular marker of the mating type A1 is hda1 protein or a gene encoding the protein;

[0008] And / or the molecular marker of the mating type A2 is hda2 protein or a gene encoding the protein;

[0009] and / or the molecular marker of the mating type B1 is prb1 protein or a gene encoding the protein;

[0010] and / or the molecular marker of the mating type B2 is the prb2 protein or a gene encoding the protein;

[0011] The amino acid sequence of the hda1 protein is shown in SEQ ID No. 1; the amino acid sequence of the hda2 protein is shown in SEQ ID No. 5; the amino acid sequence of the prb1 protein is shown in SEQ ID No. 9; and the amino acid sequence of the prb2 protein is shown in SEQ ID No. 13.

[0012] Furthermore, the present invention provides:

[0013] The nucleotide sequence of the gene encoding hda1 protein is shown in SEQ ID No. 2;

[0014] The nucleotide sequence of the gene encoding hda2 protein is shown in SEQ ID No. 6;

[0015] The nucleotide sequence of the gene encoding the prb1 protein is shown in SEQ ID No. 10;

[0016] The nucleotide sequence of the gene encoding the prb2 protein is shown in SEQ ID No.14.

[0017] As an embodiment, the present invention provides a product for detecting the molecular marker, wherein the product includes a reagent, a test paper or a kit, and the product detects whether the molecular marker exists.

[0018] As a more specific embodiment, the present invention provides a kit comprising primers for amplifying the molecular marker.

[0019] Furthermore, the present invention provides:

[0020] Primers for amplifying hda1 gene fragments are shown in SEQ ID No. 3 and / or 4;

[0021] Primers for amplifying the hda2 gene fragment are shown in SEQ ID No. 7 and / or 8;

[0022] Primers for amplifying the prb1 gene fragment are shown in SEQ ID No. 11 and / or 12;

[0023] The primers for amplifying the prb2 gene fragment are shown in SEQ ID No. 15 and / or 16.

[0024] Those skilled in the art know that, when the target gene is determined, different amplification primers can be designed according to professional primer design software to improve specificity.

[0025] Furthermore, the present invention provides the use of the molecular marker, the product or the kit in any of the following (1) to (6):

[0026] (1) Application in identifying or assisting in identifying the mating type of Agrocybe poplarii;

[0027] (2) Application in identifying or assisting in identifying the mating type of Agrocybe monokeratinosa;

[0028] (3) Application in identifying or assisting in identifying the mating type of the asporous mononuclear Agrocybe poplarii;

[0029] (4) Application of screening mating type A1B1, A2B2, A1B2 or A2B1 in mononuclear Agrocybe poplarii;

[0030] (5) Application of molecular marker-assisted selection in breeding of Agrocybe poplar;

[0031] (6) Application in identifying or assisting in identifying the relationship of Agrocybe poplarii.

[0032] The molecular markers provided by the present invention can be detected for their presence or absence at the protein level or the gene level. In specific applications of breeding of Agrocybe chinensis, by identifying the presence or absence of mating types, in some embodiments, on the one hand, based on the conservatism of the marker gene, the Agrocybe chinensis with the marker gene has a closer genetic relationship or the same genetic background; on the other hand, it can be used to distinguish between mononuclear and dinuclear Agrocybe chinensis, such as if all four mating types are present, it is dinuclear, and if only two are present, it is mononuclear; on the other hand, among the mononuclear Agrocybe chinensis, it can be further used to screen mononuclear Agrocybe chinensis with mating types of A1B1, A2B2, A1B2 or A2B1; on the other hand, it can screen mononuclear Agrocybe chinensis with special traits, such as the screening of mononuclear Agrocybe chinensis with a spore-free type provided in the embodiments of the present invention.

[0033] In one embodiment, the present invention provides a method for identifying the mating type of Agrocybe poplarii, comprising the following steps:

[0034] S1: Extract DNA from the sample to be tested;

[0035] S2: Using the DNA of the sample to be tested as a template, PCR amplification is performed using the primers in the kit to obtain an amplified product. If the amplified product contains a target gene fragment, the sample has the corresponding mating type.

[0036] In one embodiment, the present invention provides a method for identifying the mating type of Agrocybe poplar and willow. A sample to be tested is taken and the molecular marker of the Agrocybe poplar and willow mating type is detected. When the test result shows the presence of molecular marker protein, the sample has the corresponding mating type.

[0037] In one embodiment, a method for identifying the kinship of Agrocybe poplarii is provided, wherein a sample to be tested is taken, and the molecular marker is tested. Samples containing the molecular marker in the test results have a closer kinship.

[0038] In one embodiment, a method for screening mononuclear Agrocybe poplarii with a mating type of A1B1 or A2B2,

[0039] The following steps are involved:

[0040] S1: Extract DNA from the sample to be tested;

[0041] S2: Using the DNA of the sample to be tested as a template, PCR amplification is performed using the primers in the kit to obtain an amplified product;

[0042] S3: If the hda1 and prb1 gene fragments are present in the amplified product, and the hda2 and prb2 gene fragments are absent, the sample is judged to be an A1B1 mating type mononuclear Agrocybe poplarii;

[0043] S4: If the hda2 and prb2 gene fragments are present in the amplified product, and the hda1 and prb1 gene fragments are absent, the sample is judged to be an A2B2 mating type mononuclear Agrocybe poplarii.

[0044] The effects achieved by the present invention are:

[0045] Agrocybe poplar has a typical quadrupolar mating system of basidiomycetes; the present invention systematically provides marker genes of different mating types A1, A2, B1, and B2 of Agrocybe poplar strains through genome sequencing, and simultaneously designs primers to perform PCR amplification. By sequencing the PCR products, gene sequence fragments of different mating types of the strains are obtained, providing a more accurate and convenient method for the typing and identification of the mating types of Agrocybe poplar.

[0046] The present invention establishes a method for identifying the mating type of Agrocybe poplar and willow. In the breeding of Agrocybe poplar and willow, the mating type typing can be identified by molecular detection means. The mating type of the monokaryotic strain is identified for screening new varieties of Agrocybe poplar and willow aseptic strains, and candidate strains are provided, thereby shortening the breeding process and accelerating the selection of excellent germplasm.

[0047] The present invention establishes a method for identifying the mating type of Agrocybe poplar and willow, which can be used for identifying kinship or genetic background in a large population, or for identifying the kinship between the Agrocybe poplar and willow strain YAASM0711 and other Agrocybe poplar and willow strains specifically provided in the embodiments of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Figure 1 shows the mating type identification results of the F1 generation of Agrocybe yangliu. As shown in the figure, strains with mating type A1B1 are 8, 9, 12, 13, 14, and 20; strains with mating type A1B2 are 1, 2, 10, 15, and 18; strains with mating type A2B1 are 3, 4, 5, 6, and 19; and strains with mating type A2B2 are 7, 11, 16, and 17.

[0049] Figure 2 Morphological characteristics of the fruiting bodies of Agrocybe yangliu. (A) Fruiting body of a spore-bearing strain; (B) Fruiting body of an aspore-bearing strain; (C) The gills of a spore-bearing strain are brown, with brown spores attached; (D) The gills of an aspore-bearing strain are white, with no spores attached. DETAILED DESCRIPTION

[0050] The technical scheme of the present invention is described in detail below in conjunction with the accompanying drawings and Examples, but the present invention is not limited to the scope of the embodiments. Unless otherwise specified, the experiments and methods described in the examples were carried out basically according to conventional methods well known in the art and described in various references, and the reagents and raw materials used in the present invention were all commercially available. Where specific conditions are not specified in the examples, they were carried out according to conventional conditions or conditions recommended by the manufacturer. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0051] Example 1

[0052] The present invention screened out the mating type gene in the A1 mating type locus in the Agrocybe poplar strain through genome sequencing analysis. hda1, Mating-type genes in the A2 mating-type locus hda2 , mating type gene at the B1 mating type locus prb1 , the mating type gene at the B2 mating type locus prb2 .

[0053] Specifically, the mating type gene information of mating type A1, A2, B1, and B2 is as follows:

[0054] The amino acid sequence of hda1 protein is shown in SEQ ID No. 1; the nucleotide sequence of hda1 gene is shown in SEQ ID No. 2

[0055] The amino acid sequence of hda2 protein is shown in SEQ ID No. 5; the nucleotide sequence of hda2 gene is shown in SEQ ID No. 6

[0056] The amino acid sequence of the prb1 protein is shown in SEQ ID No. 9; the nucleotide sequence of the prb1 gene is shown in SEQ ID No. 10

[0057] The amino acid sequence of the prb2 protein is shown in SEQ ID No. 13; the nucleotide sequence of the prb2 gene is shown in SEQ ID No. 14.

[0058] Primer pairs were designed to amplify four mating-type gene fragments (Table 1). Primer pair A1F / A1R was used to amplify a 663-bp fragment of the hda1 gene; primer pair A2F / A2R was used to amplify a 988-bp fragment of the hda2 gene; primer pair B1F / B1R was used to amplify a 906-bp fragment of the prb1 mating-type gene; and primer pair B2F / B2R was used to amplify an 1140-bp fragment of the prb2 mating-type gene.

[0059] A 25 µL PCR reaction system was used, consisting of 12.5 µL of 2× Taq Plus Master Mix II, 1 µL of upstream primer (10 µM), 1 µL of downstream primer (10 µM), 1 µL of DNA template (approximately 100 ng), and 9.5 µL of ddH₂O. The PCR amplification protocol consisted of 35 cycles of initial denaturation at 95°C for 3 min, denaturation at 95°C for 30 s, annealing at 59°C for 20 s, and extension at 72°C for 1 min, followed by a final extension at 72°C for 10 min.

[0060] Table 1 Primer pairs for mating type gene amplification

[0061]

[0062] The spore-deficient monokaryotic strain T32 used in the present invention has the full name: Agrocybe truncatum ( Agrocybe salicacicola ) T32, preserved in Guangdong Provincial Microbial Culture Collection Center, with the preservation number GDMCC NO: 63850. For detailed information on this strain, please refer to: CN117384767A A spore-deficient willow Agrocybe and its application.

[0063] The monokaryotic strains T1, T130 and T32 have the same origin, which is the F1 generation monokaryotic strains isolated from the same wild-type Agrocybe poplarii strain YAASM0711.

[0064] In a specific breeding process for Agrocybe poplarii, laboratory-maintained, spore-deficient, monokaryotic strains of Agrocybe poplarii exhibited two mating types: monokaryotic strains T32 (A1B1) and T130 (A2B2). To identify new aspore-deficient strains of Agrocybe poplarii, we used the wild-type monokaryotic strain T1 (A2B2) of Agrocybe poplarii strain YAASM0711 and the spore-deficient monokaryotic strain T32 (A1B1) as parents. F1 generation monokaryotic populations were obtained, with mating types of A1B1, A2B2, A1B2, or A2B1. Mating type testing of the F1 monokaryotic strains using four pairs of mating type primers (Table 1) identified monokaryotic strains with mating types of A1B1 and A2B2.

[0065] Figure 1 The mating type identification results of some F1 generation strains are shown, among which the mating type is A1B1, including strains 8, 9, 12, 13, 14 and 20, the mating type is A1B2, including strains 1, 2, 10, 15 and 18, the mating type is A2B1, including strains 3, 4, 5, 6 and 19, and the mating type is A2B2, including strains 7, 11, 16 and 17. By detecting the molecular markers provided by the present invention, the mononuclear Agrocybe poplar willow strains can be quickly and accurately typed, and strains of the target mating types A1B1 and A2B2 can be obtained.

[0066] The F1 generation monokaryotic strain with mating type A1B1 was hybridized with the asporous monokaryotic strain T130 (A2B2) to produce mushrooms; the F1 generation monokaryotic strain with mating type A2B2 was hybridized with the asporous monokaryotic strain T32 (A1B1) to produce mushrooms, and two types of mushrooms, spores and aspors, were obtained ( Figure 2 The fruiting experiment ultimately yielded 67 spore-bearing strains and 96 aspore-bearing strains. The aspore-bearing strains with better mushroom types were selected for mycelial differentiation and secondary fruiting experiments, yielding multiple stable aspore-bearing binucleate strains, providing candidate strains for obtaining new aspore-bearing varieties of Agrocybe truncatula in the future.

[0067] The poplar and willow Agrocybe has a typical quadripolar mating system of basidiomycetes; the present invention systematically provides marker genes of different mating types A1, A2, B1, and B2 of the poplar and willow Agrocybe strain through genome sequencing, and simultaneously designs primers for PCR amplification, wherein primers A1F and A1R amplify the mating type gene hda1 in the A1 mating type site, primers A2F and A2R amplify the mating type gene hda2 in the A2 mating type site, primers B1F and B1R amplify the mating type gene prb1 in the B1 mating type site, and primer pair B2F / B2R amplifies the mating type gene prb2 in the B2 mating type site. By sequencing the PCR products, sequence fragments of different mating type genes of the strain are obtained, providing a more accurate and convenient method for the typing and identification of the mating type of the poplar and willow Agrocybe.

[0068] The foregoing description shows and describes several preferred embodiments of the present invention. However, as previously stated, it should be understood that the present invention is not limited to the forms disclosed herein and should not be construed as excluding other embodiments. Rather, the present invention is applicable to various other combinations and modifications and can be modified within the scope of the present invention through the above teachings or the skills or knowledge in the relevant field. Modifications and variations made by those skilled in the art that do not depart from the spirit and scope of the present invention are intended to be protected by the appended claims.

Claims

1. A molecular marker for the mating type of Agrocybe poplarii, wherein the mating type is A1, A2, B1 and B2, characterized in that: The molecular marker of the mating type A1 is hda1 protein or a gene encoding the protein; The molecular marker of the mating type A2 is hda2 protein or a gene encoding the protein; The molecular marker of the mating type B1 is the prb1 protein or a gene encoding the protein; The molecular marker of the mating type B2 is the prb2 protein or a gene encoding the protein; The amino acid sequence of the hdal protein is shown in SEQ ID No. 1; the amino acid sequence of the hdal protein is shown in SEQ ID No. 5; the amino acid sequence of the prb1 protein is shown in SEQ ID No. 9; and the amino acid sequence of the prb2 protein is shown in SEQ ID No.

13.

2. The molecular marker according to claim 1, characterized in that The nucleotide sequence of the gene encoding hda1 protein is shown in SEQ ID No. 2; The nucleotide sequence of the gene encoding hda2 protein is shown in SEQ ID No. 6; The nucleotide sequence of the gene encoding the prb1 protein is shown in SEQ ID No. 10; The nucleotide sequence of the gene encoding the prb2 protein is shown in SEQ ID No.

14.

3. Detection of the molecularly labeled product according to claim 1 or 2, characterized in that: The product includes a reagent, a test paper or a kit, and the product detects whether the molecular marker exists.

4. A kit, characterized in that The kit contains primers for amplifying the molecular marker according to claim 2.

5. The kit according to claim 4, characterized in that The primers for amplifying the hda1 gene fragment are shown in SEQ ID Nos. 3 and 4; The primers for amplifying the hda2 gene fragment are shown in SEQ ID Nos. 7 and 8; The primers for amplifying the prb1 gene fragment are shown in SEQ ID Nos. 11 and 12; The primers for amplifying the prb2 gene fragment are shown in SEQ ID No. 15 and 16.

6. Use of the molecular marker according to claim 1 or 2, the product according to claim 3, or the kit according to claim 4 or 5 in any of the following (1) to (2): (1) Application in identifying the mating type of Agrocybe poplarii; (2) Screening the mating type of A1B1, A2B2, A1B2 or A2B1 for application in mononuclear willow Agaricus truncatus.

7. A method for identifying the mating type of Agrocybe poplarii, characterized in that: The following steps are involved: S1: Extract DNA from the sample to be tested; S2: Using the DNA of the sample to be tested as a template, PCR amplification is performed using the primers in the kit according to claim 5 to obtain an amplified product. If the amplified product contains a target gene fragment, the sample has the corresponding mating type.

8. A method for identifying the mating type of Agrocybe poplarii, characterized in that: Take the sample to be tested and detect the mating type molecular marker of the Agrocybe poplar and willow oxalis as claimed in claim 1. When the test result shows the presence of the molecular marker protein, the sample has the corresponding mating type.

9. A method for screening mononuclear Agrocybe poplarii with mating type A1B1 or A2B2, characterized in that: The following steps are involved: S1: Extract DNA from the sample to be tested; S2: Using the DNA of the sample to be tested as a template, performing PCR amplification using the primers in the kit according to claim 5 to obtain an amplified product; S3: If the hda1 and prb1 gene fragments are present in the amplified product, and the hda2 and prb2 gene fragments are absent, the sample is judged to be an A1B1 mating type mononuclear Agrocybe poplarii; S4: If the hda2 and prb2 gene fragments are present in the amplified product, and the hda1 and prb1 gene fragments are absent, the sample is judged to be an A2B2 mating type mononuclear Agrocybe poplarii.

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