Specific primer pair and identification method for identifying mating types of monokaryotic strains of stropharia rugoso-annulata

Abstract

The invention discloses a specific primer pair for identifying mating types of monokaryotic strains of stropharia rugoso-annulata and an identification method, and belongs to the field of molecular detection. The mating type identification primer based on the stropharia rugoso-annulata can be used for performing mating type identification on the monokaryotic strain of the stropharia rugoso-annulata, and the identification method is simple, economic and efficient; by adopting the detection method provided by the invention, according to the identification result of the mating type of the monokaryotic strain, the assembled hybrid combination can be successfully hybridized, and the locking combination is detected, which shows that the detection accuracy of identifying the mating type of the stropharia rugosoannulata by the primer is high; by utilizing the mating type site specific primer combination, A1 or A2, B1 or B2 can be specifically detected, the variety of mating types can be clearly distinguished and judged, and the specificity is relatively high.

Description

Technical Field

[0001] This invention relates to the field of molecular detection, specifically to a specific primer pair combination and identification method for identifying the mating type of a monokaryotic strain of *Agaricus bisporus*. Background Technology

[0002] Giant king mushroom ( Stropharia rugosoannulata *Stropharia macrocarpa* is an important fungus with edible, medicinal, and ecological value. Due to its rich nutrition and unique flavor, it has become a significant economic species in the edible fungi industry. In the production of *Stropharia macrocarpa*, hybridization breeding is a key method for cultivating new varieties. *Stropharia macrocarpa* is a tetrapolar heterothallic fungus, and its mating type is controlled by two mating type factors, A and B. These factors follow the laws of segregation and independent assortment during meiosis, producing four different mating types. Therefore, obtaining *Stropharia macrocarpa* monokaryotes and accurately identifying their mating types is an important foundation for conducting hybridization breeding.

[0003] Currently, hybridization breeding of *Stropharia macrocarpa* mainly involves multiple rounds of pairing and hybridization of monokaryotic strains, with microscopic examination of clamp connections to determine successful hybridization. Monokaryotic cells of *Stropharia macrocarpa* are typically obtained through methods such as protoplast uninucleation or basidiospore isolation. However, using monokaryotic strains with unknown mating types for pairing tests is cumbersome, time-consuming, and inefficient.

[0004] With the development of genome sequencing technology, the mating type gene sequences of various edible fungi have been deciphered. Studies have shown that there are a large number of single nucleotide polymorphisms (SNPs) in the A and B mating type loci regions. Based on these loci, specific primers can be designed to achieve accurate identification of mating types. For example, invention patent CN120310957A discloses a molecular marker for identifying the mating type of *Agaricus bisporus* and its application. Using the molecular marker provided by this invention, mating type classification can be identified by molecular detection methods in the breeding of *Agaricus bisporus*, shortening the breeding process and accelerating the selection of superior germplasm. In addition, invention patent CN115838821A discloses a three-primer composition and identification method for identifying the mating type of *Agaricus bisporus* W192 single-spore strain. Compared with conventional methods for identifying the mating type of single-spore strains of *Agaricus bisporus*, this invention has the advantages of shorter time consumption, higher accuracy, simpler operation, lower cost, no need for enzyme digestion, and no need for fruiting. The method of this invention can rapidly identify heterokine or homokine sterile strains and their mating types in single-spore strains of Agaricus bisporus W192, which can greatly improve the efficiency of Agaricus bisporus hybridization breeding.

[0005] The above is currently the most accurate method for identifying mating types in edible fungi, laying the foundation for guiding molecular breeding of *Agaricus bisporus*. Summary of the Invention

[0006] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a specific primer pair combination, identification method and application for identifying mating types of monokaryotic strains of Agaricus macrocarpa.

[0007] The specific technical solution of the present invention is as follows: This invention provides specific primer pairs for identifying the mating type of monokaryotic strains of *Agaricus bisporus*. The specific primer pairs include primer pairs specifically amplifying the A1 mating type site, the A2 mating type site, the B1 mating type site, and the B2 mating type site of *Agaricus bisporus*. The primer pair specifically amplifying the A1 mating type site of *Pleurotus ostreatus* includes the following primers: A1-F: 5'-CGGCTCTACATCAACTACTC-3', A1-R: 5'-GTTCACGTCGTCTATGCATC-3'; The primer pair specifically amplifying the A2 mating type site of *Pleurotus ostreatus* includes the following primers: A2-F: 5'-GGCTGTCGAAAGTAATGGAG-3', A2-R: 5'-CTCTTGCCTCAAAACTTGG-3'; The primer pair specifically amplifying the B1 mating type site of *Pleurotus ostreatus* includes the following primers: B1-F: 5'-GACTAACCTCTGCCATGATG-3', B1-R: 5'-GTTCTTTTTGGCCTTCCG-3'; The primer pair specifically amplifying the B2 mating type site of *Pleurotus ostreatus* includes the following primers: B2-F: 5'-GCCTTCATCGCGAATTTGTG-3', B2-R: 5'-CACCAGAAGAGCATCATAGC-3'.

[0008] The present invention also provides a product for identifying the mating type of a monokaryotic strain of *Agaricus bisporus*, comprising the above-mentioned specific primer pair.

[0009] The present invention also provides the application of the specific primer pair or the reagent or kit in mating types of Pleurotus ostreatus monokaryotic strains.

[0010] This invention also provides a method for identifying the mating type of a monokaryotic strain of *Agaricus macrocarpa*, using the specific primer pair or the reagent or kit described above for molecular identification of the *Agaricus macrocarpa* monokaryotic strain.

[0011] The identification method includes the following steps: (1) Extract genomic DNA from the monokaryotic strain of *Agaricus macrocarpa* to be tested; (2) The genomic DNA was amplified by PCR using the specific primer pairs to obtain the amplification products; (3) The mating type of the giant puffball mushroom monokaryotic strain was analyzed and identified by amplifying the mating type sites corresponding to the fragment length.

[0012] Further, in step (2), the amplification conditions are as follows: PCR amplification system: 25 μL Green Taq Mix, 2 μL 10μM upstream primer, 2 μL 10μM downstream primer, 1 μL genomic DNA, and sterile ultrapure water to 50 μL.

[0013] PCR amplification conditions: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 45 s, 33 cycles; 72℃ extension for 5 min; 4℃ storage.

[0014] Further, in step (3), the mating type of the tested *Agaricus bisporus* monokaryotic strain is determined based on the PCR amplification results, wherein, If primers A1-F and A1-R amplify a DNA fragment of 998 bp in length, it can be determined that the tested *Agaricus bisporus* monokaryotic strain has an A1 mating type site. If primers A2-F and A2-R amplify a DNA fragment of 1096 bp in length, it can be determined that the tested *Agaricus bisporus* monokaryotic strain has the A2 mating type site. If primers B1-F and B1-R amplify a DNA fragment of 868 bp in length, it can be determined that the tested *Agaricus bisporus* monokaryotic strain has a B1 mating type site. If primers B2-F and B2-R amplify a DNA fragment of 714 bp, it can be determined that the tested *Agaricus bisporus* monokaryotic strain has the B2 mating type site.

[0015] If a DNA fragment of the appropriate length is not obtained, it can be determined that the tested *Agaricus macrocarpa* monokaryotic strain does not possess the corresponding mating type site.

[0016] Furthermore, in step (3), the mating type of the *Agaricus bisporus* monokaryotic strain to be tested is the mating type site combination corresponding to the length of the amplified fragment of the specific primer pair.

[0017] This invention also provides the application of the specific primer pairs, the reagents or kits, or the identification methods in the breeding and / or identification of *Agaricus bisporus* species.

[0018] The beneficial effects of this invention are: (1) Using the mating type identification primers based on Pleurotus ostreatus of the present invention, mating type identification of Pleurotus ostreatus monokaryotic strains can be performed. The identification method is simple, economical and efficient. (2) Using the detection method of the present invention, based on the results of the mating type identification of mononuclear strains, all the hybrid combinations can be successfully hybridized and clamp connections are detected, indicating that the primers of this application have high accuracy in identifying mating types of Pleurotus ostreatus. (3) Using the mating type site specific primer combination of the present invention, A1 or A2, B1 or B2 can be specifically detected, and the types of mating types can be clearly distinguished and judged, which has strong specificity. Attached Figure Description

[0019] Figure 1 The mating type genes of the A1, A2, B1, and B2 strains of *Stropharia macrocarpa* were identified, and the amino acid sequences of the mating type genes of *Stropharia macrocarpa* A1, A2, B1, and B2 were compared with the amino acid sequences of the mating type sites of *Lentinula edodes*.

[0020] Figure 2 Amplification results of mating types A and B in the binucleate strain of *Agaricus bisporus*, where A1: amplification results of primers A1-F and A1-R; A2: amplification results of primers A2-F and A2-R; B1: amplification results of primers B1-F and B1-R; B2: amplification results of primers B2-F and B2-R; M: standard DNA molecular weight (5000 bp, 3000 bp, 2000 bp, 1000 bp, 750 bp, and 500 bp).

[0021] Figure 3 PCR identification of mating types of monokaryotic strains of *Agaricus bisporus*, where A1: amplification results of primers A1-F and A1-R; A2: amplification results of primers A2-F and A2-R; B1: amplification results of primers B1-F and B1-R; B2: amplification results of primers B2-F and B2-R; M: standard DNA molecular weight (5000 bp, 3000 bp, 2000 bp, 1000 bp, 750 bp, and 500 bp).

[0022] Figure 4 Hybrid combination of monokaryotic strains of *Agaricus bisporus*. Detailed Implementation

[0023] The present invention will be further explained below with reference to specific embodiments.

[0024] Example 1: Design of mating-specific primers for Pleurotus ostreatus A1, B1, A2, and B2. Using BLASTn, amino acid sequences from the mating type A site HD1 region (GenBank: HQ201372) of *Lentinula edodes* strain SR18 (National Genome Science Data Center, Accession No.: GWHHCOY00000000.1) and the mating type B site LEBup1 region (GenBank: JX297330) of *Lentinula edodes* strain L26P2 were compared. Highly conserved amino acid sequences were found in *Lentinula edodes* strains A1 (Hap1_0G08305), A2 (Hap2_0G09718), B1 (Hap1_0G00090), and B2 (Hap2_0G06454). Figure 1 The A1 mating type gene is 2058 bp in length, and its nucleotide sequence is shown in SEQ ID NO.1; the A2 mating type gene is 1988 bp in length, and its nucleotide sequence is shown in SEQ ID NO.2; the B1 mating type gene is 1490 bp in length, and its nucleotide sequence is shown in SEQ ID NO.3; the B2 mating type gene is 1777 bp in length, and its nucleotide sequence is shown in SEQ ID NO.4.

[0025] Based on the above mating type gene nucleotide sequence, a primer combination was designed to specifically amplify the mating type of *Pleurotus ostreatus*: Primer pair for specific amplification of the A1 mating type site of *Pleurotus ostreatus*: A1-F: 5'-CGGCTCTACATCAACTACTC-3', A1-R: 5'-GTTCACGTCGTCTATGCATC-3'; It can specifically amplify a 998 bp DNA fragment from the A1 mating type site of *Pleurotus ostreatus*. Primer pair for specific amplification of the A2 mating type site of *Pleurotus ostreatus*: A2-F: 5'-GGCTGTCGAAAGTAATGGAG-3', A2-R: 5'-CTCTTGCCTCAAAACTTGG-3'; It can specifically amplify a 1096 bp DNA fragment from the A2 mating site of *Stropharia macrocarpa*. Primer pair for specific amplification of the B1 mating type site of *Pleurotus ostreatus*: B1-F: 5'-GACTAACCTCTGCCATGATG-3', B1-R: 5'-GTTCTTTTTGGCCTTCCG-3'; It can specifically amplify an 868 bp DNA fragment of the B1 mating type site possessed by *Pleurotus ostreatus*. Primer pair for specific amplification of the B2 mating type site of *Pleurotus ostreatus*: B2-F: 5'-GCCTTCATCGCGAATTTGTG-3', B2-R: 5'-CACCAGAAGAGCATCATAGC-3'; It can specifically amplify a 714 bp DNA fragment of the B2 mating type site of *Pleurotus ostreatus*.

[0026] The genomic DNA of *Agaricus bisporus* strains was detected by PCR using four pairs of primers: A1-F and A1-R, A2-F and A2-R, B1-F and B1-R, and B2-F and B2-R. PCR amplification was performed using GreenTaq Mix premixed reagent (catalog number: P131) from Novizan Biotechnology Co., Ltd.

[0027] PCR amplification system: 25 μL Green Taq Mix, 2 μL 10 μM upstream primer, 2 μL 10 μM downstream primer, 1 μL genomic DNA, add sterile ultrapure water to 50 μL.

[0028] PCR amplification conditions: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 45 s, 33 cycles; 72℃ extension for 5 min; 4℃ storage.

[0029] Electrophoresis: PCR products were electrophoresed on a 1% agarose gel at 135 V for 20 min, and photographed using a gel imaging system.

[0030] A DNA fragment of 998 bp was obtained by amplification using A1-F and A1-R; a DNA fragment of 1096 bp was obtained by amplification using A2-F and A2-R; a DNA fragment of 868 bp was obtained by amplification using B1-F and B1-R; and a DNA fragment of 714 bp was obtained by amplification using B2-F and B2-R. Figure 2 ).

[0031] Genomic DNA from unidentified mating type monokaryotic strains SR18-4, A3, D2, C3, and F8 isolated from the binuclear strain SR18 (a Zhejiang Province certified variety, approved by the Zhejiang Provincial Crop Variety Approval Committee in April 2023 (Approval No.: Zherenjun 2023003)) of *Pleurotus ostreatus* was analyzed by PCR. SR18-4 amplified using A1-F and A1-R amplification yielded a 998 bp DNA fragment, but A2-F and A2-R amplification did not yield a 1096 bp DNA fragment, therefore SR18-4 possesses the A1 mating type locus. SR18-4 amplified using B1-F and B1-R amplification yielded an 868 bp DNA fragment, but B2-F and B2-R amplification did not yield a 714 bp DNA fragment, therefore SR18-4 possesses the B1 mating type locus. Therefore, the mating type of SR18-4 can be determined to be A1B1. Similarly, based on the amplified DNA bands, the mating type of A3 is A2B2, the mating type of D2 is A1B2, the mating type of C3 is A2B1, and the mating type of F8 is A2B1. Figure 3 ).

[0032] Example 2: Hybridization verification of different mating type monokaryotic hyphae identified using mating type-specific primers from *Agaricus bisporus*. As described above, PCR testing showed that the mating type of SR18-4 was A1B1, the mating type of A3 was A2B2, the mating type of D2 was A1B2, the mating type of C3 was A2B1, and the mating type of F8 was A2B1.

[0033] Five known mating types of *Agaricus bisporus* monokaryotic mycelia (SR18-4, A3, D2, C3, and F8), identified by PCR, were paired and inoculated for confrontation culture. Mycelia at the interface were then observed under a microscope. The results showed that numerous clamp connections were observed in the pairings of SR18-4 and A3, D2 and C3, and D2 and F8, demonstrating affinity hybridization between the paired monokaryotic strains, consistent with the PCR results of mating type detection. Figure 4 ).

[0034] The above embodiments are only used to understand the technical solutions of this application and do not limit the scope of protection of this application.

Claims

1. A specific primer pair for identifying the mating type of a monokaryotic strain of *Agaricus bisporus*, characterized in that, The specific primer pairs include primer pairs specifically amplifying the A1 mating type site of *Pleurotus ostreatus*, primer pairs specifically amplifying the A2 mating type site of *Pleurotus ostreatus*, primer pairs specifically amplifying the B1 mating type site of *Pleurotus ostreatus*, and primer pairs specifically amplifying the B2 mating type site of *Pleurotus ostreatus*, wherein... The primer pair specifically amplifying the A1 mating type site of *Pleurotus ostreatus* includes the following primers: A1-F: 5'-CGGCTCTACATCAACTACTC-3', A1-R: 5'-GTTCACGTCGTCTATGCATC-3'; The primer pair specifically amplifying the A2 mating type site of *Pleurotus ostreatus* includes the following primers: A2-F: 5'-GGCTGTCGAAAGTAATGGAG-3', A2-R: 5'-CTCTTGCCTCAAAACTTGG-3'; The primer pair specifically amplifying the B1 mating type site of *Pleurotus ostreatus* includes the following primers: B1-F: 5'-GACTAACCTCTGCCATGATG-3', B1-R: 5'-GTTCTTTTTGGCCTTCCG-3'; The primer pair specifically amplifying the B2 mating type site of *Pleurotus ostreatus* includes the following primers: B2-F: 5'-GCCTTCATCGCGAATTTGTG-3', B2-R: 5'-CACCAGAAGAGCATCATAGC-3'.

2. A product for identifying the mating type of a monokaryotic strain of *Agaricus bisporus*, characterized in that, Includes the specific primer pair described in claim 1.

3. The use of the specific primer pair of claim 1 or the reagent or kit of claim 2 in the mating type of Pleurotus ostreatus monokaryotic strain.

4. A method for identifying the mating type of a monokaryotic strain of *Agaricus macrocarpa*, characterized in that, Molecular identification of *Agaricus macrocarpa* monokaryotic strains was performed using the specific primer pair described in claim 1 or the reagent or kit described in claim 2.

5. The identification method as described in claim 4, characterized in that, Includes the following steps: (1) Extract genomic DNA from the monokaryotic strain of *Agaricus macrocarpa* to be tested; (2) The genomic DNA was amplified by PCR using the specific primer pair described in claim 1 to obtain the amplification products; (3) The mating type of the giant puffball mushroom monokaryotic strain was analyzed and identified by amplifying the mating type sites corresponding to the fragment length.

6. The identification method as described in claim 5, characterized in that, In step (2), the amplification conditions are as follows: PCR amplification system: 25 μL Green Taq Mix, 2 μL 10 μM upstream primer, 2 μL 10 μM downstream primer, 1 μL genomic DNA, and sterile ultrapure water to 50 μL; PCR amplification conditions: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 45 s, 33 cycles; 72℃ extension for 5 min; store at 4℃.

7. The identification method as described in claim 5, characterized in that, In step (3), if primers A1-F and A1-R amplify a DNA fragment of 998 bp in length, it can be determined that the tested *Agaricus bisporus* monokaryotic strain has an A1 mating type site. If primers A2-F and A2-R amplify a DNA fragment of 1096 bp in length, it can be determined that the tested *Agaricus bisporus* monokaryotic strain has the A2 mating type site. If primers B1-F and B1-R amplify a DNA fragment of 868 bp in length, it can be determined that the tested *Agaricus bisporus* monokaryotic strain has a B1 mating type site. If primers B2-F and B2-R amplify a DNA fragment of 714 bp, it can be determined that the tested *Agaricus bisporus* monokaryotic strain has the B2 mating type site.

8. The identification method as described in claim 5, characterized in that, In step (3), the mating type of the *Agaricus bisporus* monokaryotic strain to be tested is the mating type site combination corresponding to the length of the amplified fragment of the specific primer pair.

9. The application of the specific primer pair of claim 1, the reagent or kit of claim 2, or the identification method of any one of claims 4-8 in the breeding and / or identification of Pleurotus ostreatus.

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