Primer and method for rapidly identifying mating types of phlebopus portentosus homonuclear strains

Abstract

The invention provides a primer and a method for identifying the mating type of a homonuclear strain of phlebopus portentosus, relates to the technical field of biology, and can rapidly and accurately identify the mating type of the homonuclear strain of phlebopus portentosus so as to improve the working efficiency of crossbreeding. The primers comprise one or more pairs of a first primer pair, a second primer pair, a third primer pair, a fourth primer pair, a fifth primer pair, a sixth primer pair, a seventh primer pair, an eighth primer pair, a ninth primer pair and a tenth primer pair, wherein 10 pairs of primers respectively have sequences shown as SEQ ID NO.1 to SEQ ID NO.20 in a sequence table. The 10 pairs of primers or a part of the primers are mainly used for performing amplification sequencing on genome specific areas of a to-be-detected homonuclear strain and a parent heterokaryotic strain of the homonuclear strain, and the mating type of the homonuclear strain is judged by utilizing the SNP molecular marker in the primers.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular, to a primer and method for rapidly identifying the mating type of homokaryotic strains of Phlebopus portentosus. Background Art

[0002] Phlebopus portentosus is currently the only bolete species that has achieved mass production worldwide and has been factory-cultivated. The artificially cultivated Phlebopus portentosus has a smooth taste and unique flavor, and is deeply loved by consumers. Some recent studies have also found that Phlebopus portentosus is rich in various beneficial components and has various activities such as antioxidant, anti-cancer, anti-viral, immune-enhancing, and neuroprotective activities, indicating that Phlebopus portentosus has great economic value and development potential.

[0003] Since the cultivation period of Phlebopus portentosus is relatively short, the related work on strain breeding still needs to be vigorously developed to support the increasing market demand and the development of artificial cultivation technology. Among them, cross-breeding is one of the important contents in the breeding work of Phlebopus portentosus. The prerequisite for cross-breeding is the identification of the mating type of homokaryotic strains. The traditional method is to carry out pairwise confrontation cultivation and microscopic examination of clamp connections. This method has a large workload, a long time-consuming, and is prone to errors when the observation is incomplete, reducing the efficiency of cross-breeding. Therefore, developing a method for rapidly identifying the mating type of homokaryotic strains of Phlebopus portentosus has become an important means to improve its breeding efficiency. Summary of the Invention

[0004] In view of this, the embodiments of the present invention provide a primer and method for identifying the mating type of homokaryotic strains of Phlebopus portentosus, which can rapidly and accurately identify the mating type of homokaryotic strains of Phlebopus portentosus, thereby improving the efficiency of cross-breeding work.

[0005] To achieve the above object, the present invention mainly provides the following technical solutions:

[0006] On the one hand, the present invention provides a primer for rapidly identifying the mating type of homokaryotic strains of Phlebopus portentosus, characterized in that it includes: one or more pairs selected from the first primer pair, the second primer pair, the third primer pair, the fourth primer pair, the fifth primer pair, the sixth primer pair, the seventh primer pair, the eighth primer pair, the ninth primer pair, and the tenth primer pair;

[0007] The first primer pair has the sequences shown in SEQ ID NO.1 and SEQ ID NO.2 in the sequence listing;

[0008] The second primer pair has the sequences shown in SEQ ID NO.3 and SEQ ID NO.4 in the sequence listing;

[0009] The third primer pair has the sequences shown in SEQ ID NO.5 and SEQ ID NO.6 in the sequence listing;

[0010] The fourth primer pair has the sequences shown in SEQ ID NO.7 and SEQ ID NO.8 in the sequence listing;

[0011] The fifth primer pair has the sequences shown in SEQ ID NO.9 and SEQ ID NO.10 in the sequence listing;

[0012] The sixth primer pair has the sequences shown in SEQ ID NO.11 and SEQ ID NO.12 in the sequence listing;

[0013] The seventh primer pair has the sequences shown in SEQ ID NO.13 and SEQ ID NO.14 in the sequence listing;

[0014] The eighth primer pair has the sequences shown in SEQ ID NO.15 and SEQ ID NO.16 in the sequence listing

[0015] The ninth primer pair has the sequences shown in SEQ ID NO.17 and SEQ ID NO.18 in the sequence listing

[0016] The tenth primer pair has the sequences shown in SEQ ID NO.19 and SEQ ID NO.20 in the sequence listing.

[0017] On the other hand, the present invention provides a kit for rapidly identifying the mating type of homokaryotic strains of Pulveroboletus ravenelii, and the kit includes any one or more pairs of the above primers.

[0018] On the other hand, the present invention provides a test strip for rapidly identifying the mating type of homokaryotic strains of Pulveroboletus ravenelii, and the test strip includes any one or more pairs of the above primers.

[0019] On the other hand, the present invention provides a method for rapidly identifying the mating type of homokaryotic strains of Pulveroboletus ravenelii, specifically including the following steps:

[0020] S1: Collect heterokaryotic strains of Pulveroboletus ravenelii, and obtain corresponding homokaryotic strains through the germination of sexual spores;

[0021] S2: Collect the mycelia of the heterokaryotic strains and homokaryotic strains in step S1, and extract genomic DNA from the mycelia.

[0022] S3: Use the 10 pairs of primers in claim 1 to perform PCR amplification and sequencing on the mycelial DNA of the heterokaryotic strains.

[0023] S4: According to the results of PCR amplification and sequencing of the heterokaryotic strain in step S3, select at least one pair of primer pairs with obvious SNPs from the first to fifth primer pairs as the molecular marker primers for identifying the mating type gene A locus, and select at least one pair of primer pairs with obvious SNPs from the sixth to tenth primer pairs as the molecular marker primers for identifying the mating type gene B locus.

[0024] S5: Use the primer pairs selected from the first to fifth primer pairs in step S4 to perform PCR amplification and sequencing for identifying the mating type gene A locus on the homokaryotic strain to be tested, use the primer pairs selected from the sixth to tenth primer pairs in step S4 to perform PCR amplification and sequencing for identifying the mating type gene B locus on the homokaryotic strain to be tested, and perform mating type gene typing on the homokaryotic strain to be tested according to the SNP distribution in the sequencing results.

[0025] On the other hand, in the method for quickly identifying the mating type of homokaryotic strains of Phlebopus portentosus in the present invention, the genomic DNA extraction from the mycelium is carried out by using a high-efficiency plant genomic DNA extraction kit to extract genomic DNA from the mycelium.

[0026] On the other hand, in the method for quickly identifying the mating type of homokaryotic strains of Phlebopus portentosus in the present invention, the genomic DNA extraction from the mycelium is carried out by the CTAB method; the specific steps for extracting genomic DNA by the CTAB method are as follows:

[0027] T1: Add quartz sand and 250 μL of 2×CTAB lysis buffer to the mycelium of the strain to be tested, grind it to a homogeneous paste, and then add 600 μL of 2×CTAB lysis buffer, and invert and mix well.

[0028] T2: Place it in a water bath at 65 °C for 1 hour, and turn it over every 10 minutes.

[0029] T3: Add 300 μL each of phenol and chloroform, invert it multiple times, and centrifuge at 13000 rpm for 10 minutes; then collect the upper aqueous phase.

[0030] T4: Repeat step S3 multiple times until there is no precipitate at the two-phase interface.

[0031] T5: Add an equal volume of chloroform for extraction once, and collect the upper aqueous phase.

[0032] T6: Add 0.5–1 times the volume of pre-cooled isopropanol or 2 times the volume of absolute ethanol, centrifuge at 13000 rpm for 10 minutes at 4 °C, and discard the supernatant.

[0033] T7: Add 1 mL of pre-cooled 75% ethanol, invert it multiple times, centrifuge at 13000 rpm for 10 minutes, discard the supernatant, and repeat twice.

[0034] T8: Dry it at room temperature or in a vacuum system;

[0035] T9: Add 50 μL of sterile water and dissolve it at room temperature, then store it at -20 °C for later use.

[0036] On the other hand, in the method for rapidly identifying the mating type of homokaryotic strains of Boletellus obscurebrownioides of the present invention, the reaction system for PCR amplification includes: 45 μl of PCR Mix, 2 μl of forward primer, 2 μl of reverse primer, and 1 μl of DNA template.

[0037] On the other hand, in the method for rapidly identifying the mating type of homokaryotic strains of Boletellus obscurebrownioides of the present invention, the reaction system for PCR amplification is: 1 μL of a 50 ng / μL template, 5 μL of 25 mM MgCl2, 1 μL of 10 mM dNTPs, 1 μL of each 6 μM primer, 0.2 μL of 5 U / μL DNA Taq enzyme, 5 μL of 10×PCR reaction buffer, and 35.8 μL of deionized water.

[0038] On the other hand, in the method for rapidly identifying the mating type of homokaryotic strains of Boletellus obscurebrownioides of the present invention, the reaction program for PCR amplification is: pre-denaturation at 94 - 98 °C for 2 min, followed by denaturation at 94 - 98 °C for 10 - 30 s, primer annealing at the primer annealing temperature for 10 - 30 s, extension at 72 °C for 10 - 30 s, for a total of 35 cycles, and finally extension at 72 °C for 5 - 10 min.

[0039] Among them, the annealing temperatures of each primer from SEQ ID NO.1 to SEQ ID NO.20 are 58.0 °C, 57.7 °C, 56.0 °C, 54.4 °C, 55.3 °C, 54.9 °C, 57.3 °C, 55.4 °C, 54.2 °C, 55.4 °C, 55.8 °C, 56.3 °C, 54.9 °C, 56.3 °C, 58.1 °C, 59.9 °C, 58.5 °C, 56.1 °C, 54.7 °C, 57.1 °C, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 UPGMA tree typing of the amplified sequences of the second primer pair from the HZ19110 homokaryotic strain

[0041] Figure 2 UPGMA tree typing of the amplified sequences of the seventh primer pair from the HZ19110 homokaryotic strain

[0042] Figure 3 UPGMA tree typing of the amplified sequences of the fifth primer pair from the HZ20029 homokaryotic strain

[0043] Figure 4 UPGMA tree typing of the amplified sequences of the tenth primer pair from the HZ20029 homokaryotic strain Detailed implementation manners

[0044] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

[0045] The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels without special instructions.

[0046] Example 1: Primer design

[0047] Based on genomic data, the mating type genes of Pulveroboletus ravenelii were annotated and studied in populations. It was found that the bata-Flanking gene closely linked to the mating type gene A was relatively conserved, but still contained a large number of SNP sites. Among the mating type genes B, STE3.2 was relatively conserved. Therefore, the bata-Flanking and STE3.2 gene sequences were extracted from the genomic data of Pulveroboletus ravenelii, and Primer Premier 5 was used for primer design. The selected primer sequences were aligned with the genomic sequences using the local blastn program to ensure that there was only one pairing site for the primer in the genome. Finally, 5 pairs of primers (the first to fifth primer pairs in the present invention) for amplifying the bata-Flanking gene region and 5 pairs of primers (the sixth to tenth primer pairs in the present invention) for the STE3.2 gene region were determined (Table 1).

[0048] Table 1. Primer sets for mating type identification of Pulveroboletus ravenelii

[0049]

[0050] Example 2: Mating type identification of homokaryotic strains produced from the heterokaryotic strain HZ19110

[0051] The test strains included: the heterokaryotic strain HZ19110 of Pulveroboletus ravenelii, which was isolated and purified from wild fruiting bodies collected from Nandaohe, Pu'er City, Yunnan Province in 2019, and 17 homokaryotic strains germinated from sexual spores produced from the heterokaryotic strain HZ19110.

[0052] Extraction of genomic DNA: Collect the mycelia of the test strains. Use a high-efficiency plant genomic DNA extraction kit ( Hi-Pure Plant Genomic DNAKit TSP102-200, Beijing Tsingke Biotechnology Co., Ltd.) to extract genomic DNA from the above mycelia.

[0053] In addition, genomic DNA extraction from the above mycelium can also be carried out by the CTAB method; the specific steps for genomic DNA extraction by the CTAB method are as follows:

[0054] T1: Add quartz sand and 250 μL of 2×CTAB lysis buffer to the mycelium of the strain to be tested, grind it to a homogeneous paste, and then add 600 μL of 2×CTAB lysis buffer, and invert and mix well;

[0055] T2: Place it in a water bath at 65 °C for 1 hour, and turn it over every 10 minutes;

[0056] T3: Add 300 μL each of phenol and chloroform, invert it multiple times, and centrifuge at 13000 rpm for 10 minutes; then collect the upper aqueous phase;

[0057] T4: Repeat step S3 multiple times until there is no precipitate at the two-phase interface;

[0058] T5: Extract once with an equal volume of chloroform, and collect the upper aqueous phase;

[0059] T6: Add 0.5–1 times the volume of pre-cooled isopropanol or 2 times the volume of absolute ethanol, centrifuge at 13000 rpm for 10 minutes at 4 °C, and discard the supernatant;

[0060] T7: Add 1 mL of pre-cooled 75% ethanol, invert it multiple times, centrifuge at 13000 rpm for 10 minutes, discard the supernatant, and repeat twice;

[0061] T8: Place it at room temperature or in a vacuum system for drying;

[0062] T9: Add 50 μL of sterile water and dissolve it at room temperature, and store it at -20 °C for later use.

[0063] PCR amplification and sequencing of the heterokaryotic mycelium: Using the 10 pairs of primers of the present invention, PCR amplification and sequencing were performed on the mycelium DNA of the HZ19110 heterokaryotic strain. The 50 μl PCR amplification reaction system includes: GoldMix Ver.2( T6Super PCR Mix Ver.2 (1.1×) TSE102 Beijing Tsingke Biotechnology Co., Ltd.) 45 μl, forward primer 2 μl, reverse primer 2 μl, DNA template 1 μl.

[0064] In addition, the PCR amplification reaction system can also be: 1 μL of 50 ng / μL template, 5 μL of 25 mM MgCl2, 1 μL of 10 mM dNTPs, 1 μL each of 6 μM primers, 0.2 μL of 5 U / μL DNA Taq enzyme, 5 μL of 10×PCR reaction buffer, and 35.8 μL of deionized water.

[0065] The PCR amplification reaction procedure was as follows: pre-denaturation at 94 - 98°C for 2 min, followed by denaturation at 94 - 98°C for 10 - 30 s, primer annealing at 10 - 30 s, extension at 72°C for 10 - 30 s, for a total of 35 cycles, and finally extension at 72°C for 5 - 10 min.

[0066] Among them, for each primer from SEQ ID NO.1 to SEQ ID NO.20, the annealing temperatures were 58.0°C, 57.7°C, 56.0°C, 54.4°C, 55.3°C, 54.9°C, 57.3°C, 55.4°C, 54.2°C, 55.4°C, 55.8°C, 56.3°C, 54.9°C, 56.3°C, 58.1°C, 59.9°C, 58.5°C, 56.1°C, 54.7°C, 57.1°C, respectively.

[0067] The obtained product was sent to Beijing Tsingke Biotechnology Co., Ltd. (Kunming Branch) for sequencing.

[0068] PCR amplification and sequencing of homokaryotic mycelia: SNP sites of the sequencing peak map of the heterokaryotic strain were examined. Finally, the second primer pair was selected to genotype its A site, and the seventh primer pair was used to genotype its B site. PCR amplification and sequencing were performed on the homokaryotic mycelia of the mating type to be identified. The specific method was the same as the PCR amplification and sequencing method of the heterokaryotic mycelia in this example.

[0069] Identification of the mating type of homokaryotic strains: After manually examining the sequencing peak map of the homokaryotic mycelia obtained in this example, the sequencing sequences amplified by the second primer pair and the seventh primer pair were respectively imported into the Clustal_X software for alignment, and the miscellaneous peak regions at both ends were trimmed. Then, the aligned file was imported into the MEGA software for UPGMA tree construction (attached Figure 1 、 2 ), and the following genotyping results (Table 2) were obtained from the figure:

[0070] Table 2 Mating type genotyping results of each homokaryotic strain produced from HZ19110

[0071] Mating type Strain A1B1 HZ19110-60, HZ19110-82, HZ19110-43, HZ19110-41, HZ19110-21 A2B2 HZ19110-11, HZ19110-40, HZ19110-49, HZ19110-90 A1B2 HZ19110-18, HZ19110-3, HZ19110-30, HZ19110-47 A2B1 HZ19110-35, HZ19110-63, HZ19110-26, HZ19110-10

[0072] The homokaryotic strains produced from HZ19110 in this example were paired pairwise for confrontation culture experiments, and clamp connections were examined under the microscope to verify the accuracy of the present invention. The results are shown in Table 3 (for ease of tabulation, the strain numbers in the following table are only represented by the last digits). Thus, it can be seen that the method used in the present invention can accurately and quickly identify the mating type of homokaryotic strains of Boletus obscure-venosus.

[0073] Table 3 Confrontation culture results of homokaryotic strains produced from HZ19110

[0074]

[0075]

[0076] Note: "+" indicates that clamp connections can be observed in the hyphae after confrontation culture; "-" indicates that there are no clamp connections after confrontation culture; " / " indicates that no pairing is required or it has been recorded in other positions in the table.

[0077] Example 3: Identification of mating types of homokaryotic strains derived from the heterokaryotic strain HZ20029

[0078] The tested strains include: the heterokaryotic strain HZ20029 of Phlebopus portentosus, which was isolated and purified from wild fruit bodies collected from Dongfeng Farm, Jinghong City, Yunnan Province in 2020, and 13 homokaryotic strains germinated from sexual spores derived from the heterokaryotic strain HZ20029.

[0079] Extraction of genomic DNA: The specific method is the same as that for DNA extraction in Example 1.

[0080] PCR amplification and sequencing of heterokaryotic mycelia: The PCR amplification system and amplification reaction procedure are the same as those in Example 1.

[0081] PCR amplification and sequencing of homokaryotic mycelia: Check the SNP sites of the sequencing peak map of the heterokaryotic strain. Finally, select the fifth primer pair to type its A site and the tenth primer pair to type its B site. Perform PCR amplification and sequencing on the homokaryotic mycelia whose mating types are to be identified. The specific method is the same as that for PCR amplification and sequencing of heterokaryotic mycelia in this example.

[0082] Identification of mating types of homokaryotic strains: After manually checking the sequencing peak map of the homokaryotic mycelia obtained in this example, import the sequencing sequences amplified by the fifth primer pair and the tenth primer pair into the Clustal_X software for alignment, and cut off the miscellaneous peak regions at both ends. Then import the alignment file into the MEGA software to construct a UPGMA tree (attached Figure 3 、 4 ), and the following typing results (Table 4) can be obtained from the figure:

[0083] Table 4 Typing results of mating types of each homokaryotic strain derived from HZ20029

[0084]

[0085] Pair the homokaryotic strains derived from HZ20029 in this example in pairs, conduct confrontation culture tests, and examine the clamp connections under the microscope to verify the accuracy of the present invention. The results are shown in Table 5 (for the convenience of tabulation, the strain numbers in the following tables are only represented by the last digits). It can be seen from this that the method used in the present invention can accurately and quickly identify the mating types of homokaryotic strains of Phlebopus portentosus.

[0086] Table 5 Results of confrontation culture of isonuclear strains from HZ19110

[0087]

[0088]

[0089] Note: "+" indicates that clamp connections can be observed in the mycelium after confrontation culture; "-" indicates that there are no clamp connections after confrontation culture; " / " indicates that pairing is not required or has been recorded in other positions in the table.

[0090] The above are only the preferred embodiments of the present invention, and do not impose any formal limitations on the present invention. Any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A primer for rapidly identifying the mating type of a homokaryon strain of Boletus edulis, characterized in that: include: one or more pairs of the first primer pair, the second primer pair, the third primer pair, the fourth primer pair, the fifth primer pair, the sixth primer pair, the seventh primer pair, the eighth primer pair, the ninth primer pair, and the tenth primer pair; The first primer pair has the sequences shown in SEQ ID NO.1 and SEQ ID NO.2 in the sequence list; The second primer pair has the sequences shown as SEQ ID NO.3 and SEQ ID NO.4 in the sequence listing; The third primer pair has the sequences shown in SEQ ID NO.5 and SEQ ID NO.6 in the sequence listing; The fourth primer pair has the sequences shown in SEQ ID NO.7 and SEQ ID NO.8 in the sequence listing; The fifth primer pair has the sequences shown in SEQ ID NO.9 and SEQ ID NO.10 in the sequence listing; The sixth primer pair has the sequences shown in SEQ ID NO.11 and SEQ ID NO.12 in the sequence listing; The seventh primer pair has the sequences shown in SEQ ID NO.13 and SEQ ID NO.14 in the sequence listing; The eighth primer pair has the sequences shown in SEQ ID NO.15 and SEQ ID NO.16 in the sequence list. The ninth primer pair has the sequences shown in SEQ ID NO. 17 and SEQ ID NO. 18 in the sequence list. The tenth primer pair has the sequences shown as SEQ ID NO. 19 and SEQ ID NO. 20 in the sequence listing.

2. A kit for rapidly identifying the mating type of a homokaryon strain of Boletus edulis, characterized in that: The kit comprises any one or more pairs of primers as claimed in claim 1.

3. A test strip for rapid identification of the mating type of a homokaryon strain of Boletus edulis, characterized in that: The test strip comprises any one or more pairs of primers as described in claim 1.

4. A method for rapidly identifying the mating type of a homokaryon strain of Boletus edulis, characterized in that: The following steps are involved: S1: Collect heterokaryotic strains of Boletus edulis and obtain the corresponding homokaryotic strains through sexual spore germination; S2: Collect the mycelia of the heterokaryotic strain and the homokaryotic strain in step S1, and extract genomic DNA from the mycelia. S3: Using the 10 primer pairs in claim 1, PCR amplification and sequencing of the mycelial DNA of the heterokaryotic strain. S4: Based on the results of PCR amplification and sequencing of the heterokaryotic strain in step S3, select at least one primer pair with obvious SNP from the first to the fifth primer pairs as molecular marker primers for identifying the mating type gene A site, and select at least one primer pair with obvious SNP from the sixth to the tenth primer pairs as molecular marker primers for identifying the mating type gene B site. S5: Use the primer pairs selected from the first to the fifth primer pairs in step S4 to perform PCR amplification and mating type gene A site identification sequencing on the homokaryotic strain to be tested, use the primer pairs selected from the sixth to the tenth primer pairs in step S4 to perform PCR amplification and mating type gene B site identification sequencing on the homokaryotic strain to be tested, and perform mating type genotyping on the homokaryotic strain to be tested according to the SNP distribution in the sequencing results.

5. The method for rapid identification of the mating type of a homokaryon strain of Boletus edulis according to claim 4, characterized in that: The method of extracting genomic DNA from mycelium is to extract genomic DNA from mycelium using a high-efficiency plant genomic DNA extraction kit.

6. The method for rapid identification of the mating type of a homokaryon strain of Boletus edulis according to claim 4, characterized in that: The genomic DNA extraction from mycelium is performed by the CTAB method; the specific steps of the CTAB method for extracting genomic DNA are as follows: T1: Add quartz sand and 250 μL of 2×CTAB lysis solution to the mycelium of the strain to be tested, grind to a homogenous state, then add 600 μL of 2×CTAB lysis solution, and mix by inversion; T2: Place in a 65°C water bath for 1 hour and flip every 10 minutes; T3: Add 300 μL of phenol and 300 μL of chloroform, invert several times, and centrifuge at 13,000 rpm for 10 minutes; then collect the upper aqueous phase; T4: Repeat step S3 multiple times until there is no precipitation on the interface between the two phases; T5: Add an equal volume of chloroform to extract once and collect the upper aqueous phase; T6: Add 0.5–1 volume of pre-cooled isopropanol or 2 volumes of anhydrous ethanol, centrifuge at 13,000 rpm for 10 minutes at 4°C, and discard the supernatant; T7: Add 1 mL of pre-cooled 75% ethanol, invert several times, centrifuge at 13,000 rpm for 10 minutes, discard the supernatant, and repeat twice; T8: Dry at room temperature or in a vacuum system; T9: Add 50 μL sterile water to dissolve at room temperature and store at -20°C for later use.

7. The method for rapidly identifying the mating type of a homokaryon strain of Boletus edulis according to claim 4, characterized in that: The PCR amplification reaction system includes: 45 μl of PCR Mix, 2 μl of forward primer, 2 μl of reverse primer, and 1 μl of DNA template.

8. The method for rapid identification of the mating type of a homokaryon strain of Boletus edulis according to claim 4, characterized in that: The reaction system of the PCR amplification is: 1 μL of 50 ng / μL template, 5 μL of 25 mM MgCl2, 1 μL of 10 mM dNTPs, 1 μL of each 6 μM primer, 0.2 μL of 5 U / μL DNA Taq enzyme, 5 μL of 10× PCR reaction buffer, and 35.8 μL of deionized water.

9. The method for rapidly identifying the mating type of a homokaryon strain of Boletus edulis according to any one of claims 7-8, characterized in that: The reaction procedure of the PCR amplification is: pre-denaturation at 94-98°C for 2 minutes, followed by denaturation at 94-98°C for 10-30 seconds, primer annealing temperature for 10-30 seconds, extension at 72°C for 10-30 seconds, a total of 35 cycles, and finally extension at 72°C for 5-10 minutes. Among them, the annealing temperatures of each primer from SEQ ID NO.1 to SEQ ID NO.20 are 58.0℃, 57.7℃, 56.0℃, 54.4℃, 55.3℃, 54.9℃, 57.3℃, 55.4℃, 54.2℃, 55.4℃, 55.8℃, 56.3℃, 54.9℃, 56.3℃, 58.1℃, 59.9℃, 58.5℃, 56.1℃, 54.7℃, and 57.1℃, respectively.

Images