Method, specific primer pair and kit for rapidly identifying single-core and double-core edible mushrooms and application

Abstract

The invention belongs to the technical field of biology, and particularly relates to a method, a specific primer pair and a kit for rapidly identifying single-core and double-core edible mushrooms and application. According to the method for rapidly identifying the single-core and double-core edible mushrooms, specific primers meeting PCR requirements are designed according to conserved genes of the edible mushrooms to be detected, PCR amplification is conducted through the specific primers, obtained amplification products are sequenced, and the single-core and double-core edible mushrooms are judged by observing a sequencing peak map. The method is simple and rapid, the identification result is accurate and reliable, and the method can be used for screening hybrid seeds in the edible mushroom breeding process, and is particularly suitable for screening edible mushroom hybrid strains with unobvious locking combination. The specific primer pair provided by the invention is used for rapidly identifying single-core and double-core stemless ganoderma lucidum, is high in specificity and good in sensitivity, and can be used for a breeding process of a stemless ganoderma lucidum hybrid variety.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for rapidly identifying mononuclear and dinuclear edible fungi, a specific primer pair, a kit and applications. Background Art

[0002] During the sexual reproduction of edible fungi, only when a monokaryotic mycelium fuses with another compatible monokaryotic mycelium with a different nucleus to form a binucleate mycelium can hybridization be completed and offspring with new genetic characteristics be produced. Through monokaryotic and binucleate identification, it is possible to accurately determine whether the hyphae fusion has successfully formed a binucleate, thereby screening out effective hybrid combinations and avoiding the waste of time and resources caused by ineffective hybridization.

[0003] The existing technologies for identifying mononuclear and dinuclear edible fungi all have some limitations: 1. Microscope observation method: The morphological characteristics and cell structure of the hyphae are directly observed under an optical microscope to identify mononuclear and dinuclear. The dinuclear hyphae usually have a lock-like joint structure, while the mononuclear hyphae do not. Its limitations are: it requires professional personnel to operate and judge, which is highly subjective and requires high experience and skills of the operator; the microscopic examination process is time-consuming and inefficient, and it is difficult to quickly detect a large number of samples. 2. Fluorescence staining method: The hyphae are stained with fluorescent dyes, and the binuclear hyphae and mononuclear hyphae are identified by the difference in their binding ability or fluorescence response to the fluorescent dye. Its limitations are: the selection of fluorescent dyes and staining conditions need to be optimized, otherwise the staining effect and accuracy may be affected; a fluorescent microscope is required, and the equipment cost is high; the observation and analysis of the stained samples also requires professionals, and there is also a certain degree of subjectivity. 3. Physiological and biochemical methods, such as growth rate determination method: binuclear hyphae usually grow faster than mononuclear hyphae. The growth rate of the tested hyphae is measured by culturing the tested hyphae under the same culture conditions to indirectly judge the mononuclear and dinuclear. Its limitations are: the growth rate of hyphae is affected by many environmental factors, such as temperature, humidity, culture medium composition, etc., and the stability and repeatability of the experimental results may be poor; the growth rate differences of different species or strains may not be obvious, and it is difficult to accurately distinguish between mononuclear and binuclear hyphae. 4. Enzyme activity assay: binuclear hyphae and mononuclear hyphae may differ in the activity of certain enzymes, and the activity of specific enzymes is measured to identify mononuclear and binuclear hyphae. Its limitations are: the enzyme activity assay method is relatively complex and requires specific enzyme activity detection kits or instruments and equipment; enzyme activity is also affected by environmental factors and culture conditions, and the accuracy and reliability of the results need to be improved.

[0004] In recent years, molecular biological methods have been increasingly used in the identification of monokaryotic and dikaryotic hyphae: for example, PCR amplification technology is used to design specific primers for PCR amplification based on the differences in gene sequences between dikaryotic hyphae and monokaryotic hyphae, and the size, quantity or sequence analysis of the amplified products are used to determine monokaryotic and dikaryotic hyphae; DNA molecular marker technology, such as restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), simple sequence repeats (SSR), amplified fragment length polymorphism (AFLP), etc., detects polymorphisms at the DNA molecular level to identify monokaryotic and dikaryotic hyphae. Since monokaryotic hyphae hybridization can produce different hybrid offspring, the above PCR amplification technology has the problem of inaccurate judgment; DNA molecular marker technology has the defect that the detection and result judgment methods are relatively complicated. Summary of the invention

[0005] In view of the above technical problems, the present invention provides a method for quickly identifying mononuclear and dinuclear edible fungi, a specific primer pair, a kit and an application. The method provided in the present application determines dinuclear and mononuclear edible fungi by observing whether there are overlapping peaks in the sequencing peak graph. The method is simple, rapid, easy to operate, and the identification results are accurate and reliable. It can be used for the screening of hybrids in the process of edible fungi breeding.

[0006] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions: The first aspect of the present invention provides a method for rapidly identifying monokaryotic and dikaryotic edible fungi: specific primers that meet the requirements of PCR amplification are designed according to the conserved genes of the edible fungi to be tested, wherein the conserved genes are rRNA genes, glutamate synthase genes or ura3 genes; PCR amplification is performed using the specific primers using the genomic DNA of the edible fungi to be tested as a template, and the obtained amplification products are sequenced. If overlapping peaks appear in the sequencing peak graph, the mycelium of the edible fungi is determined to be dikaryotic mycelium, otherwise it is monokaryotic mycelium.

[0007] The present invention designs specific primers for rRNA gene, glutamate synthase gene or ura3 gene, and after PCR amplification, the mycelium can be judged to be mononuclear or dinuclear by observing the sequencing peak graph. Compared with the existing microscope observation method, fluorescent staining method, physiological and biochemical method and enzyme activity determination method, the method has low requirements on operators and equipment, is not affected by human subjective judgment, mycelium growth rate and environment, has high accuracy and sensitivity of identification results, and is easy to operate; compared with the molecular biology method, the method can accurately identify various hybrid offspring of mononuclear mycelium, and the detection and judgment process are simple and easy. When using the method for identification, it only needs to observe whether the sequencing peak graph has overlapping peaks to judge the mononuclear or dinuclear, and does not need to judge the result from the obvious degree of the electrophoresis band, so the sensitivity is higher.

[0008] The method can be used for rapid identification of hybrid varieties of edible fungi, and is helpful for rapid screening of effective hybrid combinations. Currently, there is no report on the identification of mononuclear and dinuclear edible fungi by the overlapping peak phenomenon in the sequencing peak graph. The present invention provides a simple and easy new idea for the rapid identification of mononuclear and dinuclear edible fungi.

[0009] Preferably, the edible fungi include Agrocybe aegerita, Ganoderma lucidum and Pleurotus ostreatus.

[0010] The second aspect of the present invention provides the application of the above method in screening hybrids during the edible fungus breeding process.

[0011] The third aspect of the present invention provides the use of the above method in identifying mono- and binucleate sessile Ganoderma lucidum, wherein the conserved gene is an rRNA gene, a glutamate synthase gene or a ura3 gene.

[0012] A fourth aspect of the present invention provides a method for rapidly identifying single-core and double-core sessile Ganoderma lucidum ( Ganoderma resinaceum ), consisting of primer ura3-F and primer ura3-R, or consisting of primer 18s rRNA-F and primer 28s rRNA-R, or consisting of primer GOGAT-F and primer GOGAT-R; The primer ura3-F is a single-stranded DNA molecule as shown in SEQ ID NO.1, or a DNA molecule having the same function as the single-stranded DNA molecule shown in SEQ ID NO.1 after one or more nucleotides are replaced and / or deleted and / or added; The primer ura3-R is a single-stranded DNA molecule as shown in SEQ ID NO.2, or a DNA molecule having the same function as the single-stranded DNA molecule as shown in SEQ ID NO.2 after one or more nucleotides are replaced and / or deleted and / or added; The primer 18s rRNA-F is a single-stranded DNA molecule as shown in SEQ ID NO.3, or a DNA molecule in which one or more nucleotides are substituted and / or deleted and / or added to the single-stranded DNA molecule as shown in SEQ ID NO.3 and which has the same function as the single-stranded DNA molecule as shown in SEQ ID NO.3; The primer 28s rRNA-R is a single-stranded DNA molecule as shown in SEQ ID NO.4, or a DNA molecule having the same function as the single-stranded DNA molecule as shown in SEQ ID NO.4 after one or more nucleotides are replaced and / or deleted and / or added; The primer GOGAT-F is a single-stranded DNA molecule as shown in SEQ ID NO.5, or a DNA molecule having the same function as the single-stranded DNA molecule as shown in SEQ ID NO.5 after one or more nucleotides are replaced and / or deleted and / or added; The primer GOGAT-R is a single-stranded DNA molecule as shown in SEQ ID NO.6, or a DNA molecule in which one or several nucleotides are substituted and / or deleted and / or added to the single-stranded DNA molecule as shown in SEQ ID NO.6 and which has the same function as the single-stranded DNA molecule as shown in SEQ ID NO.6.

[0013] The sequence of SEQ ID NO.1 is: 5´-TTGAACCAATTGACGGGTGT-3´; The sequence of SEQ ID NO.2 is: 5´-AGCGCTTTACTTGGCAGATT-3´; The sequence of SEQ ID NO.3 is: 5'-CTTGTCTCAAAGATTAAGCCATGCAT-3'; The sequence of SEQ ID NO.4 is: 5´-TCGGCAGGAACCAGCTACTA-3´; The sequence of SEQ ID NO.5 is: 5´-ATTATGGCGAATTTGAGGAGGA-3´; The sequence of SEQ ID NO.6 is: 5´-CAAAGACGGGAGGATAGAAGTATAAA-3´.

[0014] The specific primer pair consisting of primer ura3-F and primer ura3-R is a primer pair designed for PCR amplification of the ura3 gene of sessile Ganoderma lucidum, the specific primer pair consisting of primer 18s rRNA-F and primer 28s rRNA-R is a primer pair designed for PCR amplification of the rRNA gene of sessile Ganoderma lucidum, and the specific primer pair consisting of primer GOGAT-F and primer GOGAT-R is a primer pair designed for PCR amplification of the glutamate synthase gene of sessile Ganoderma lucidum. After PCR amplification using the above primer pair with sessile Ganoderma lucidum genomic DNA as a template, by observing the peak overlap in the sequencing peak graph of the amplified product, it can be quickly and accurately identified whether the mycelium of the sessile Ganoderma lucidum to be tested is binucleate or mononucleate.

[0015] Currently, there is no method for judging mononuclear and dinuclear sessile Ganoderma by observing the overlapping peak phenomenon in the sequencing peak graph. The present invention provides a new method for rapid judgment of mononuclear and dinuclear sessile Ganoderma and provides a specific primer pair.

[0016] Experimental verification has shown that the above-mentioned specific primer pairs have high specificity and sensitivity for the identification of mono- and di-nucleate sessile Ganoderma lucidum. The operation is simple and fast, and has low requirements on the operator's experience and skills, equipment conditions, mycelium growth conditions, environment, etc., and can be used in the breeding process of sessile Ganoderma lucidum hybrid varieties.

[0017] Preferably, the primer ura3-F is a single-stranded DNA molecule as shown in SEQ ID NO.1.

[0018] Preferably, the primer ura3-R is a single-stranded DNA molecule as shown in SEQ ID NO.2.

[0019] Preferably, the primer 18s rRNA-F is a single-stranded DNA molecule as shown in SEQ ID NO.3.

[0020] Preferably, the primer 28s rRNA-R is a single-stranded DNA molecule as shown in SEQ ID NO.4.

[0021] Preferably, the primer GOGAT-F is a single-stranded DNA molecule as shown in SEQ ID NO.5.

[0022] Preferably, the primer GOGAT-R is a single-stranded DNA molecule as shown in SEQ ID NO.6.

[0023] The fifth aspect of the present invention provides the use of the above-mentioned specific primer pair in the preparation of a kit for rapid identification of mono- and binucleate sessile Ganoderma lucidum.

[0024] The sixth aspect of the present invention provides a kit for rapid identification of mono- and binucleate sessile Ganoderma lucidum, the kit comprising the following reagents: a primer solution of the above-mentioned specific primer pair, DNA polymerase, dNTPs and a buffer.

[0025] Exemplarily, the kit includes the following reagents: a primer solution of the specific primer pair and 2×TOROBlue Flash KOD Dye Mix.

[0026] The seventh aspect of the present invention provides a method for rapidly identifying single-core and double-core sessile Ganoderma lucidum, the specific steps comprising: S1. Extracting genomic DNA from the sessile Ganoderma mycelium to be tested; S2. Using the genomic DNA extracted in S1 as a template, perform PCR amplification using the above-mentioned specific primer pair or kit; sequence the amplified product, and if overlapping peaks appear in the sequencing peak graph, determine that the sessile Ganoderma mycelium is a binucleate mycelium, otherwise it is a monokaryotic mycelium.

[0027] Preferably, the reaction system for PCR amplification is:

[0028] The reaction conditions of the PCR amplification are:

[0029] An eighth aspect of the present invention provides the use of the above-mentioned specific primer pair, kit or method in screening sessile Ganoderma lucidum hybrids during the sessile Ganoderma lucidum breeding process.

[0030] Compared with the prior art, the present invention has the following beneficial effects: The present invention determines whether the di-monokaryotic edible fungi have overlapping peaks by observing whether the sequencing peak graph has overlapping peaks. The invention has the advantages of being simple and rapid, and has low requirements on operators and equipment. The invention is not affected by human subjective judgment, mycelium growth rate and environment, and the identification result is accurate and reliable. The invention can be used for screening hybrids in the edible fungus breeding process, and is particularly suitable for screening hybrid strains of edible fungi with unclear lock-shaped union.

[0031] The specific primer pair provided by the present invention has strong specificity and high sensitivity. The primer pair can be used to quickly and accurately identify single-core and double-core sessile Ganoderma lucidum through PCR amplification and observation of sequencing peak graphs, providing a new method for the rapid judgment of single-core and double-core sessile Ganoderma lucidum, and can be used in the breeding process of sessile Ganoderma lucidum hybrid varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The dikaryotic strain with lock-like union in Example 8 of the present invention; Figure 2 It is a monokaryotic strain without lock-like union in Example 8 of the present invention; Figure 3 The peak diagram is obtained after sequencing the amplified products obtained by PCR amplification of different ribotype strains in Example 8 of the present invention using the specific primer pairs in Example 1; Figure 4 The peak graph of different karyotype strains obtained after sequencing the amplification products obtained by PCR amplification of different karyotype strains using the specific primer pairs in Example 2 in Example 8 of the present invention; Figure 5 The peak graph of different ribotype strains obtained after sequencing the amplified products obtained by PCR amplification of different ribotype strains in Example 8 of the present invention using the specific primer pairs in Example 3; Figure 6 The electrophoresis diagram of the amplified products obtained by PCR amplification of different ribotype strains in Example 8 of the present invention using the specific primer pairs in Example 1; Figure 7The electrophoresis diagram of the amplified products obtained by PCR amplification of different ribotype strains in Example 8 of the present invention using the specific primer pair of Example 2; Figure 8 This is an electrophoresis diagram of the amplified products obtained by PCR amplification of different ribotype strains in Example 8 of the present invention using the specific primer pairs in Example 3. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work fall within the scope of protection of the present invention.

[0034] At present, the identification of single-core and double-core edible fungi has high requirements on operators and equipment, is affected by human subjective judgment, mycelium growth rate and environment, and has inaccurate judgment results or cumbersome detection and judgment methods. It is not convenient to apply it to the breeding process of edible fungi hybrid varieties.

[0035] In view of the problems existing in the prior art, an embodiment of the present invention provides a method for quickly identifying monokaryotic and dikaryotic edible fungi: specific primers that meet the requirements of PCR amplification are designed according to the conserved genes of the edible fungi to be tested, and the conserved genes are rRNA genes, glutamate synthase genes or ura3 genes; the genomic DNA of the edible fungi to be tested is used as a template, PCR amplification is performed using the specific primers, and the obtained amplification products are sequenced. If overlapping peaks appear in the sequencing peak graph, the edible fungus mycelium is determined to be dikaryotic mycelium, otherwise it is monokaryotic mycelium.

[0036] The embodiment of the present invention also provides the application of the above method in identifying single-core and double-core sessile Ganoderma lucidum.

[0037] The embodiments of the present invention also provide a specific primer pair for rapidly identifying mono- and di-nucleate sessile Ganoderma lucidum, which is composed of primer ura3-F and primer ura3-R, or primer 18s rRNA-F and primer 28s rRNA-R, or primer GOGAT-F and primer GOGAT-R.

[0038] The embodiment of the present invention also provides a kit for rapid identification of mono- and binucleate sessile Ganoderma lucidum, comprising the following reagents: primer solution of the above-mentioned specific primer pair, DNA polymerase, dNTPs, and buffer.

[0039] The embodiment of the present invention also provides a method for rapidly identifying single-core and double-core sessile Ganoderma lucidum, the specific steps comprising: S1. Extracting genomic DNA from the sessile Ganoderma mycelium to be tested; S2. Using the genomic DNA extracted in S1 as a template, perform PCR amplification using the above-mentioned specific primer pair or kit; sequence the amplified product, and if overlapping peaks appear in the sequencing peak graph, determine that the sessile Ganoderma mycelium is a binucleate mycelium, otherwise it is a monokaryotic mycelium.

[0040] The following is further described with reference to specific embodiments.

[0041] The sources or components of each culture medium involved in the following examples are: PDB medium (potato dextrose liquid medium): purchased from Tianjin Guangfu Technology Development Co., Ltd. YMG protoplast regeneration medium: weigh 4 g Yeast Extract (Oxoid, UK), 10 g Malt Extract (Oxoid, UK), 4 g anhydrous glucose (Shanghai Sinopharm Chemical Reagent Co., Ltd.), 109.3 g mannitol, add appropriate amount of deionized water, stir thoroughly and adjust the volume to 1 L, and sterilize at 121℃ for 20 minutes.

[0042] The experimental methods in the following examples, unless otherwise specified, are all conventional methods in the art, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, are all obtained from commercial sources.

[0043] Example 1 This embodiment provides a specific primer pair for rapid identification of mono- and di-nucleate sessile Ganoderma lucidum, which consists of primer ura3-F (sequence shown in SEQ ID NO.1) and primer ura3-R (sequence shown in SEQ ID NO.2), as shown in Table 1.

[0044] Table 1 Primers and their sequences

[0045] Example 2 This example provides a specific primer pair for rapid identification of mono- and di-nucleate sessile Ganoderma lucidum, which consists of primer 18srRNA-F (sequence shown in SEQ ID NO.3) and primer 28s rRNA-R (sequence shown in SEQ ID NO.4), as shown in Table 2.

[0046] Table 2 Sequences of primers 18s rRNA-F and 28s rRNA-R

[0047] Example 3 This example provides a specific primer pair for rapid identification of mono- and di-nucleate sessile Ganoderma lucidum, which consists of primer GOGAT-F (sequence shown in SEQ ID NO.5) and primer GOGAT-R (sequence shown in SEQ ID NO.6), as shown in Table 3.

[0048] Table 3 Sequences of primers GOGAT-F and GOGAT-R

[0049] Example 4 This example provides a kit for rapid identification of mono- and binucleate sessile Ganoderma lucidum, which is composed of the specific primer pair of Example 1 and 2×TOROBlue Flash KOD Dye Mix.

[0050] Example 5 This example provides a kit for rapid identification of mono- and di-nucleate sessile Ganoderma lucidum, which is composed of the specific primer pair of Example 2 and 2×TOROBlue Flash KOD Dye Mix.

[0051] Example 6 This example provides a kit for rapid identification of mono- and di-nucleate sessile Ganoderma lucidum, which is composed of the specific primer pair of Example 3 and 2×TOROBlue Flash KOD Dye Mix.

[0052] Example 7 This embodiment provides a method for rapidly identifying single-core and double-core sessile Ganoderma lucidum.

[0053] 1. Use the fungal DNA miniprep kit to extract genomic DNA.

[0054] 2. PCR amplification was performed using genomic DNA as a template, and the amplification primers used were the specific primer pair shown in Table 1 of Example 1, the specific primer pair shown in Table 2 of Example 2, or the specific primer pair shown in Table 3 of Example 3. The reaction system is shown in Table 4, and the reaction conditions are shown in Table 5.

[0055] Table 4 PCR amplification reaction system

[0056] The PCR instrument was set up with the following reaction conditions: Table 5 PCR instrument setting reaction conditions

[0057] The amplified products were sequenced and the sequencing peak graph was observed. If overlapping peaks appeared in the sequencing peak graph, the sessile Ganoderma mycelium was determined to be a binucleate mycelium, otherwise it was a monokaryon mycelium.

[0058] Example 8 The present invention verifies the method provided in Example 7 using sessile Ganoderma lucidum of karyotype A and karyotype B and binucleate sessile Ganoderma lucidum obtained by hybridization of the two karyotypes. The details are as follows: 1. Preparation of protoplasts of binucleate sessile Ganoderma lucidum strain Prepare 2% wt lytic enzyme solution and filter with 0.22μm microporous filter to sterilize. Inoculate binucleate sessile Ganoderma lucidum strain (wild isolate) in PDB medium, culture at 25℃ for 7d, pick the mycelium and wash it 3 times with 0.6mol / L mannitol solution (osmotic pressure stabilizer), centrifuge at 6000r / min for 10min, and discard the supernatant. Add 2% lytic enzyme solution at a ratio of 0.2g wet mycelium to 1mL enzyme solution, shake it thoroughly, break up the mycelium, and enzymolyze it in a constant temperature water bath at 31℃ for 3.5h. During the enzymolysis process, shake it gently every 0.5h. After the enzymolysis is completed, remove the mycelium fragments with a 0.45µm microporous filter membrane, centrifuge the filtrate at 4000r / min for 10min, discard the supernatant, and obtain the protoplast precipitate. Redissolve the protoplast precipitate in 0.6mol / L mannitol solution, centrifuge it at 4000r / min for 10min, repeat 3 times, and completely remove the enzyme solution. The protoplasts were added into an appropriate amount of 0.6 mol / L mannitol solution to prepare a protoplast suspension.

[0059] The above experimental process was repeated three times, and the number of protoplasts in each ml of suspension was calculated using a hemocytometer.

[0060] 2. Protoplast regeneration and microscopic examination Prepare YMG protoplast regeneration medium and dilute the protoplast stock solution to 10 7 / mL, culture 100μL on each YMG protoplast regeneration medium, invert and culture at 25℃, pick out the regenerated colonies and add them to the YMG protoplast regeneration medium after most of the single colonies have grown, culture the inserts for 7 days, remove the inserts with hyphae and examine them under a microscope to compare with the starting strain to see if they contain lock-like union (see Figure 1 , Figure 2 ).

[0061] 3. Extraction of genomic DNA Use the Fungal DNA Mini-Extraction Kit (Guangzhou Meiji Biotechnology Co., Ltd.) to extract the DNA of the regenerated mycelium. The specific steps are as follows: 1) The YMG protoplast regeneration medium inoculated with the regenerated colony in step 2 and cultured for 7 days was placed in a -80°C refrigerator for 24 hours, and then the Ganoderma lucidum hyphae were scraped with a weighing spoon and transferred to a mortar, and liquid nitrogen was immediately added to grind the Ganoderma lucidum hyphae into fine powder.

[0062] 2) Transfer the ground sample to a 1.5 ml centrifuge tube, immediately add 360 µL Buffer STE and 40 µL Buffer SDS to the sample, vortex to fully disperse the sample, and treat at 65°C for 15 minutes.

[0063] 3) Add 10 µL RNase A to the lysate, vortex to mix, and let stand at room temperature for 15-30 minutes to digest and remove RNA.

[0064] 4) Add 140 µL Buffer PS to the lysate. Vortex for 30 seconds and place on ice for 10 minutes.

[0065] 5) Centrifuge at 13000g for 5 minutes at room temperature. Carefully transfer 400µL of supernatant to a new centrifuge tube.

[0066] 6) Add 600 µL Buffer PBD (diluted with anhydrous ethanol) to the sample. Vortex mix for 30 seconds. If obvious flocculent precipitate appears, use a pipette to break up the precipitate as much as possible.

[0067] 7) Place the DNA binding column in a collection tube, transfer the general mixture to the column, and centrifuge at 8000g for 60 seconds.

[0068] 8) Discard the filtrate and return the column to the collection tube. Transfer the remaining mixture to the column. Centrifuge at 8000g for 60 seconds.

[0069] 9) Discard the filtrate and put the column back into the collection tube. Add 600µL Buffer GW2 (diluted with anhydrous ethanol) to the column. Centrifuge at 8000g for 60 seconds.

[0070] 10) Repeat step 9.

[0071] 11) Discard the filtrate and put the column back into the collection tube. Centrifuge at 10,000 g for 2 minutes to remove the residual ethanol in the column.

[0072] 12) Transfer the column to a new 1.5 ml centrifuge tube. Add 30 µL of preheated 65°C Buffer AE to the center of the column membrane. Let stand at room temperature for 2 minutes. Centrifuge at 10,000 g for 1 minute.

[0073] 13) Discard the DNA binding column and store the DNA at -80°C.

[0074] (4) PCR amplification and sequencing verification PCR amplification was performed using genomic DNA as a template. The primers used for amplification were primer ura3-F and primer ura3-R in Example 1, or primer 18s rRNA-F and primer 28s rRNA-R in Example 2, or primer GOGAT-F and primer GOGAT-R in Example 3. The reaction system was shown in Table 4 in Example 7, and the reaction conditions were shown in Table 5 in Example 7. The amplified product obtained by PCR amplification using the specific primer pair in Example 1 was sequenced to obtain the following sequence: Figure 3 As shown in the peak diagram, the specific primer pair of Example 2 was used to perform PCR amplification to obtain the amplified product, which was sequenced to obtain the following Figure 4 The peak diagram shown in the figure, the specific primer pair of Example 3 was used to perform PCR amplification to obtain the amplified product, and the following was obtained after sequencing: Figure 5 The electropherogram shown. Figure 3~Figure 5 It can be seen that there are single base mutations (marked with blue bars) between monokaryotic strains of different karyotypes. If there are overlapping peaks, it is a binuclear strain. If there are different single peaks, it indicates a different karyotype (A / B).

[0075] The electrophoresis of PCR products is shown in Figure 6~Figure 8 As shown in the figure, no mixed bands appeared in the amplified products of monokaryotic and dikaryotic mycelia, indicating that the primer pairs provided in Examples 1 to 3 have excellent specificity. In addition, the amplified products of monokaryotic and dikaryotic mycelia all showed clear bands at the same position, indicating that the method of using PCR amplification technology to identify monokaryotic and dikaryotic mycelia through electrophoresis bands has the problem of inaccurate judgment.

[0076] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for rapidly identifying mononuclear and dinuclear edible fungi, characterized in that: Specific primers that meet the requirements of PCR amplification are designed according to the conserved genes of the edible fungi to be tested, wherein the conserved genes are rRNA genes, glutamate synthase genes or ura3 genes; the genomic DNA of the edible fungi to be tested is used as a template, PCR amplification is performed using the specific primers, and the obtained amplification products are sequenced. If overlapping peaks appear in the sequencing peak graph, the edible fungus mycelium is determined to be a binucleate mycelium, otherwise it is a monokaryon mycelium.

2. The method according to claim 1, characterized in that The edible fungi include Agrocybe edulis, Ganoderma lucidum and Pleurotus ostreatus.

3. Application of the method according to claim 1 or 2 in screening hybrids during edible fungus breeding.

4. A specific primer pair for rapid identification of mononuclear and dinuclear sessile Ganoderma lucidum, characterized in that: It consists of primer ura3-F and primer ura3-R, or primer 18s rRNA-F and primer 28s rRNA-R, or primer GOGAT-F and primer GOGAT-R; The primer ura3-F is a single-stranded DNA molecule as shown in SEQ ID NO.1, or a DNA molecule having the same function as the single-stranded DNA molecule shown in SEQ ID NO.1 after one or more nucleotides are replaced and / or deleted and / or added; The primer ura3-R is a single-stranded DNA molecule as shown in SEQ ID NO.2, or a DNA molecule having the same function as the single-stranded DNA molecule as shown in SEQ ID NO.2 after one or more nucleotides are replaced and / or deleted and / or added; The primer 18s rRNA-F is a single-stranded DNA molecule as shown in SEQ ID NO.3, or a DNA molecule in which one or more nucleotides are substituted and / or deleted and / or added to the single-stranded DNA molecule as shown in SEQ ID NO.3 and which has the same function as the single-stranded DNA molecule as shown in SEQ ID NO.3; The primer 28s rRNA-R is a single-stranded DNA molecule as shown in SEQ ID NO.4, or a DNA molecule having the same function as the single-stranded DNA molecule as shown in SEQ ID NO.4 after one or more nucleotides are replaced and / or deleted and / or added; The primer GOGAT-F is a single-stranded DNA molecule as shown in SEQ ID NO.5, or a DNA molecule having the same function as the single-stranded DNA molecule as shown in SEQ ID NO.5 after one or more nucleotides are replaced and / or deleted and / or added; The primer GOGAT-R is a single-stranded DNA molecule as shown in SEQ ID NO.6, or a DNA molecule in which one or several nucleotides are substituted and / or deleted and / or added to the single-stranded DNA molecule as shown in SEQ ID NO.6 and which has the same function as the single-stranded DNA molecule as shown in SEQ ID NO.

6.

5. The specific primer pair according to claim 4, characterized in that: The primer ura3-F is a single-stranded DNA molecule as shown in SEQ ID NO.1; and / or The primer ura3-R is a single-stranded DNA molecule as shown in SEQ ID NO.2; and / or The primer 18s rRNA-F is a single-stranded DNA molecule as shown in SEQ ID NO.3; and / or The primer 28s rRNA-R is a single-stranded DNA molecule as shown in SEQ ID NO.4; and / or The primer GOGAT-F is a single-stranded DNA molecule as shown in SEQ ID NO.5; and / or The primer GOGAT-R is a single-stranded DNA molecule as shown in SEQ ID NO.

6.

6. Use of the specific primer pair according to claim 4 or 5 in preparing a kit for rapid identification of mono- and binucleate sessile Ganoderma lucidum.

7. A kit for rapid identification of single-core and double-core sessile Ganoderma lucidum, characterized in that: The method comprises the following reagents: a primer solution of the specific primer pair according to claim 4 or 5, a DNA polymerase, dNTPs, and a buffer.

8. A method for rapid identification of single-core and double-core sessile Ganoderma lucidum, characterized in that: The specific steps include: S1. Extracting genomic DNA from the sessile Ganoderma mycelium to be tested; S2. Using the genomic DNA extracted from S1 as a template, PCR amplification is performed using the specific primer pair described in claim 4 or 5 or the kit described in claim 7; sequencing the amplified product, and if overlapping peaks appear in the sequencing peak graph, the sessile Ganoderma mycelium is determined to be a binucleate mycelium, otherwise it is a monokaryotic mycelium.

9. The method according to claim 8, characterized in that The reaction system of the PCR amplification is: The reaction conditions of the PCR amplification are:

10. Use of the specific primer pair according to claim 4 or 5, the kit according to claim 7, or the method according to claim 8 or 9 in screening sessile Ganoderma lucidum hybrids during the breeding process of sessile Ganoderma lucidum.

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