A three-prime primer composition and identification method for identifying the mating type of single spore strains of Agaricus bisporus W192

Abstract

The present invention discloses a three - primer composition and an identification method for identifying the mating type of single - spore strains of Agaricus bisporus W192, which relates to the technical field of molecular marker - assisted breeding of edible fungi. The primer composition includes: a first primer shown in SEQ ID NO.1, a second primer shown in SEQ ID NO.2, and a third primer shown in SEQ ID NO.3. Compared with the conventional method for identifying the mating type of single - spore strains of Agaricus bisporus, the present invention has the advantages of short time - consumption, high accuracy, simple operation, low cost, no need for enzyme digestion, and no need for fruiting. The method of the present invention can quickly identify heterokaryotic or homokaryotic sterile strains and their mating types among the single - spore strains of Agaricus bisporus W192 strain, and can greatly improve the efficiency of cross - breeding of Agaricus bisporus.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular marker-assisted breeding of edible fungi, and in particular, to a three-primer composition and an identification method for identifying the mating type of single-spore strains of Agaricus bisporus W192. Background Art

[0002] W192 is a high-yield and high-quality variety of Agaricus bisporus independently selected and bred in China, and has become the leading variety of Agaricus bisporus in China. In industrial production, W192 has replaced some foreign high-yield varieties.

[0003] The technical bottleneck of Agaricus bisporus hybrid breeding mainly focuses on the difficulty of obtaining infertile homokaryotic strains. There are two reasons: on the one hand, the proportion of homokaryotic sterile single-spore strains of Agaricus bisporus is very small, only about 3%, and they germinate later and grow slower than heterokaryotic fertile strains; on the other hand, the fertile heterokaryotic mycelia of Agaricus bisporus do not show clamp connections, and it is difficult to distinguish homokaryotic and heterokaryotic strains by microscopic examination like secondary heterothallic edible fungi such as Lentinula edodes and Flammulina velutipes. The acquisition of homokaryotic sterile single-spore strains and the identification of their mating types are one of the most crucial basic tasks in the genetics and hybrid breeding of Agaricus bisporus. The mating types of homokaryotic sterile single-spore strains of Agaricus bisporus are divided into A+ and A-. After obtaining homokaryotic sterile single-spore strains, strains of different mating types can hybridize to form fertile heterokaryotic strains. The uncertainty of the mating type undoubtedly reduces the hybridization success rate and increases the labor, material and time costs of breeding.

[0004] The traditional method for obtaining homokaryotic sterile single-spore strains of Agaricus bisporus is mainly to observe whether they can produce mushrooms after fruiting cultivation. If they can produce mushrooms normally, then they are considered heterokaryotic fertile strains; if they cannot produce mushrooms normally, then they may be homokaryotic sterile single-spore strains, or they may not be, but rather other reasons such as mycelial degeneration and cultivation conditions cause the failure to produce mushrooms. The whole cycle takes at least 3 months, which is not only time-consuming and laborious, but also the obtained homokaryotic sterile single-spore strains are inaccurate, and the mating type cannot be identified. At present, the mating type of homokaryotic sterile single-spore strains of Agaricus bisporus is mainly identified by molecular marker methods. However, most methods have poor identification effects and low efficiency, and some methods also require enzymatic digestion of PCR amplification products, which is not simple and efficient enough.

[0005] The hybrid breeding of Agaricus bisporus mainly includes four steps: obtaining homokaryotic strains, identifying mating types, hybrid pairing, and cultivating mushrooms. It often requires a large amount of manpower and material resources, with a large workload and a long time-consuming. The first two steps affect the subsequent hybrid pairing and mushroom cultivation, and determine the success rate and cost of the entire hybrid breeding. Whether using traditional methods or existing molecular methods, the efficiency cannot be effectively improved.

[0006] In view of this, the present invention is specifically proposed. Summary of the Invention

[0007] The object of the present invention is to provide a three - primer composition and an identification method for identifying the mating type of the homokaryotic single - spore strain of Agaricus bisporus W192, so as to solve the problem of difficult mating type identification.

[0008] The present invention is implemented as follows:

[0009] The present invention provides a three - primer composition for identifying the mating type of the homokaryotic single - spore strain of Agaricus bisporus W192, which includes: a first primer shown in SEQ ID NO.1, a second primer shown in SEQ ID NO.2, and a third primer shown in SEQ ID NO.3.

[0010] The inventors developed the first primer and the second primer for the SNP sites scaffold_1:900669 and scaffold_1:900649 in the mating factor A+ and A− regions of the homokaryotic sterile single - spore strain of Agaricus bisporus, and designed the third primer at scaffold_1:900873.

[0011] The first primer is a forward primer named Mats2F1, and its sequence of SEQ ID NO.1 is as follows: 5’ - GTGAGGCAAGATTTCG - 3’.

[0012] The second primer is also a forward primer named Mats2F2, and its sequence of SEQ ID NO.2 is as follows: 5’ - GAGCTCCTCACTGATCC - 3’.

[0013] The third primer is a reverse primer named Mats2R, and its sequence of SEQ ID NO.3 is as follows: 5’ - GCATTGTGTATGAGGAAGG - 3’.

[0014] The amplification length of the first primer and the third primer is 204bp, which is used to detect the A+ factor, and the amplification length of the second primer and the third primer is 224bp, which is used to detect the A− factor.

[0015] The inventors found that by using the above - mentioned primer composition for amplification, it can quickly identify whether the single - spore strain of Agaricus bisporus W192 is a heterokaryotic or homokaryotic sterile single - spore strain, and at the same time identify its mating type.

[0016] The present invention also provides a reagent or kit for identifying the mating type of the homokaryotic sterile single - spore strain of Agaricus bisporus, which includes the above - mentioned three - primer composition.

[0017] It should be noted that the forms of the reagent include, but are not limited to: freeze - dried powder, granule, solution, suspension, emulsion, semi - emulsion, etc.

[0018] In a preferred embodiment of the application of the present invention, the kit further includes a PCR premix, and the PCR premix includes a PCR Buffer and an enzyme mixture.

[0019] PCR buffer refers to a solution that can meet the requirements of the PCR reaction, including components such as dNTP, metal ions, stabilizers, etc., and can be selected from commercially available PCR buffers.

[0020] The enzyme mixture includes substances such as Taq enzyme.

[0021] In other embodiments, the kit further includes a positive standard.

[0022] The present invention also provides a molecular marker, which includes the above-mentioned three-primer composition.

[0023] The present invention also provides a method for identifying the mating type of the homokaryotic sterile strain of Agaricus bisporus W192, which includes the following steps: simultaneously performing PCR amplification on the DNA of the sample to be tested using the above three primers; if the PCR amplification product of the three-primer composition is a 204bp fragment, it is determined as a homokaryotic sterile strain with a mating type of A+; if the PCR amplification product of the three-primer composition is a 224bp fragment, it is determined as a homokaryotic sterile strain with a mating type of A−; if the PCR amplification product of the three-primer composition has two fragments of 204bp and 224bp at the same time, it is determined as a heterokaryotic strain with a mating type of A+A−.

[0024] Compared with the existing identification methods, the method provided by the present invention is easy to operate, and the present invention has the advantages of short time consumption, high accuracy, easy operation, low cost, no need for enzyme digestion, and no need for fruiting. By using the identification method provided by the present invention, heterokaryotic or homokaryotic sterile strains and their mating types can be quickly identified among the monosporic strains of Agaricus bisporus W192 strain, which can greatly improve the efficiency of crossbreeding of Agaricus bisporus.

[0025] In a preferred embodiment of the application of the present invention, the conditions for PCR amplification include: pre-denaturation at 94-95°C for 1-5 min; denaturation at 94-95°C for 0.5-1 min, the annealing temperature of the primers is 55-58°C, the annealing time is 40-45 s, extension at 70-72°C for 0.5-1 min, and 30-40 cycles. Using the above PCR amplification conditions is beneficial to quickly and accurately identify the homokaryotic sterile strain and determine its mating type.

[0026] In a preferred embodiment of the application of the present invention, pre-denaturation at 94°C for 5 min; denaturation at 94°C for 1 min, the annealing temperature of the primers is 55°C, the annealing time is 45 s, extension at 72°C for 1 min, and 35 cycles; incubation at 72°C for 7 min.

[0027] In a preferred embodiment of the application of the present invention, the PCR amplification system is as follows: 0.25 - 1 μL of the first primer at 10 - 20 μmol / L, 0.25 - 1 μL of the second primer at 10 - 20 μmol / L, 0.25 - 2 μL of the third primer at 10 - 20 μmol / L; 5 - 10 μL of PCR buffer, 0.2 - 1 μL of the strain template DNA, and the rest is water.

[0028] The PCR amplification system is as follows: 1 μL of the first primer at 10 μmol / L, 0.5 μL of the second primer at 10 μmol / L, 2 μL of the third primer at 10 μmol / L; 10 μL of PCR buffer, 1 μL of the strain template DNA, and the rest is water.

[0029] The PCR amplification system can be set according to actual amplification needs, such as a 20 μL system.

[0030] In other embodiments, the above identification method further includes the steps of mycelium culture and genomic DNA extraction. The mycelium culture specifically includes transferring the single spore strain of the Agaricus bisporus W192 strain to a potato dextrose agar medium (PDA) and culturing it statically in the dark at 24°C - 25°C.

[0031] The preparation of genomic DNA can choose the mycelium rapid extraction method or conventional extraction methods, such as the CTAB method, etc.

[0032] The above method further includes electrophoretic identification of the amplified product, specifically including adding a fluorescent dye to the amplified product or gel for identification of the amplified product. The amplified product can also be subjected to sequencing identification.

[0033] In addition, the present invention also provides a test strip, on which the above three - primer composition is coated.

[0034] The present invention also provides the application of the above three - primer composition in the preparation of reagents, kits or test strips.

[0035] The present invention has the following beneficial effects:

[0036] The present invention provides a three - primer composition and an identification method for identifying the mating type of the single spore strain of Agaricus bisporus W192. Compared with the conventional methods for identifying the mating type of single spore strains of Agaricus bisporus in the prior art, the primers provided by the present invention have the advantages of short time - consuming, high accuracy, simple operation, low cost, no need for enzyme digestion, and no need for fruiting. It can be used to develop corresponding detection reagents or kits to meet the needs of quickly identifying heterokaryotic or homokaryotic sterile single spore strains and their mating types. The proposal of the present invention helps to improve the efficiency of cross - breeding and shorten the cost of cross - breeding.

[0037] By using the identification method provided by the present invention, heterokaryotic or homokaryotic sterile strains and their mating types can be rapidly identified among the single-spore strains of the Agaricus bisporus W192 strain, which can greatly improve the efficiency of Agaricus bisporus cross-breeding.

[0038] By using the identification method of the present invention, the time required for conventional identification can be shortened from 3 months to 1 week, and heterokaryotic strains that do not produce fruiting bodies can be excluded. Brief Description of the Drawings

[0039] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention, and thus should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0040] Figure 1 Amplification map of single-spore strains numbered 1-25 of "W192" in Example 1 of the present invention;

[0041] Figure 2 Amplification map of single-spore strains numbered 1-25 of "AB2410" in Example 2 of the present invention. Detailed Embodiments

[0042] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those conditions not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0043] The features and properties of the present invention will be further described in detail below in conjunction with the embodiments.

[0044] Example 1

[0045] This example provides a three-prime primer composition for identifying the mating type of single-spore strains of Agaricus bisporus W192 and a method for identifying the mating type of single-spore strains of Agaricus bisporus W192. The W192 strain is preserved in the Edible Fungi Sub-center of the China Center for Agricultural Culture Collection. Specifically, it includes the following steps carried out in sequence:

[0046] (1) Mycelium culture: Transfer 418 single-spore strains of the W192 strain to a petri dish filled with potato dextrose agar medium (PDA), and culture them statically in the dark at 25 °C on a 90 cm diameter petri dish.

[0047] (2) Rapid preparation of genomic DNA: After culturing the mycelium for 15 days, add 100 μL of TE buffer to each sample tube in the 96-well plate. Use the tip of an inoculation needle to pick a small amount of mycelium from each monokaryotic strain and suspend it in the sample tube respectively. Record the numbers well. After treating the 96-well plate at 98 °C for 5 min with a PCR instrument, immediately place it on ice for cooling and reserve for later use.

[0048] In the embodiments of the present invention, the method for rapidly preparing DNA from mycelium can achieve rapid identification of a large number of mycelia. In addition, in other embodiments, conventional DNA extraction methods such as CTAB can also be used to extract mycelial DNA. In the embodiments of the present invention, the extraction of genomic DNA can reasonably adjust the number of times and purity of DNA extraction according to needs.

[0049] (3) Detection of the mating type of the strain: Perform PCR amplification on the DNA extracted in step (2).

[0050] The PCR amplification system is as follows: The total volume is 20 μL, including: Premix Taq TM 10 μL, 1 μL of Mats2F1 primer at 10 μmol / L, 0.5 μL of Mats2F2 primer, 2 μL of Mats2R primer, 1 μL of the extracted strain template DNA, and 6.5 μL of ddH2O;

[0051] PCR reaction conditions: 95 °C for 5 min; 94 °C for 1 min, the annealing temperature of the primer is 55 °C, the annealing time is 45 s for each, 72 °C for extension for 1 min, 35 cycles; 72 °C for incubation for 5 min. The specific information of the primer group is shown in Table 1.

[0052] In Table 1, Mats2F1 and Mats2F2 respectively represent 2 forward primers among 3 primers in 1 group, and Mats2R represents the reverse primer among 3 primers in 1 group.

[0053] Table 1: List of detailed primer information

[0054]

[0055] (4) Electrophoresis detection: Subject the product obtained by PCR amplification in step (3) to 2.5% agarose gel electrophoresis (previously add 1 / 10000 SYBR Green I staining agent to the agarose gel), with a voltage of 90 V and electrophoresis for 4 h. Observe and record the results with a photography system.

[0056] All monosporic strains were subjected to PCR amplification using a three - primer composition. By recording the molecular weight size and the presence or absence of amplified bands for each pair of primers, the mating type of each strain was determined. If the PCR amplification product was a 204 - bp fragment, the strain was determined to be a homokaryotic sterile strain with a mating type of A+; if the PCR amplification product was a 224 - bp fragment, the strain was determined to be a homokaryotic sterile strain with a mating type of A−; if the PCR amplification product had both 204 - bp and 224 - bp fragments, the strain was determined to be a heterokaryotic strain with a mating type of A+A−.

[0057] The electrophoresis pattern was referred to Figure 1 as shown Figure 1 It represents the electrophoresis pattern after amplification using the three - primer composition. The electrophoresis results showed that among 418 monosporic strains, the proportion of homokaryotic sterile monosporic strains was 3.59%, and the ratio of the two mating types A+ and A− was 5:10.

[0058] Since in this example, the DNA prepared from the mycelium in batches and quickly was directly subjected to PCR amplification, there was a situation where the quality of a small amount of prepared DNA was unstable and affected the amplification results. To ensure obtaining all the detection results, some DNA with poor amplification effects needed to be prepared and amplified repeatedly.

[0059] Example 2

[0060] This example provides a three - primer composition and an identification method for identifying the mating type of monosporic strains of Agaricus bisporus AB2410 (heterokaryotic monosporic strains of the W192 strain), specifically including the following steps carried out in sequence:

[0061] (1) Mycelium culture: 435 monosporic strains of AB2410 were transferred to plates containing potato dextrose agar medium (PDA) and cultured statically in the dark at 25°C on plates with a diameter of 90 cm.

[0062] (2) Rapid preparation of genomic DNA: After 15 days of mycelium culture, 100 μL of TE buffer was added to each sample tube in a 96 - well plate. A small amount of mycelium was picked from each monokaryotic strain with the tip of an inoculation needle and suspended in the sample tube respectively. After recording the numbers, the 96 - well plate was treated at 98°C for 5 min using a PCR instrument and then immediately placed on ice for cooling and standby.

[0063] (3) Detection of the mating type of the strain: The DNA extracted in step (2) was subjected to PCR amplification.

[0064] The PCR amplification system was: the total volume was 20 μL, including: Premix Taq TM1 μL of 10 μmol / L Mats2F1 primer, 0.5 μL of Mats2F2 primer, 2 μL of Mats2R primer, 1 μL of the extracted strain template DNA, 6.5 μL of ddH2O;

[0065] PCR reaction conditions: 95°C for 5 min; 94°C for 1 min, the annealing temperature of the primers is 55°C, the annealing time is 45 s each, extension at 72°C for 1 min, 35 cycles; incubation at 72°C for 5 min. The specific information of the primer set is shown in Table 1.

[0066] (4) Electrophoresis detection: The product obtained by PCR amplification in step (3) is subjected to 2.5% agarose gel electrophoresis (previously adding 1 / 10000 SYBR Green I staining agent to the agarose gel), voltage 90 V, electrophoresis for 4 h, and the results are observed and recorded using a photography system.

[0067] All monosporic strains were subjected to PCR amplification using a three-primer composition. By recording the molecular weight size and the presence or absence of the amplified bands of each pair of primers, the mating type of each strain was determined. If the PCR amplification product is a 204-bp fragment, the strain is determined to be a homokaryotic sterile strain with a mating type of A+; if the PCR amplification product is a 224-bp fragment, the strain is determined to be a homokaryotic sterile strain with a mating type of A−; if the PCR amplification product has both 204-bp and 224-bp fragments, the strain is determined to be a heterokaryotic strain with a mating type of A+A−.

[0068] The electrophoresis pattern is referred to Figure 2 as shown Figure 2 and represents the electrophoresis pattern after amplification using the three-primer composition. The electrophoresis results show that the proportion of homokaryotic sterile strains among 435 monosporic strains is 4.19%, and the ratio of the two mating types A+ and A− is 1:1.7.

[0069] Since in this example, the DNA prepared from the mycelia in batches and quickly was directly subjected to PCR amplification, there was a situation where the quality of a small amount of the prepared DNA was unstable and affected the amplification results. To ensure obtaining all the detection results, some DNA with poor amplification effects needed to be prepared and amplified repeatedly.

[0070] In summary, the present invention provides a three-prime primer composition and an identification method for identifying the mating type of single-spore strains of Agaricus bisporus W192. Compared with the conventional method for identifying the mating type of homokaryotic sterile single-spore strains of Agaricus bisporus in the prior art, the present invention has the advantages of short time consumption, high accuracy, simple operation, low cost, no need for enzyme digestion, and no need for fruiting. By using the identification method provided by the present invention, heterokaryotic or homokaryotic sterile strains and their mating types can be quickly identified among the single-spore strains of Agaricus bisporus W192, which can greatly improve the efficiency of crossbreeding of Agaricus bisporus. Using the identification method of the present invention, the time required for conventional identification can be shortened from 3 months to 1 week, and heterokaryotic strains that do not fruit can be excluded.

[0071] The foregoing is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A three - primer composition for identifying the mating type of single - spore strains of Agaricus bisporus W192, characterized in that, It includes: a first primer as shown in SEQ ID NO.1, a second primer as shown in SEQ ID NO.2, and a third primer as shown in SEQ ID NO.3; using the three primers to simultaneously perform PCR amplification on the DNA of the sample to be tested; if the PCR amplification product of the three-primer composition is a 204bp fragment, it is determined as a homokaryotic sterile strain with mating type A+; if the PCR amplification product of the three-primer composition is a 224bp fragment, it is determined as a homokaryotic sterile strain with mating type A−; if the PCR amplification product of the three-primer composition has two fragments of 204bp and 224bp simultaneously, it is determined as a heterokaryotic strain with mating type A+A−.

2. A reagent or kit for identifying the mating type of single spore strains of Agaricus bisporus W192, characterized in that, It includes the three-primer composition described in claim 1.

3. The reagent or kit according to claim 2, characterized in that, The kit further includes a PCR premix, and the PCR premix includes a PCR Buffer and an enzyme mixture.

4. A method for identifying the mating type of single-spore strains of Agaricus bisporus W192, comprising the following steps: using the three-primer composition of claim 1 to simultaneously perform PCR amplification on the DNA of the sample to be tested; if the PCR amplification product of the three-primer composition is a 204bp fragment, then it is determined as a homokaryotic sterile strain with a mating type of A+; if the PCR amplification product of the three-primer composition is a 224bp fragment, it is determined as a homokaryotic sterile strain with mating type A−; if the PCR amplification product of the three-primer composition has two fragments of 204bp and 224bp simultaneously, it is determined as a heterokaryotic strain with mating type A+A−.

5. The method according to claim 4, wherein The conditions for the PCR amplification include: pre-denaturation at 94 - 95°C for 1 - 5 min; denaturation at 94 - 95°C for 0.5 - 1 min, the annealing temperature of the primers is 55 - 58°C, the annealing time is 40 - 45 s, extension at 70 - 72°C for 0.5 - 1 min, for 30 - 40 cycles.

6. The method according to claim 5, wherein Pre-denaturation at 94°C for 5 min; denaturation at 94°C for 1 min, the annealing temperature of the primers is 55°C, the annealing time is 45 s, extension at 72°C for 1 min, for 35 cycles; incubation at 72°C for 5 min.

7. The method according to claim 4, characterized in that The system for the PCR amplification is: 0.25 - 1 μL of the first primer at 10 - 20 μmol / L, 0.25 - 1 μL of the second primer at 10 - 20 μmol / L, 0.25 - 2 μL of the third primer at 10 - 20 μmol / L; 5 - 10 μL of the PCR buffer, 0.2 - 1 μL of the strain template DNA, and the rest is water.

8. The method according to claim 7, characterized in that, The system for the PCR amplification is: 1 μL of the first primer at 10 μmol / L, 0.5 μL of the second primer at 10 μmol / L, 2 μL of the third primer at 10 μmol / L; 10 μL of the PCR buffer, 1 μL of the strain template DNA, and the rest is water.

9. A test strip, characterized in that, The test strip is coated with the three-primer composition described in claim 1.

10. Use of a three-primer composition as described in claim 1 in the preparation of reagents, kits or test strips.

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