CN115838821A - Three-primer composition for identifying mating type of agaricus bisporus W192 single spore strain and identification method - Google Patents

Abstract

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

Description

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

technical field

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

Background technique

W192 is a high-yield and high-quality Agaricus bisporus variety independently selected and bred by my country. It has become the leading variety of Agaricus bisporus in my country. In factory production, W192 has replaced some foreign high-yield varieties.

The technical bottleneck of Agaricus bisporus hybrid breeding mainly lies in the difficulty in obtaining infertile homokaryotic strains. There are two reasons: on the one hand, the homokaryotic sterile monosporus strains of Agaricus bisporus account for a very small proportion, only about 3%, and they germinate later and grow slower than the heterokaryotic fertile strains; on the other hand, Agaricus bisporus can The heterokaryotic hyphae cultivated in this study do not appear lock-like joints, and it is difficult to distinguish homokaryotic and heterokaryotic strains by microscopic examination, just like the secondary heterothallic joint edible fungi such as shiitake mushrooms and enoki mushrooms. The acquisition of S. monosporus strains and the identification of their mating types are one of the most critical basic tasks in the genetics and cross-breeding of Agaricus bisporus. The mating types of the homosterile monosporus strains of Agaricus bisporus are divided into A+ and A-. After the homogeneous sterile monospore strains are obtained, the strains of different mating types can be hybridized to form fertile heterokaryotic strains. The mating type is uncertain. , undoubtedly reduces the success rate of hybridization, and increases the manpower, material resources and time costs of breeding.

The traditional method to obtain Agaricus bisporus S. monosporus is mainly to observe whether it can produce fruit after cultivation. If it can produce fruit normally, then it is considered to be a heterokaryotic fertile strain; if it cannot produce fruit normally, then it may be a homokaryotic sterile monospora strain, or it may not be due to mycelial degradation, cultivation conditions and other reasons Cause no mushrooms. The whole cycle is at least 3 months, which is not only time-consuming and labor-intensive, but also the obtained homosteric monospore strains are not accurate, and the mating type cannot be identified. At present, molecular markers are mainly used to identify the mating type of Agaricus bisporus strains. However, most of the methods have poor identification effect and low efficiency, and some methods also need to digest PCR amplification products, which is not simple and efficient enough.

Agaricus bisporus hybrid breeding is mainly divided into four steps: acquisition of isokaryotic strains, identification of mating type, hybridization pairing, and cultivation of mushrooms. It often requires a lot of manpower and material resources, a large workload, and a long time-consuming process. The first two steps affect the subsequent cross-breeding and cultivation, and determine the success rate and cost of the entire cross-breeding. Neither the traditional method nor the existing molecular method can effectively improve its efficiency.

In view of this, the present invention is proposed.

Contents of the invention

The purpose of the present invention is to provide a three-primer composition and an identification method for identifying the mating type of Agaricus bisporus W192 monosporus strain to solve the problem of mating type identification.

The present invention is achieved like this:

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

The inventor developed the first primer and the second primer at the SNP site scaffold_1:900669 and scaffold_1:900649 of the mating factor A+ and A-regions of Agaricus bisporus strain A+ and A-, and designed a primer at scaffold_1:900873 third primer.

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

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

The third primer is a reverse primer named Mats2R, whose sequence of SEQ ID NO.3 is as follows: 5'-GCATTGTGTATGAGGAAGG-3'.

The amplification length of the first primer and the third primer is 204bp, and is used for detecting A+ factor, and the amplification length of the second primer and the third primer is 224bp, and is used for detecting A-factor.

The inventors found that the single-spore strain of the Agaricus bisporus strain W192 can be quickly identified as a heterokaryotic or homokaryotic sterile single-spore strain through amplification by using the above primer composition, and its mating type can be identified at the same time.

The present invention also provides a reagent or a kit for identifying the mating type of Agaricus bisporus isosterile monosporus strain, which comprises the above-mentioned three-primer composition.

It should be noted that the form of the reagent includes but is not limited to: freeze-dried powder, granule, solution, suspension, emulsion, half-emulsion, etc.

In a preferred embodiment of the application of the present invention, the kit further includes a PCR premix, which includes PCRBuffer and an enzyme mixture.

PCR buffer refers to a solution capable of satisfying the PCR reaction, including dNTPs, metal ions, stabilizers and other components, which can be selected from commercially available PCR buffers.

The enzyme mixture includes substances such as Taq enzyme.

In other embodiments, the kit also includes a positive standard.

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

The present invention also provides a method for identifying the mating type of Agaricus bisporus W192 monosporus strain, which includes the following steps: using the above 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 compositions is 204bp fragment, it is judged that the mating type is a homokaryotic sterile strain of A+; If the PCR amplification product has two fragments of 204bp and 224bp at the same time, it can be judged that the mating type is a heterokaryotic strain of A+A-.

Compared with the existing identification methods, the method provided by the invention is easy to operate, and the invention has the advantages of short time-consuming, high accuracy, simple operation, low cost, no need for enzymatic cutting, and no need for fruiting. By adopting the identification method provided by the invention, heterokaryotic or homokaryotic sterile strains and their mating types can be quickly identified in the monospore strain of Agaricus bisporus W192 strain, which can greatly improve the efficiency of Agaricus bisporus hybrid breeding.

In a preferred embodiment of the application of the present invention, the conditions for PCR amplification include: pre-denaturation at 94-95°C for 1-5min; denaturation at 94-95°C for 0.5-1min, the annealing temperature of the primers is 55-58°C, and the annealing time is 40-45s, 70-72°C extension for 0.5-1min, 30-40 cycles. Adoption of the above PCR amplification conditions is conducive to rapid and accurate identification of synkaryotic sterile strains and determination of their mating types.

In a preferred embodiment of the application of the present invention, pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 1 minute, primer annealing temperature at 55°C, annealing time of 45s, extension at 72°C for 1 minute, 35 cycles, and incubation at 72°C for 7 minutes.

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

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

The PCR amplification system can be set according to actual amplification needs, for example, a 20 μL system.

In other embodiments, the above identification method also includes the steps of mycelia culture and genomic DNA extraction. The mycelia culture specifically includes transferring the monospore strain of Agaricus bisporus W192 strain to potato dextrose agar medium (PDA), and cultivating statically in the dark at 24°C to 25°C.

Genomic DNA can be prepared by rapid mycelium extraction method or conventional extraction method, such as CTAB method.

The above method also includes identifying the amplified product by electrophoresis, specifically including adding a fluorescent dye to the amplified product or gel to identify the amplified product. The amplified products can also be identified by sequencing.

In addition, the present invention also provides a test strip coated with the above-mentioned three-primer composition.

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

The present invention has the following beneficial effects:

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

By adopting the identification method provided by the invention, heterokaryotic or homokaryotic sterile strains and their mating types can be quickly identified in the monospore strain of Agaricus bisporus W192 strain, which can greatly improve the efficiency of Agaricus bisporus hybrid breeding.

Utilizing the identification method of the invention, the time required for conventional identification of three months can be shortened to one week, and heterokaryotic strains without fruiting can be excluded.

Description of drawings

In order to illustrate the technical solutions of the embodiments of the present invention more clearly, the accompanying drawings used 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 It should be regarded as a limitation on the scope, and those skilled in the art can also obtain other related drawings based on these drawings without creative work.

Fig. 1 is the single spore strain amplification pattern of No. 1-25 of "W192" in Example 1 of the present invention;

Fig. 2 is the amplification pattern of single-spore strains numbered 1-25 of "AB2410" in Example 2 of the present invention.

Detailed ways

In order to make the purpose, 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. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased from the market.

The characteristics and performance of the present invention will be described in further detail below in conjunction with the examples.

Example 1

This example provides a three-primer composition for identifying the mating type of Agaricus bisporus W192 single-spore strain and a method for identifying the mating type of Agaricus bisporus W192 single-spore strain. Specifically, the following steps are carried out in sequence:

(1) Mycelia culture: 418 monospore strains of the W192 strain were transferred to a plate equipped with potato dextrose agar medium (PDA), and cultured statically on a plate with a diameter of 90 cm at 25° C. in the dark.

(2) Rapid preparation of genomic DNA: After 15 days of mycelia culture, add 100 μLTE buffer to each sample tube of a 96-well plate, pick a small amount of mycelium from each monokaryon strain with the tip of an inoculation needle, and suspend them in In the sample tube, record the serial number, process the 96-well plate at 98°C for 5 minutes with a PCR machine, and immediately place it on ice to cool it for later use.

In the embodiment of the present invention, the method of rapidly preparing DNA from mycelium can realize rapid identification of large quantities of mycelium. In addition, in other embodiments, conventional DNA extraction methods such as CTAB can also be used to extract mycelial DNA. In the extraction of genomic DNA in the embodiment of the present invention, the frequency and purity of DNA extraction can be reasonably adjusted as required.

(3) Detecting the mating type of the strain: performing PCR amplification on the DNA extracted in step (2).

The PCR amplification system is: a total volume of 20 μL, including: Premix Taq ^TM 10 μL, 10 μmol/L Mats2F1 primer 1 μL, Mats2F2 primer 0.5 μL, Mats2R primer 2 μL, extracted strain template DNA 1 μL, ddH ₂ O 6.5 μL;

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 45s, 72°C for 1 min, 35 cycles; 72°C for 5 min. The specific information of the primer set is shown in Table 1.

In Table 1, Mats2F1 and Mats2F2 respectively represent two forward primers in one set of three primers, and Mats2R represents one reverse primer among one set of three primers.

Table 1: List of primer details

(4) Electrophoresis detection: the product obtained by PCR amplification in step (3) is subjected to 2.5% agarose gel electrophoresis (adding 1/10000 SYBR Green I dye to the agarose gel in advance), voltage 90V, electrophoresis 4h, use the camera system to observe and record the results.

All monospore strains were amplified by PCR using a three-primer composition, and the mating type of each strain was judged by recording the molecular weight of the band amplified by each pair of primers and whether the band was present or not. If the PCR amplification product is a 204bp fragment, it is judged that the strain is a homokaryotic sterile strain whose mating type is A+; If the PCR amplification product has two fragments of 204bp and 224bp at the same time, it is judged that the strain is a heterokaryotic strain with a mating type of A+A-.

The electropherogram is shown in FIG. 1 , and FIG. 1 shows the electropherogram after amplification using the three-primer composition. The results of electrophoresis showed that the proportion of monospores was 3.59% among the 418 monospores, and the ratio of the two mating types A+ and A- was 5:10.

Since in this example, the DNA rapidly prepared by mycelia in batches is directly amplified by PCR, the quality of a small amount of prepared DNA may not be stable and affect the amplification results. Repeat preparations and amplifications were performed.

Example 2

The present embodiment provides a three-primer composition and an identification method for identifying the mating type of Agaricus bisporus AB2410 (heterokaryonospora strain of W192 bacterial strain) monospore strain, specifically comprising the following steps carried out in sequence:

(1) Mycelia culture: 435 monospore strains of AB2410 were transferred to a plate equipped with potato dextrose agar medium (PDA), and cultured on a plate with a diameter of 90 cm at 25° C. in the dark.

(2) Rapid preparation of genomic DNA: After 15 days of mycelia culture, add 100 μLTE buffer to each sample tube of a 96-well plate, pick a small amount of mycelium from each monokaryon strain with the tip of an inoculation needle, and suspend them in In the sample tube, record the serial number, process the 96-well plate at 98°C for 5 minutes with a PCR machine, and immediately place it on ice to cool it for later use.

(3) Detecting the mating type of the strain: performing PCR amplification on the DNA extracted in step (2).

The PCR amplification system is: a total volume of 20 μL, including: Premix Taq ^TM 10 μL, 10 μmol/L Mats2F1 primer 1 μL, Mats2F2 primer 0.5 μL, Mats2R primer 2 μL, extracted strain template DNA 1 μL, ddH ₂ O 6.5 μL;

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 45s, 72°C for 1 min, 35 cycles; 72°C for 5 min. The specific information of the primer set is shown in Table 1.

(4) Electrophoresis detection: the product obtained by PCR amplification in step (3) is subjected to 2.5% agarose gel electrophoresis (adding 1/10000 SYBR Green I dye to the agarose gel in advance), voltage 90V, electrophoresis 4h, use the camera system to observe and record the results.

All monospore strains were amplified by PCR using a three-primer composition, and the mating type of each strain was judged by recording the molecular weight of the band amplified by each pair of primers and whether the band was present or not. If the PCR amplification product is a 204bp fragment, it is judged that the strain is a homokaryotic sterile strain whose mating type is A+; If the PCR amplification product has two fragments of 204bp and 224bp at the same time, it is judged that the strain is a heterokaryotic strain with a mating type of A+A-.

The electropherogram is shown in FIG. 2 , and FIG. 2 shows the electropherogram after amplification using the three-primer composition. The results of electrophoresis showed that the ratio of the homokaryotic sterile strains among the 435 monospore strains was 4.19%, and the ratio of the two mating types A+ and A- was 1:1.7.

Since in this example, the DNA rapidly prepared by mycelia in batches is directly amplified by PCR, the quality of a small amount of prepared DNA may not be stable and affect the amplification results. Repeat preparations and amplifications were performed.

In summary, the present invention provides a three-primer composition and identification method for identifying the mating type of Agaricus bisporus W192 monosporus strain. Compared with the conventional method for identifying the mating type of Agaricus bisporus isogeneous sterile monosporus strain in the prior art, the present invention has the advantages of short time consumption, high accuracy, simple operation, low cost, no need for enzymatic cutting, and no need for fruiting. advantage. By adopting the identification method provided by the invention, heterokaryotic or homokaryotic sterile strains and their mating types can be quickly identified in the monospore strain of Agaricus bisporus W192 strain, which can greatly improve the efficiency of Agaricus bisporus hybrid breeding. Utilizing the identification method of the invention, the time required for conventional identification of three months can be shortened to one week, and heterokaryotic strains without fruiting can be excluded.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims (10)

1. A three-primer composition for identifying the mating type of Agaricus bisporus W192 monosporus strain, characterized in that it comprises: the first primer shown in SEQ ID NO.1, the first primer shown in SEQ ID NO.2 Two primers and a third primer shown in SEQ ID NO.3. 2. A reagent or kit for identifying the mating type of Agaricus bisporus W192 monosporus strain, characterized in that it comprises the three-primer composition according to claim 1. 3. The reagent or kit according to claim 2, characterized in that, the kit also includes a PCR premix, and the PCR premix includes PCR Buffer and an enzyme mixture. 4. A molecular marker, characterized in that it comprises the three-primer composition according to claim 1. 5. A method for identifying the mating type of Agaricus bisporus W192 monosporus strain, comprising the steps of: using the three-primer composition of claim 1 to carry out PCR amplification of the DNA of the sample to be tested simultaneously; if the three-primer composition PCR amplification If the product is a 204bp fragment, it is judged that the mating type is a homokaryotic sterile strain of A+; if the PCR amplification product of the three-primer composition is a 224bp fragment, then it is judged that the mating type is a homokaryotic sterile strain of A-; if the three primers If the PCR amplification product of the composition has two fragments of 204bp and 224bp at the same time, it can be judged that the mating type is a heterokaryotic strain of A+A-. 6. The method according to claim 5, wherein the conditions for the PCR amplification include: 94-95°C pre-denaturation for 1-5min; 94-95°C denaturation for 0.5-1min, the annealing temperature of the primers is 55- 58°C, annealing time 40-45s, 70-72°C extension 0.5-1min, 30-40 cycles. 7. The method according to claim 6, characterized in that, pre-denaturation at 94°C for 5min; denaturation at 94°C for 1min, the annealing temperature of the primers is 55°C, the annealing time is 45s, the extension at 72°C for 1min, 35 cycles; 72 ℃ for 5 minutes. 8. The method according to claim 5, wherein the PCR amplification system is: 0.25-1 μL of the first primer of 10-20 μmol/L, 0.25-1 μL of the second primer of 10-20 μmol/L, 0.25-2 μL of the third primer of 10-20 μmol/L; 5-10 μL of PCR buffer, 0.2-1 μL of strain template DNA, the rest is water; Preferably, the PCR amplification system is: 1 μL of 10 μmol/L first primer, 0.5 μL of 10 μmol/L second primer, 2 μL of 10 μmol/L third primer; 10 μL of PCR buffer, 1 μL of strain template DNA , and the rest is water. 9. A test strip, characterized in that the three primer compositions of claim 1 are coated on the test strip. 10. The application of a three-primer composition as claimed in claim 1 in the preparation of reagents, test kits or test strips.

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