CN115838821A - Three-primer composition for identifying mating type of agaricus bisporus W192 single spore strain and identification method - Google Patents
Three-primer composition for identifying mating type of agaricus bisporus W192 single spore strain and identification method Download PDFInfo
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Abstract
The invention discloses a three-primer composition for identifying the mating type of a single spore strain of Agaricus bisporus W192 and an identification method, and relates to the technical field of molecular marker assisted breeding of edible fungi. The primer composition comprises: a first primer shown as SEQ ID NO.1, a second primer shown as SEQ ID NO.2 and a third primer shown as SEQ ID NO. 3. Compared with the conventional method for identifying the mating type of the agaricus bisporus single spore strain, the method has the advantages of short time consumption, high accuracy, simple and convenient operation, low cost, no enzyme digestion and no fruiting. The method of the invention can rapidly identify heterokaryon or homokaryon sterile strains and mating types thereof in the single spore strain of the agaricus bisporus W192 strain, and can greatly improve the efficiency of cross breeding of the agaricus bisporus.
Description
Technical Field
The invention relates to the technical field of edible fungus molecular marker assisted breeding, in particular to a three-primer composition for identifying the mating type of a single spore strain of agaricus bisporus W192 and an identification method.
Background
W192 is a high-yield and high-quality agaricus bisporus variety independently selected and bred in China, becomes the current variety of agaricus bisporus in China, and in industrial production, W192 replaces part of foreign high-yield varieties.
The technical bottleneck of cross breeding of agaricus bisporus is mainly focused on that non-fertile homokaryon strains are difficult to obtain. The reason is twofold: on one hand, the same-nucleus sterile single-spore strain of the agaricus bisporus accounts for a very small amount, only accounts for about 3 percent, and germinates late and grows slowly compared with a heteronuclear fertile strain; on the other hand, the mycelium of the heterokaryon which can be bred by the agaricus bisporus does not have the locked combination, and the homokaryon and heterokaryon strains are difficult to be distinguished by microscopic examination like the secondary heterokaryon combined edible fungi such as shiitake mushroom, flammulina velutipes and the like. The acquisition of homogenic sterile single spore strains and the identification of mating types are one of the most key basic works of agaricus bisporus heredity and cross breeding. The mating types of the homonuclear sterile monospore strains of the agaricus bisporus are divided into A + and A-, after the homonuclear sterile monospore strains are obtained, strains with different mating types can be hybridized to form fertile heteronuclear strains, the mating types are uncertain, the success rate of hybridization is undoubtedly reduced, and the labor, material and time costs of breeding are increased.
The traditional method for obtaining the same-core sterile single-spore strain of the agaricus bisporus is mainly used for observing whether the agaricus bisporus can grow after fruiting cultivation. If the mushroom can be normally grown, the strain is considered to be a heterocaryic fertile strain; if the fruiting cannot be normally carried out, the strain may be the homonuclear sterile monospore strain, or the fruiting cannot be caused by other reasons such as hypha degeneration, cultivation conditions and the like instead of the homonuclear sterile monospore strain. The whole period is at least 3 months, not only the time and the labor are consumed, but also the obtained homonuclear sterile monospore strain is inaccurate, and the mating type can not be identified. At present, a molecular marker method is mainly adopted for identifying the mating type of the agaricus bisporus homonuclear sterile single spore strains. However, most methods have poor identification effect and low efficiency, and some methods also need enzyme digestion on PCR amplification products, which is not simple and efficient enough.
The cross breeding of the agaricus bisporus is mainly divided into four steps of homonuclear strain obtaining, mating type identification, cross pairing and mushroom cultivation, and usually a large amount of manpower and material resources are required to be invested, so that the workload is large and the time consumption is long. The former two steps affect the subsequent hybridization pairing and mushroom cultivation, and determine the success rate and cost of the whole hybridization breeding. The efficiency cannot be improved practically by either the conventional method or the conventional molecular method.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a three-primer composition for identifying the mating type of a single spore strain of agaricus bisporus W192 and an identification method so as to solve the problem of mating type identification.
The invention is realized by the following steps:
the invention provides a three-primer composition for identifying mating types of a single spore strain of Agaricus bisporus W192, which comprises the following components: a first primer shown as SEQ ID NO.1, a second primer shown as SEQ ID NO.2 and a third primer shown as SEQ ID NO. 3.
The inventors developed a first primer and a second primer at the SNP sites scaffold _1 and scaffold _ 1.
The first primer is a forward primer, named Mats2F1, and has the sequence of SEQ ID NO.1 as follows: 5 'GTGAGGCAAGATTTCG-3'.
The second primer, also the forward primer, was named Mats2F2, and its sequence of SEQ ID NO.2 is as follows: 5 'GAGCTCCTCACTGATCC-3'.
The third primer is a reverse primer and is named as Mats2R, and the sequence of SEQ ID NO.3 is as follows: 5 'GCATTGTGTGTATGAGGAAGG + 3'.
The amplification length of the first primer and the third primer is 204bp and is used for detecting A + factors, and the amplification length of the second primer and the third primer is 224bp and is used for detecting A-factors.
The inventor finds that the single spore strain of the agaricus bisporus W192 strain can be rapidly identified as heterokaryotic or homokaryotic sterile single spore strain by amplification by using the primer composition, and the mating type of the single spore strain can be identified at the same time.
The invention also provides a reagent or a kit for identifying the mating type of the agaricus bisporus homonuclear sterile single spore strain, which comprises the three primer compositions.
The form of the reagent includes, but is not limited to: lyophilized powder, granule, solution, suspension, emulsion, semi-emulsion, etc.
In a preferred embodiment of the present invention, the kit further comprises a PCR premix, wherein the PCR premix comprises a PCR Buffer and an enzyme mixture.
The PCR buffer is a solution capable of satisfying PCR reaction, comprises dNTP, metal ions, a stabilizer and the like, and can be selected from commercially available PCR buffers.
The enzyme mixture includes Taq enzyme.
In other embodiments, the kit further comprises a positive standard.
The invention also provides a molecular marker which comprises the three-primer composition.
The invention also provides a method for identifying the mating type of the agaricus bisporus W192 monospore strain, which comprises the following steps: performing PCR amplification on the DNA of a sample to be detected by using the three primers; if the PCR amplification product of the three-primer composition is a 204bp fragment, judging the homonuclear sterile strain with the mating type of A +; if the PCR amplification product of the three-primer composition is a 224bp fragment, judging the homonuclear sterile strain with the 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, the mating type of the heterokaryotic strain is judged to be A + A-.
Compared with the existing identification method, the method provided by the invention is simple and convenient to operate, and has the advantages of short time consumption, high accuracy, simplicity and convenience in operation, low cost, no need of enzyme digestion and no need of fruiting. By adopting the identification method provided by the invention, heterokaryon or homokaryon sterile strains and mating types thereof can be quickly identified in the monospore strain of the agaricus bisporus W192 strain, and the cross breeding efficiency of the agaricus bisporus can be greatly improved.
In a preferred embodiment of the present invention, the PCR amplification conditions include: pre-denaturation at 94-95 deg.C for 1-5min; denaturation at 94-95 deg.C for 0.5-1min, annealing at 55-58 deg.C for 40-45s, extension at 70-72 deg.C for 0.5-1min, and 30-40 cycles. The PCR amplification condition is favorable for rapidly and accurately identifying the homonuclear sterile strain and judging the mating type of the homonuclear sterile strain.
In the preferred embodiment of the present invention, the pre-denaturation is carried out at 94 ℃ for 5min; denaturation at 94 ℃ for 1min, annealing temperature of the primers is 55 ℃, annealing time is 45s, extension at 72 ℃ is 1min, and 35 cycles are performed; keeping the temperature at 72 ℃ for 7min.
In a preferred embodiment of the present invention, the PCR amplification system is: 0.25-1 mu L of first primer of 10-20 mu mol/L, 0.25-1 mu L of second primer of 10-20 mu mol/L, and 0.25-2 mu L of third primer of 10-20 mu mol/L; 5-10 μ L of PCR buffer,0.2-1 μ L of strain template DNA, and the balance of water.
The PCR amplification system is as follows: 1 mu L of 10 mu mol/L first primer, 0.5 mu L of 10 mu mol/L second primer and 2 mu L of 10 mu mol/L third primer; 10. Mu.L of PCR buffer, 1. Mu.L of strain template DNA, and the balance water.
The PCR amplification system can be set according to the actual amplification requirement, for example, 20. Mu.L system.
In other embodiments, the above-described identification method further comprises the steps of hyphal culture and extraction of genomic DNA. Wherein the hypha culture comprises transferring the single spore strain of Agaricus bisporus W192 strain into Potato Dextrose Agar (PDA) culture medium, and standing and culturing at 24-25 deg.C in dark place.
The preparation of the genomic DNA can be carried out by a hyphal rapid extraction method or a conventional extraction method such as a CTAB method.
The method also comprises the step of carrying out electrophoretic identification on the amplified product, and specifically comprises the step of adding a fluorescent dye into the amplified product or gel to carry out identification on the amplified product. The amplified product can also be subjected to sequencing identification.
In addition, the invention also provides a test strip, and the three-primer composition is coated on the test strip.
The invention also provides application of the three-primer composition in preparation of a reagent, a kit or a test strip.
The invention has the following beneficial effects:
the invention provides a three-primer composition for identifying the mating type of a single spore strain of Agaricus bisporus W192 and an identification method. Compared with the conventional method for identifying the mating type of the agaricus bisporus monospore strain in the prior art, the primer provided by the invention has the advantages of short time consumption, high accuracy, simplicity and convenience in operation, low cost, no need of enzyme digestion and no need of fruiting. The kit can be used for developing corresponding detection reagents or kits to meet the requirements of rapidly identifying heterokaryon or homokaryon sterile monospore strains and mating types thereof, and the method is beneficial to improving the efficiency of cross breeding and shortening the cost of cross breeding.
By adopting the identification method provided by the invention, heterokaryon or homokaryon sterile strains and mating types thereof can be quickly identified in the monospore strain of the agaricus bisporus W192 strain, and the cross breeding efficiency of the agaricus bisporus can be greatly improved.
By using the identification method of the present invention, the time of 3 months required for conventional identification can be shortened to 1 week, and heterokaryotic strains which do not produce mushrooms can be excluded.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is an amplification map of the monospore strain Nos. 1 to 25 of "W192" in example 1 of the present invention;
FIG. 2 is an amplification map of the single spore strains numbered 1 to 25 of "AB2410" in example 2 of the present invention.
Detailed Description
In order to make the objects, 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. The examples, in which specific conditions are not specified, were carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
The embodiment provides a three-primer composition for identifying the mating type of a single spore strain of Agaricus bisporus W192 and a method for identifying the mating type of the single spore strain of Agaricus bisporus W192, wherein the strain W192 is preserved in the edible fungus center of the China agricultural microbiological culture Collection center. The method specifically comprises the following steps of:
(1) Hypha culture: 418 single spore strains of the W192 strain were transferred to a plate containing Potato Dextrose Agar (PDA) and cultured on a plate 90cm in diameter in a dark place at 25 ℃.
(2) Rapid preparation of genomic DNA: after hyphae are cultured for 15 days, 100 mu L of TE buffer is added into each sample tube of a 96-well plate, a small amount of hyphae are picked from each monokaryon strain by using the tip of an inoculating needle, suspended in the sample tubes respectively, recorded and numbered, and immediately placed on ice for cooling after the 96-well plate is treated for 5min at 98 ℃ by a PCR instrument for later use.
In the embodiment of the invention, the method for quickly preparing DNA by hyphae can realize the quick identification of a large batch of hyphae, and in addition, in other embodiments, the extraction of hyphae DNA can also be realized by conventional DNA extraction methods such as CTAB and the like. The extraction of the genome DNA in the embodiment of the invention can reasonably adjust the times and purity of extracting the DNA according to the requirement.
(3) Detecting mating types of the strains: and (3) carrying out PCR amplification on the DNA extracted in the step (2).
The PCR amplification system is as follows: total volume 20 μ L, including: premix Taq TM 10 μ L,1 μ L of 10 μmol/L Mats2F1 primer, 0.5 μ L of Mats2F2 primer, 2 μ L of Mats2R primer, 1 μ L of extracted strain template DNA, ddH 2 O 6.5μL；
And (3) PCR reaction conditions: 5min at 95 ℃;94 ℃ for 1min, the annealing temperature of the primer is 55 ℃, the annealing time is 45s, the extension is 1min at 72 ℃, and 35 cycles are carried out; keeping the temperature at 72 ℃ for 5min. The specific information of the primer set is shown in Table 1.
Mats2F1 and Mats2F2 in Table 1 represent 2 forward primers in 1 set of 3 primers, respectively, and Mats2R represents a reverse primer in 1 set of 3 primers.
Table 1: detailed information List of primers
(4) And (3) electrophoresis detection: and (4) carrying out 2.5% agarose gel electrophoresis on the product obtained by PCR amplification in the step (3) (1/10000 SYBR Green I staining agent is added into the agarose gel in advance), carrying out electrophoresis for 4h at a voltage of 90V, observing by using a photographic system, and recording the result.
PCR amplification was performed on all single spore strains using a three primer combination, and the mating type of each strain was judged by recording the size of the amplified bands for each primer pair and the absence of bands. If the PCR amplification product is a 204bp fragment, judging the strain to be a homonuclear sterile strain with the mating type of A +; if the PCR amplification product is a 224bp fragment, judging the strain to be a homonuclear sterile strain with the mating type of A-; if the PCR amplification product has two fragments of 204bp and 224bp at the same time, the strain is judged to be a heterokaryotic strain with the mating type of A + A-.
FIG. 1 shows an electrophoretogram after amplification using a three-primer composition, and FIG. 1 shows an electrophoretogram after amplification using a three-primer composition. The electrophoresis results show that: the ratio of homogenic sterile single spore strains in 418 single spore strains was 3.59%, wherein the ratio of 2 mating types A + and A-was 5:10.
in this embodiment, DNA prepared by hypha in a batch and rapidly is directly subjected to PCR amplification, so that the quality of a small amount of prepared DNA is unstable, which affects the amplification result, and in order to ensure that all detection results are obtained, some DNA with poor amplification effect needs to be repeatedly prepared and amplified.
Example 2
The embodiment provides a three-primer composition for identifying the mating type of a single spore strain of agaricus bisporus AB2410 (an heterokaryotic strain of a W192 strain) and an identification method, and the three-primer composition specifically comprises the following steps of:
(1) Hypha culture: 435 single spore strains of AB2410 were transferred to a plate containing Potato Dextrose Agar (PDA) and cultured on a plate 90cm in diameter in a dark place at 25 ℃.
(2) Rapid preparation of genomic DNA: after hyphae are cultured for 15 days, 100 mu L of TE buffer is added into each sample tube of a 96-well plate, a small amount of hyphae are picked from each monokaryon strain by using the tip of an inoculating needle, suspended in the sample tubes respectively, recorded and numbered, and immediately placed on ice for cooling after the 96-well plate is treated for 5min at 98 ℃ by a PCR instrument for later use.
(3) Detecting mating types of the strains: and (3) carrying out PCR amplification on the DNA extracted in the step (2).
The PCR amplification system is as follows: total volume 20 μ L, including: premix Taq TM 10 μ L,1 μ L of 10 μmol/L Mats2F1 primer, 0.5 μ L of Mats2F2 primer, 2 μ L of Mats2R primer, 1 μ L of extracted strain template DNA, ddH 2 O 6.5μL；
And (3) PCR reaction conditions: 5min at 95 ℃;94 ℃ for 1min, the annealing temperature of the primer is 55 ℃, the annealing time is 45s, the extension is 1min at 72 ℃, and 35 cycles are carried out; keeping the temperature at 72 ℃ for 5min. The specific information of the primer set is shown in Table 1.
(4) And (3) electrophoresis detection: and (3) carrying out 2.5% agarose gel electrophoresis (adding 1/10000 SYBR Green I stain into the agarose gel in advance) on the product obtained by PCR amplification in the step (3), carrying out electrophoresis for 4h at a voltage of 90V, observing by using a photographic system, and recording the result.
PCR amplification was performed on all single spore strains using a three primer combination, and the mating type of each strain was judged by recording the size of the amplified bands for each primer pair and the absence of bands. If the PCR amplification product is a 204bp fragment, judging the strain to be a homonuclear sterile strain with the mating type of A +; if the PCR amplification product is a 224bp fragment, judging the strain to be a homonuclear sterile strain with the mating type of A-; if the PCR amplification product has two fragments of 204bp and 224bp at the same time, the strain is judged to be a heterokaryotic strain with the mating type of A + A-.
FIG. 2 shows an electrophoretogram, and FIG. 2 shows an electrophoretogram after amplification using a three-primer composition. The electrophoresis results show that: the proportion of homonuclear sterile strains in 435 single-spore strains is 4.19%, wherein the proportion of 2 mating types A + and A-is 1:1.7.
in this embodiment, DNA prepared by hypha in a batch and rapidly is directly subjected to PCR amplification, so that the quality of a small amount of prepared DNA is unstable, which affects the amplification result, and in order to ensure that all detection results are obtained, some DNA with poor amplification effect needs to be repeatedly prepared and amplified.
In conclusion, the invention provides a three-primer composition for identifying the mating type of a single spore strain of Agaricus bisporus W192 and an identification method. Compared with the conventional method for identifying the mating type of the agaricus bisporus homokaryon sterile monospore strain in the prior art, the method has the advantages of short time consumption, high accuracy, simple and convenient operation, low cost, no need of enzyme digestion and no need of fruiting. By adopting the identification method provided by the invention, heterokaryon or homokaryon sterile strains and mating types thereof can be quickly identified in the monospore strain of the agaricus bisporus W192 strain, and the cross breeding efficiency of the agaricus bisporus can be greatly improved. By using the identification method of the invention, the time of 3 months required by conventional identification can be shortened to 1 week, and heterokaryotic strains which can not produce mushroom can be excluded.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A three-primer composition for identifying the mating type of a single spore strain of Agaricus bisporus W192, which is characterized by comprising: a first primer shown as SEQ ID NO.1, a second primer shown as SEQ ID NO.2 and a third primer shown as SEQ ID NO. 3.
2. A reagent or a kit for identifying the mating type of agaricus bisporus W192 monospore strain, which comprises the three-primer composition of claim 1.
3. The reagent or kit of claim 2, wherein the kit further comprises a PCR premix comprising a PCR Buffer and an enzyme cocktail.
4. A molecular tag comprising the three-primer composition of claim 1.
5. A method for identifying the mating type of a single spore strain of Agaricus bisporus W192, which comprises the following steps: simultaneously performing PCR amplification on DNA of a sample to be tested by using the three-primer composition of claim 1; if the PCR amplification product of the three-primer composition is a 204bp fragment, judging the homonuclear sterile strain with the mating type of A +; if the PCR amplification product of the three-primer composition is a 224bp fragment, judging the homonuclear sterile strain with the 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, the mating type of the heterokaryotic strain is judged to be A + A-.
6. The method of claim 5, wherein the PCR amplification conditions comprise: pre-denaturation at 94-95 deg.C for 1-5min; denaturation at 94-95 deg.C for 0.5-1min, annealing at 55-58 deg.C for 40-45s, extension at 70-72 deg.C for 0.5-1min, and 30-40 cycles.
7. The method of claim 6, wherein the pre-denaturation is carried out at 94 ℃ for 5min; denaturation at 94 ℃ for 1min, annealing temperature of the primers at 55 ℃, annealing time of 45s, extension at 72 ℃ for 1min, and 35 cycles; keeping the temperature at 72 ℃ for 5min.
8. The method of claim 5, wherein the PCR amplification system is: 0.25-1 mu L of first primer of 10-20 mu mol/L, 0.25-1 mu L of second primer of 10-20 mu mol/L, and 0.25-2 mu L of third primer of 10-20 mu mol/L; 5-10 mu L of PCR buffer,0.2-1 mu L of strain template DNA and the balance of water;
preferably, the PCR amplification system is as follows: 1 mu L of 10 mu mol/L first primer, 0.5 mu L of 10 mu mol/L second primer and 2 mu L of 10 mu mol/L third primer; 10. Mu.L of PCR buffer, 1. Mu.L of strain template DNA, and the balance water.
9. A test strip, wherein the three-primer composition of claim 1 is coated on the test strip.
10. Use of the three-primer composition of claim 1 in the preparation of a reagent, kit or strip.
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| CN118460784A (en) * | 2024-07-11 | 2024-08-09 | 鲁东大学 | Molecular marker assisted breeding method for identifying agaricus bisporus spore-free sites |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN118460784B (en) * | 2024-07-11 | 2024-09-27 | 鲁东大学 | Molecular marker assisted breeding method for identifying agaricus bisporus spore-free sites |
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