CN117737287A - Primer group for identifying mating type of agrocybe cylindracea AS-5 mononucleosome strain, identification method and application - Google Patents
Primer group for identifying mating type of agrocybe cylindracea AS-5 mononucleosome strain, identification method and application Download PDFInfo
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- CN117737287A CN117737287A CN202311785356.6A CN202311785356A CN117737287A CN 117737287 A CN117737287 A CN 117737287A CN 202311785356 A CN202311785356 A CN 202311785356A CN 117737287 A CN117737287 A CN 117737287A
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- 238000001962 electrophoresis Methods 0.000 claims abstract description 7
- 239000003147 molecular marker Substances 0.000 claims abstract description 6
- 244000045069 Agrocybe aegerita Species 0.000 claims description 23
- 108020004414 DNA Proteins 0.000 claims description 19
- 238000012408 PCR amplification Methods 0.000 claims description 14
- 238000012360 testing method Methods 0.000 claims description 9
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- 238000009402 cross-breeding Methods 0.000 abstract description 3
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- 239000001965 potato dextrose agar Substances 0.000 description 4
- 238000009396 hybridization Methods 0.000 description 3
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- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention discloses a primer group for identifying mating type of agrocybe cylindracea (Gantea AS-5) mononuclear body strains, an identification method and application thereof, and relates to the technical field of edible fungus molecular marker assisted breeding. The identification method specifically comprises the following steps: hypha culture, rapid preparation of genome DNA, PCR detection of mating, and electrophoresis detection to determine the result. Compared with the conventional method for identifying the mating type of the mononuclear body strain, the method has the advantages of short detection time, high accuracy and simplicity and convenience in operation. By using the identification method provided by the invention, the double-nucleus or single-nucleus strain and mating type thereof can be rapidly identified, the identification time is greatly shortened, the false detection rate is reduced, and the efficiency of agrocybe cylindracea cross breeding is improved.
Description
Technical Field
The invention relates to the technical field of edible fungus molecular marker assisted breeding, in particular to a primer group for identifying mating type of agrocybe cylindracea 'Gancha AS-5' mononuclear body strains, an identification method and application.
Background
Acquisition of monokaryons and mating type identification are important bases for agrocybe cylindracea cross breeding and genetic research work. Mating is a key regulatory factor in the sexual reproduction process of edible fungi, determines the affinities between two mononucleoid parents for hybridization, and only the affinities between the compatible mononucleoids can be hybridized to form a dinuclear offspring.
The identification of the traditional agrocybe cylindracea mononuclear bodies is generally judged by microscopic examination to confirm whether the lock-shaped combination exists, and the mating identification of the mononuclear bodies is judged by microscopic observation of the lock-shaped combination after the opposite culture of the mononuclear bodies. Many single-nucleus species are often involved in cross breeding, and many rounds of pairing are often labor intensive and time consuming. In addition, in the microscopic examination process, because of factors such as the formation time of the lock-shaped combination, the time for picking the colony, different positions and the like, the manual false detection is easy to occur, and the subsequent test is influenced. The molecular marker assisted selection has the advantages of simple operation, short time consumption, high accuracy and the like, and is a main method adopted in the mating identification of the edible fungus mononuclear body strain in recent years. At present, agrocybe cylindracea mononuclear strain and mating type identification thereof are not carried out by adopting a molecular marking method.
In view of this, the present invention has been made.
Disclosure of Invention
The first aim of the invention is to provide a primer group and an identification method for identifying the mating type of the agrocybe cylindracea 'Gancha AS-5' mononuclear body strain, so AS to quickly and accurately identify the mating type of the agrocybe cylindracea 'Gancha AS-5' mononuclear body strain.
The second object of the invention is to provide an application of a primer group for identifying mating type monokaryon strain of agrocybe cylindracea 'Gancha AS-5' in molecular markers, kits and test strips.
The invention is realized in the following way:
the invention provides a primer group for identifying mating type agrocybe cylindracea (Gansu tea AS-5) mononucleosome strains, which consists of a first primer pair and a second primer pair;
the nucleotide sequences of the first primer pair are shown as SEQ ID NO.1 and SEQ ID NO.2, and are as follows:
SEQ ID NO.1matA_F：5’-AGTGGGCATCCATCAGTGAG-3’；
SEQ ID NO.2matA_R：5’-TCTTTCGGGCTTTTTGCACTC-3’；
the nucleotide sequences of the second primer pair are shown as SEQ ID NO.3 and SEQ ID NO.4, and are:
SEQ ID NO.3matB_F：5’-GCTTCGCATCTTAATCCTGG-3’；
SEQ ID NO.4matB_R：5’-CGGCGTCACAATACGCATA-3’。
the agrocybe cylindracea AS-5 strain has a preservation unit name of China general microbiological culture Collection center, a preservation unit address of Beichen Xiyun No.1 and No.3 in the Korean area of Beijing, a preservation number of CGMCC No.40980, a preservation classification named Chaxingu (Agrocybe chaxingu) and a preservation date of 2023, 11, 15 days.
The invention also provides a method for identifying mating type of agrocybe cylindracea AS-5 mononuclear body strain, which comprises the following steps: PCR amplification is carried out on the DNA template of the sample to be detected by using the first primer pair and the second primer pair respectively,
if the product band amplified by the first primer pair is 131bp and the product band amplified by the second primer pair is 101bp, judging that the mating type is A1B1;
if the product band amplified by the first primer pair is 131bp and the product band amplified by the second primer pair is 84bp, judging that the mating type is A1B2;
if the product band amplified by the first primer pair is 141bp and the product band amplified by the second primer pair is 101bp, judging that the mating type is A2B1;
if the product band amplified by the first primer pair is 141bp and the product band amplified by the second primer pair is 84bp, judging that the mating type is A2B2;
if the first primer pair is amplified and has bands of 131bp and 141bp at the same time or the second primer pair is amplified and has bands of 101bp and 84bp at the same time, and the A factor and the B factor have 2 bands at the same time, judging that mating is a binuclear body.
Further, the above identification method further comprises a step of hypha culture and a step of extracting genomic DNA. Wherein the mycelium culture comprises transferring agrocybe cylindracea "Gancha AS-5" mononuclear body strain into potato dextrose agar medium PDA, and culturing at 25-28deg.C in dark place.
The genome extraction step comprises: when the colony grows to the size of 2-3 cm in diameter, a small amount of hypha is picked up by a sterilized gun head or an inoculating needle, the hypha is suspended in a 0.2ml centrifuge tube provided with 100 mu L TE buffer, and the mycelium is immediately placed on ice for cooling after being cracked for 5min at 95 ℃ on a PCR instrument for standby.
The amplification length of the first primer pair is 131bp and 141bp respectively for detecting the factor A, and the amplification length of the second primer pair is 84bp and 101bp respectively for detecting the factor B.
In a preferred embodiment of the invention, the DNA template of the sample to be tested is genomic DNA of the sample to be tested.
In a preferred embodiment of the invention, the method further comprises performing electrophoretic identification of the amplified product; preferably, the electrophoretic identification comprises adding a fluorescent dye to the amplification product or gel for identification of the amplification product or sequencing the amplification product.
In a preferred embodiment of the present invention, the PCR amplification conditions of the first primer pair and the second primer pair are: pre-denaturation at 94-98 ℃ for 1-5min, denaturation at 94-95 ℃ for 0.5-1.0min, annealing at 55-58 ℃ for 30s-60s, extension at 70-72 ℃ for 30-60s,30-40 cycles, and final extension at 70-72 ℃ for 5-10min.
Preferably, the PCR amplification conditions of the first primer pair and the second primer pair are as follows: pre-denaturation at 94℃for 5min, denaturation at 94℃for 30s, annealing at 58℃for 30s, extension at 72℃for 1min,35 cycles, and final extension at 72℃for 7min.
In a preferred embodiment of the invention, the PCR amplification system is: 10-20. Mu. Mol/L of the first primer pair 0.5-2. Mu.L (0.25-1. Mu.L each of forward and reverse primers), or 10-20. Mu. Mol/L of the second primer pair 0.5-2. Mu.L (0.25-1. Mu.L each of forward and reverse primers); 5-10 mu L of PCR buffer,1-2 mu L of sample DNA template to be detected, and the balance of ddH 2 O。
Preferably, the PCR amplification system is: 10. Mu. Mol/L of the first primer pair 2. Mu.L (1. Mu.L each of the forward and reverse primers), or 10. Mu. Mol/L of the second primer pair 2. Mu.L (1. Mu.L each of the forward and reverse primers), 10. Mu.L of PCR buffer, 2. Mu.L of DNA template of the sample to be tested, 6. Mu.L of ddH 2 O。
The system of PCR amplification may be set according to the actual amplification requirements, for example, a 20. Mu.L system.
The invention also provides a reagent or a kit prepared by using the primer group, wherein the reagent or the kit comprises a detection reagent, and the detection reagent comprises a first primer pair and a second primer pair.
The invention also provides a test strip prepared by using the primer group, and the test strip is coated with the first primer pair or the second primer pair.
The invention provides a molecular marker prepared by using the primer set, which comprises the first primer pair and the second primer pair.
The invention has the following beneficial effects:
the invention provides a primer group for identifying mating type agrocybe cylindracea AS-5 mononuclear body strain, an identification method and application. Compared with the conventional method for identifying the mating type of the mononuclear body in the prior art, the method has the advantages of simplicity and convenience in operation, short detection time and high accuracy. The identification method provided by the invention can greatly shorten the identification time of the mating type of the single-core body of the strain, and the traditional identification method generally takes about 2 months, and can be shortened to 1 week by using the identification method provided by the invention. Can also eliminate the double-nucleus bodies mixed in the conventional false detection, and obviously improve the hybridization breeding efficiency of agrocybe cylindracea.
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In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the amplification patterns of the mating type factor A of 24 putative spore monokaryon strains of the "Gancha tea AS-5" in example 1 of the present invention (1 indicates that the size of the amplified product is 131bp, which represents A1; 2 indicates that the size of the amplified product is 141bp, which represents A2);
FIG. 2 shows the amplification patterns of mating type factor B of 24 putative spore mononuclear body strains of "Gancha tea AS-5" in example 1 of the present invention (wherein 1 indicates that the size of the amplified product is 84bp, representing B2; 2 indicates that the size of the amplified product is 101bp, representing B1);
FIG. 3 is a microscopic view of the lock-like association structure characteristic of the binuclear body in example 1 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1
The embodiment provides a method for identifying the mating type of agrocybe cylindracea (Gancha tea AS-5) single-nucleus strain, wherein the agrocybe cylindracea (Gancha tea AS-5) strain is preserved in China center for culture collection of microorganisms (CGMCC NO. 40980) with a preservation date of 2023, 11 months and 15 days.
The method specifically comprises the following steps of:
(1) Hypha culture: 468 putative spore mononuclear strains of Agrocybe cylindracea "Gancha tea AS-5" were transferred to plates with potato dextrose agar PDA medium and grown in a 90mm diameter plate at 25℃in a dark place.
(2) Rapid preparation of genomic DNA: after 7 days of mycelium culture, 100 mu L of TE buffer is added into each sample tube of a 96-well plate, a small amount of mycelium is picked from the tip of an inoculating needle on each mononuclear body strain, and is respectively suspended in the sample tubes, the serial numbers are recorded, the 96-well plate is treated for 5 minutes at 95 ℃ by a PCR instrument, and the 96-well plate is immediately placed on ice for cooling for standby.
The method for rapidly preparing DNA by using hyphae in the embodiment of the invention can realize rapid identification of hyphae in a large scale, and in other embodiments, DNA extraction methods such as CTAB and the like can also be used for extracting hyphae DNA. The times and the purity of the extracted DNA can be reasonably adjusted according to the needs in the extraction of the genome DNA in the embodiment of the invention.
(3) Detecting mating type of the strain: and (3) carrying out PCR amplification on the DNA extracted in the step (2).
The PCR amplification system is as follows: a total volume of 20 μl, comprising: 2 XSanTaq PCR mix 10. Mu.L, 10. Mu. Mol/L forward and reverse primers 1. Mu.L each, extracted strain template DNA 2. Mu.L, ddH 2 O 6μL；
PCR reaction conditions for the first primer pair and the second primer pair: 94 ℃ for 5min;94 ℃ for 30s, the annealing temperature is 58 ℃, the annealing time is 30s, and the annealing time is 72 ℃ and extends for 1min for 35 cycles; extending at 72℃for 7min. Specific information on the primer set is shown in Table 1.
In Table 1, matA-F and matB-F represent forward primers of the first primer pair and the second primer pair, respectively, and matA-R and matB-R represent reverse primers of the first primer pair and the second primer pair.
TABLE 1 detailed information list of PCR primers
(4) And (3) electrophoresis detection: and (3) carrying out 3% agarose gel electrophoresis (Gold View coloring agent of 1/20000 is added in the agarose gel in advance) on the product obtained by the PCR amplification in the step (3), carrying out 110V voltage electrophoresis for 1h, and observing and recording the result by a photographic system.
The 2 pairs of primers in this example were PCR primers developed based on Indel sites of agrocybe cylindracea "Gancha AS-5" mating factors A and B region.
And (3) carrying out PCR amplification on all the mononuclear body strains by adopting 2 pairs of primers, and judging the mating type of each mononuclear body by recording the molecular weight of each pair of primer amplified bands and the existence of the bands. If the amplification products of the first primer pair and the second primer pair are 131bp and 101bp respectively, judging that the mating type is A1B1; if the amplification products of the first primer pair and the second primer pair are 131bp and 84bp respectively, judging that the mating type is A1B2; if the amplification products of the first primer pair and the second primer pair are 141bp and 101bp respectively, judging that the mating type is A2B1; if the amplification products of the first primer pair and the second primer pair are 141bp and 84bp respectively, judging that the mating type is A2B2; if the first primer pair is amplified and has bands of 131bp and 141bp at the same time or the second primer pair is amplified and has bands of 101bp and 84bp at the same time, and the A factor and the B factor have 2 bands at the same time, judging that mating is a binuclear body.
The electrophoresis pattern is shown with reference to fig. 1 and 2, and the electrophoresis result is that: there were a total of 420 spore mononucleosome strains among 468 putative mononucleosome strains, of which the ratio of four mating types A1B1, A2B2, A1B2, A2B1 was 30.00:16.43:18.33:35.24.
there are 48 strains, and when 2 pairs of primers are used for amplifying products, the A factor has 131bp and 141bp bands at the same time or the B factor has 101bp and 84bp bands at the same time, and the A factor and the B factor have 2 bands at the same time.
Further, microscopic examination of the mycelia of binuclear body was confirmed, and the microscopic examination result is shown in FIG. 3. In fig. 3, the arrows show the characteristic lock association phenomenon of the binuclear body, whereas the mononuclear body does not have this structure. Thus, these 48 strains were judged to be each of the binuclear strains.
In this example, the DNA prepared rapidly in batch from hyphae is directly subjected to PCR amplification, so that the quality of the DNA prepared in small amounts is unstable and affects the amplification result, and in order to ensure that all the detection results are obtained, the DNA with poor partial amplification effect needs to be repeatedly prepared and amplified. 25 strains are randomly selected from the mononuclear body group of each mating type to be matched with an compatible combination for hybridization one by one so as to verify the reliability of the molecular marker identification result. The test microscopic examination result shows that 150 pairs of hybrid combinations have the generation of the dual-nucleosome, namely mating type compatible of two paired single-nucleosome, and the PCR identification result is completely accurate.
In summary, the invention provides a primer group for identifying mating type of agrocybe cylindracea AS-5 mononuclear body strain, an identification method and application thereof. Compared with the conventional method for identifying the mating type of the mononuclear body in the prior art, the method has the advantages of simplicity and convenience in operation, short detection time and high accuracy. The identification method provided by the invention can greatly shorten the identification time of the mating type of the single-nucleosome of the strain, and can also eliminate the double-nucleosome mixed in the conventional false detection. By using the identification method of the present invention, the time of 1-2 months required for conventional identification can be shortened to 1 week, and false detection of the binuclear body can be eliminated.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. A primer set for identifying mating type of agrocybe cylindracea 'Gancha AS-5' mononucleosome strain, which is characterized by comprising a first primer pair and a second primer pair;
the nucleotide sequences of the first primer pair are shown as SEQ ID NO.1 and SEQ ID NO.2, and are as follows:
SEQ ID NO.1matA_F：5’-AGTGGGCATCCATCAGTGAG-3’；
SEQ ID NO.2matA_R：5’-TCTTTCGGGCTTTTTGCACTC-3’；
the nucleotide sequences of the second primer pair are shown as SEQ ID NO.3 and SEQ ID NO.4, and are:
SEQ ID NO.3matB_F：5’-GCTTCGCATCTTAATCCTGG-3’；
SEQ ID NO.4matB_R：5’-CGGCGTCACAATACGCATA-3’。
2. a method for identifying mating type of mononucleosome strain of agrocybe cylindracea "gan tea AS-5", comprising the steps of: performing PCR amplification on a DNA template of a sample to be tested by using the first primer pair and the second primer pair respectively;
if the product band amplified by the first primer pair is 131bp and the product band amplified by the second primer pair is 101bp, judging that the mating type is A1B1;
if the product band amplified by the first primer pair is 131bp and the product band amplified by the second primer pair is 84bp, judging that the mating type is A1B2;
if the product band amplified by the first primer pair is 141bp and the product band amplified by the second primer pair is 101bp, judging that the mating type is A2B1;
if the product band amplified by the first primer pair is 141bp and the product band amplified by the second primer pair is 84bp, judging that the mating type is A2B2;
if the first primer pair is amplified and has bands of 131bp and 141bp at the same time or the second primer pair is amplified and has bands of 101bp and 84bp at the same time, and the A factor and the B factor have 2 bands at the same time, judging that mating is a binuclear body.
3. The method of claim 2, wherein the DNA template of the test sample is genomic DNA of the test sample.
4. The method according to claim 2, wherein the method further comprises: and carrying out electrophoresis identification on the amplified product, wherein the electrophoresis identification comprises the steps of adding fluorescent dye into the amplified product or gel for identification of the amplified product, or carrying out sequencing identification on the amplified product.
5. The method of claim 2, wherein the PCR amplification conditions of the first primer pair and the second primer pair are: pre-denaturation at 94-98 ℃ for 1-5min, denaturation at 94-95 ℃ for 0.5-1.0min, annealing temperature of 55-58 ℃, annealing time of 30-60s, extension at 70-72 ℃ for 30-60s,30-40 cycles, and final extension at 70-72 ℃ for 5-10min.
6. The method of claim 5, wherein the PCR amplification conditions of the first primer pair and the second primer pair are: pre-denaturation at 94℃for 5min, denaturation at 94℃for 30s, annealing at 58℃for 30s, extension at 72℃for 1min,35 cycles, and final extension at 72℃for 7min.
7. A reagent or kit for identifying mating type of agrocybe cylindracea "Gancha AS-5" mononucleosome strain, wherein the reagent or kit comprises a detection reagent comprising the first primer pair and the second primer pair according to claim 1.
8. A test strip prepared by using the primer set as claimed in claim 1, wherein the test strip is coated with a first primer pair and a second primer pair.
9. A molecular marker for identifying a mononucleosome strain of agrocybe cylindracea "gan tea AS-5", comprising a first primer pair and a second primer pair according to claim 1.
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