A primer set for identifying the mating type of the monokaryon strain of Agrocybe gancha AS-5, an identification method and its application
By using specific primer sets for PCR amplification and electrophoresis detection, the mating type of "Gancha AS-5" mononuclear strain of tea tree mushroom "Gancha AS-5" was quickly and accurately identified, solving the problem of time-consuming and easy to detect in traditional methods, and improving the efficiency of hybrid breeding.
Patent Information
- Application Number
- CN202311785356.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-25
- Publication Date
- 2025-08-12
- Estimated Expiration
- 2043-12-25
AI Technical Summary
In the prior art, the identification of mating type of the tea tree mushroom mononuclear strain is time-consuming and prone to artificial mistesting, which affects the efficiency of hybrid breeding.
A specific primer set (first primer pair and second primer pair) was used for PCR amplification, combined with electrophoresis detection, and the mating type of the tea tree mushroom "Gancha AS-5" mononuclear strain was quickly identified. The primer set was shown by the nucleotide sequence of SEQ ID NO.1-4.
The identification time is shortened, the identification accuracy is improved, the efficiency of hybrid breeding of tea tree mushrooms is significantly improved, and the dinucleos mixed with routine missed detection is eliminated.
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Figure CN117737287B_ABST
Abstract
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 primer set, an identification method and an application for identifying the mating type of a monokaryon strain of Agrocybe oleracea "Gancha AS-5". Background Art
[0002] Obtaining monokaryons and identifying their mating types are crucial foundations for hybrid breeding and genetic research in Agrocybe oleracea. Mating type is a key regulatory factor in the sexual reproduction of edible fungi, determining the compatibility between the two monokaryon parents involved in the hybridization process. Only compatible monokaryons can hybridize to form dikaryon offspring.
[0003] Traditionally, the identification of Agrocybe tumefaciens monokaryons is usually determined by microscopic examination to confirm the presence of a lock-like union, while the identification of the monokaryon mating type is determined by microscopic observation of the lock-like union after culturing the monokaryons in pairs. Hybrid breeding often involves a large number of monokaryons, and multiple rounds of pairing are usually labor-intensive and time-consuming. In addition, during the microscopic examination process, due to factors such as the formation time of the lock-like union, the time and location of colony picking, etc., human misdetection is prone to occur, which affects subsequent experiments. Molecular marker-assisted selection has the advantages of simple operation, short time consumption, and high accuracy. It is the main method used in recent years for the identification of the mating type of edible fungi monokaryon strains. At present, there is no method using molecular markers to identify Agrocybe tumefaciens monokaryon strains and their mating types.
[0004] In view of this, the present invention is proposed. Summary of the Invention
[0005] The first purpose of the present invention is to provide a primer set and identification method for identifying the mating type of the monokaryon strain of Agrocybe tumefaciens "Gancha AS-5", so as to quickly and accurately identify the mating type of the monokaryon strain of Agrocybe tumefaciens "Gancha AS-5".
[0006] The second object of the present invention is to provide a primer set for identifying the mating type of the monokaryon strain of Agrocybe aegerita “Gancha AS-5” and its use in molecular markers, kits and test strips.
[0007] The present invention is achieved in that:
[0008] The present invention provides a primer set for identifying the mating type of the monokaryon strain of Agrocybe tumefaciens "Gancha AS-5", wherein the primer set consists of a first primer pair and a second primer pair;
[0009] The nucleotide sequences of the first primer pair are shown in SEQ ID NO.1 and SEQ ID NO.2, which are:
[0010] SEQ ID NO.1matA_F: 5'-AGTGGGCATCCATCAGTGAG-3';
[0011] SEQ ID NO.2matA_R: 5'-TCTTTCGGGCTTTTGCACTC-3';
[0012] The nucleotide sequences of the second primer pair are shown in SEQ ID NO.3 and SEQ ID NO.4, which are:
[0013] SEQ ID NO.3matB_F: 5'-GCTTCGCATCTTAATCCTGG-3';
[0014] SEQ ID NO. 4matB_R: 5'-CGGCGTCACAATACGCATA-3'.
[0015] The tea tree mushroom strain "Gancha AS-5" described in the present invention is deposited in the General Microbiology Center of the China Culture Collection Administration, the address of the depository is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit number is CGMCCNO.40980, the deposit classification name is Agrocybe chaxingu, and the deposit date is November 15, 2023.
[0016] The present invention also provides a method for identifying the mating type of the monokaryon strain of Agrocybe aegerita "Gancha AS-5", which comprises: using a first primer pair and a second primer pair to perform PCR amplification on a DNA template of a sample to be tested,
[0017] If the product band amplified by the first primer pair is 131 bp and the product band amplified by the second primer pair is 101 bp, the mating type is determined to be A1B1;
[0018] If the product band amplified by the first primer pair is 131 bp and the product band amplified by the second primer pair is 84 bp, the mating type is determined to be A1B2;
[0019] If the product band amplified by the first primer pair is 141 bp and the product band amplified by the second primer pair is 101 bp, the mating type is determined to be A2B1;
[0020] If the product band amplified by the first primer pair is 141 bp and the product band amplified by the second primer pair is 84 bp, the mating type is determined to be A2B2;
[0021] If the first primer pair amplifies bands of 131 bp and 141 bp simultaneously, or the second primer pair amplifies bands of 101 bp and 84 bp simultaneously, and both A and B factors have two bands simultaneously, the mating type is determined to be dikaryon.
[0022] Furthermore, the identification method further includes the steps of mycelial culture and genomic DNA extraction, wherein the mycelial culture comprises transferring the monokaryotic strain of Agrocybe aegerita "Gancha AS-5" to a potato dextrose agar (PDA) medium and culturing the mixture in a dark environment at 25-28°C.
[0023] The genome extraction step includes: when the colony grows to a diameter of 2-3 cm, a small amount of hyphae is picked up with a sterilized pipette tip or inoculation needle, the hyphae are suspended in a 0.2 ml centrifuge tube containing 100 μL TE buffer, and the hyphae are lysed at 95°C for 5 minutes on a PCR instrument, and then immediately placed on ice for cooling before use.
[0024] The amplification lengths of the first primer pair of the present invention are 131 bp and 141 bp, respectively, for detecting factor A, and the amplification lengths of the second primer pair are 84 bp and 101 bp, respectively, for detecting factor B.
[0025] In a preferred embodiment of the invention, the DNA template of the sample to be tested is the genomic DNA of the sample to be tested.
[0026] In a preferred embodiment of the invention, the method further comprises performing electrophoresis identification on the amplified product; preferably, the electrophoresis identification comprises adding a fluorescent dye to the amplified product or gel to identify the amplified product, or performing sequencing identification on the amplified product.
[0027] In a preferred embodiment of the present invention, the PCR amplification conditions for the first primer pair and the second primer pair are: pre-denaturation at 94-98°C for 1-5 min, denaturation at 94-95°C for 0.5-1.0 min, annealing temperature at 55-58°C, annealing time for 30s-60s, extension at 70-72°C for 30-60s, 30-40 cycles, and final extension at 70-72°C for 5-10 min.
[0028] Preferably, the PCR amplification conditions of the first primer pair and the second primer pair are: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing temperature at 58°C for 30 s, extension at 72°C for 1 min, 35 cycles, and final extension at 72°C for 7 min.
[0029] In a preferred embodiment of the present invention, the PCR amplification system is: 0.5-2 uL of the first primer pair (0.25-1 uL each of the forward and reverse primers) at 10-20 μmol / L, or 0.5-2 uL of the second primer pair (0.25-1 uL each of the forward and reverse primers) at 10-20 μmol / L; 5-10 μL of PCR buffer, 1-2 μL of the test sample DNA template, and the rest ddH2O.
[0030] Preferably, the PCR amplification system is: 2 μL of 10 μmol / L first primer pair (1 μL each of forward and reverse primers), or 2 μL of 10 μmol / L second primer pair (1 μL each of forward and reverse primers), 10 μL of PCR buffer, 2 μL of the test sample DNA template, and 6 μL of ddH2O.
[0031] The PCR amplification system can be set according to actual amplification needs, such as a 20 μL system.
[0032] The present invention also provides a reagent or a kit prepared using the primer set, wherein the reagent or the kit comprises a detection reagent, and the detection reagent comprises a first primer pair and a second primer pair.
[0033] The present invention also provides a test strip prepared using the primer set, wherein the test strip is coated with the first primer pair or the second primer pair.
[0034] The present invention provides a molecular marker prepared using the primer set, which comprises the first primer pair and the second primer pair.
[0035] The present invention has the following beneficial effects:
[0036] The present invention provides a primer set for identifying the mating type of the monokaryon strain "Gancha AS-5" of Agrocybe tumefaciens, an identification method and an application. Compared with the conventional monokaryon mating type identification method in the prior art, the present invention has the advantages of simple operation, short detection time and high accuracy. The identification method provided by the present invention can greatly shorten the identification time of the monokaryon mating type of the strain. The traditional identification method usually takes about 2 months, but the identification method of the present invention can shorten it to 1 week. It can also eliminate the binuclear bodies that are mixed in by conventional misdetection, and significantly improve the efficiency of hybrid breeding of Agrocybe tumefaciens. BRIEF DESCRIPTION OF THE DRAWINGS
[0037] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.
[0038] Figure 1 This is the amplification map of the mating type A factor of 24 random hypothetical spore monokaryon strains of "Gancha AS-5" in Example 1 of the present invention (1 in the figure indicates an amplification product size of 131 bp, representing A1; 2 in the figure indicates an amplification product size of 141 bp, representing A2);
[0039] Figure 2This is a B-factor amplification map of 24 random hypothetical spore monokaryon strains of the "Gancha AS-5" in Example 1 of the present invention (1 in the figure indicates an amplification product size of 84 bp, representing B2; 2 in the figure indicates an amplification product size of 101 bp, representing B1);
[0040] Figure 3 This is a microscopic image of the lock-like joint structure unique to the binuclear body in Example 1 of the present invention. DETAILED DESCRIPTION
[0041] 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 are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.
[0042] The features and performance of the present invention are further described in detail below with reference to the embodiments.
[0043] Example 1
[0044] This embodiment provides a method for identifying the mating type of the monokaryon strain of Agrocybe tumefaciens "Gancha AS-5". The Agrocybe tumefaciens "Gancha AS-5" strain described in the present invention is deposited with the Institute of Agricultural Applied Microbiology, Jiangxi Academy of Agricultural Sciences, on November 15, 2023, and is deposited in the General Microbiology Center of the China Culture Collection Administration Committee with a deposit number of CGMCC NO.40980.
[0045] Specifically, the following steps are performed in sequence:
[0046] (1) Mycelial culture: 468 hypothetical spore monokaryon strains of Agrocybe aegerita “Gancha AS-5” were transferred to a plate containing potato dextrose agar (PDA) medium and cultured at 25°C in the dark on a 90 mm diameter plate.
[0047] (2) Rapid preparation of genomic DNA: After 7 days of mycelial culture, add 100 μL of TE buffer to each sample tube in a 96-well plate. Use the tip of an inoculation needle to pick a small amount of mycelium from each monokaryon strain and suspend them in the sample tubes. Record the numbers and treat the 96-well plate at 95°C for 5 minutes using a PCR instrument. Immediately place the plate on ice to cool for later use.
[0048] The method for rapidly preparing DNA from mycelium in the embodiments of the present invention can achieve rapid identification of large quantities of mycelium. In addition, in other embodiments, DNA extraction methods such as CTAB can also be used to extract mycelial DNA. The number of extractions and purity of genomic DNA in the embodiments of the present invention can be reasonably adjusted as needed.
[0049] (3) Detecting the mating type of the strain: The DNA extracted in step (2) was amplified by PCR.
[0050] The PCR amplification system was as follows: total volume 20 μL, including: 2× SanTaq PCR mix 10 μL, 1 μL each of 10 μmol / L forward primer and reverse primer, 2 μL of extracted strain template DNA, and 6 μL of ddH2O;
[0051] PCR reaction conditions for the first and second primer pairs were: 94°C for 5 min, 94°C for 30 s, annealing temperature at 58°C for 30 s, extension at 72°C for 1 min, 35 cycles, and extension at 72°C for 7 min. Detailed information about the primer sets is shown in Table 1.
[0052] In Table 1, matA-F and matB-F represent the forward primers of the first primer pair and the second primer pair, respectively, and matA-R and matB-R represent the reverse primers of the first primer pair and the second primer pair.
[0053] Table 1 PCR primer details list
[0054]
[0055] (4) Electrophoresis detection: The product obtained by PCR amplification in step (3) was subjected to 3% agarose gel electrophoresis (1 / 20000 Gold View dye was added to the agarose gel in advance) at a voltage of 110 V for 1 h. The results were observed and recorded using a photographic system.
[0056] The two pairs of primers in this example are PCR primers developed based on the Indel sites in the mating factor A and B regions of the Agrocybe tumefaciens “Gancha AS-5”.
[0057] PCR amplification was performed on all monokaryon strains using two primer pairs. The mating type of each monokaryon was determined by recording the molecular weight and presence of the amplified bands for each primer pair. If the amplified products of the first and second primer pairs were 131 bp and 101 bp, respectively, the mating type was determined to be A1B1; if the amplified products of the first and second primer pairs were 131 bp and 84 bp, respectively, the mating type was determined to be A1B2; if the amplified products of the first and second primer pairs were 141 bp and 101 bp, respectively, the mating type was determined to be A2B1; if the amplified products of the first and second primer pairs were 141 bp and 84 bp, respectively, the mating type was determined to be A2B2; if the first primer pair amplified both 131 bp and 141 bp bands, or the second primer pair amplified both 101 bp and 84 bp bands, and if two bands were present for both factors A and B, the mating type was determined to be dikaryon.
[0058] Electropherogram reference Figure 1 and Figure 2 As shown, the electrophoresis results showed that there were 420 spore monokaryon strains among the 468 putative monokaryon strains, among which the ratios of the four mating types A1B1, A2B2, A1B2, and A2B1 were 30.00:16.43:18.33:35.24.
[0059] For 48 strains, when two pairs of primers were used to amplify the products, factor A had bands of 131bp and 141bp at the same time, factor B had bands of 101bp and 84bp at the same time, and both factors A and B had two bands at the same time.
[0060] Further confirmation was made by microscopic examination of binuclear hyphae. The results of microscopic examination were referred to Figure 3 shown. Figure 3 In the figure, the arrows indicate the lock-like union characteristic of dikaryons, which is absent in monokaryons. Therefore, all 48 strains were determined to be dikaryotic.
[0061] Because DNA rapidly prepared from batches of hyphae was directly subjected to PCR amplification in this example, there was a risk that the quality of a small amount of prepared DNA would be unstable, affecting the amplification results. To ensure complete test results, some DNA with poor amplification required repeated preparation and amplification. Twenty-five strains were randomly selected from the monokaryon population of each mating type to form compatible combinations and perform one-on-one hybridization to verify the reliability of the molecular marker identification results. Microscopic examination revealed that all 150 hybridization combinations produced dikaryon offspring, indicating that the mating types of the two paired monokaryons were compatible, and the PCR identification results were completely accurate.
[0062] In summary, the present invention provides a primer set, identification method, and application for identifying the mating type of the monokaryon strain of Agrocybe aegerita "Gancha AS-5". Compared with the conventional monokaryon mating type identification methods in the prior art, the present invention has the advantages of simple operation, short detection time, and high accuracy. The identification method provided by the present invention can greatly shorten the identification time of the monokaryon mating type of the strain, and can also eliminate the binuclear bodies that are mistakenly detected by conventional methods. 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 the binuclear bodies that are mistakenly detected can be eliminated.
[0063] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.
Claims
1. A primer set for identifying the mating type of the monokaryon strain of Agrocybe tumefaciens "Gancha AS-5", characterized in that: The primer set consists of a first primer pair and a second primer pair; The nucleotide sequences of the first primer pair are shown in SEQ ID NO.1 and SEQ ID NO.2, which are: SEQ ID NO.1 matA_F:5 ， -AGTGGGCATCCATCAGTGAG-3 ， ; SEQ ID NO.2 matA_R:5 ， -TCTTTCGGGCTTTTTGCACTC-3 ， ; The nucleotide sequences of the second primer pair are shown in SEQ ID NO.3 and SEQ ID NO.4, which are: SEQ ID NO.3 matB_F:5 ， -GCTTCGCATCTTAATCCTGG-3 ， 100. SEQ ID NO.4 matB_R:5 ， -CGGCGTCACAATAGCATA-3 ， 。 2. A method for identifying the mating type of the monokaryon strain of Agrocybe aegerita "Gancha AS-5", characterized in that: The method comprises the following steps: using the first primer pair and the second primer pair as claimed in claim 1 to perform PCR amplification on the DNA template of the sample to be tested; If the product band amplified by the first primer pair is 131 bp and the product band amplified by the second primer pair is 101 bp, the mating type is determined to be A1B1; If the product band amplified by the first primer pair is 131 bp and the product band amplified by the second primer pair is 84 bp, the mating type is determined to be A1B2; If the product band amplified by the first primer pair is 141 bp and the product band amplified by the second primer pair is 101 bp, the mating type is determined to be A2B1; If the product band amplified by the first primer pair is 141 bp and the product band amplified by the second primer pair is 84 bp, the mating type is determined to be A2B2; If the first primer pair amplifies both 131 bp and 141 bp bands or the second primer pair amplifies both 101 bp and 84 bp bands, and both A and B factors have two bands, the mating type is determined to be dikaryotic; The amplification lengths of the first primer pair are 131 bp and 141 bp, respectively, for detecting factor A; the amplification lengths of the second primer pair are 84 bp and 101 bp, respectively, for detecting factor B.
3. The method according to claim 2, characterized in that The DNA template of the sample to be tested is the genomic DNA of the sample to be tested.
4. The method according to claim 2, characterized in that The method further comprises: performing electrophoresis identification on the amplified product, wherein the electrophoresis identification comprises adding a fluorescent dye to the amplified product or gel to identify the amplified product, or performing sequencing identification on the amplified product.
5. The method according to claim 2, characterized in that The PCR amplification conditions of the first primer pair and the second primer pair are as follows: pre-denaturation at 94-98°C for 1-5 min, denaturation at 94-95°C for 0.5-1.0 min, annealing temperature at 55-58°C for 30-60 s, extension at 70-72°C for 30-60 s, 30-40 cycles, and final extension at 70-72°C for 5-10 min.
6. The method according to claim 5, wherein The PCR amplification conditions for the first primer pair and the second primer pair were as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing temperature at 58°C for 30 s, extension at 72°C for 1 min, 35 cycles, and final extension at 72°C for 7 min.
7. A reagent or kit for identifying the mating type of the monokaryon strain of Agrocybe tumefaciens "Gancha AS-5", characterized in that: The reagent or kit includes a detection reagent, and the detection reagent includes the first primer pair and the second primer pair as claimed in claim 1.
8. A test strip prepared using the primer set according to claim 1, characterized in that: The test strip is coated with a first primer pair and a second primer pair.