Primer combination and method for identifying mating types of mononuclear bodies of same strain of hericium erinaceus
By using InDel primer combination for PCR amplification and electrophoresis analysis, the problem of low mating type identification efficiency of Monocytic cervix was solved, and fast and accurate identification results were achieved.
Patent Information
- Application Number
- CN202510600240.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-30
- Publication Date
- 2025-08-01
AI Technical Summary
The method for identifying mononuclear mating in the prior art is inefficient, time-consuming and prone to human errors, and it is necessary to develop more efficient and accurate identification methods.
Two pairs of InDel primer combinations were used to molecularly identify the mononucleosomes of the same strain of cerves, and the mating type was determined by PCR amplification and electrophoresis.
The identification time is greatly shortened, the detection results are highly accurate, and manual mis-checking is avoided. The total time is less than one week, which significantly improves efficiency and accuracy compared with traditional methods.
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Figure CN120400408A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biotechnology, and particularly relates to an identification primer combination and an identification method for the mating type of mononuclears of the same strain of Hericium erinaceus. Background Art
[0002] The mononuclear mycelium of Hericium erinaceus is an important breeding material. Compatible mononuclear mycelia can form dikaryotic mycelia only after pairing, and then form fruiting bodies. Hericium erinaceus is a tetrapolar heterothallic edible mushroom. The mononuclear compatibility is jointly controlled by A mating type and B mating type loci distributed on different chromosomes. As long as one of the A and B mating type locus genotypes is the same, the mononuclear mycelia cannot pair. Only mononuclears with different genotypes at both the A and B mating type loci can be compatible to form dikaryotic offspring. Therefore, constructing a mononuclear material population and identifying the mating type of mononuclears are one of the important tasks in the breeding of Hericium erinaceus.
[0003] At present, the main method for identifying the mating type of Hericium erinaceus is to combine mononuclears in pairs for mating and microscopically examine the presence or absence of clamp connections. However, during the breeding process, a large number of combinations of mononuclears need to be mated. In this process, the workload of manual microscopic examination is huge, the efficiency is low, the time consumption is long, and human errors are prone to occur. Therefore, developing a more efficient and accurate identification method to replace the manual mating type identification is a technical problem to be solved in this field. Summary of the Invention
[0004] Aiming at the problems existing in the prior art, the purpose of the present invention is to provide an identification primer combination and an identification method for the mating type of mononuclears of the same strain of Hericium erinaceus, which greatly shortens the identification time, can exclude the dikaryons with artificial misdetection and the mixed mononuclears, and has high accuracy of detection results.
[0005] The identification primer combination for the mating type of mononuclears of the same strain of Hericium erinaceus described in the present invention includes two pairs of InDel primer combinations, and their sequences are respectively:[[]]
[0006] The first pair of InDel primers:
[0007] Upstream primer:
[0008] 5'-GGGCAAACTCAAGGAAGAACC-3';
[0009] Downstream primer: 5'-CGAAGACTCATCCACGCACC-3';
[0010] The second pair of InDel primers:
[0011] Upstream primer: 5'-CAGACGGAGAAAGACGACAGT-3';
[0012] Downstream primer: 5'-GACGCAGGTCAGGATACAAAG-3'.
[0013] The method for identifying the mating type of mononuclears of the same Hericium erinaceus strain in the present invention uses the above two pairs of InDel primer combinations to perform molecular identification on the mating type of mononuclears of the same Hericium erinaceus strain.
[0014] Specifically, the method for identifying the mating type of mononuclears of the same Hericium erinaceus strain includes the following steps:
[0015] (1) Extract the mycelial DNA of the mononuclears of the Hericium erinaceus strain;
[0016] (2) Use the two pairs of InDel primer combinations to perform PCR amplification on the mycelial DNA respectively to obtain amplification products;
[0017] (3) Electrophorese the amplification products, and analyze and identify the mating type of the mononuclears of the Hericium erinaceus strain through the genotypes corresponding to the amplified fragment lengths.
[0018] Preferably, the Hericium erinaceus strains include Hericium erinaceus 911, wild Hericium erinaceus in Qingzhou, Hericium erinaceus 920, Hericium erinaceus King, Hericium erinaceus No. 6, Hericium erinaceus 4903, Hericium erinaceus 4916, Hericium erinaceus 3003, Hericium erinaceus with long spines, Hericium erinaceus Jie No. 2, Hericium erinaceus 99, H8, Hericium erinaceus with long and sharp spines, and Hericium erinaceus in Central China.
[0019] Preferably, in step (2), the PCR system used for PCR amplification is: 5 μL of PCR premix enzyme, 0.5 μL each of the upstream primer and the downstream primer at 10 μmol / L, 1 μL of the mycelial DNA of the mononuclears of the Hericium erinaceus strain extracted, and supplemented with ddH2O to 10 μL.
[0020] Preferably, in step (2), the reaction program for PCR amplification is: preheat at 95°C for 5 min, denature at 95°C for 30 s, anneal the first pair of InDel primers at 58°C for 30 s, anneal the second pair of InDel primers at 55°C for 30 s, extend at 72°C for 40 s, 36 cycles, and keep warm at 72°C for 7 min.
[0021] Preferably, in step (3), the PCR amplification products are electrophoresed on a 3% agarose gel.
[0022] In step (3), the genotypes corresponding to the amplified fragment lengths are as follows: the amplified fragment length of the first pair of InDel primers is 282 bp, and the genotype is A1; the amplified fragment length of the first pair of InDel primers is 264 bp, and the genotype is A2; the amplified fragment length of the second pair of InDel primers is 198 bp, and the genotype is B1; the amplified fragment length of the second pair of InDel primers is 186 bp, and the genotype is B2.
[0023] In step (3), the mating type of the monokaryon of the Hericium erinaceus strain is the genotype combination corresponding to the amplified fragment lengths of the first pair of InDel primers and the amplified fragment lengths of the second pair of InDel primers.
[0024] For example, when identifying the Hericium erinaceus "Hericium 911" strain, if the amplified product of the first pair of InDel primers is 282 bp and the amplified product of the second pair of InDel primers is 198 bp, it indicates that its mating type is A1B1; when the amplified product of the first pair of InDel primers is 282 bp and the amplified product of the second pair of InDel primers is 186 bp, the mating type is A1B2; when the amplified product of the first pair of InDel primers is 264 bp and the amplified product of the second pair of InDel primers is 198 bp, its mating type is A2B1; when the amplified product of the first pair of InDel primers is 264 bp and the amplified product of the second pair of InDel primers is 186 bp, its mating type is A2B2; when the amplified products of the first pair of InDel primers of 282 bp, 300 bp and 264 bp exist simultaneously, and the amplified products of the second pair of InDel primers of 198 bp and 186 bp exist simultaneously, its mating type is the dikaryon of A1A2B1B2; when the products amplified by the first pair of InDel primers and the second pair of InDel primers are two products of one pair and one product of the other pair, it is determined that there are two kinds of monokaryons mixed in the mycelium.
[0025] The principle of action of the two pairs of InDel primer combinations of the present invention is as follows: for the dikaryon strains that can amplify two bands of different sizes using both the first pair of InDel primers and the second pair of InDel primers, the mating types of their monokaryon strains can be distinguished. The strains that only amplify two bands with the first pair of InDel primers are only suitable for distinguishing the mating type of monokaryon A, and the strains that only amplify two bands with the second pair of InDel primers are only suitable for distinguishing the mating type of monokaryon B. If the two pairs of InDel primers can only amplify one band, it indicates that this InDel primer combination is not suitable for distinguishing the mating type of monokaryons.
[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0027] The present invention adopts the molecular identification method of InDel primers. Compared with the existing identification methods of monokaryon mating types, it greatly shortens the identification time. The time required for the identification itself is only one day, and the mycelium culture only requires 5 - 6 days. The time required for the identification itself is only one day, and the total duration is less than one week, while the traditional identification method takes about two months. Moreover, since the InDel primer molecular identification method does not rely on manual microscopy, it avoids manual misdetection, thus ensuring the accuracy of the detection results and having a very broad application prospect. Description of the Drawings
[0028] Figure 1This is an electrophoresis diagram of the amplification products of the first pair of InDel primers of the Hericium erinaceus strain "Hericium erinaceus 911" and its spore monokaryotes numbered 1-23 in Example 1;
[0029] Figure 2 This is an electrophoresis diagram of the amplification products of the second pair of InDel primers of the Hericium erinaceus strain "Hericium erinaceus 911" and its spore monokaryons numbered 1-23 in Example 1;
[0030] Figure 3 This is a microscope observation of the lock-like joint structure unique to the binucleate body of Hericium erinaceus;
[0031] Figure 4 This is the electrophoresis diagram of the amplification products of the first pair of InDel primers of the Hericium erinaceus "Monkey 3003" strain and its spore monokaryons numbered 1-24 in Example 2;
[0032] Figure 5 This is the electrophoresis diagram of the amplification products of the second pair of InDel primers of the Hericium erinaceus "Monkey 3003" strain and its spore monokaryotes numbered 1-24 in Example 2;
[0033] Figure 6 The electrophoresis diagram of the amplification products of 14 Hericium erinaceus binuclear strains A mating type primers (the first pair of InDel primers);
[0034] Figure 7 The electrophoresis diagram of the amplification products of 14 Hericium erinaceus binuclear strains B mating type primers (the second pair of InDel primers);
[0035] In the figure, the lengths of the amplified fragments represented by M are 100 bp, 250 bp, and 500 bp from bottom to top. DETAILED DESCRIPTION
[0036] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described below with reference to specific embodiments.
[0037] Example 1
[0038] The monokaryon mating type of the collected Hericium erinaceus strain "Hericium erinaceus 911" was identified by the following method:
[0039] (1) Extraction of mycelial DNA:
[0040] Inoculate the spore mononucleate of the Hericium erinaceus strain "Hericium erinaceus 911" on a PDA plate (potato - dextrose - agar medium), and culture it at a constant temperature of 26°C in the dark. When the spore mononucleate colony grows to about 1 cm in diameter, preserve the spore mononucleate and scrape the mycelium. After picking the obtained "Hericium 911" spore mononucleate mycelium, add it to TE buffer, place it in a PCR instrument, treat it at 95°C for 5 min, and then immediately place it on ice and pipette it up and down 20 times to quickly obtain genomic DNA.
[0041] Use a Thermo Scientific NanoDrop spectrophotometer to detect the rapidly extracted DNA. If the pure DNA is between 1.8 - 2.0, it is considered that the DNA quality is qualified and subsequent experiments can be carried out.
[0042] (2) PCR amplification:
[0043] The sequences of the two pairs of InDel primer combinations used are as follows:
[0044] The first pair of InDel primers:
[0045] Forward primer: 5'-GGGCAAACTCAAGGAAGAACC-3';
[0046] Reverse primer: 5'-CGAAGACTCATCCACGCACC-3';
[0047] The second pair of InDel primers:
[0048] Forward primer: 5'-CAGACGGAGAAAGACGACAGT-3';
[0049] Reverse primer: 5'-GACGCAGGTCAGGATACAAAG-3'.
[0050] Use the above two pairs of InDel primer combinations to perform PCR amplification on the mycelium DNA respectively. The PCR system for PCR amplification is as follows: 5 μL of PCR premix enzyme, 0.5 μL each of 10 μmol / L forward primer and reverse primer, 1 μL of the mycelium DNA of the mononucleate of the Hericium erinaceus strain extracted, and make up to 10 μL with ddH2O. The reaction program for PCR amplification is: preheat at 95°C for 5 min, denature at 95°C for 30 s, anneal the first pair of InDel primers at 58°C for 30 s, anneal the second pair of InDel primers at 55°C for 30 s, extend at 72°C for 40 s, for 36 cycles, and incubate at 72°C for 7 min.
[0051] Details of the PCR amplification primer combinations are shown in Table 1. Among them, MatA-F is the upstream primer of the first pair of primers, MatA-R is the downstream primer of the first pair of primers, MatB-F is the upstream primer of the second pair of primers, and MatB-R is the downstream primer of the second pair of primers.
[0052] Table 1 Information of PCR Amplification Primer Combinations
[0053]
[0054] (3) Electrophoresis of amplification products:
[0055] Electrophorese the PCR amplification products on a 3% agarose gel, and after completion, observe and photograph with a gel imager.
[0056] The electrophoresis diagrams of the amplification products of the two pairs of InDel primers of the Hericium erinaceus strain "Hericium erinaceus 911" and its spore mononuclears numbered 1-23 in this example are respectively as Figure 1-2 shown.
[0057] From Figure 1 it can be seen that the mating types of numbers 1, 2, 3, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 19, 20, 21, 23 are A1, and the rest are A2; from Figure 2 it can be seen that the mating types of numbers 1, 4, 7, 12, 19, 22 are B1, and the rest are B2; therefore, it can be concluded that the mating types of numbers 1, 7, 12, 19 are A1B1, the mating types of numbers 2, 3, 8, 9, 10, 13, 14, 15, 16, 17, 20, 21, 23 are A1B2, the mating types of numbers 4, 22 are A2B1, and the mating types of numbers 5, 6, 11, 18 are A2B2.
[0058] Figure 1-2 All use the dikaryon strain "Hericium 911" (CK) as a control. In the results, the sizes of the bands amplified by the two pairs of primers are consistent with those of the control strain and the amplification fragment lengths. Therefore, the two pairs of primers can be used for the mating type identification of the spore mononuclear population of this strain.
[0059] To further verify the accuracy of the molecular identification results of the present invention, randomly select 5 strains from each mating type of mononuclear population to form compatible combinations and hybridize them one by one. The microscopic observation results show that all 10 pairs of hybrid combinations have dikaryon offspring, indicating that the mating types of the two paired mononuclears are compatible, and further indicating that the identification results of the present invention are completely accurate.
[0060] Example 2
[0061] Identify the mating type of the mononuclear body of the Hericium erinaceus strain "Hericium 3003" collected, and the method is the same as that in Example 1.
[0062] The electrophoretic results of the amplification products of two pairs of InDel primers for the spore mononucleates numbered 1 - 23 of the Hericium erinaceus strain "Hou 3003" in this example are respectively as follows Figure 4-5 shown.
[0063] From Figure 4 it can be seen that the mating types of No. 1, 3, 7, 12, 14, 15, 16 are A1, the mating types of No. 2, 4, 5, 6, 8, 10, 11, 13, 17, 18, 19, 21, 22, 23, 24 are A2, and double bands appear for No. 9 and 20; from Figure 5 it can be seen that the mating types of No. 4, 5, 6, 12, 14, 16 are B1, the mating types of No. 1, 2, 7, 10, 11, 15, 17, 18, 19, 21, 22, 23, 24 are B2, and triple bands appear for No. 3, 8, 9, 13, 20; therefore, it can be concluded that the mating types of No. 12, 14, 16 are A1B1, the mating types of No. 1, 7, 15 are A1B2, the mating types of No. 4, 5, 6 are A2B1, the mating types of No. 2, 10, 11, 17, 18, 19, 21, 22, 23, 24 are A2B2, No. 3 is a mixture of mononucleates of A1B1 and A1B2, No. 8 and 13 are mixtures of mononucleates of A2B1 and A2B2, and No. 9 and 20 are binucleates of A1A2B1B2.
[0064] Figure 4-5 The sizes of the bands amplified by the two pairs of primers in
[0065] are consistent with the amplified fragment lengths of the binucleates of No. 9 and 20. Therefore, the two pairs of primers can be used for the mating type identification of the spore mononuclear population of this strain.
[0066] Example 3
[0067] Continue to identify the mating types of mononucleates of various Hericium erinaceus strains by using the two pairs of InDel primer combinations and methods described in Example 1. The Hericium erinaceus strains include Hericium 911, Qingzhou wild Hericium erinaceus, Hericium 920, Hericium king, Hericium erinaceus No. 6, Hericium 4903, Hericium 4916, Hou 3003, Hericium echinatus, Houjie No. 2, Hericium 99, H8, Hericium echinatus with long spines, Hericium erinaceus in Central China.
[0068] The electrophoresis diagrams of the amplification products of the mating type primers (the first pair of InDel primers) and B mating type primers (the second pair of InDel primers) of the 14 Hericium erinaceus dikaryotic strains in this example are respectively as follows Figure 6-7 shown; the Hericium erinaceus strains numbered 1-14 in the figure are in turn: 1, Hericium erinaceus 911; 2, wild Hericium erinaceus in Qingzhou; 3, Hericium erinaceus 920; 4, Hericium erinaceus king; 5, Hericium erinaceus No. 6; 6, Hericium erinaceus 4903; 7, Hericium erinaceus 4916; 8, Hericium erinaceus 3003; 9, Hericium erinaceus with long spines; 10, Hericium erinaceus Jie No. 2; 11, Hericium erinaceus 99; 12, H8; 13, Hericium erinaceus with long spines; 14, Hericium erinaceus in Central China.
[0069] It can be seen from Figure 6-7 that among the amplification results of a total of 11 strains numbered 1, 3, 4, 5, 6, #7, 8, 9, 10, 13, and 14, both pairs of primers can amplify two bands, indicating that the two mononuclei in the dikaryon of these strains are heterozygous at both the A locus and the B locus in the markers involved in the present invention, and there are obvious fragment length differences, which can be used to identify mononuclei of different mating types; while for the strains numbered 11 and 12, the first pair of primers can amplify two bands, and the second pair of primers can only amplify a single band, indicating that the strain is heterozygous at the A locus, while there is no fragment length difference at the B locus, and only the A mating type can be identified; for the strain numbered 2, the first pair of primers can only amplify a single band, and the second pair of primers amplify a double band, indicating that there is no fragment length difference at the A locus and the B locus is heterozygous.
[0070] The present invention provides an InDel primer combination and an identification method for identifying the mating type of Hericium erinaceus mononuclei. Compared with the existing identification methods for the mating type of mononuclei, the present invention adopts the molecular identification means of InDel primers, which greatly shortens the identification time. The time required for the identification itself is only one day, and only 5-6 days are required for mycelium culture. The total duration is less than one week, while the traditional identification method takes about two months. Moreover, the traditional method is carried out by manual microscopic examination, which is prone to misidentification and has a high error rate. Since the InDel primer molecular identification means does not rely on manual microscopic examination, it ensures the accuracy of the detection results and can exclude the dikaryons with manual misdetection and the mixed mononuclei, and has a very broad application prospect.
Claims
1. A primer combination for identifying the mating type of mononuclears of the same strain of Hericium erinaceus, characterized in that: It includes two pairs of InDel primer combinations, and their sequences are respectively: The first pair of InDel primers: Forward primer: 5'-GGGCAAACTCAAGGAAGAACC-3'; Reverse primer: 5'-CGAAGACTCATCCACGCACC-3'; The second pair of InDel primers: Forward primer: 5'-CAGACGGAGAAAGACGACAGT-3'; Reverse primer: 5'-GACGCAGGTCAGGATACAAAG-3'.
2. A method for identifying the mating type of mononuclears of the same strain of Hericium erinaceus, characterized in that: Use the two pairs of InDel primer combinations described in Claim 1 to conduct molecular identification on the monokaryotic mating type of the same strain of Hericium erinaceus.
3. The identification method of the mating type of the mononuclear body of the same strain of Hericium erinaceus according to claim 2, wherein: It includes the following steps: (1) Extract the mycelial DNA of the monokaryotic of the Hericium erinaceus strain; (2) Use the two pairs of InDel primer combinations to perform PCR amplification on the mycelial DNA respectively to obtain amplification products; (3) Electrophorese the amplification products, and analyze and identify the monokaryotic mating type of the Hericium erinaceus strain through the genotypes corresponding to the amplified fragment lengths.
4. The identification method of the mating type of the mononuclear body of the same Hericium erinaceus strain according to claim 2 or 3, characterized in that: The Hericium erinaceus strains include Hericium 911, Qingzhou wild Hericium erinaceus, Hericium 920, Hericium king, Hericium erinaceus No. 6, Hericium 4903, Hericium 4916, Monkey 3003, Hericium echinulata, Hericium jie No. 2, Hericium 99, H8, Spiny long Hericium erinaceus, Central China Hericium erinaceus.
5. The identification method of the mating type of single nuclei of the same strain of Hericium erinaceus according to claim 3, characterized in that: In step (2), the PCR system used for PCR amplification is: 5 μL of PCR premix enzyme, 0.5 μL each of 10 μmol / L forward primer and reverse primer, 1 μL of the mycelial DNA of the monokaryotic of the Hericium erinaceus strain, and supplemented with ddH2O to 10 μL.
6. The method for identifying the mating type of mononuclear bodies of the same strain of Hericium erinaceus according to claim 3, characterized in that: In step (2), the reaction program for PCR amplification is: preheat at 95°C for 5 min, denature at 95°C for 30 s, anneal the first pair of InDel primers at 58°C for 30 s, anneal the second pair of InDel primers at 55°C for 30 s, extend at 72°C for 40 s, 36 cycles, and incubate at 72°C for 7 min.
7. The method for identifying the mating type of mononuclear bodies of the same Hericium erinaceus strain according to claim 3, characterized in that: In step (3), electrophorese the PCR amplification products through 3% agarose gel.
8. The method for identifying the mating type of mononucleate of the same strain of Hericium erinaceus according to claim 3, characterized in that: In step (3), the genotypes corresponding to the amplified fragment lengths are as follows: the amplified fragment length of the first pair of InDel primers is 282 bp, and the genotype is A1; the amplified fragment length of the first pair of InDel primers is 264 bp, and the genotype is A2; the amplified fragment length of the second pair of InDel primers is 198 bp, and the genotype is B1; the amplified fragment length of the second pair of InDel primers is 186 bp, and the genotype is B2.
9. The identification method of the mating type of the mononuclear body of the same strain of Hericium erinaceus according to claim 3, characterized in that: In step (3), the mating type of the monokaryotic of the Hericium erinaceus strain is the genotype combination corresponding to the amplified fragment lengths of the first pair of InDel primers and the second pair of InDel primers.