Primer combination and method for identifying mating types of monokaryons of oyster mushroom strains
By using InDel primer combination for PCR amplification and electrophoresis analysis, the inefficiency and false detection of mononuclear mating type identification of oyster mushroom strains was solved, and rapid and accurate mating type identification was achieved.
Patent Information
- Application Number
- CN202510599388.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-11
- Publication Date
- 2025-08-08
AI Technical Summary
The method of identifying mononuclear mating types of oyster mushroom strains in the prior art is inefficient, the workload of artificial microscopy is large and prone to misdetection, making it difficult to quickly and accurately determine the mating types.
Two pairs of InDel primer combinations were used to molecularly identify the mononucleosomes of the oyster mushroom strain, and the mating type was determined by PCR amplification and electrophoresis analysis of the amplified fragment length.
It significantly shortens the identification time, improves the accuracy of the detection results, and can eliminate artificial mis-tested di-core bodies and mixed mononuclear bodies, and has high accuracy in the identification results.
Smart Images
Figure CN120442840A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and particularly relates to a primer combination and an identification method for the monokaryon mating type of an oyster mushroom strain. Background Art
[0002] Monokaryons are crucial intermediate materials for hybrid breeding research in Pleurotus ostreatus. Compatible monokaryon hyphae must pair to form dikaryons, and subsequently, fruiting bodies. Pleurotus ostreatus is a quaternary heterothallic edible fungus. Monokaryon affinity is controlled by the A and B mating-type loci, located on different chromosomes. If only one of the A and B genotype loci is identical, monokaryon mycelia are incompatible. Only monokaryon mycelia with different genotypes at both the A and B loci can form dikaryon progeny. Therefore, obtaining monokaryons of Pleurotus ostreatus strains and identifying their mating types are crucial aspects of hybrid breeding.
[0003] Currently, the main method for identifying the mating type of Pleurotus ostreatus involves culturing monokaryons in pairs and then manually observing them under a microscope to determine whether they exhibit a lock-like union. However, the hybridization process requires culturing and combining a large number of monokaryons. Manual microscopic examination is labor-intensive, inefficient, time-consuming, and prone to human error. Therefore, developing a more accurate and rapid identification method to replace manual mating type identification is a technical problem to be solved in this field. Summary of the Invention
[0004] In view of the problems existing in the prior art, the purpose of the present invention is to provide a primer combination and identification method for the mating type of the monokaryon of Pleurotus ostreatus strain, which greatly shortens the identification time, can eliminate the dikaryons and mixed monokaryons that are mistakenly detected by humans, and has high accuracy of the detection results.
[0005] The primer combination for identifying the monokaryon mating type of the Pleurotus ostreatus strain of the present invention comprises two pairs of InDel primer combinations, the sequences of which are:
[0006] The first pair of InDel primers:
[0007] Upstream primer: 5′-AGCATGAATGTCGAGGAAGG-3′;
[0008] Downstream primer: 5′-CACTTTCCGTCGCGTATTTTA-3′;
[0009] The second pair of InDel primers:
[0010] Upstream primer: 5′-TACCCCTTCTGTGACAACCA-3′;
[0011] Downstream primer: 5′-GCCATAGAAGCACTCGACTT-3′.
[0012] The method for identifying the monokaryon mating type of the Pleurotus ostreatus strain of the present invention uses the above two pairs of InDel primer combinations to perform molecular identification of the monokaryon mating type of the Pleurotus ostreatus strain.
[0013] Specifically, the method for identifying the monokaryon mating type of the Pleurotus ostreatus strain comprises the following steps:
[0014] (1) Extracting mycelial DNA from the monokaryon of Pleurotus ostreatus strains;
[0015] (2) Two pairs of InDel primers were used to perform PCR amplification on mycelial DNA to obtain amplified products;
[0016] (3) The amplified products were subjected to electrophoresis, and the genotype corresponding to the length of the amplified fragment was used to analyze and identify the monokaryon mating type of the Pleurotus ostreatus strain.
[0017] Preferably, the oyster mushroom strains include JC-3015, 8129, Xiaohei Ping, Teping No. 8, Suyan No. 1, Shuangkang (Hei Ping), Nongping 10, Nongfeng No. 5, Xintai-3, Zaoyou No. 8, Zaoqiu 903, Youping 88, Nongping 21, Kangbing 265, Hei Mudan, Gaochan 8105, Defeng No. 5, Chunqiu Kangwang, YH-3, and TB-10.
[0018] Preferably, in step (2), the PCR system used for PCR amplification is: 10 μL of PCR premixed enzyme, 1 μL of 10 μmol / L upstream primer and downstream primer, 2 μL of mycelial DNA extracted from the monokaryon of the Pleurotus ostreatus strain, and ddH2O added to make up to 20 μL.
[0019] Preferably, in step (2), the reaction procedure of PCR amplification is: preheating at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 40 s, 35 cycles, insulation at 72°C for 10 min, and storage at 4°C.
[0020] Preferably, in step (3), the PCR amplification product is subjected to electrophoresis on a 3% agarose gel at a voltage of U=110 V for 1 h.
[0021] Further preferably, in step (3), 4S Green Plus non-toxic nucleic acid dye is added to the agarose gel.
[0022] In step (3), the genotypes corresponding to the lengths of the amplified fragments are as follows: the length of the fragment amplified by the first pair of InDel primers is 217 bp, and the genotype is A1; the length of the fragment amplified by the first pair of InDel primers is 206 bp, and the genotype is A2; the length of the fragment amplified by the second pair of InDel primers is 329 bp, and the genotype is B1; the length of the fragment amplified by the second pair of InDel primers is 220 bp, and the genotype is B2.
[0023] In step (3), the mating type of the monokaryon of the Pleurotus ostreatus strain is a genotype combination corresponding to the length of the fragment amplified by the first pair of InDel primers and the length of the fragment amplified by the second pair of InDel primers.
[0024] For example, when identifying the Pleurotus ostreatus "Xintai-3" strain, if the length of the fragment amplified by the first pair of InDel primers is 217 bp and the length of the fragment amplified by the second pair of InDel primers is 329 bp, then its mating type is A1B1; when the length of the fragment amplified by the first pair of InDel primers is 217 bp and the length of the fragment amplified by the second pair of InDel primers is 220 bp, then its mating type is A1B2; when the length of the fragment amplified by the first pair of InDel primers is 206 bp and the length of the fragment amplified by the second pair of InDel primers is 329 bp, then its mating type is A2B1; when the length of the fragment amplified by the first pair of InDel primers is 206 bp and the length of the fragment amplified by the second pair of InDel primers is 329 bp, then its mating type is A2B1; when the length of the fragment amplified by the first pair of InDel primers is 217 bp and the length of the fragment amplified by the second pair of InDel primers is 220 bp, then its mating type is A1B2 .... When the first pair of InDel primers amplifies a fragment of 206 bp in length and the second pair of InDel primers amplifies a fragment of 220 bp in length, the mating type is A2B2. When the first pair of InDel primers amplifies fragments of 217 bp and 206 bp in length, and the second pair of InDel primers amplifies fragments of 329 bp and 220 bp in length, the mating type is a dikaryon (A1A2B1B2). When one of the first and second InDel primer pairs amplifies two fragments of the same length and the other amplifies only one fragment of the same length, the strain is judged to be a mixture of two monokaryons.
[0025] The working principle of the two-pair InDel primer combination of the present invention is that for dikaryon strains where both the first and second pairs of InDel primers can amplify two bands of different sizes, the mating types of their monokaryon strains can be distinguished. Strains where only the first pair of InDel primers amplifies two bands are only suitable for distinguishing the monokaryon A mating type, while strains where only the second pair of InDel primers amplifies two bands are only suitable for distinguishing the monokaryon B mating type. If both pairs of InDel primers amplify only one band in a strain, this InDel primer combination is not suitable for distinguishing the monokaryon mating type.
[0026] Compared with the prior art, the present invention has the following beneficial effects:
[0027] The present invention uses a molecular identification method using InDel primers. Compared with existing monokaryon mating type identification methods, the identification time is greatly shortened. The identification itself takes only one day, and mycelial culture only takes 5-6 days, with a total duration of less than one week. Traditional identification methods require at least one to two months and are performed through manual microscopy, which is prone to misidentification and has a high error rate. However, since the InDel primer molecular identification method does not rely on manual microscopy, it ensures the accuracy of the test results and can eliminate manually misidentified dikaryons and mixed monokaryons, which has a very broad application prospect. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Figure 1 This is the electrophoresis diagram of the amplification products of the first pair of InDel primers of spore monokaryons numbered 1-24 of the Pleurotus ostreatus "Xintai-3" strain in Example 1;
[0029] Figure 2 This is the electrophoresis diagram of the amplification products of the second pair of InDel primers of spore monokaryons numbered 1-24 of the Pleurotus ostreatus "Xintai-3" strain in Example 1;
[0030] Figure 3 This is the electrophoresis diagram of the amplification products of the first pair of InDel primers of spore monokaryons numbered 1-22 of the Pleurotus ostreatus "8129" strain in Example 2;
[0031] Figure 4 This is the electrophoresis diagram of the amplification products of the second pair of InDel primers of spore monokaryons numbered 1-22 of the Pleurotus ostreatus "8129" strain in Example 2;
[0032] Figure 5 The electrophoresis diagram of the amplified products of 40 Pleurotus ostreatus binucleate strains using the mating type primers A (the first pair of InDel primers);
[0033] Figure 6 This is the electrophoresis diagram of the amplification products of 40 Pleurotus ostreatus binucleate strains B mating type primers (the second pair of InDel primers). DETAILED DESCRIPTION
[0034] 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.
[0035] Example 1
[0036] The monokaryon mating type of the collected Pleurotus ostreatus "Xintai-3" strain was identified as follows:
[0037] (1) Extraction of mycelial DNA:
[0038] The spore monokaryons of the Pleurotus ostreatus "Xintai-3" strain were inoculated on a PDA plate with a diameter of 9 cm and cultured at a constant temperature of 25°C in the dark. When the colony grew to a diameter of about 1 cm, a small amount of mycelium was picked up with an inoculation needle and placed in a 96-well plate filled with 100uL 1×TE buffer. The plate was placed in a PCR instrument and maintained at 95°C for 5 minutes and 12°C for 30 seconds. The plate was then immediately placed on ice for cooling and repeatedly aspirated 20 times with a gun to quickly obtain genomic DNA.
[0039] To ensure the optimal use of DNA and the accuracy of the test results, DNA was prepared and used immediately. The UV absorbance of DNA at wavelengths of 260 nm and 280 nm was detected using a UV spectrophotometer. When the OD260 / 280 value was between 1.8 and 2.0, the DNA was of good purity and could be used for subsequent PCR amplification experiments.
[0040] (2) PCR amplification:
[0041] The sequences of the two pairs of InDel primer combinations used are:
[0042] The first pair of InDel primers:
[0043] Upstream primer: 5′-AGCATGAATGTCGAGGAAGG-3′;
[0044] Downstream primer: 5′-CACTTTCCGTCGCGTATTTTA-3′;
[0045] The second pair of InDel primers:
[0046] Upstream primer: 5′-TACCCCTTCTGTGACAACCA-3′;
[0047] Downstream primer: 5′-GCCATAGAAGCACTCGACTT-3′.
[0048] The mycelial DNA was PCR amplified using the above two pairs of InDel primer combinations. The PCR system for PCR amplification was as follows: 20 μL system, 10 μL of high-efficiency PCR premix enzyme (2×SanTaq PCR Mix premix, containing blue dye), 1 μL of 10 μmoL / L upstream primer and 1 μL of downstream primer, 2 μL of mycelial DNA extracted from the monokaryon of the Pleurotus ostreatus strain, and ddH2O was added to make up to 20 μL; the reaction procedure for PCR amplification was as follows: preheating at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 40 s, 35 cycles, insulation at 72°C for 10 min, and storage at 4°C.
[0049] The detailed information of the PCR amplification primer combination is shown in Table 1, where matAF is the upstream primer of the first pair of InDel primers, matAR is the downstream primer of the first pair of InDel primers, matBF is the upstream primer of the second pair of InDel primers, and matBR is the downstream primer of the second pair of InDel primers.
[0050] Table 1 PCR amplification primer combination information
[0051]
[0052] (3) Electrophoresis of amplified products:
[0053] The PCR amplification products were electrophoresed on a 3% agarose gel (4S Green Plus non-toxic nucleic acid dye was first added to the gel) at a voltage of U=110V for 1 h. After completion, the gel was observed and photographed using a gel imager.
[0054] The electrophoresis results of the amplification products of the two pairs of InDel primers of the spore monokaryons numbered 1-24 of the Pleurotus ostreatus "Xintai-3" strain in this example are as follows: Figure 1-2 shown.
[0055] from Figure 1 It can be seen that the mating type of strains numbered 1-4, 6, 7, 9, 11, 12, 14, 15, and 17 is A1, and the mating type of the remaining strains is A2; Figure 2 It can be seen that the mating type of strains numbered 3, 4, 6, 9, 11-13, 16, 17, 20, 21, and 23 is B1, and the mating type of the remaining strains is B2. Therefore, it can be concluded that among these 24 strains, the mating type is A1B1 in strains 3, 4, 6, 9, 11, 12, and 17, the mating type is A1B2 in strains 1, 2, 7, 14, and 15, the mating type is A2B1 in strains 13, 16, 20, 21, and 23, and the mating type is A2B2 in strains 5, 8, 10, 18, 19, 22, and 24.
[0056] In order to further verify the accuracy of the molecular identification results of the present invention, 5 strains were randomly selected from the monokaryon population of each mating type to form compatible combinations and hybridized one-to-one. The results of microscopic observation showed that all 10 hybrid combinations had dikaryon offspring, indicating that the mating types of the two paired monokaryons are compatible, and further indicating that the identification results of the present invention are completely accurate.
[0057] Example 2
[0058] The monokaryon mating type of the collected Pleurotus ostreatus "8129" strain was identified using the same method as in Example 1.
[0059] The electrophoresis of the amplification products of the two pairs of InDel primers of the spore monokaryons numbered 1-24 of the Pleurotus ostreatus "8129" strain in this example is as follows: Figure 3-4 shown.
[0060] from Figure 3 It can be seen that the mating type of strains numbered 1, 2, 5, 8, 9, 10, 12, 14, 17, and 21 is A1, and the mating type of the remaining strains is A2; Figure 4 It can be seen that the mating type of strains numbered 1, 4, 5, 18, 19, 20, 21, and 22 is B1, and the mating type of the remaining strains is B2. Therefore, it can be concluded that among these 22 strains, the mating type is A1B1 for strains 1, 5, and 21, the mating type is A1B2 for strains 2, 8, 9, 10, 12, 14, and 17, the mating type is A2B1 for strains 4, 18, 19, 20, and 22, and the mating type is A2B2 for strains 3, 6, 7, 11, 13, 15, and 16.
[0061] In order to further verify the accuracy of the molecular identification results of the present invention, 5 strains were randomly selected from the monokaryon population of each mating type to form compatible combinations and hybridized one-to-one. The results of microscopic observation showed that all 10 hybrid combinations had dikaryon offspring, indicating that the mating types of the two paired monokaryons are compatible, and further indicating that the identification results of the present invention are completely accurate.
[0062] Example 3
[0063] The mating types of the 40 collected Pleurotus ostreatus strains were identified using the same method as in Example 1.
[0064] The electrophoretic patterns of the amplified products of the A mating type primer (the first pair of InDel primers) and the B mating type primer (the second pair of InDel primers) of the 40 dikaryotic strains of Pleurotus ostreatus in this example are as follows: Figure 5-6 As shown; the oyster mushroom strains No. 1-40 in the figure are: 1. Xuemei F2; 2. Daye 39; 3. JC-3015; 4. G20; 5. June Hui; 6. 8129; 7. Zhongshu No. 10; 8. Zaoqiu Gaofeng; 9. Zaoguan No. 1; 10. Yupingwang; 11. Xinnong No. 1; 12. Xinke 205; 13. Xiaoheiping; 14. Xiahui No. 1; 15. Teping No. 8; 16. Suyan No. 1; 17. Shuangkang (Heiping); 18. Nongping 10; 19. Nongfeng No. 5; 20. Xintai 21. Pleurotus eryngii; 22. Zaoyou 8; 23. Early Autumn 903; 24. Youping 88; 25. Wenquan 1; 26. 16-3; 27. Nongping 21; 28. Lushanping 1-2; 29. Kejia 1; 30. Disease-resistant 265; 31. Black Peony; 32. High Temperature 908; 33. Gaoping 3; 34. High Yield 8105; 35. Fuzhouxia 200; 36. Fuxia 200; 37. Defeng 5; 38. Spring and Autumn Anti-King; 39. YH-3; 40. TB-10.
[0065] from Figure 5-6 It can be seen that in the amplification results of 13 strains numbered 3, 6, 15, 17, 19, 20, 22, 23, 24, 27, 30, 34, and 37, two pairs of InDel primers can amplify two bands, indicating that the two monokaryons in the dikaryons of these strains are in a heterozygous state at both sites A and B of the markers involved in the present invention, and there are obvious differences in fragment lengths, which can be used to identify monokaryons of different mating types;
[0066] The second pair of InDel primers for strains numbered 13, 16, 18, and 31 amplified two bands, while the first pair of primers only amplified one band, indicating that the strains were heterozygous at the B site and had no fragment length difference at the A site, thus only the B mating type could be identified.
[0067] For strains numbered 1, 2, 4, 5, 7, 8, 9, 10, 11, 12, 14, 21, 25, 26, 28, 29, 32, 33, 35, 36, 38, 39, and 40, both pairs of InDel primers could only amplify one band. Therefore, there was no difference in fragment length at the two marker sites, and this InDel primer combination could not be used for mating type identification.
Claims
1. A primer combination for identifying the monokaryon mating type of a Pleurotus ostreatus strain, characterized by: It includes two pairs of InDel primer combinations, whose sequences are: The first pair of InDel primers: Upstream primer: 5′-AGCATGAATGTCGAGGAAGG-3′; Downstream primer: 5′-CACTTTCCGTCGCGTATTTTA-3′; The second pair of InDel primers: Upstream primer: 5′-TACCCCTTCTGTGACAACCA-3′; Downstream primer: 5′-GCCATAGAAGCACTCGACTT-3′.
2. A method for identifying the monokaryon mating type of a Pleurotus ostreatus strain, characterized by: The two pairs of InDel primers according to claim 1 are used to perform molecular identification of the monokaryon mating type of the Pleurotus ostreatus strain.
3. The method for identifying the monokaryon mating type of a Pleurotus ostreatus strain according to claim 2, wherein: The following steps are involved: (1) Extracting mycelial DNA from the monokaryon of Pleurotus ostreatus strains; (2) Two pairs of InDel primers were used to perform PCR amplification on mycelial DNA to obtain amplified products; (3) The amplified products were subjected to electrophoresis, and the genotype corresponding to the length of the amplified fragment was used to analyze and identify the monokaryon mating type of the Pleurotus ostreatus strain.
4. The method for identifying the monokaryon mating type of a Pleurotus ostreatus strain according to claim 2 or 3, wherein: The oyster mushroom strains include JC-3015, 8129, Xiaohei Ping, Teping No. 8, Suyan No. 1, Shuangkang (Hei Ping), Nongping No. 10, Nongfeng No. 5, Xintai-3, Zaoyou No. 8, Zaoqiu 903, Youping 88, Nongping 21, Anti-disease 265, Black Peony, High-yield 8105, Defeng No. 5, Chunqiu Kangwang, YH-3, and TB-10.
5. The method for identifying the monokaryon mating type of a Pleurotus ostreatus strain according to claim 3, wherein: In step (2), the PCR system used for PCR amplification is: 10 μL of PCR premixed enzyme, 1 μL of 10 μmol / L upstream primer and downstream primer, 2 μL of mycelial DNA extracted from the monokaryon of the Pleurotus ostreatus strain, and ddH2O added to make up to 20 μL.
6. The method for identifying the monokaryon mating type of Pleurotus ostreatus strain according to claim 3, wherein: In step (2), the reaction procedure of PCR amplification is as follows: preheating at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 40 s, 35 cycles, insulation at 72°C for 10 min, and storage at 4°C.
7. The method for identifying the monokaryon mating type of a Pleurotus ostreatus strain according to claim 3, wherein: In step (3), the PCR amplification product was electrophoresed on a 3% agarose gel at a voltage of U=110 V for 1 h.
8. The method for identifying the monokaryon mating type of a Pleurotus ostreatus strain according to claim 7, wherein: In step (3), 4S Green Plus non-toxic nucleic acid dye is added to the agarose gel.
9. The method for identifying the monokaryon mating type of a Pleurotus ostreatus strain according to claim 3, wherein: In step (3), the genotypes corresponding to the lengths of the amplified fragments are as follows: the length of the fragment amplified by the first pair of InDel primers is 217 bp, and the genotype is A1; the length of the fragment amplified by the first pair of InDel primers is 206 bp, and the genotype is A2; the length of the fragment amplified by the second pair of InDel primers is 329 bp, and the genotype is B1; the length of the fragment amplified by the second pair of InDel primers is 220 bp, and the genotype is B2.
10. The method for identifying the monokaryon mating type of a Pleurotus ostreatus strain according to claim 3, wherein: In step (3), the mating type of the monokaryon of the Pleurotus ostreatus strain is a genotype combination corresponding to the length of the fragment amplified by the first pair of InDel primers and the length of the fragment amplified by the second pair of InDel primers.