CN114540535B - Molecular marker for rapidly identifying auricularia polytricha and application thereof - Google Patents
Molecular marker for rapidly identifying auricularia polytricha and application thereof Download PDFInfo
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- 240000005710 Auricularia polytricha Species 0.000 title claims abstract description 65
- 235000000024 Auricularia polytricha Nutrition 0.000 title claims abstract description 65
- 239000003147 molecular marker Substances 0.000 title claims abstract description 18
- 239000002773 nucleotide Substances 0.000 claims abstract description 7
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 7
- AWXGSYPUMWKTBR-UHFFFAOYSA-N 4-carbazol-9-yl-n,n-bis(4-carbazol-9-ylphenyl)aniline Chemical compound C12=CC=CC=C2C2=CC=CC=C2N1C1=CC=C(N(C=2C=CC(=CC=2)N2C3=CC=CC=C3C3=CC=CC=C32)C=2C=CC(=CC=2)N2C3=CC=CC=C3C3=CC=CC=C32)C=C1 AWXGSYPUMWKTBR-UHFFFAOYSA-N 0.000 claims abstract description 4
- 101000837344 Homo sapiens T-cell leukemia translocation-altered gene protein Proteins 0.000 claims abstract description 4
- 102100028692 T-cell leukemia translocation-altered gene protein Human genes 0.000 claims abstract description 4
- 235000000023 Auricularia auricula Nutrition 0.000 claims description 16
- 241001149430 Auricularia auricula-judae Species 0.000 claims description 16
- 241000221377 Auricularia Species 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 13
- 230000003321 amplification Effects 0.000 claims description 12
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- 238000001962 electrophoresis Methods 0.000 claims description 3
- 238000011161 development Methods 0.000 abstract description 3
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 108020004414 DNA Proteins 0.000 description 14
- 241000233866 Fungi Species 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 3
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
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- 238000012408 PCR amplification Methods 0.000 description 2
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- 238000002955 isolation Methods 0.000 description 2
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- JEOQACOXAOEPLX-WCCKRBBISA-N (2s)-2-amino-5-(diaminomethylideneamino)pentanoic acid;1,3-thiazolidine-4-carboxylic acid Chemical compound OC(=O)C1CSCN1.OC(=O)[C@@H](N)CCCN=C(N)N JEOQACOXAOEPLX-WCCKRBBISA-N 0.000 description 1
- 241001057324 Auricularia villosula Species 0.000 description 1
- 241000221454 Auriculariaceae Species 0.000 description 1
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241001480058 Quercus glauca Species 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
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- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- ZZUFCTLCJUWOSV-UHFFFAOYSA-N furosemide Chemical compound C1=C(Cl)C(S(=O)(=O)N)=CC(C(O)=O)=C1NCC1=CC=CO1 ZZUFCTLCJUWOSV-UHFFFAOYSA-N 0.000 description 1
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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Abstract
The application discloses a molecular marker for rapidly identifying auricularia polytricha and application thereof, and belongs to the technical field of molecular markers. The molecular marked nucleotide is shown as SEQ ID NO:1, the 120 th bit of the sequence is C, and the 130 th to 133 th bits are TCTA; the molecular marker is used for designing a specific primer pair, and the nucleotide sequence of the specific primer pair is shown as SEQ ID NO: 5-6. The application can distinguish Auricularia polytricha from other Auricularia polytricha by utilizing the specific primer pair, which provides technical support for definitely identifying artificial cultivars of Auricularia polytricha, auricularia polytricha and the like, and is beneficial to the protection and sustainable development of wild resources of Auricularia polytricha.
Description
Technical Field
The application relates to the technical field of molecular markers, in particular to a molecular marker for rapidly identifying auricularia polytricha and application thereof.
Background
Auricularia polytricha is an approximate species of Auricularia polytricha, belonging to Auricularia of Auriculariaceae of Auricularia, and is a wild fungus for both medicine and food, and is mainly distributed in tropical subtropical region. Currently, most of short auricularia polytricha circulated in the market is a collected wild dry product, is very similar to black auricularia polytricha in appearance, is difficult to distinguish, and seriously influences the protection and sustainable development of short auricularia polytricha resources.
Although the ISSR analysis technology can distinguish different species of Auricularia, the technology also has the defects of unstable band, uncertain characteristic band sequence and the like, and a plurality of primers are often needed for distinguishing, so that the process is complicated. Therefore, it is necessary to develop a molecular marker capable of simply, rapidly and clearly identifying the artificially cultivated species of auricularia polytricha, auricularia auricula and the like, and rapidly distinguishing auricularia polytricha.
Disclosure of Invention
The application aims to provide a molecular marker for rapidly identifying auricularia polytricha and application thereof, so as to solve the problems in the prior art, and the purpose of specifically identifying auricularia polytricha is realized by screening the molecular marker of auricularia polytricha and designing specific primers so as to accurately distinguish auricularia polytricha from other auricularia polytricha of auricularia.
In order to achieve the above object, the present application provides the following solutions:
the application provides a molecular marker for identifying Auricularia polytricha, and the nucleotide of the molecular marker is shown as SEQ ID NO: 1. the 120 th bit of the sequence is shown as C, and the 130 th to 133 th bits are shown as TCTA.
The application also provides an amplification primer pair for identifying the auricularia polytricha molecular marker, and the nucleotide sequence of the amplification primer pair is shown as SEQ ID NO: 5-6.
The application also provides a kit comprising the primer pair.
The application also provides a method for identifying Auricularia polytricha, which comprises the following steps:
and amplifying the target fragment by using the genomic DNA of the auricularia auricula to be detected as a template and using the primer pair, detecting by electrophoresis, and judging whether the auricularia auricula is short or not according to the detected result.
Preferably, the nucleotide sequence of the target fragment is shown as SEQ ID NO:2 or comprises the sequence set forth in SEQ ID NO:2, and a gene fragment having the sequence shown in seq id no.
Preferably, the amplification reaction system is: 2X Flash Hot Star Master Mix. Mu.L, ddH 2 O12. Mu.L, 1. Mu.L each of the upstream primer F/downstream primer R, 1. Mu.L of R-primer and 1. Mu.L of DNA template.
Preferably, the amplification reaction procedure is: pre-denaturing for 3min at 95℃and denaturing for 30s at 95 ℃; annealing at 56 ℃ for 30s; extending at 72 ℃ for 30s;29 cycles, 7min at 72 ℃.
The application also provides application of the molecular marker or the primer pair or the kit or the method in breeding the Auricularia polytricha variety.
The application discloses the following technical effects:
according to the application, the molecular marker capable of identifying the auricularia polytricha is obtained through screening, a pair of specific primers is designed by utilizing mutation sites of the molecular marker, 293bp specific target fragments can be amplified specifically by utilizing the pair of specific primers, and the auricularia polytricha is distinguished from other auricularia polytricha of auricularia, so that technical support is provided for definitely identifying artificial cultivars such as auricularia polytricha and auricularia auricula, and the method is beneficial to the protection and sustainable development of wild resources of auricularia polytricha.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the results of identifying different Auricularia auricula varieties by using the molecular markers of the present application; 1: black fungus 916;2: black fungus No. Gui Yun; 3: auricularia polytricha 193;4: auricularia polytricha Ty081;5: auricularia polytricha 12;6: auricularia auricula 504;7: auricularia polytricha TE011;8: auricularia auricula 503;9: black Auricularia auricular (L.) Underspra (L.) Unders; 10: auricularia polytricha 1413;11: auricularia polytricha 1416:12: black fungus "New family No. 4".
Detailed Description
Various exemplary embodiments of the application will now be described in detail, which should not be considered as limiting the application, but rather as more detailed descriptions of certain aspects, features and embodiments of the application.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the application. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the application described herein without departing from the scope or spirit of the application. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present application. The specification and examples of the present application are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
Example 1 isolation and identification of Auricularia polytricha Ty081
(1) Fruiting body acquisition
The plant is collected on cyclobalanopsis glauca in Guangxi Zhuang nationality, bai-color city, tianlin county, badu county and six chapter villages on 5 months and 6 days.
(2) Tissue isolation and purification
Washing impurities on the fruiting body with flowing water, and wiping off water. Placing in 75% alcohol, and sterilizing for 3-5min. Cleaning surface with sterilized water for 3-5 times on an ultra clean bench, air drying, cutting fruiting body with a blade, tearing the inner layer of fruiting body, cutting into blocks of 0.5-1cm, attaching the inner layer on PDA separation and purification culture medium culture dish, dark culturing at 25deg.C for 3-5 days, removing the contaminated culture dish, and picking up the culture dish with no pollution and good growth to obtain strain. And then the pure strain of Auricularia polytricha is screened out by using a PDA culture medium through a conventional separation and purification method.
(3) Identification of strains
DNA is extracted from the fruiting body to be tested and the corresponding mycelium according to a CTAB method, PCR amplification and ITS sequence determination are carried out, and pure culture obtained by separation is determined to be the auricularia polytricha mycelium through analysis of the Blast comparison samples ITS and auricularia polytricha (Auricularia villosula) identifiers=525/528 (99%), caps=1/528 (0%).
Biological traits: the fruiting body size is 6.33cm x 7.46cm; the ear base is small, and the ear piece is thinner; fruiting body is flaky, clustered, and has obvious wrinkles. The fruiting body has elasticity, colloid, translucency, discoid shape, yellow brown color, and dry and thin innovation, and is grey brown or dark brown. The sterile mask is soft and has no marrow layer in cross section.
The separated Auricularia polytricha is preserved in China general microbiological culture Collection center (CGMCC) in the 2018 01 month 29 day, and the preservation address is CGMCC No.15278 of China academy of sciences of national academy of sciences of China No. 3 of the North-West-Lu No.1 of the Korean area of Beijing.
Example 2 Auricularia polytricha molecular marker screening
Extracting DNA of Auricularia polytricha, auricularia polytricha and Auricularia polytricha (see table 1), and performing PCR amplification reaction by using the following general primers:
ITS1(F-primer，SEQ ID NO：3)：5’-TCCGTAGGTGAACCTGCGG-3’；
ITS4(R-primer，SEQ ID NO：4)：5’-TCCTCCGCTTATTGATATGC-3’。
amplification reaction system: 2X Flash Hot Star Master Mix. Mu.L, ddH 2 O12. Mu.L, F-primer 1. Mu.L, R-primer 1. Mu.L and DNA template 1. Mu.L.
Amplification reaction procedure: pre-denaturing for 3min at 95℃and denaturing for 30s at 95 ℃; annealing at 56 ℃ for 30s; extending at 72 ℃ for 30s;29 cycles, 7min at 72 ℃.
The PCR product obtained by amplifying the conditions is used for sequencing the amplified gene fragment (shown as SEQ ID NO: 1), and the sequencing result shows that: the Auricularia polytricha ITS sequence 120 is C, 130-133 is TCTA, and is different from the sequences of other Auricularia polytricha varieties such as Auricularia polytricha, auricularia polytricha and the like, and the mutation is generated. Therefore, a specific primer for identifying the auricularia polytricha is designed by the mutation site:
an upstream primer F (SEQ ID NO: 5): 5'-CGAAAGTCCAGAATGTGATCTA-3';
the downstream primer R (SEQ ID NO: 6): 5'-AGCACTCTAAAAGCGCCAGCTAATG-3'.
SEQ ID NO:1 is as follows:
GCCTTAGGCCTTGCCTTCAGCTGTGCGCTTCGGCTGCACGCTGGATAAAATCTCACACCT GTGCACCTTTTCGGTTGCGGTTTCGGCCGCTTCCGCTTTTACATTCAACCACGAAAGTCCAGAATGTGATCTAAACTATAAAGTAACAACTTTCAACAACGGATCTCTTGGCTCTCGCATCGATGAAG AACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACCTTGCGCTCCTTGGTATTCCATGGAGCATGCCTGTTTGAGTGTCACGTAAACCCTCACCC CTGCGATGTAACAGTCGCGTGCGGTGGACTTGGACTGTGCCGTAATCGGCTGGTCTTGAAATGCATTAGCTGGCGCTTTTAGAGTGCTGGGCGAACGGTGTGATAATTATCTGCGCCGACTGCCCTGG GCCTCTTCAGCGGCGCTGCTTACAGCCGTCCCTCGTGGACAACTATTTTAAAGCTTGGCCTCAA ATCA。
example 3 application of Auricularia polytricha molecular marker
1. Material
Different kinds of agaric shown in table 1 are selected as the public materials.
TABLE 1 Auricularia auricula variety
2. Specificity of Auricularia auricula molecular markers
Extracting the DNA of the different agaric in the table 1 by using a fungus DNA extraction kit (OMEGA), and amplifying by using the DNA as a template and using the following specific primers and reaction conditions, wherein the specific primers are as follows:
an upstream primer F5'-CGAAAGTCCAGAATGTGATCTA-3';
the downstream primer R is 5'-AGCACTCTAAAAGCGCCAGCTAATG-3'.
Amplification reaction system: 2X Flash Hot Star Master Mix. Mu.L, ddH 2 O12. Mu.L, 1. Mu.L each of the upstream primer F/downstream primer R, 1. Mu.L of R-primer and 1. Mu.L of DNA template.
Amplification reaction procedure: pre-denaturing for 3min at 95℃and denaturing for 30s at 95 ℃; annealing at 56 ℃ for 30s; extending at 72 ℃ for 30s;29 cycles, 7min at 72 ℃.
Meanwhile, the universal primers and the amplification conditions are used for amplifying different auricularia auricula varieties in table 1 to serve as a control. Universal primer:
ITS1(F-primer)：5’-TCCGTAGGTGAACCTGCGG-3’；
ITS4(R-primer)：5’-TCCTCCGCT TATTGATATGC-3’。
the gene fragment (shown as SEQ ID NO:2, 293 bp) obtained by amplification with the specific primer and the gene fragment (shown as SEQ ID NO:1, 512 bp) obtained with the universal primer were respectively subjected to electrophoresis detection, and the results are shown as figure 1.
SEQ ID NO:2 is as follows:
CGAAAGTCCAGAATGTGATCTAAACTATAAAGTAACAACTTTCAACAACGGATCTCTTGG CTCTCGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACCTTGCGCTCCTTGGTATTCCATGGAGCATGCCTGTTTGAGTGT CACGTAAACCCTCACCCCTGCGATGTAACAGTCGCGTGCGGTGGACTTGGACTGTGCCGTAATC GGCTGGTCTTGAAATGCATTAGCTGGCGCTTTTAGAGTGCT。
3. results and analysis
As can be seen from FIG. 1, all the Auricularia auricular varieties can be amplified by using the universal primers, and different Auricularia auricular varieties cannot be distinguished, but only Auricularia auricular short Auricularia auricular with the specific primers designed by the application can be amplified, and Auricularia auricular short Auricularia auricular and other Auricularia auricular varieties can be distinguished.
The above embodiments are only illustrative of the preferred embodiments of the present application and are not intended to limit the scope of the present application, and various modifications and improvements made by those skilled in the art to the technical solutions of the present application should fall within the protection scope defined by the claims of the present application without departing from the design spirit of the present application.
Sequence listing
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<120> molecular marker for rapidly identifying Auricularia polytricha and application thereof
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gccttaggcc ttgccttcag ctgtgcgctt cggctgcacg ctggataaaa tctcacacct 60
gtgcaccttt tcggttgcgg tttcggccgc ttccgctttt acattcaacc acgaaagtcc 120
agaatgtgat ctaaactata aagtaacaac tttcaacaac ggatctcttg gctctcgcat 180
cgatgaagaa cgcagcgaaa tgcgataagt aatgtgaatt gcagaattca gtgaatcatc 240
gaatctttga acgcaccttg cgctccttgg tattccatgg agcatgcctg tttgagtgtc 300
acgtaaaccc tcacccctgc gatgtaacag tcgcgtgcgg tggacttgga ctgtgccgta 360
atcggctggt cttgaaatgc attagctggc gcttttagag tgctgggcga acggtgtgat 420
aattatctgc gccgactgcc ctgggcctct tcagcggcgc tgcttacagc cgtccctcgt 480
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cgaaagtcca gaatgtgatc taaactataa agtaacaact ttcaacaacg gatctcttgg 60
ctctcgcatc gatgaagaac gcagcgaaat gcgataagta atgtgaattg cagaattcag 120
tgaatcatcg aatctttgaa cgcaccttgc gctccttggt attccatgga gcatgcctgt 180
ttgagtgtca cgtaaaccct cacccctgcg atgtaacagt cgcgtgcggt ggacttggac 240
tgtgccgtaa tcggctggtc ttgaaatgca ttagctggcg cttttagagt gct 293
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tccgtaggtg aacctgcgg 19
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tcctccgctt attgatatgc 20
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cgaaagtcca gaatgtgatc ta 22
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
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agcactctaa aagcgccagc taatg 25
Claims (3)
1. An application of molecular markers in breeding Auricularia polytricha variety is characterized in that,
the molecular marked nucleotide is shown as SEQ ID NO: 1. the 120 th bit of the sequence is C, and 130 th to 133 th bits are TCTA;
the Auricularia polytricha varieties are Auricularia polytricha Ty081 and Auricularia polytricha TE011;
the breeding refers to the breeding of the auricularia polytricha variety from auricularia 916, auricularia auricula "Gui Yun No. 3", auricularia polytricha 193, auricularia polytricha Ty081, auricularia polytricha TE011, auricularia polytricha "black 3" and auricularia polytricha "new family No. 4";
the breeding method comprises the following steps: designing a primer by using the molecular marker, amplifying a target fragment by using the genomic DNA of the auricularia auricula to be detected as a template and utilizing the designed primer, and finally detecting by electrophoresis, if the 293bp strip can be amplified, identifying as auricularia polytricha Ty081 or auricularia polytricha TE011, and if the 293bp strip cannot be amplified, identifying as auricularia auricula 916, auricularia auricula Gui Yun No. 3, auricularia polytricha 193, auricularia auricula "black 3" or auricularia auricula "new family No. 4";
the nucleotide sequence of the primer is shown as SEQ ID NO: 5-6.
2. The use according to claim 1, wherein the amplification reaction system is: 2X FlashHot Star Master Mix. Mu.L, ddH 2 O12. Mu.L, 1. Mu.L each of the upstream primer F/the downstream primer R, and 1. Mu.L each of the DNA template.
3. The use according to claim 2, wherein the amplification reaction procedure is: pre-denaturing for 3min at 95℃and denaturing for 30s at 95 ℃; annealing at 56 ℃ for 30s; extending at 72 ℃ for 30s; 29. the cycle was terminated at 72℃for 7min.
Priority Applications (1)
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| CN101798601A (en) * | 2010-04-28 | 2010-08-11 | 黑龙江省科学院微生物研究所 | Molecular marker identification method of black fungus strain and specific molecular marker primers of black fungus strain HW 5 |
| CN111778351A (en) * | 2020-07-31 | 2020-10-16 | 广西壮族自治区农业科学院微生物研究所 | Specific molecular marker primer and kit for black fungus strain and application of specific molecular marker primer and kit |
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| CN101798601A (en) * | 2010-04-28 | 2010-08-11 | 黑龙江省科学院微生物研究所 | Molecular marker identification method of black fungus strain and specific molecular marker primers of black fungus strain HW 5 |
| CN111778351A (en) * | 2020-07-31 | 2020-10-16 | 广西壮族自治区农业科学院微生物研究所 | Specific molecular marker primer and kit for black fungus strain and application of specific molecular marker primer and kit |
| CN111926107A (en) * | 2020-09-15 | 2020-11-13 | 广西壮族自治区农业科学院微生物研究所 | Molecular marker for identifying Pleurotus citrinopileatus and application thereof |
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