CN104131007A - SSR labeled fingerprint of flammulina velutipes "chuanhuang" strain and applications thereof - Google Patents
SSR labeled fingerprint of flammulina velutipes "chuanhuang" strain and applications thereof Download PDFInfo
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- CN104131007A CN104131007A CN201410318255.2A CN201410318255A CN104131007A CN 104131007 A CN104131007 A CN 104131007A CN 201410318255 A CN201410318255 A CN 201410318255A CN 104131007 A CN104131007 A CN 104131007A
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- 235000001674 Agaricus brunnescens Nutrition 0.000 claims description 49
- 230000001580 bacterial effect Effects 0.000 claims description 29
- 108700028369 Alleles Proteins 0.000 claims description 28
- 238000007639 printing Methods 0.000 claims description 3
- 230000003321 amplification Effects 0.000 abstract description 11
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 11
- 238000001514 detection method Methods 0.000 abstract description 9
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- 238000002372 labelling Methods 0.000 abstract 1
- 108020004414 DNA Proteins 0.000 description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 239000000975 dye Substances 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 238000012408 PCR amplification Methods 0.000 description 4
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 229910052709 silver Inorganic materials 0.000 description 4
- 239000004332 silver Substances 0.000 description 4
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000003042 antagnostic effect Effects 0.000 description 3
- 238000011161 development Methods 0.000 description 3
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- 229910021641 deionized water Inorganic materials 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 229920002401 polyacrylamide Polymers 0.000 description 2
- 238000011160 research Methods 0.000 description 2
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- 238000007400 DNA extraction Methods 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Substances N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 1
- 235000008935 nutritious Nutrition 0.000 description 1
- SCVFZCLFOSHCOH-UHFFFAOYSA-M potassium acetate Chemical compound [K+].CC([O-])=O SCVFZCLFOSHCOH-UHFFFAOYSA-M 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a SSR label fingerprint of a flammulina velutipes "chuanhuang" strain and applications thereof. The fingerprint is composed of six pairs of SSR labeled specific allelic sections which are developed on the basis of the genomic sequence of flammulina velutipes. The fingerprint can be used to identify the flammulina velutipes "chuanhuang" strain, and the identification comprises the following steps: carrying out SSR labeling amplification on a flammulina velutipes strain, and then comparing the obtained fingerprint with the fingerprint of flammulina velutipes "chuanhuang" strain, if the two fingerprints are identical, the flammulina velutipes strain is the "chuanhuang" strain. Compared to the conventional morphology detection, antagonism tests, and fruiting experiments, the fingerprint identification method has the advantages of short detection time, high accuracy, and good repeatability, and has the specificity on "chuanhuang" strain in collected 24 flammulina velutipes strains which are commonly cultured in China.
Description
Technical field
The invention belongs to the detection field of needle mushroom bacterial classification, particularly SSR marking fingerprint and the application of the yellow bacterial classification in a kind of needle mushroom river.
Background technology
Needle mushroom [Flammulina velutipes (Fr.) Sing.] has another name called golden mushroom, plain mushroom, structure bacterium, QINGKANGJUN, hair handle money bacterium, in classification, belongs to Agaricales Bai Mo section flammule Pseudomonas.Needle mushroom is widely distributed, nutritious, is edible mushrooms and medicinal fungus that a kind of economic worth is very high, is also one of natural health care extremely selling well on market.Needle mushroom is that Chinese Main Cultivation edible mushrooms and modern factoriesization are produced one of Pollution In Edible Mushrooms, and within 2013, domestic batch production day output reaches 2700 tons.
The contribution rate of good quality strain in needle mushroom per unit area yield and quality is very important, and this has determined the critical role of needle mushroom bacterial classification in needle mushroom industry.Within 1999, China has signed the international new variety of plant protection method of < < > >; this not only requires us to respect other national kind intellecture property; also want the kind intellecture property of our country oneself of more protection simultaneously; in order to set up edible mushrooms new variety resignation system, really protect the kind property right of China; the cultivar identification technology of necessary model maturation, for new variety registration lays the foundation.Especially Japan comes into effect < < seedling method amendment > > on April 1st, 2004, and the production of China edible mushrooms especially needle mushroom has been formed to great threat.With regard to domestic, the phenomenon of golden mushroom plantation bacterial classification " different name of the same race, xenogenesis is of the same name ", has brought great loss not only to mushroom agriculture, has affected their cultivation enthusiasm, has also greatly affected the development of Chinese needle mushroom industry; And, along with the appearance of large-scale factory culture mode, more and more higher to the requirement of golden mushroom plantation bacterial strain quality, need that development is more easy, identification of strains technology fast and accurately, with guarantee every batch with kind all accurate.
In the face of this situation, just need us further to accelerate the research of strain identification technology, in the research of needle mushroom, set up more efficiently strain identification system.
Summary of the invention
Technical problem to be solved by this invention is to provide SSR marking fingerprint and construction process and the application of the yellow bacterial classification in a kind of needle mushroom river, this finger printing is compared with conventional morphologic detection, antagonistic effect, fruiting experiment, have detection time short, accuracy is high, reproducible advantage.
The SSR marking fingerprint of the yellow bacterial classification in a kind of needle mushroom of the present invention river, this finger printing is comprised of 6 pairs of SSR marks, is based on needle mushroom genome simple repeated sequence fragment SSR primers development, and amplification banding pattern is good, the SSR primer that repeatability is high, mark details are as table 1.
The list of table 1 SSR mark details
The application of the SSR marking fingerprint of the yellow bacterial classification in a kind of needle mushroom of the present invention river, 6 pairs of SSR primers that utilize the exploitation of needle mushroom genome simple repeated sequence fragment, by the SSR primer banding pattern of 24 parts of domestic main golden mushroom plantation kinds of collecting is increased, the present invention has determined quantity the numbering (table 2) of the allele that 6 pairs of SSR primers amplify in the yellow bacterial classification in needle mushroom river, by the numbering combination of different SSR alleles, can effectively identify the yellow bacterial classification (Fig. 2-7) in river.By contrast 50bp DNA ladder, can determine the relative molecular weight of the allele of each SSR primer amplification, there is the bacterial classification of the special SSR allele combination of the yellow bacterial classification in river, be the yellow bacterial classification in needle mushroom river, the numbering of this bacterial classification is combined as: (1+2) 12 (1+2) (1+2+3+5) (2+3).
The allele information summary sheet of table 2 SSR primer amplification
beneficial effect
The present invention compares with conventional morphologic detection, antagonistic effect, fruiting experiment, has detection time short, and accuracy is high, reproducible advantage.Detecting required time only needs 3～4 days, and conventional antagonistic effect required time at least needs two time-of-weeks, and fruiting experiment needs the time of at least 2 months; The method has the specificity of the yellow bacterial classification in river in the main cultivation bacterial classification of 24 needle mushroom of collected China, has a good application prospect.
Accompanying drawing explanation
Fig. 1 is needle mushroom river yellow bacterial classification SSR marking fingerprint, and wherein M is 50bp DNA ladder, digitized representation be 6 pairs of SSR primers used, C is blank, the stable and special SSR allele that arrow refers to the yellow bacterial classification in needle mushroom river combines;
Fig. 2 is the amplification collection of illustrative plates of primers F v_fp0001 in selected golden mushroom plantation material;
Fig. 3 is the amplification collection of illustrative plates of primers F v_fp0002 in selected golden mushroom plantation material;
Fig. 4 is the amplification collection of illustrative plates of primers F v_fp0003 in selected golden mushroom plantation material;
Fig. 5 is the amplification collection of illustrative plates of primers F v_fp0004 in selected golden mushroom plantation material;
Fig. 6 is the amplification collection of illustrative plates of primers F v_fp0005 in selected golden mushroom plantation material;
Fig. 7 is the amplification collection of illustrative plates of primers F v_fp0006 in selected golden mushroom plantation material.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read the content of the present invention's instruction, these equivalent form of values fall within the application's appended claims limited range equally.
Embodiment 1
(1) mycelium culture: needle mushroom bacterial classification is transferred in potato glucose substratum, and lucifuge shake-flask culture at 19 ℃～21 ℃, collects mycelia after 3d～4d;
(2) extraction of genomic dna: extract the genomic dna of above-mentioned mycelia by CTAB (cetyl trimethylammonium bromide) method, ultraviolet spectrophotometry detects total genomic dna concentration and purity, and the concentration of adjusting sample DNA is consistent;
The genomic dna technique that CTAB method is extracted mycelia comprises:
1. by the needle mushroom mycelia grind into powder of liquid-nitrogen freeze drying, add 2 * CTAB extract of 65 ℃ of preheatings, in 65 ℃ insulation 45～60min, or jog mix;
2. 12000rpm, 4 ℃ of centrifugal 20min, get supernatant liquor;
3. in above-mentioned supernatant liquor, adding volume ratio is the mixed solution of phenol, chloroform and the primary isoamyl alcohol of 25:24:1, mixes gently 10min, and 12000rpm, 4 ℃ of centrifugal 10min get supernatant liquor and move in centrifuge tube;
4. in above-mentioned centrifuge tube, adding volume ratio is chloroform and the primary isoamyl alcohol mixed solution of 24:1, mixes gently 10min, and 12000rpm, 4 ℃ of centrifugal 10min get supernatant liquor and move in another centrifuge tube;
5. the Virahol that adds-20 ℃ of precoolings of 2/3 volume (with respect to the supernatant liquor in step (4)) in above-mentioned centrifuge tube, shakes 5min gently, and 8000rpm, 4 ℃ of centrifugal 10min, remove supernatant;
6. the precipitation obtaining is washed 2～3 times to each 8000rpm, the centrifugal 5min of room temperature with 75vol% ethanol and 10mmol/L Potassium ethanoate;
7. the 95vol% ethanol that adds precooling, turns upside down gently, and the centrifugal 10min of 8000rpm room temperature, abandons ethanol, and vacuum is drained or naturally dried;
8. add 100 μ L10 * TE (Tris/EDTA) damping fluids, beat gently and make resolution of precipitate;
9. add the RNaseA of 1 μ L10mg/mL in 37 ℃ of water-bath 1h, remove RNA;
10. the DNA extraction thing obtaining is standby in-20 ℃ of refrigerator storages;
(3) detection of SSR molecule marker: the pcr amplification that the DNA of said extracted is carried out to gene SSR mark;
Pcr amplification system is: cumulative volume 20 μ L, comprising: 10 * PCR buffer2 μ L, 25mmol/L MgCl
22 μ L, 10mmol/L dNTP0.4 μ L, 5U/ μ L Taq DNA enzyme 0.2 μ L, each 1.5 μ L of 10 μ mol/L SSR mark forward primers and reverse primer cumulative volume, the template DNA 2 μ L that concentration 20ng～30ng/ μ L extracts, ddH
2o10.4 μ L;
PCR reaction conditions: 94 ℃ of 5min; 94 ℃ of 30second, 55 ℃ of 30second, 72 ℃ of 30second, 30 circulations; 72 ℃ of 5min.
(4) electrophoresis detection: the product that above-mentioned pcr amplification is obtained and 12 μ L sample loading buffers mix, in 95 ℃ of sex change 5min, finishes to be placed on cooling 3min in mixture of ice and water, point sample 3 μ L electrophoresis on denaturing polyacrylamide gel, the concentration of volume percent of denaturing polyacrylamide gel is 6%, electrophoretic buffer is 1 * TBE, voltage 1000v, electric current 200mA, power 200W, electrophoresis 45min, silver dyes, colour developing, takes pictures, analytical results.
The concrete technology that silver dyes is to unload and take apart sheet glass after electrophoresis completes, after offset plate unloads, with deionized water, wash 5-8 second, after draining away the water, in silver-colored dye liquor (1.5 grams Silver Nitrate and 1500ml water), lucifuge silver dyes 8 minutes, after silver dyes, with deionized water, wash 5-8 second, drain away the water, put into developing solution (16 grams of sodium hydroxide, 1000ml water and 8ml formaldehyde), be readable to the rear taking-up washing of developing.
Adopt 6 pairs of SSR primer pair Strains of Flammulina velutipes to carry out pcr amplification, by contrast 50bp DNA ladder, can determine quantity and the relative molecular weight of the allele of each SSR primer amplification, find and meet numbering and be combined as: (1+2) (1+2+3+5) bacterial classification of (2+3) of 12 (1+2), can determine that this bacterial classification is the yellow bacterial classification in needle mushroom river, as shown in arrow mark in Fig. 1.For the accuracy that guarantees to identify, suggestion carries out repeating for three times experiment.
Claims (3)
1. the SSR marking fingerprint of the yellow bacterial classification in a needle mushroom river, it is characterized in that: this finger printing is combined by the special allele of 6 pairs of SSR marks based on the exploitation of needle mushroom genome sequence, 6 couples of SSR are marked at the allele information increasing on the yellow bacterial classification in needle mushroom river and are specially:
The quantity of the allele of Fv_fp0001 is 2, and allele 1 is 700bp with respect to the molecular weight of 50bp DNA ladder; Allele 2 is 680bp with respect to the molecular weight of 50bp DNA ladder;
The quantity of the allele of Fv_fp0002 is 1, and allele 1 is 1300bp with respect to the molecular weight of 50bp DNA ladder,
The quantity of the allele of Fv_fp0003 is 1, and allele 2 is 680bp with respect to the molecular weight of 50bp DNA ladder;
The quantity of the allele of Fv_fp0004 is 2, and allele 1 is 1250bp with respect to the molecular weight of 50bp DNA ladder; Allele 2 is 1200bp with respect to the molecular weight of 50bp DNA ladder;
The quantity of the allele of Fv_fp0005 is 4, and allele 1 is 1100bp with respect to the molecular weight of 50bp DNA ladder, and allele 2 is 800bp with respect to the molecular weight of 50bp DNA ladder; Allele 3 is 530bp with respect to the molecular weight of 50bp DNA ladder, and allele 5 is 480bp with respect to the molecular weight of 50bp DNA ladder;
The quantity of the allele of Fv_fp0006 is 2, and allele 2 is 370bp with respect to the molecular weight of 50bp DNA ladder; Allele 3 is 360bp with respect to the molecular weight of 50bp DNA ladder.
2. the SSR marking fingerprint of the yellow bacterial classification in a kind of needle mushroom according to claim 1 river, is characterized in that: the special numbering of described 6 pairs of SSR mark alleles is combined as: (1+2) 12 (1+2) (1+2+3+5) (2+3).
3. an application for the SSR marking fingerprint of the yellow bacterial classification in needle mushroom as claimed in claim 1 river, is characterized in that: be applied to identify the special allelic variation combination of the yellow bacterial classification in needle mushroom river and identify that the yellow bacterial classification in needle mushroom river is with respect to the specificity of other Varieties in Flammulina velutipes.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410318255.2A CN104131007B (en) | 2014-07-04 | 2014-07-04 | The SSR marker finger printing of a kind of Flammulina velutiper (Fr.) Sing river Huang strain and application |
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| CN201410318255.2A CN104131007B (en) | 2014-07-04 | 2014-07-04 | The SSR marker finger printing of a kind of Flammulina velutiper (Fr.) Sing river Huang strain and application |
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| CN104131007A true CN104131007A (en) | 2014-11-05 |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105316329A (en) * | 2015-11-20 | 2016-02-10 | 中国科学院昆明植物研究所 | Flammulina velutipes SSR molecular marker and corresponding primers and application thereof |
| CN105506124A (en) * | 2016-01-12 | 2016-04-20 | 浙江省农业科学院 | SSR labeling primer of flammulina velutipes F101 culture and fingerprint application thereof |
| CN106754978A (en) * | 2016-12-26 | 2017-05-31 | 上海市农业科学院 | A kind of specific molecular marker of bacterial strains of asparagus NG1 92 and its preparation method and application |
| CN112921113A (en) * | 2021-03-29 | 2021-06-08 | 昆明理工大学 | Application of gene TPS2 in detection of flammulina velutipes-derived components |
| CN112980994A (en) * | 2021-04-09 | 2021-06-18 | 上海市农业科学院 | Identification method of SSR marker fingerprint of needle mushroom strain and construction method and application thereof |
| CN113186327A (en) * | 2021-04-08 | 2021-07-30 | 上海市农业科学院 | Identification method of microsatellite DNA marker fingerprint of flammulina velutipes FC89 strain, construction method and application thereof |
| CN113186328A (en) * | 2021-04-09 | 2021-07-30 | 上海市农业科学院 | Identification method of microsatellite DNA marker fingerprint of flammulina velutipes xujin 18 strain and construction method and application thereof |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105316329A (en) * | 2015-11-20 | 2016-02-10 | 中国科学院昆明植物研究所 | Flammulina velutipes SSR molecular marker and corresponding primers and application thereof |
| CN105316329B (en) * | 2015-11-20 | 2018-09-11 | 中国科学院昆明植物研究所 | Needle mushroom SSR molecular marker and its corresponding primer and application |
| CN105506124A (en) * | 2016-01-12 | 2016-04-20 | 浙江省农业科学院 | SSR labeling primer of flammulina velutipes F101 culture and fingerprint application thereof |
| CN105506124B (en) * | 2016-01-12 | 2019-05-31 | 浙江省农业科学院 | The SSR label primer and its finger-print application of needle mushroom F101 strain |
| CN106754978A (en) * | 2016-12-26 | 2017-05-31 | 上海市农业科学院 | A kind of specific molecular marker of bacterial strains of asparagus NG1 92 and its preparation method and application |
| CN112921113A (en) * | 2021-03-29 | 2021-06-08 | 昆明理工大学 | Application of gene TPS2 in detection of flammulina velutipes-derived components |
| CN112921113B (en) * | 2021-03-29 | 2024-05-24 | 昆明理工大学 | Application of gene TPS2 in detecting needle mushroom source component |
| CN113186327A (en) * | 2021-04-08 | 2021-07-30 | 上海市农业科学院 | Identification method of microsatellite DNA marker fingerprint of flammulina velutipes FC89 strain, construction method and application thereof |
| CN112980994A (en) * | 2021-04-09 | 2021-06-18 | 上海市农业科学院 | Identification method of SSR marker fingerprint of needle mushroom strain and construction method and application thereof |
| CN113186328A (en) * | 2021-04-09 | 2021-07-30 | 上海市农业科学院 | Identification method of microsatellite DNA marker fingerprint of flammulina velutipes xujin 18 strain and construction method and application thereof |
| CN113186328B (en) * | 2021-04-09 | 2023-02-28 | 上海市农业科学院 | Identification method of microsatellite DNA marker fingerprint of flammulina velutipes xujin 18 strain and construction method and application thereof |
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| Publication number | Publication date |
|---|---|
| CN104131007B (en) | 2016-08-24 |
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