CN104131007B - The SSR marker finger printing of a kind of Flammulina velutiper (Fr.) Sing river Huang strain and application - Google Patents
The SSR marker finger printing of a kind of Flammulina velutiper (Fr.) Sing river Huang strain and application Download PDFInfo
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Abstract
The present invention relates to SSR marker finger printing and the application of a kind of Flammulina velutiper (Fr.) Sing river Huang strain, this finger printing is combined by the special allele of 6 SSR marker to developing based on Flammulina velutiper (Fr.) Sing genome sequence.Application includes: Needle mushroom strain is carried out SSR marker amplification, is compareed with finger printing by the banding pattern obtained, and consistent with this finger printing is river Huang strain.The present invention, compared with routine morphological detection, antagonistic effect, fruiting experiment, has that the detection time is short, accuracy is high, the advantage of favorable repeatability, has the specificity of river Huang strain in the main cultivation Needle mushroom strain of 24, China collected.
Description
Technical field
The invention belongs to the detection field of Needle mushroom strain, the SSR marker particularly to a kind of Flammulina velutiper (Fr.) Sing river Huang strain refers to
Stricture of vagina collection of illustrative plates and application.
Background technology
Flammulina velutiper (Fr.) Sing [Flammulina velutipes (Fr.) Sing.] has another name called Flammulina velutipes, Flammulina velutipes (Fr.) Sing, structure bacterium, Russulaalutacea[Agari-cusalutaveaPers., Mao Bing
Money bacterium, belongs to gill fungi whitish eye mushroom section flammule Pseudomonas in classification.Flammulina velutiper (Fr.) Sing is widely distributed, nutritious, is a kind of economical
It is worth the highest edible fungi and medicinal fungus, is also one of natural health care of extremely selling well on market.Flammulina velutiper (Fr.) Sing is that China is main
Wanting culturing edible fungus and modern factories metaplasia to produce one of Pollution In Edible Mushrooms, within 2013, country facilities day output reaches 2700
Ton.
Good quality strain contribution rate in Flammulina velutiper (Fr.) Sing per unit area yield and quality is held the balance, and which dictates that Needle mushroom strain is at acupuncture needle
Critical role in mushroom industry.Within 1999, China endorsed " International Plant New variety protection method ", and this does not require nothing more than us and respects
The kind intellectual property of other country, also to strengthen protecting the kind intellectual property of our country oneself, in order to set up food simultaneously
The kind property right of China is really protected, it is necessary to initially set up the cultivar identification technology of maturation by bacterium new varieties resignation system, for
New varieties registration lays the foundation.Especially Japan comes into effect " seedling method amendment " on April 1st, 2004, outstanding to China's edible fungi
It is that the production of Flammulina velutiper (Fr.) Sing constitutes threat greatly.For domestic, golden mushroom plantation strain " homonymus or synonymum, xenogenesis is of the same name "
Phenomenon, bring greatly loss not only to mushroom agriculture, have impact on their cultivation enthusiasm, the most greatly have impact on China's gold
The development of pin mushroom industry；And, along with the appearance of large-scale factory culture mode, golden mushroom plantation bacterial strain quality is wanted
Ask more and more higher, need that development is the easiest, identification of strains technology fast and accurately, to ensure the most accurate by kind of every batch
Errorless.
In the face of this situation, it is necessary to we further speed up the research of strain identification technology, in the research of Flammulina velutiper (Fr.) Sing
Set up more efficiently strain identification system.
Summary of the invention
The technical problem to be solved be to provide a kind of Flammulina velutiper (Fr.) Sing river Huang strain SSR marker finger printing and
Construction method and application, this finger printing, compared with routine morphological detection, antagonistic effect, fruiting experiment, has the detection time
Short, accuracy is high, reproducible advantage.
The SSR marker finger printing of a kind of Flammulina velutiper (Fr.) Sing river Huang strain of the present invention, this finger printing is by 6 pairs of SSR marker groups
Becoming, be that amplification banding pattern is good based on Flammulina velutiper (Fr.) Sing genome simple repeated sequence fragment SSR primers development, the high SSR of repeatability draws
Thing, labelling details such as table 1.
Table 1 SSR marker details list
The application of the SSR marker finger printing of a kind of Flammulina velutiper (Fr.) Sing river Huang strain of the present invention, is to utilize Flammulina velutiper (Fr.) Sing genome
6 pairs of SSR primers of simple repeated sequence fragment exploitation, by the SSR to the 24 parts of domestic main golden mushroom plantation kinds collected
Primer banding pattern expands, and present invention determine that the quantity of the allele that 6 pairs of SSR primers amplify in the Huang strain of Flammulina velutiper (Fr.) Sing river also
Numbering (table 2), can effectively identify river Huang strain (Fig. 2-7) by the numbering combination of different SSR alleles.By comparison 50bp
DNA ladder can determine that the relative molecular weight of the allele of each SSR primer amplification, there is river Huang strain special SSR equipotential plate
The strain of Duan Zuhe, is i.e. Flammulina velutiper (Fr.) Sing river Huang strain, and the numbering of this strain 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, compared with routine morphological detection, antagonistic effect, fruiting experiment, has the detection time short, and accuracy is high,
Reproducible advantage.Detection required time has only to 3～4 days, and the antagonistic effect required time of routine at least needs two weeks
Time, fruiting experiment then needs the time of at least 2 months；The method has in collected 24 Flammulina velutiper (Fr.) Sing main cultivation strains of China
There is the specificity of river Huang strain, have a good application prospect.
Accompanying drawing explanation
Fig. 1 is Flammulina velutiper (Fr.) Sing river Huang strain SSR marker finger printing, and wherein M is 50bp DNA ladder, digitized representation
Being used 6 pair SSR primers, C is blank, the stable and special SSR etc. of arrow referred to Flammulina velutiper (Fr.) Sing river Huang strain
Position fragment combination；
Fig. 2 is primers F v_fp0001 AFLP system in selected golden mushroom plantation material；
Fig. 3 is primers F v_fp0002 AFLP system in selected golden mushroom plantation material；
Fig. 4 is primers F v_fp0003 AFLP system in selected golden mushroom plantation material；
Fig. 5 is primers F v_fp0004 AFLP system in selected golden mushroom plantation material；
Fig. 6 is primers F v_fp0005 AFLP system in selected golden mushroom plantation material；
Fig. 7 is primers F v_fp0006 AFLP system in selected golden mushroom plantation material.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that these embodiments are merely to illustrate the present invention
Rather than restriction the scope of the present invention.In addition, it is to be understood that after having read the content that the present invention lectures, people in the art
The present invention can be made various changes or modifications by member, and these equivalent form of values fall within the application appended claims equally and limited
Scope.
Embodiment 1
(1) mycelia culture: Needle mushroom strain be transferred in potato dextrose medium, at 19 DEG C～21 DEG C, lucifuge is shaken
Bottle is cultivated, and collects mycelia after 3d～4d；
(2) extraction of genomic DNA: extract the genome of above-mentioned mycelia by CTAB (cetyl trimethylammonium bromide) method
DNA, ultraviolet spectrophotometry detection total genomic dna concentration and purity, the concentration adjusting sample DNA is consistent；
CTAB method is extracted the genomic DNA technique of mycelia and is included:
1. by the Flammulina velutipes mycelium grind into powder of liquid-nitrogen freeze drying, 2 × CTAB extract of 65 DEG C of preheatings is added, in
65 DEG C insulation 45～60min, or jog mixing；
2. 12000rpm, 4 DEG C of centrifugal 20min, take supernatant；
3. in above-mentioned supernatant, add the mixed liquor that volume ratio is the phenol of 25:24:1, chloroform and isoamyl alcohol, mix gently
10min, 12000rpm, 4 DEG C of centrifugal 10min, take supernatant and move in centrifuge tube；
4. in above-mentioned centrifuge tube, add chloroform and the isoamyl alcohol mixed liquor that volume ratio is 24:1, mix 10min gently,
12000rpm, 4 DEG C of centrifugal 10min, take supernatant and move in another centrifuge tube；
5. in above-mentioned centrifuge tube, add the isopropyl of-20 DEG C of pre-coolings of 2/3 volume (supernatant relative in step (4))
Alcohol, is shaken gently for 5min, 8000rpm, 4 DEG C of centrifugal 10min, removes supernatant；
6. the precipitation 75vol% ethanol obtained and 10mmol/L potassium acetate are washed 2～3 times, each 8000rpm, room
The centrifugal 5min of temperature；
7. adding the 95vol% ethanol of pre-cooling, turn upside down gently, 8000rpm room temperature is centrifuged 10min, abandons ethanol, vacuum
Drain or naturally dry；
8. add 100 μ L10 × TE (Tris/EDTA) buffer, beat gently and make resolution of precipitate；
9. the RNaseA of 1 μ L10mg/mL is added in 37 DEG C of water-bath 1h, removal RNA；
10. the DNA extraction thing obtained is preserved standby in-20 DEG C of refrigerators；
(3) detection of SSR molecular marker: the DNA of said extracted is carried out the PCR amplification of gene SSR marker；
PCR amplification system is: cumulative volume 20 μ L, including: 10 × PCR buffer2 μ L, 25mmol/L MgCl22 μ L,
10mmol/L dNTP0.4 μ L, 5U/ μ L Taq DNA enzymatic 0.2 μ L, 10 μm ol/L SSR marker forward primers and reverse primer are total
The template DNA 2 μ L, ddH that each 1.5 μ L of volume, concentration 20ng～30ng/ μ L extract2O10.4μL；
PCR reaction condition: 94 DEG C of 5min；94 DEG C of 30second, 55 DEG C of 30second, 72 DEG C of 30second, 30 circulations；
72℃5min。
(4) electrophoresis detection: the product above-mentioned PCR amplification obtained and the mixing of 12 μ L sample loading buffers, in 95 DEG C of degeneration
5min, terminates to be placed in mixture of ice and water cooling 3min, point sample 3 μ L electrophoresis on denaturing polyacrylamide gel, and degeneration gathers
The concentration of volume percent of acrylamide gel is 6%, and electrophoretic buffer is 1 × TBE, voltage 1000v, electric current 200mA, power
200W, electrophoresis 45min, silver staining, colour developing, take pictures, analysis result.
The concrete technology of silver staining is to unload and take apart glass plate after electrophoresis completes, and after offset plate unloads, washes with deionized water
The 5-8 second, after draining away the water, lucifuge silver staining 8 minutes in silver staining liquid (1.5 grams of silver nitrate and 1500ml water), after silver staining, spend from
The sub-water washing 5-8 second, drain away the water, put in developer solution (16 grams of sodium hydroxide, 1000ml water and 8ml formaldehyde), to development
It is readable after taking out washing.
Use 6 to Strains of Flammulina velutipes, SSR primer is carried out PCR amplification, be can determine that respectively by comparison 50bp DNA ladder
The quantity of the allele of SSR primer amplification and relative molecular weight, find and meet numbering and be combined as: (1+2) 12 (1+2) (1+2+3
+ 5) strain of (2+3), i.e. can determine that this strain is Flammulina velutiper (Fr.) Sing river Huang strain, as shown in arrow mark in Fig. 1.For ensureing to identify
Accuracy, it is proposed that carry out three times repeating experiment.
Claims (1)
1. the method identifying Flammulina velutiper (Fr.) Sing river Huang strain, it specifically comprises the following steps that
(1) mycelia culture: Needle mushroom strain is transferred in potato dextrose medium, lucifuge shaking flask training at 19 DEG C～21 DEG C
Support, after 3d～4d, collect mycelia；
(2) extraction of genomic DNA: extract the genomic DNA of above-mentioned mycelia by CTAB method, ultraviolet spectrophotometry detects total base
Because of group DNA concentration and purity, the concentration adjusting sample DNA is consistent；
CTAB method is extracted the genomic DNA technique of mycelia and is included:
1. by the Flammulina velutipes mycelium grind into powder of liquid-nitrogen freeze drying, 2 × CTAB extract of 65 DEG C of preheatings is added, in 65 DEG C
Insulation 45～60min, or jog mixing；
2. 12000rpm, 4 DEG C of centrifugal 20min, take supernatant；
3. in above-mentioned supernatant, add the mixed liquor that volume ratio is the phenol of 25:24:1, chloroform and isoamyl alcohol, mix gently
10min, 12000rpm, 4 DEG C of centrifugal 10min, take supernatant and move in centrifuge tube；
4. in above-mentioned centrifuge tube, add chloroform and the isoamyl alcohol mixed liquor that volume ratio is 24:1, mix 10min gently,
12000rpm, 4 DEG C of centrifugal 10min, take supernatant and move in another centrifuge tube；
5. in above-mentioned centrifuge tube, add the isopropanol of-20 DEG C of pre-coolings of 2/3 volume, be shaken gently for 5min, 8000rpm, 4 DEG C from
Heart 10min, removes supernatant；
6. the precipitation 75vol% ethanol obtained and 10mmol/L potassium acetate are washed 2～3 times, each 8000rpm, room temperature from
Heart 5min；
7. adding the 95vol% ethanol of pre-cooling, turn upside down gently, 8000rpm room temperature is centrifuged 10min, abandons ethanol, and vacuum is drained
Or naturally dry；
8. add 100 μ L 10 × TE buffer, beat gently and make resolution of precipitate；
9. the RNaseA of 1 μ L 10mg/mL is added in 37 DEG C of water-bath 1h, removal RNA；
10. the DNA extraction thing obtained is preserved standby in-20 DEG C of refrigerators；
(3) detection of SSR molecular marker: the DNA of said extracted is carried out the PCR amplification of gene SSR marker；
PCR amplification system is: cumulative volume 20 μ L, including: 10 × PCR buffer 2 μ L, 25mmol/L MgCl22 μ L, 10mmol/
LdNTP 0.4 μ L, 5U/ μ L Taq DNA enzymatic 0.2 μ L, 10 μm ol/L SSR marker forward primers and reverse primer cumulative volume are each
The template DNA 2 μ L, ddH that 1.5 μ L, concentration 20ng～30ng/ μ L extract2O 10.4μL；
PCR reaction condition: 94 DEG C of 5min；94 DEG C of 30second, 55 DEG C of 30second, 72 DEG C of 30second, 30 circulations；72℃
5min；
6 pairs of SSR primers are as follows:
The positive/negative of Fv_fp0001 is respectively as follows: to primer
AGAGCTGTCCATGTCTCGG/AGCGCTCGGTTTGATGAAC；
The positive/negative of Fv_fp0002 is respectively as follows: to primer
CTAGCCATGCAAAGGCGTC/CCAGCATGCGGTTTAGTCG；
The positive/negative of Fv_fp0003 is respectively as follows: to primer
CAGCTTTGAACAGGCGTCC/CTCCAAGCCAACACCTTGC；
The positive/negative of Fv_fp0004 is respectively as follows: to primer
GGCAGACGAGTGTGCTTTC/ATCAGCCTCGTTCTCCGTG；
The positive/negative of Fv_fp0005 is respectively as follows: to primer
TGGAAAGACAGGCTCTCGG/CGTACTTTCCTTGGTGCGG；
The positive/negative of Fv_fp0006 is respectively as follows: to primer
TGGAAAGGGATCGGTGCTG/GTGTTGCGCAGATGTGGAG；
(4) electrophoresis detection: the product above-mentioned PCR amplification obtained and the mixing of 12 μ L sample loading buffers, in 95 DEG C of degeneration 5min, knot
Bundle is placed in mixture of ice and water cooling 3min, point sample 3 μ L electrophoresis on denaturing polyacrylamide gel, denaturing polyacrylamide
The concentration of volume percent of gel is 6%, and electrophoretic buffer is 1 × TBE, voltage 1000v, electric current 200mA, power 200W, electricity
Swimming 45min, silver staining, colour developing, take pictures, analysis result；
The concrete technology of silver staining is to unload and take apart glass plate after electrophoresis completes, and after offset plate unloads, washes 5-8 with deionized water
Second, after draining away the water, lucifuge silver staining 8 minutes in silver staining liquid, after silver staining, wash the 5-8 second with deionized water, drain away the water, put
Enter in developer solution, be readable after taking out washing to development；
Use 6 to Strains of Flammulina velutipes, SSR primer is carried out PCR amplification, can determine that each SSR by comparison 50bp DNA ladder
The quantity of the allele of primer amplification and relative molecular weight, find and meet numbering and be combined as: (1+2) 12 (1+2) (1+2+3+5)
(2+3) strain, i.e. can determine that this strain is Flammulina velutiper (Fr.) Sing river Huang strain.
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| 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 |
| 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 |
| CN112921113B (en) * | 2021-03-29 | 2024-05-24 | 昆明理工大学 | Application of gene TPS2 in detecting needle mushroom source component |
| CN113186327B (en) * | 2021-04-08 | 2023-03-24 | 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 |
| CN112980994B (en) * | 2021-04-09 | 2023-03-24 | 上海市农业科学院 | Identification method of SSR marker fingerprint of needle mushroom 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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| CN102851364A (en) * | 2012-08-29 | 2013-01-02 | 上海市农业科学院 | SSR fingerprint of Cr02 strain of Lentinus edodes, and construction method and application thereof |
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| CN102851364A (en) * | 2012-08-29 | 2013-01-02 | 上海市农业科学院 | SSR fingerprint of Cr02 strain of Lentinus edodes, and construction method and application thereof |
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| 23个金针菇菌株的SRAP分析;贾定洪等;《西南农业学报》;20111231;第24卷(第5期);第1871-1874页 * |
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