CN105950777A - Method for rapidly and accurately authenticating and evaluating black fungus germplasm resources and black 29 variety specific molecular identity card - Google Patents
Method for rapidly and accurately authenticating and evaluating black fungus germplasm resources and black 29 variety specific molecular identity card Download PDFInfo
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Abstract
The invention discloses a method for rapidly and accurately authenticating and evaluating black fungus germplasm resources and a black 29 variety specific molecular identity card. Black fungus varieties are studied through SSR molecular markers, the study result is analyzed through resource feature analysis software, the study result is quantified, a character string type result is obtained, and a black fungus variety molecular identity card specific fingerprint spectrum is established. Variety features are digitalized through the molecular identity card, the difference between different black fungus varieties can be easily and clearly distinguished, the molecular identity card can be used for authentication and genetic relationship analysis of the black fungus varieties, and an effective scientific basis is provided for intellectual property protection of the black fungus varieties. When the black fungus varieties are authenticated and evaluated, whether the black fungus varieties are authorized black fungus varieties or not can be confirmed only by carrying out comparative analysis on the black fungus varieties and the existing specific molecular identity card. The time for authentication and evaluation of the black fungus germplasm resources is greatly shortened, the cost for authentication and evaluation of the black fungus germplasm resources is greatly reduced, and the efficiency of for authentication and evaluation of the black fungus varieties is improved.
Description
Technical field
The present invention relates to a kind of Auricularia germplasm resource evaluation authentication method and structure in the method
The black 29 varietY specificity molecular identity cards built.
Background technology
Heilongjiang Province ranks first in the country as Auricularia main producing region, Auricularia yield, eats according to China
Adding up with bacterium association, within 2014, Heilongjiang Province's Auricularia plants 63.5 hundred million bags, total output 316.44
Ten thousand tons, the output value 166.7 hundred million yuan.Heilongjiang Province's temperature is low, day and night temperature big, the sunshine-duration is long
The Auricularia darkish complexion meat making production Deng unique climatic characteristic is thick, quality better.Along with Heilongjiang Province
The development of Auricularia industry, the demand for Auricularia strain is increasing.But, market pair
Chaotic in Auricularia variety managements, mutually introduce a fine variety frequent and autonomous between different strain manufacturing enterprise
Name, causes Auricularia strain market " synonym, homonym " phenomenon generally to exist,
This brings to the intellectual property protection of Auricularia kind, strain quality management and new varieties authorization
The biggest difficulty.
In order to protect excellent Auricularia germ plasm resource, need Auricularia kind is carried out science mirror
Determine and evaluate.But lack the identification means that are concisely and efficiently at present, therefore in the urgent need to developing
A kind of easy, strain identification assessment technique and standard method fast and accurately, to ensure to produce
Accurate with plant.
Heilongjiang Province came into effect the authorization of Auricularia kind, Auricularia authorization product from 2008
The distinctiveness appraisal planted mainly is commented by economical character, RAPD and ISSR method
Valency is identified.
Economical character mainly according to the DUS of phenotype analytical (Distinctness, Uniformity and
Stability) test is identified, but, due to Auricularia sporophore shape simple in construction,
Under different cultivation modes, different way to manages, the form of Auricularia sporophore, color and luster,
There is larger difference in yield, and the qualification of Part Traits is easily affected by human factors.Molecular marker
Method overcomes economical character to be affected by anthropic factor, but RAPD is single primer molecule mark
Note, amplification is not sufficiently stable, and repeatability is poor.ISSR is also single primer molecule labelling,
Although improve annealing temperature, overcome the deficiency of RAPD, but at Auricularia variety evaluation
During qualification, result is vulnerable to operate the impact of batch, and the repeatability of result is poor.Use in pairs
SRAP and the TRAP molecular marker repeatability of primer is preferable, but for edible fungi kind matter
Use the method time, genetic diversity is relatively low, need a large amount of primer to could effectively distinguish, work
Work amount is big and loaded down with trivial details.And, when carrying out the characterization and evaluation of new varieties, need whenever there being new varieties
When wanting characterization and evaluation, being required for again being analyzed the kind of characterization and evaluation, workload is big,
It is unfavorable for the quick and precisely characterization and evaluation of new varieties.
Simple repeated sequence (SSR) is the tandem sequence repeats sequence with 2～6 bases as repetitive
Row, are widely present in the genome of higher organism.SSR molecular marker meets Variety identification
Basic norm, i.e. environmental stability, inter-variety variance recognizability, minimum product intraspecific variablity
With experimental result reliability.Therefore, SSR molecular marker has in Auricularia cultivar identification is evaluated
There is good application prospect.
Summary of the invention
It is an object of the invention to by SSR molecular marker, Auricularia kind be studied, build
The method of vertical a kind of quick and precisely characterization and evaluation Auricularia germ plasm resource and black 29 varietY specificities
Molecular identity is demonstrate,proved, and uses resource characteristic analysis software result of study, result of study is quantified,
Draw the result of character string forms, build Auricularia kind molecular identity card specificity fingerprint image
Spectrum.Specific molecular identity card, varietal characteristic digitized, so can be distinguished simply
The different interracial differences of Auricularia, this specific molecular identity card can be used for Auricularia kind
Authenticity and genetic affinity analysis, the intellectual property protection for Auricularia kind has provided
The scientific basis of effect.And, when Auricularia cultivar identification is evaluated, it is only necessary to be detected black
Auricularia kind is experiment material, operates according to Auricularia germplasm identification evaluation methodology, passes through
With the comparative analysis of existing specific molecular identity card, can determine whether it is by authorization
Auricularia kind.This greatly reduces time and the expense that Auricularia germplasm identification is evaluated
With, improve the efficiency that Auricularia cultivar identification is evaluated, and degree of accuracy is higher.
It is an object of the invention to be achieved through the following technical solutions:
A kind of method of quick and precisely characterization and evaluation Auricularia germ plasm resource, comprises the steps:
(1) female kind activates
Mother is planted and returns to room temperature state from preservation state, mother is planted and is transferred to the training of cPDA solid
Supporting ware central authorities, 25 DEG C of constant temperature lucifuges are cultivated 8d and are activated standby.
(2) liquid culture
Punch at identical cell age with 0.5cm card punch during inoculation, in inoculation conical flask, every bottle
Equipped with liquid PD culture medium 200mL in conical flask, each conical flask inoculates 10 inoculation blocks,
120r/min, 25 DEG C of constant temperature lucifuges cultivate 10d.
(3) mycelia is collected
Liquid shaking bottle mycelium four layers of filtered through gauze, the mycelium distilled water flushing after filtration
Surface impurity, the mycelium of collection saves backup in-20 DEG C.
(4) total DNA extraction
Use plant genome DNA to extract test kit and extract STb gene.
(5) STb gene quality testing
DYY-12 type electrophresis apparatus is used to detect with 1.0% agarose gel electrophoresis.
Use UV757CRT UV spectrophotometer measuring STb gene concentration, measure A260
Value and A280 value, it is desirable to A280/A260 is to can be used for follow-up test operation when 1.8 to 2.0,
Diluted sample is to 50mg/ μ L, and-20 DEG C save backup.
(6) SSR primer
SSR primer is as shown in table 1.
Table 1SSR primer table
(7) SSR amplification
Amplification system: 2.0 μ L 10 × Ex Taq Buffer (Takara), 2.0 μ L dNTP (0.2
Mmol/L), 1.0 μ L forward primer (0.5 μm ol/L), 1.0 μ L downstream primers (0.5
μm ol/L), 0.5U archaeal dna polymerase (Takara), 1.0 μ L template DNAs (50ng/ μ L),
ddH2O mends to 20 μ L.
Amplification program: 94 DEG C of denaturations 5min；94 DEG C of degeneration 30s, 54～60 DEG C of renaturation
30s, 72 DEG C extend 2min, 35 circulations；72 DEG C extend 7min, annealing temperature such as table
Shown in 1.
Electrophoretic image: use BIO-RAD gel imaging system take pictures and record electrophoresis result.
(8) data statistic analysis
The electrophoresis result of SSR amplified production is carried out manpower comparing to, correction, has band to be designated as 1,
It is designated as 0 without band, builds initial 0,1 data matrix.
(9) resource characteristic analysis software
Resource characteristic is used to analyze software I D Analysis software according to SSR primer numbers sequentially,
Build the specific molecular identity card of Auricularia germ plasm resource.
(10) Auricularia cultivar identification is evaluated
When Auricularia kind needs characterization and evaluation, with Auricularia kind to be detected for experiment material
Material, perform step (1)-(9), by with existing specific molecular identity card comparative analysis,
The Auricularia kind by authorization can be determined whether it is.
A kind of black 29 varietY specificity molecular identity cards, its construction method is as follows:
(1) female kind activates
Black 29 female kinds are returned to room temperature state from preservation state, mother is planted and is transferred to cPDA
Solid culture ware central authorities, 25 DEG C of constant temperature lucifuges are cultivated 8d and are activated standby.
(2) liquid culture
Punch at identical cell age with 0.5cm card punch during inoculation, in inoculation conical flask, every bottle
Equipped with liquid PD culture medium 200mL in conical flask, each conical flask inoculates 10 inoculation blocks,
120r/min, 25 DEG C of constant temperature lucifuges cultivate 10d.
(3) mycelia is collected
Liquid shaking bottle mycelium four layers of filtered through gauze, the mycelium distilled water flushing after filtration
Surface impurity, the mycelium of collection saves backup in-20 DEG C.
(4) total DNA extraction
Use plant genome DNA to extract test kit and extract STb gene.
(5) STb gene quality testing
DYY-12 type electrophresis apparatus is used to detect with 1.0% agarose gel electrophoresis.
Use UV757CRT UV spectrophotometer measuring STb gene concentration, measure A260
Value and A280 value, it is desirable to A280/A260 is to can be used for follow-up test operation when 1.8 to 2.0,
Diluted sample is to 50mg/ μ L, and-20 DEG C save backup.
(6) SSR primer
SSR primer is as shown in table 1.
(7) SSR amplification
Amplification system: 2.0 μ L 10 × Ex Taq Buffer (Takara), 2.0 μ L dNTP (0.2
Mmol/L), 1.0 μ L forward primer (0.5 μm ol/L), 1.0 μ L downstream primers (0.5
μm ol/L), 0.5U archaeal dna polymerase (Takara), 1.0 μ L template DNAs (50ng/ μ L),
ddH2O mends to 20 μ L.
Amplification program: 94 DEG C of denaturations 5min；94 DEG C of degeneration 30s, 54～60 DEG C of renaturation
30s, 72 DEG C extend 2min, 35 circulations；72 DEG C extend 7min, annealing temperature such as table
Shown in 1.
Electrophoretic image: use BIO-RAD gel imaging system take pictures and record electrophoresis result.
(8) data statistic analysis
The electrophoresis result of SSR amplified production is carried out manpower comparing to, correction, has band to be designated as 1,
It is designated as 0 without band, builds initial 0,1 data matrix.
(9) resource characteristic analysis software
Resource characteristic is used to analyze software I D Analysis software according to SSR primer numbers sequentially,
Building the specific molecular identity card of black 29, its special molecular identity card is
111010110011011110101011。
The present invention establishes the standard that a kind of germplasm identification of Auricularia fast and accurately is evaluated
Method, the molecular identity card that each Auricularia breed formation is unique, to building Auricularia product
Plant molecular identity card specificity finger printing.When Auricularia kind needs characterization and evaluation, only need
Will be with Auricularia kind to be detected as experiment material, according to Auricularia germplasm identification evaluation side
Method operates, and by the comparative analysis with existing specific molecular identity card, can determine if
For by the Auricularia kind of authorization.This greatly reduces Auricularia germplasm identification evaluation
Time and expense, improve the efficiency that Auricularia cultivar identification is evaluated, and degree of accuracy is higher.
Specific molecular identity card, varietal characteristic digitized, uses resource characteristic analysis software to obtain
Go out the result of character string forms, so can distinguish interracial difference simply, its
In molecular data can be used for authenticity and the genetic affinity analysis of Auricularia kind, for black wood
The intellectual property protection of ear kind provides effective scientific basis.
Detailed description of the invention
Below technical scheme is further described, but is not limited thereto, all
It is technical solution of the present invention to be modified or equivalent, without deviating from the technology of the present invention side
The spirit and scope of case, all should contain in protection scope of the present invention.
Detailed description of the invention one: present embodiments provide for the one black wood of quick and precisely characterization and evaluation
The method of ear germ plasm resource, specifically comprises the following steps that
(1) female kind activates
Preparation cPDA solid medium, culture medium prescription: Rhizoma Solani tuber osi 20% (with diffusion juice),
Magnesium sulfate 0.05%, potassium dihydrogen phosphate 0.2%, agar 2%.Mother is planted and recovers from preservation state
To room temperature state, mother is planted and is transferred to cPDA solid culture ware central authorities, 25 DEG C of constant temperature lucifuge trainings
Support 8d activation standby.
(2) liquid culture
Punch at identical cell age with 0.5cm card punch during inoculation, in inoculation conical flask, every bottle
Equipped with liquid PD culture medium 200mL in conical flask, each conical flask inoculates 10 inoculation blocks,
Using ZHWY-211B shaking table, 120r/min, 25 DEG C of constant temperature lucifuges cultivate 10d.
(3) mycelia is collected
Liquid shaking bottle mycelium four layers of filtered through gauze, the mycelium distilled water flushing after filtration
Surface impurity, the mycelium of collection saves backup in-20 DEG C.
(4) total DNA extraction
Use plant genome DNA to extract test kit (DP305-03, sky root is biochemical) to extract
STb gene.
(5) STb gene quality testing
DYY-12 type electrophresis apparatus is used to detect with 1.0% agarose gel electrophoresis, it is desirable to extraction
STb gene electrophoretic band is clear, and concentration is bigger.
Use UV757CRT UV spectrophotometer measuring STb gene concentration, measure A260
Value and A280 value, it is desirable to A280/A260 is to can be used for follow-up test operation when 1.8 to 2.0,
Diluted sample is to 50mg/ μ L, and-20 DEG C save backup.
(6) SSR primer
From other kinds of edible fungi screening application effect preferable SSR primer, from SSR with
In power traction thing, screening application effect preferable SSR primer, utilizes Auricularia SSR in NCBI
Sequential design SSR primer.Wherein filter out 8 pairs of polymorphism height, expand stable, reproducible
SSR primer, can be used for the quick and precisely characterization and evaluation of Auricularia germ plasm resource.Filter out
8 pairs of primers are as shown in table 1.
Table 1SSR primer table
(7) SSR amplification
Amplification system: 2.0 μ L 10 × Ex Taq Buffer (Takara), 2.0 μ L dNTP (0.2
Mmol/L), 1.0 μ L forward primer (0.5 μm ol/L), 1.0 μ L downstream primers (0.5
μm ol/L), 0.5U archaeal dna polymerase (Takara), 1.0 μ L template DNAs (50ng/ μ L),
ddH2O mends to 20 μ L.
Amplification program: 94 DEG C of denaturations 5min；94 DEG C of degeneration 30s, 54～60 DEG C of renaturation
30s, 72 DEG C extend 2min, 35 circulations；72 DEG C extend 7min.Amplified reaction exists
S1000TMCarry out on Thermal Cycler (BIO-RAD) instrument.Concrete return of goods temperature such as table
Shown in 1.
Electrophoretic image: use BIO-RAD gel imaging system take pictures and record electrophoresis result.
(8) data statistic analysis
The electrophoresis result of SSR amplified production is carried out manpower comparing to, correction, has band to be designated as 1,
It is designated as 0 without band, builds initial 0,1 data matrix.
(9) resource characteristic analysis software
Resource characteristic is used to analyze software I D Analysis software according to SSR primer numbers sequentially,
Build the specific molecular identity card of Auricularia germ plasm resource.
In present embodiment, build the specific molecular identity card examination used of Auricularia germ plasm resource
Test material as shown in table 2,8 strain Auricularia kind altogether.All of Auricularia kind in table 2
The most by authorization, for Heilongjiang Province's Auricularia main black fungus cultivation kind.Based on present embodiment
The method provided has constructed this 8 strain Auricularia varietY specificity molecular identity card, and is formed black
Auricularia specific molecular identity card finger printing.
Table 2 Auricularia kind
(10) new varieties characterization and evaluation
When Auricularia kind needs characterization and evaluation, with Auricularia kind to be detected for experiment material
Material, perform step (1)-(9), by with existing specific molecular identity card comparative analysis,
The Auricularia kind by authorization can be determined whether it is.
Detailed description of the invention two: present embodiments provide for black 29 varietY specificity molecular identity
Card, its construction method is as follows:
(1) female kind activates
Black 29 female kinds are returned to room temperature state from preservation state, mother is planted and is transferred to cPDA
Solid culture ware central authorities, 25 DEG C of constant temperature lucifuges are cultivated 8d and are activated standby.
(2) liquid culture
Punch at identical cell age with 0.5cm card punch during inoculation, in inoculation conical flask, every bottle
Equipped with liquid PD culture medium 200mL in conical flask, each conical flask inoculates 10 inoculation blocks,
Using ZHWY-211B shaking table, 120r/min, 25 DEG C of constant temperature lucifuges cultivate 10d.
(3) mycelia is collected
Liquid shaking bottle mycelium four layers of filtered through gauze, the mycelium distilled water flushing after filtration
Surface impurity, the mycelium of collection saves backup in-20 DEG C.
(4) total DNA extraction
Use plant genome DNA to extract test kit (DP305-03, sky root is biochemical) to extract
STb gene.
(5) STb gene quality testing
DYY-12 type electrophresis apparatus is used to detect with 1.0% agarose gel electrophoresis, it is desirable to extraction
STb gene electrophoretic band is clear, and concentration is bigger.
Use UV757CRT UV spectrophotometer measuring STb gene concentration, measure A260
Value and A280 value, it is desirable to A280/A260 is to can be used for follow-up test operation when 1.8 to 2.0,
Diluted sample is to 50mg/ μ L, and-20 DEG C save backup.
(6) SSR primer
Primer is as shown in table 1.
(7) SSR amplification
Amplification system: 2.0 μ L 10 × Ex Taq Buffer (Takara), 2.0 μ L dNTP (0.2
Mmol/L), 1.0 μ L forward primer (0.5 μm ol/L), 1.0 μ L downstream primers (0.5
μm ol/L), 0.5U archaeal dna polymerase (Takara), 1.0 μ L template DNAs (50ng/ μ L),
ddH2O mends to 20 μ L.
Amplification program: 94 DEG C of denaturations 5min；94 DEG C of degeneration 30s, 54～60 DEG C of renaturation
30s, 72 DEG C extend 2min, 35 circulations；72 DEG C extend 7min.Amplified reaction exists
S1000TMCarry out on Thermal Cycler (BIO-RAD) instrument.Concrete return of goods temperature such as table
Shown in 1.
Electrophoretic image: use BIO-RAD gel imaging system take pictures and record electrophoresis result.
(8) data statistic analysis
The electrophoresis result of SSR amplified production is carried out manpower comparing to, correction, has band to be designated as 1,
It is designated as 0 without band, builds initial 0,1 data matrix.
(9) resource characteristic analysis software
Resource characteristic is used to analyze software I D Analysis software according to SSR primer numbers sequentially,
Building the specific molecular identity card of black 29 kinds, its specific molecular identity card is
111010110011011110101011.It is embodied as: the first, second of pair of primers
Amplified band is all had with the 3rd allele site；First allele of second pair of primer
Site does not has amplified band, and there is amplified band in second allele site of second pair of primer；
First allele site of the 3rd pair of primer does not has amplified band, the second of the 3rd pair of primer
Amplified band is had with the 3rd allele site；First and second equipotentials of the 4th pair of primer
Gene loci does not has amplified band, and there is amplification article in the 3rd allele site of the 4th pair of primer
Band；The first of 5th pair of primer and the 3rd allele site have amplified band, the 5th to drawing
Second allele site of thing does not has amplified band；First, second He of the 6th pair of primer
All there is amplified band in 3rd allele site；The first of 7th pair of primer and the 3rd equipotential
Gene loci does not has amplified band, the second of the 7th pair of primer and the 4th allele site equal
There is amplified band；First allele site of the 8th pair of primer does not has amplified band, and the 8th
Second and the 3rd allele site to primer all have amplified band.
Black 29 Auricularia kinds are the excellent black of Institute of Microbiology, Heilongjiang Academy of Sciences's selection-breeding
Auricularia kind, is the most extensively cultivated.2008, black 29 passed through National Agricultural skill
Art extension service center is assert, assert that numbered state product recognize bacterium 2007018.Now, black 29
The cultivation amount in area is still growing on and on northeastward, is deeply favored by plantation family, it has also become northeast always
The first-selected kind of area cultivation of auricularia auricula, achieves good economic benefit and social benefit.But
It is during cultivation of auricularia auricula is planted, the problems such as repeatedly strain infringement to occur, and can not lead to always
Cross one method fast and accurately black 29 kind intellectual properties are effectively protected.Therefore originally
Embodiment constructs the molecular identity card of black 29 varietY specificities, by black 29 Auricularia kinds
Feature digitized, forms the form of character string, and simple and clear distinguishes with other Auricularia kinds
Open, to can effectively protect black 29 Auricularia kinds.
Claims (7)
1. the method for a quick and precisely characterization and evaluation Auricularia germ plasm resource, it is characterised in that
Described method step is as follows:
(1) female kind activates
Mother is planted and returns to room temperature state from preservation state, mother is planted and is transferred to the training of cPDA solid
Supporting ware central authorities, 25 DEG C of constant temperature lucifuges are cultivated 8d and are activated standby；
(2) liquid culture
Punch at identical cell age with 0.5cm card punch during inoculation, in inoculation conical flask, every bottle
Equipped with liquid PD culture medium 200mL in conical flask, each conical flask inoculates 10 inoculation blocks,
120r/min, 25 DEG C of constant temperature lucifuges cultivate 10d；
(3) mycelia is collected
Liquid shaking bottle mycelium four layers of filtered through gauze, the mycelium distilled water flushing after filtration
Surface impurity, the mycelium of collection saves backup in-20 DEG C；
(4) total DNA extraction
Use plant genome DNA to extract test kit and extract STb gene；
(5) STb gene quality testing
DYY-12 type electrophresis apparatus is used to detect with 1.0% agarose gel electrophoresis；
Use UV757CRT UV spectrophotometer measuring STb gene concentration, measure A260
Value and A280 value, diluted sample is to 50mg/ μ L, and-20 DEG C save backup；
(6) SSR primer
SSR primer is as shown in table 1:
Table 1 SSR primer table
(7) SSR amplification
Amplification system: 2.0 μ L 10 × Ex Taq Buffer, 2.0 μ L 0.2mmol/L dNTP,
1.0 μ L 0.5 μm ol/L forward primer, 1.0 μ L 0.5 μm ol/L trip primers, 0.5U DNA gathers
Synthase, 1.0 μ L 50ng/ μ L template DNAs, ddH2O mends to 20 μ L；
Amplification program: 94 DEG C of denaturations 5min；94 DEG C of degeneration 30s, 54～60 DEG C of renaturation
30s, 72 DEG C extend 2min, 35 circulations；72 DEG C extend 7min, annealing temperature such as table
Shown in 1；
Electrophoretic image: use BIO-RAD gel imaging system take pictures and record electrophoresis result；
(8) data statistic analysis
The electrophoresis result of SSR amplified production is carried out manpower comparing to, correction, has band to be designated as 1,
It is designated as 0 without band, builds initial 0,1 data matrix；
(9) resource characteristic analysis software
Resource characteristic is used to analyze software I D Analysis software according to SSR primer numbers sequentially,
Build the specific molecular identity card of Auricularia germ plasm resource；
(10) new varieties characterization and evaluation
When Auricularia kind needs characterization and evaluation, with Auricularia kind to be detected for experiment material
Material, perform step (1)-(9), by with existing specific molecular identity card comparative analysis,
The Auricularia kind by authorization can be determined whether it is.
Quick and precisely characterization and evaluation Auricularia germ plasm resource the most according to claim 1
Method, it is characterised in that described A280/A260 is 1.8 to 2.0.
Quick and precisely characterization and evaluation Auricularia germ plasm resource the most according to claim 1
Method, it is characterised in that build the specific molecular identity card test used of Auricularia germ plasm resource
Material is black 29, black prestige 15, black prestige monolithic, black prestige 10, black prestige 981, S1, woods
Section 3 and Hongda 2009.
4. black 29 varietY specificity molecular identity cards, it is characterised in that described black 29 product
Species specificity molecular identity card builds as follows:
(1) female kind activates
Black 29 female kinds are returned to room temperature state from preservation state, mother is planted and is transferred to cPDA
Solid culture ware central authorities, 25 DEG C of constant temperature lucifuges are cultivated 8d and are activated standby；
(2) liquid culture
Punch at identical cell age with 0.5cm card punch during inoculation, in inoculation conical flask, every bottle
Equipped with liquid PD culture medium 200mL in conical flask, each conical flask inoculates 10 inoculation blocks,
120r/min, 25 DEG C of constant temperature lucifuges cultivate 10d；
(3) mycelia is collected
Liquid shaking bottle mycelium four layers of filtered through gauze, the mycelium distilled water flushing after filtration
Surface impurity, the mycelium of collection saves backup in-20 DEG C；
(4) total DNA extraction
Use plant genome DNA to extract test kit and extract STb gene；
(5) STb gene quality testing
DYY-12 type electrophresis apparatus is used to detect with 1.0% agarose gel electrophoresis；
Use UV757CRT UV spectrophotometer measuring STb gene concentration, measure A260
Value and A280 value, diluted sample is to 50mg/ μ L, and-20 DEG C save backup；
(6) SSR primer
SSR primer is as shown in table 1:
Table 1 SSR primer table
(7) SSR amplification
Amplification system: 2.0 μ L 10 × Ex Taq Buffer, 2.0 μ L 0.2mmol/L dNTP,
1.0 μ L 0.5 μm ol/L forward primer, 1.0 μ L 0.5 μm ol/L trip primers, 0.5U DNA gathers
Synthase, 1.0 μ L 50ng/ μ L template DNAs, ddH2O mends to 20 μ L；
Amplification program: 94 DEG C of denaturations 5min；94 DEG C of degeneration 30s, 54～60 DEG C of renaturation
30s, 72 DEG C extend 2min, 35 circulations；72 DEG C extend 7min, annealing temperature such as table
Shown in 1；
Electrophoretic image: use BIO-RAD gel imaging system take pictures and record electrophoresis result；
(8) data statistic analysis
The electrophoresis result of SSR amplified production is carried out manpower comparing to, correction, has band to be designated as 1,
It is designated as 0 without band, builds initial 0,1 data matrix；
(9) resource characteristic analysis software
Resource characteristic is used to analyze software I D Analysis software according to SSR primer numbers sequentially,
Build the specific molecular identity card of Auricularia germ plasm resource.
Black 29 varietY specificity molecular identity cards the most according to claim 4, its feature
It is that described A280/A260 is 1.8 to 2.0.
Black 29 varietY specificity molecular identity cards the most according to claim 4, its feature
Be to build test material used by the specific molecular identity card of Auricularia germ plasm resource be black 29,
Black prestige 15, black prestige monolithic, black prestige 10, black prestige 981, S1, Lin Ke No. 3 numbers and grand
2009。
Black 29 varietY specificity molecular identity cards the most according to claim 4, its feature
It is that described specific molecular identity card is 111010110011011110101011, specifically represents
For: all there is amplified band in first, second, and third allele site of pair of primers；
First allele site of second pair of primer does not has amplified band, the second of second pair of primer
There is amplified band in individual allele site；First allele site of the 3rd pair of primer does not has
Amplified band, the second of the 3rd pair of primer and the 3rd allele site have amplified band；The
First and second allele sites of four pairs of primers do not have amplified band, the 4th pair of primer
There is amplified band in 3rd allele site；The first of 5th pair of primer and the 3rd equipotential base
Because there is amplified band in site, second allele site of the 5th pair of primer does not expand article
Band；All there is amplified band in first, second, and third allele site of the 6th pair of primer；
The first of 7th pair of primer and the 3rd allele site there is no amplified band, the 7th pair of primer
Second and the 4th allele site all have amplified band；First of 8th pair of primer etc.
Position gene loci does not has amplified band, the second of the 8th pair of primer and the 3rd allele site
All there is amplified band.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
-
2016
- 2016-07-18 CN CN201610565772.9A patent/CN105950777A/en active Pending
Non-Patent Citations (3)
| Title |
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| RUI YING ZHANG ET AL.: "Development of SSR markers for typing cultivars in the mushroom Auricularia auricula-judae", 《MYCOLOGICAL PROGRESS》 * |
| 于潇 等: "黑龙江部分野生黑木耳菌株的分子ID构建", 《中国农学通报》 * |
| 冯立健: "黑龙江野生黑木耳种质资源的SSR分析", 《中国优秀硕士学位论文全文数据库（农业科技辑）》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN111778351B (en) * | 2020-07-31 | 2021-02-09 | 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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