CN108504727A - The method that high throughput sequencing technologies detect white fungus and incense ashes bacterium proportioning in white fungus strain - Google Patents
The method that high throughput sequencing technologies detect white fungus and incense ashes bacterium proportioning in white fungus strain Download PDFInfo
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- CN108504727A CN108504727A CN201810310490.3A CN201810310490A CN108504727A CN 108504727 A CN108504727 A CN 108504727A CN 201810310490 A CN201810310490 A CN 201810310490A CN 108504727 A CN108504727 A CN 108504727A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
- A01G18/40—Cultivation of spawn
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
Abstract
The invention discloses a kind of methods that high throughput sequencing technologies detect white fungus and incense ashes bacterium proportioning in white fungus strain.This method comprises the following steps：PCR amplification is carried out using the genome of the areas fungi ITS2 primer pair white fungus strain to be measured and builds library, high-flux sequence, then OTU clusterings will be carried out according to 97% or more similitude after high-flux sequence acquired results data filtering impurity elimination, compare fungi ITS sequence database, white fungus and the corresponding OTU information of incense ashes bacterium are obtained, and then realizes the quantitative analysis to the proportioning of white fungus and incense ashes bacterium in the white fungus strain to be measured.The preparation of white fungus strain sporophore growth situation under the method for the present invention combination different ratio, the influence of qualitative assessment white fungus and incense ashes bacterium different ratio to fruit-body formation, the strain in being produced for white fungus provides reliable reference standard.Especially suitable for the service of each edible mushroom and research unit the safety and stability of Edible Fungi is ensured to break through or make corresponding technical barrier.
Description
Technical field
The present invention relates to white fungus in edible mushroom especially white fungus strain and incense ashes bacterium quantitative proportioning detection field, especially relate to
And a kind of method and its application detecting white fungus and incense ashes bacterium proportioning in white fungus strain based on high throughput sequencing technologies.
Background technology
China is big country of mushroom producing, and annual output accounts for about the 3/4 of the world, and edible mushroom has occupied in China's agricultural production
The fifth-largest industry is only second to grain, vegetables, fruit tree, oil plant.White fungus is also known as tremella, and tremella is under the jurisdiction of basidiomycetes in fungi
Door (Basidiaomycota), agaric subphylum (Agaricomycotina), white fungus guiding principle (Tremellomycetes), Tremellales
(Tremellales), Tremellaceae (Tremellaceae), Tremella (Tremella) they are China tradition food medicine dual-purpose fungies, it
It is not only the good medicine having long enjoyed a good reputation in China's medical treasure-house and excellent tonic product and banquet delicacies.China is white fungus artificial cultivation
Cradle and the world's white fungus production and export most countries, white fungus gross annual output amount has been approached 400,000 tons.Wherein with Bag Material
Cultivation is that the Fujian Province of Main Patterns is the maximum province of white fungus production yields, occupies the 90% of national white fungus total output always
More than.
Different from most of edible mushroom types of production cultivation, white fungus has its very unique history of life, it must be in companion
Its history of life could be completed under the synergistic effect of raw fungi incense ashes bacterium.White fungus itself does not have decomposing lignocellulose and starch matrix
Ability, cannot individually be grown on lignocellulosic, only with incense ashes bacterium carry out symbiosis culture when could normal growth and development
And form fructification.Incense ashes bacterium acts as the role of concomitance bacterium in the incubation of white fungus, and secretion extracellular matrix degrading enzyme is to planting
Training material matrix is decomposed, and nutrition is provided for the growth and development of white fungus.Therefore the silver in the preparation process of the upper white fungus strain of production
The ratio of ear and incense ashes bacterium is very crucial, directly affects later stage Tremella fructification growth and development and its commercial value.However
In actual production process, this committed step is often replaced by experience, strain preparation person according to white fungus mycelia grow compared with
Slowly, this fast feature of incense ashes mycelia growth carries out the preparation of white fungus and incense ashes bacterium using the means of empiric observation, to make
This process is filled with uncertain and uncontrollability.With the lasting development of production, the quality of white fungus strain can not just ensure,
Often occur in actual production produce agaric rate it is low or not produce agaric phenomena such as.And due to cannot quantitatively match white fungus and incense ashes bacterium
Silk, prodigious obstacle is brought to the selection and breeding of strain especially for the wild species newly detached.Therefore, to white fungus in white fungus strain and
The quantitative detection of the proportioning of incense ashes bacterium is that a decisive ring the most key in production proposes on the basis of quantitative in reality
The strain breeding method that can be used in production, to break through the technical barrier in edible fungus species production.
High throughput sequencing technologies have the characteristics that efficient, quick, accurate, economical.Currently, technique extensive use
In the detection etc. of environmental microorganism, the mankind and plant pathogenic microorganisms.But in the context of detection of edible fungus species, do not have still
There are the correlative study using high throughput sequencing technologies and report.
Invention content
The object of the present invention is to provide one kind to be matched based on white fungus in high throughput sequencing technologies detection white fungus strain and incense ashes bacterium
The method and its application of ratio.
In a first aspect, the method that white fungus and incense ashes bacterium match in a kind of claimed detection white fungus strain.
The method of white fungus and incense ashes bacterium proportioning, is to use high-flux sequence in detection white fungus strain provided by the present invention
Method detects the proportioning of white fungus and incense ashes bacterium in white fungus strain.
Further, the method may include following steps：Using the base of the areas fungi ITS2 primer pair white fungus strain to be measured
Build library because group carrying out a PCR amplification, high-flux sequence, then by after high-flux sequence acquired results data filtering impurity elimination according to 97%
The above similitude carries out OTU clusterings, compares fungi ITS sequence database, obtains white fungus and the corresponding OTU letters of incense ashes bacterium
Breath, and then realize the quantitative analysis to the proportioning of white fungus and incense ashes bacterium in the white fungus strain to be measured.
Further, the method specifically may include following steps：
(1) genomic DNA of white fungus strain to be measured is extracted；
(2) quality testing is carried out to the genomic DNA of extraction；
(3) it uses the areas fungi ITS2 primer pair genome to carry out PCR amplification and builds library；
(4) high-flux sequence is carried out by Miseq platforms；
(5) OTU clusters point will be carried out according to 97% or more similitude after high-flux sequence acquired results data filtering impurity elimination
Analysis compares fungi ITS sequence database, obtains white fungus and the corresponding OTU information of incense ashes bacterium, and then realize to the white fungus to be measured
The quantitative analysis of the proportioning of white fungus and incense ashes bacterium in strain.
In one embodiment of the invention, in step (1), specifically sodium laurate method is used to extract the white fungus to be measured
The genomic DNA of strain.
In one embodiment of the invention, in step (3), the specific portion in the areas the fungi ITS2 primer of use
The sequence divided is SEQ ID No.1 and SEQ ID No.2.Further, used primer is the specificity with barcode
Primer.
In one embodiment of the invention, in step (5), specifically according to following standard to the high-flux sequence institute
It obtains result data and is filtered impurity elimination：A1) long 20 base below of (read) Quality of Tail value is read in filtering, and the window of 20bp is arranged
Mouthful, if the average mass values in window are less than 20, rear end base is clipped since window, 100bp is below after filtering Quality Control
It reads long (read)；A2 overlapping (overlap) relationship between long (PE reads)) is read according to double end sequencings, will read to grow in pairs
(reads) it is spliced into a sequence, minimum overlay (overlap) length is 10bp；A3 non repetitive sequence) is extracted, and removes and does not have
There is the simple sequence of repetition.
Wherein, it is exactly paired-end reads that double end sequencings, which read long (PE reads),.Reads (reading length) is high-throughput
The sequencing sequence that a reaction obtains in sequencing.In sequencing procedure, the both ends of a DNA molecular can be sequenced.First survey it
In one end, obtain a reads, then return again to the other end sequencing, obtain another reads.The two obtained
Reads is exactly PE reads.
In one embodiment of the invention, in step (5), the OTU clusters specifically are carried out using usearch methods
Analysis.The other methods outside usearch methods can certainly be used to carry out the OTU clusterings.
In one embodiment of the invention, in step (5), the fungi ITS sequence database is specially NCBI data
Library and/or UNITE databases.
Second aspect, claimed the method (white fungus and incense ashes in i.e. previously described detection white fungus strain
The method of bacterium proportioning) formation and/or development of white fungus and incense ashes bacterium different ratio to Tremella fructification in assessing white fungus strain
Influence in application.
The third aspect, claimed the method (white fungus and incense ashes in i.e. previously described detection white fungus strain
Bacterium proportioning method) instruct white fungus strain prepare in application.
Fourth aspect, a kind of claimed method for instructing white fungus strain to prepare.
The method provided by the present invention for instructing white fungus strain to prepare specifically may include method previously (i.e. institute above
State detection white fungus strain in white fungus and incense ashes bacterium proportioning method) in Overall Steps and following steps：In conjunction with white fungus strain
Middle white fungus and incense ashes bacterium different ratio carry out Tremella fructification development experiment, test white fungus and perfume (or spice) in observation white fungus strain in the process
The formation of Tremella fructification and/or developmental state under grey bacterium different ratio, to provide ginseng for the white fungus strain preparation in production
Examine standard.
A kind of specific method for preparing white fungus strain is also claimed in 5th aspect, the present invention.
The method provided by the present invention for preparing white fungus strain, specifically comprises the following steps：White fungus strain is configured to it
Middle white fungus hyphae content is higher than 1 times of incense ashes bacterium hyphae content and is less than 2 times of incense ashes bacterium hyphae contents.More specifically, being will be silver-colored
Ear strain is configured to 1.25 times that wherein white fungus hyphae content is incense ashes bacterium hyphae content.
The detection method of the present invention is sequenced the genomic DNA of white fungus strain using high throughput sequencing technologies, and
The proportioning of white fungus and incense ashes bacterium in white fungus strain is analyzed by bioinformatic analysis, in conjunction with white fungus bacterium under different ratio
Kind fructification growing state, the influence of qualitative assessment white fungus and incense ashes bacterium different ratio to fruit-body formation, in white fungus production
The preparation of strain reliable reference standard is provided.Especially suitable for the service of each edible mushroom and research unit, to break through or formulate
Go out corresponding technical barrier, ensures the safety and stability of Edible Fungi.
Description of the drawings
Fig. 1 is Tremella fructification fresh weight under different white fungus/incense ashes bacterium conditions of mixture ratios.Different lowercase letters are marked in P<
0.05 horizontal upper significant difference.
Fig. 2 is Tremella fructification dry weight under different white fungus/incense ashes bacterium conditions of mixture ratios.Different lowercase letters are marked in P<
0.05 horizontal upper significant difference.
Fig. 3 is the weight of waste mushroom packet under different white fungus/incense ashes bacterium conditions of mixture ratios.Different lowercase letters are marked in P<
0.05 horizontal upper significant difference.
Specific implementation mode
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Embodiment 1 detects the method and its answer that white fungus and incense ashes bacterium match in white fungus strain based on high throughput sequencing technologies
With
The present embodiment is using the main cultivation white fungus yellow cultivars Tr21 in Gutian as research object, 22 DEG C of picking of difference, 24 DEG C, 28 DEG C of trainings
Strain under the conditions of supporting does 3 repetitions under each temperature condition.Sample source is in December, 2017 Gutian Area, Fujian Province section peak edible mushroom
Research institute.
One, using sodium laurate method to the extracting genome DNA of white fungus strain
(1) it weighs and is mixed with the white fungus strain sample 2-3g of sawdust materials in seed bottle and is placed in mortar, be added appropriate
Liquid nitrogen, rapid grinding is uniformly to powder level.
(2) ground powder is moved into the 1.5ml centrifuge tubes after sterilizing, the sodium laurate that 750 μ l are added immediately is taken out
Buffer solution is carried, is slowly shaken up repeatedly, about 10min；
Wherein, the formula of the sodium laurate extraction buffer：Tris-Hcl(pH8.0)100mmol/L；NaCl
100mmol/L；EDTA(pH8.0)20mmol/L；Sodium laurate 1%.With method：Accurately weigh NaCl 5.844g, sodium laurate
10g is placed in 1000mL beakers, and 100ml Tris-Hcl (1M) and 40mL EDTA (0.5M), 800ml ddH is added2O is used
Salt acid for adjusting pH is settled to 1000mL, room temperature preservation after sterilizing to 8.0.
(3) use pipette that the phenol of isometric (about 700 μ l) is added into 1.5ml centrifuge tubes:Chloroform:Isoamyl alcohol (volume ratio
25:24:1) it, slowly shakes up repeatedly.
(4) 13000rpm, 4 DEG C centrifugation 20min after take out, about 700 μ l of careful Aspirate supernatant in Amoxcillin 1.5ml from
In heart pipe.
(5) 5 μ l of RNAase (20mg/ml) are added, 37 DEG C, 30min, repeats step (3), is added isometric (about 705 μ l)
Chloroform:Isoamyl alcohol (volume ratio 24:1), it slowly shakes up, 13000rpm, is taken out after 4 DEG C of centrifugation 20min repeatedly, it is careful to inhale
Take about 700 μ l of supernatant in the 1.5ml centrifuge tubes of Amoxcillin.
(6) 700 μ l isopropanols are added into above-mentioned centrifuge tube, it is slowly reverse up and down to have white flock precipitate precipitation for several times,
Pay attention to, toward a direction, preventing precipitation from scattering when reverse, is unfavorable for subsequent experimental, -20 DEG C of placement 1h.
(7) 13000rpm, 4 DEG C of centrifugation 20min, abandons supernatant.
(8) be added 100 μ l, 70% ethyl alcohol (volume fraction), repeatedly gently concussion washing, 4 DEG C, 13000rpm, at a high speed from
Heart 15min abandons supernatant, repeats this step twice.
(9) by 4 DEG C again of obtained white precipitate, the 12000rpm of short duration centrifugation several seconds, residual ethanol is blotted with pipettor.
(10) white precipitate is placed in draught cupboard makes 70% ethyl alcohol of remaining all volatilize totally overnight.
(11) it after white precipitate bleach, is added into 1.5ml centrifuge tubes in 50 μ l aqua sterilisas, is placed at room temperature for 1-2
Hour, it during which slightly plays even, DNA is made fully to dissolve.
(12) NanoDrop2000 sepectrophotofluorometers are used to carry out quality and Concentration Testing to genomic DNA.
Two, the areas fungi ITS2 PCR amplification
The DNA concentration measured according to NanoDrop2000, take the genome DNA sample of appropriate volume, total amount about 30ng into
Row PCR amplification.The primer of selection be the areas fungi ITS2 specific primer fITS7 (5 '-GTGARTCATCGAATCTTTG-3 ', i.e.,
SEQ ID No.1) and ITS4 (5 '-TCCTCCGCTTATTGATATGC-3 ', i.e. SEQ ID No.2).The primer that the present invention uses
It is the specific primer with Barcode.
PCR reaction systems are：DNA sample 30ng；1 μ l of fITS7 primers (5 μM)；1 μ l of ITS4 primers (5 μM)；BSA(2ng/
μl)3μl；2×Taq PCR MasterMix 12.5μl；ddH2O is mended to 25 μ l.
PCR amplification program is：95℃5min；35 × (95 DEG C of 45S, 58 DEG C of 50S, 72 DEG C of 45S)；72℃10min.
Each sample PCR in triplicate, will same sample PCR product mix after with the detection of 2% agarose gel electrophoresis,
Using AxyPrepDNA gel reclaims kits (AXYGEN companies) gel extraction PCR product, with 2% fine jade after Tris_HCL elutions
Sepharose electrophoresis detection.
Three, the fluorescent quantitation of PCR product
With reference to the preliminary quantitative result of electrophoresis, Caliper LabChip instruments and fluorescent quantitation reagent HT DNA 1K are used
Reagent Kit quantify PCR product, are later 20,000 requirements according to the sequencing amount of each sample, carry out corresponding proportion
Mixing.
Four, Miseq library constructions and high-flux sequence
The structure and high-flux sequence in the libraries Miseq are by Beijing Ao Weisen Gene Tech. Company Limited Illumina Miseq
(PE300) platform carries out, the data being sequenced.
Five, the bioinformatic analysis flow of sequencing result
(1) 20 base below of filtering read Quality of Tail value, is arranged the window of 20bp, if the average quality in window
Value is less than 20, and rear end base is clipped since window, filters 100bp read below after Quality Control；
(2) according to the overlap relationships between PE reads, pairs of reads is spliced into a sequence, it is minimum
Overlap length is 10bp；
(3) fastq files are renamed；
(4) fastq files are converted to fasta files；
(5) merge the fasta sequences of all samples；
(6) non repetitive sequence is extracted to optimization (sequence obtained according to step (1)-(5)), convenient for reducing analysis
Pilot process redundant computation amount；
(7) simple sequence that removal does not repeat；
(8) OTU clusters are carried out to non-duplicate (being free of simple sequence) according to 97% similitude using usearch methods；
(9) removal chimera (i.e. the sequence of different location forms chimera after connecting together)；
(10) different classes of after clustering all optimizations (sequence obtained according to step (1)-(9)) map to OTU
Representative sequence, select and represent sequence of the sequence similarity 97% or more with OTU, generate OTU tables；
(11) by the representative sequence of the different OTU classifications of 97% or more sequence similarity and NCBI and UNITE databases into
Row compares, and to obtain white fungus and the corresponding OTU information of incense ashes bacterium, and then realizes and is carried out to white fungus in white fungus strain and incense ashes bacterium
Quantitative analysis.
Six, result and analysis
1, the quantitative detection that white fungus/incense ashes bacterium matches in white fungus strain
The respective optimum growth temperature range of white fungus and incense ashes bacterium is different, therefore utilizes silver in temperature control cultivating white fungus kind
The ratio of ear and incense ashes bacterium.22 DEG C are chosen respectively, 24 DEG C, culture 7-8 days are carried out to cultivating white fungus kind under 28 DEG C of temperature conditions, often
3 repetitions are done under a temperature condition, amount to 9 samples.High-flux sequence has been carried out to the genomic DNA of this 9 strain samples,
Total to obtain 342322 sequences, average each sample obtains 38035 sequences.By the survey of 3 repeat samples of each proportioning
Sequence result and OTU units are integrated, and obtain 127028,100603,114691 sequences respectively.The sample of 3 different ratios
In processing, the content of white fungus-incense ashes bacterium composite bacteria is respectively 95.98%, 96.23% and 95.43% in white fungus strain, is occupied
Absolute advantage.Wherein 22 DEG C, 24 DEG C, ratio (the two hyphae content of white fungus and incense ashes bacterium in 28 DEG C of temperature white fungus strains
Ratio) be respectively 0.8,1.25 and 2.35.
2, the measurement of the strain fruiting body yield of different white fungus/incense ashes bacterium proportioning
Strain progress to 3 different white fungus/incense ashes bacterium proportioning (ratio of the two hyphae content) processing that step 1 measures
Produce agaric tests (the 1st kind of mix proportion scheme：The ratio of white fungus/incense ashes bacterium is 0.8；2nd kind of mix proportion scheme：The ratio of white fungus/incense ashes bacterium
It is 1.25；3rd kind of mix proportion scheme：The ratio of white fungus/incense ashes bacterium be 2.35), it is the fresh weight that measured its fructification through 30 days or so, dry
The weight of weight and waste mushroom packet.As a result as can be seen that in the 2nd kind of mix proportion scheme (i.e. white fungus/incense ashes bacterium ratio is 1.25) white fungus
The fresh weight and dry weight highest of entity, the weight of waste mushroom packet is minimum, illustrates the ratio of this proportioning pattern white fungus bacterium and incense ashes bacterium most
To be suitable, the weight in bacterium bag is fully utilized, to the fructification weight highest generated.To fructification fresh weight, dry weight
One-way analysis of variance (ANOVA) is carried out with waste mushroom packet weight to show, the 2nd kind of proportioning pattern Tremella fructification fresh weight and dry weight are equal
It is significantly higher than the 1st, 3 kind of mix proportion scheme, and waste mushroom packet weight is substantially less than the 1st, 3 kind of mix proportion scheme (Fig. 1, Fig. 2 and Fig. 3).
This illustrates that the content of white fungus mycelia is higher than incense ashes less than the content (such as the 1st kind proportioning pattern) or white fungus hyphae content of incense ashes bacterium
The fructification fresh weight that the white fungus strain of the 2 times or more (such as the 3rd kind of mix proportion scheme) of hyphae content generates is substantially lower than the 2nd kind and matches
Compare scheme.The weight of 2nd kind of proportioning pattern waste mushroom packet is minimum, corresponding with such proportioning pattern fresh weight highest, shows such match
Than bacterium bag utilization rate highest.Thus it obtains in actual production, the proportioning of white fungus bacterium and incense ashes mycelia is had in suitable model
(white fungus strain is configured to wherein white fungus hyphae content higher than 1 times of incense ashes bacterium hyphae content and less than 2 times of incense ashes bacterium bacterium in enclosing
Silk content) its fruiting body yield can highest.
<110>Institute of Microorganism, Academia Sinica；University Of Agriculture and Forestry In Fujian
<120>The method that high throughput sequencing technologies detect white fungus and incense ashes bacterium proportioning in white fungus strain
<130> GNCLN180820
<160> 2
<170> PatentIn version 3.5
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<211> 19
<212> DNA
<213> Artificial sequence
<220>
<221> misc_feature
<222> (5)..(5)
<223> r is g or a
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gtgartcatc gaatctttg 19
<210> 2
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<400> 2
tcctccgctt attgatatgc 20
Claims (10)
1. a kind of method of white fungus and incense ashes bacterium proportioning in detection white fungus strain is the method detection white fungus using high-flux sequence
The proportioning of white fungus and incense ashes bacterium in strain.
2. according to the method described in claim 1, it is characterized in that：Described method includes following steps：Using the areas fungi ITS2
The genome of primer pair white fungus strain to be measured carries out PCR amplification and builds library, high-flux sequence, then by high-flux sequence acquired results
OTU clusterings are carried out according to 97% or more similitude after data filtering impurity elimination, fungi ITS sequence database is compared, obtains silver
Ear and the corresponding OTU information of incense ashes bacterium, and then realize and the proportioning of white fungus and incense ashes bacterium in the white fungus strain to be measured is quantified
Analysis.
3. according to the method described in claim 2, it is characterized in that：Described method includes following steps：
(1) genomic DNA of white fungus strain to be measured is extracted；
(2) quality testing is carried out to the genomic DNA of extraction；
(3) it uses the areas fungi ITS2 primer pair genome to carry out PCR amplification and builds library；
(4) high-flux sequence is carried out by Miseq platforms；
(5) OTU clusterings will be carried out according to 97% or more similitude after high-flux sequence acquired results data filtering impurity elimination, it is right
Than fungi ITS sequence database, white fungus and the corresponding OTU information of incense ashes bacterium are obtained, and then realize to the white fungus strain to be measured
The quantitative analysis of the proportioning of middle white fungus and incense ashes bacterium.
4. according to the method described in claim 3, it is characterized in that：In step (1), waited for using described in the extraction of sodium laurate method
Survey the genomic DNA of white fungus strain；
And/or
In step (3), the sequence of the specificity portion in the areas the fungi ITS2 primer used is SEQ ID No.1 and SEQ
ID No.2；
And/or
It is that impurity elimination is filtered to the high-flux sequence acquired results data according to following standard in step (5)：A1) mistake
Long 20 base below of tail portion mass value is read in filter, and the window of 20bp is arranged, if the average mass values in window are less than 20, from window
Mouth starts to clip rear end base, filters 100bp reading length below after Quality Control；A2 the overlapping between length) is read according to double end sequencings
Pairs of length of reading is spliced into a sequence by relationship, and minimum overlay length is 10bp；A3 non repetitive sequence) is extracted, and removal does not have
The simple sequence repeated.
5. method according to claim 3 or 4, it is characterised in that：It is to carry out institute using usearch methods in step (5)
State OTU clusterings；
And/or
In step (5), the fungi ITS sequence database is ncbi database and/or UNITE databases.
6. any the method white fungus and incense ashes bacterium different ratio in assessing white fungus strain are real to white fungus in claim 1-5
Application in the influence of formation and/or the development of body.
7. application of any the method in instructing white fungus strain to prepare in claim 1-5.
8. a kind of method for instructing white fungus strain to prepare, including Overall Steps in any the methods of claim 1-5 and such as
Lower step：Tremella fructification development experiment is carried out in conjunction with white fungus in white fungus strain and incense ashes bacterium different ratio, is seen during experiment
The formation of Tremella fructification and/or developmental state under white fungus and incense ashes bacterium different ratio are examined in white fungus strain, in production
White fungus strain prepare provide reference standard.
9. a kind of method for preparing white fungus strain, includes the following steps：It is high that white fungus strain is configured to wherein white fungus hyphae content
In 1 times of incense ashes bacterium hyphae content and it is less than 2 times of incense ashes bacterium hyphae contents.
10. according to the method described in claim 9, it is characterized in that：Described method includes following steps：White fungus strain is prepared
At 1.25 times that wherein white fungus hyphae content is incense ashes bacterium hyphae content.
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