CN112760411A - Identification method of SSR marker fingerprint of flammulina velutipes J4137 strain and construction method and application thereof - Google Patents

Abstract

The invention discloses an identification method of an SSR marker fingerprint of a flammulina velutipes strain J4137 and a construction method and application thereof, wherein the fingerprint consists of 6 pairs of SSR markers. The construction method comprises the following steps: (1) culturing hyphae; (2) extracting genome DNA; (3) detecting SSR molecular markers; (4) and (5) detecting by capillary electrophoresis. The application comprises the following steps: and performing SSR marker amplification on the flammulina velutipes strains, comparing the obtained banding patterns with the fingerprint spectrum, and obtaining the flammulina velutipes J4137 strains if the banding patterns are consistent with the fingerprint spectrum. Compared with conventional morphological detection, antagonism test and fruiting test, the method has the advantages of short detection time, high accuracy and good repeatability, and has the specificity of the flammulina velutipes J4137 strain in the collected 105 flammulina velutipes cultivation main culture strains at home and abroad.

Description

Identification method of SSR marker fingerprint of flammulina velutipes J4137 strain and construction method and application thereof

Technical Field

The invention belongs to the technical field of detection of flammulina velutipes strains, and particularly relates to an identification method of an SSR marker fingerprint of a flammulina velutipes J4137 strain, and a construction method and application thereof.

Background

Flammulina velutipes (Flammulinaafiliformis) is a commonly cultivated edible fungus, and is generally classified into white and yellow varieties. The cultivation history is long, the total production amount is promoted year by year, and the strain is the fastest-developing and largest-scale strain in industrialized edible fungus enterprises in China at present. The daily yield of the industrial cultivation of the flammulina velutipes in China approximately accounts for 47.12 percent of the total industrial yield of the edible fungi in China. The needle mushroom is rich in nutrition, delicious in taste and high in medicinal value, is rich in various nutritional ingredients such as proteins, minerals and vitamins, has various medicinal health-care effects of resisting tumors, enhancing immunity regulation, resisting viruses, reducing blood fat, resisting fatigue, protecting liver and the like, and is popular with consumers.

The contribution rate of the high-quality strains in the yield per unit and the quality of the flammulina velutipes is significant. The industrialized culture strain of flammulina velutipes in China is mainly white strain which is bred abroad and has high first tide yield, short growth period and storage tolerance. Compared with foreign countries, the domestic needle mushroom breeding work is relatively lagged, and the reason for the small market share of the domestic strains is also caused. But compared with the current leading industrialized strains, China has rich wild and natural flammulina velutipes cultivation resources, the high-quality resources are efficiently utilized, the genetic basis of the strains is expanded, and the breeding of the domestic high-yield, high-quality and characteristic flammulina velutipes strains is facilitated. In 1999, China signed the protection law of new species of international plants, which not only required us to respect the intellectual property rights of species in other countries, but also strengthened the protection of the intellectual property rights of species in our country. In order to establish a new species registration system of edible fungi to really protect the property rights of species in China, a mature species identification technology must be established at first to lay a foundation for registering the new species. In China, the phenomenon of product homogenization caused by low diversity of needle mushroom culture strains not only brings economic loss to production enterprises, but also greatly influences the rapid development of the needle mushroom industry in China; on the other hand, the requirements of industrial cultivation modes and strain degeneration phenomena on the quality of needle mushroom cultivation strains are higher and higher, and a simpler, faster and more accurate strain identification technology needs to be developed so as to ensure that each batch of strains used is a high-quality and accurate strain.

Aiming at the current development situation of the flammulina velutipes industry, the development of an accurate and effective flammulina velutipes strain identification system by utilizing the modern molecular biology technology is an extremely important work.

Disclosure of Invention

Flammulina velutipes (Flammulinaafiliformis) J4137, which was collected at the Guangdong province microorganism culture collection center 2/8 (2021), and was collected at the Guangzhou city Mr. No. 59, 5 th floor of Michelo No. 100 college, Guangdong province microorganism research institute, and the collection number was GDMCC No. 61530.

The invention aims to solve the technical problem of providing an SSR marker fingerprint of a flammulina velutipes strain J4137 and a construction method and application thereof, wherein the fingerprint has the advantages of short detection time, high accuracy and good repeatability compared with conventional morphological detection, antagonistic test and fruiting test.

The SSR marker fingerprint of the flammulina velutipes strain J4137 consists of 6 pairs of SSR markers, SSR primers are developed based on simple repetitive sequence fragments of flammulina velutipes genomes, the SSR primers are good in amplification band type and high in repeatability, and detailed marker information is shown in a table 1:

TABLE 1 SSR tag detailed information List

The invention relates to a method for constructing an SSR marker fingerprint spectrum of a flammulina velutipes strain J4137, which comprises the following steps:

(1) hypha culture: inoculating needle mushroom strain to potato glucose agar solid culture medium (PDA), culturing at 25 deg.C for 7d, and collecting mycelium;

(2) extraction of genomic DNA: extracting the genome DNA of the hyphae by using a TaqHotStart amplification kit of TAKARA, detecting the concentration and purity of the total genome DNA by an ultraviolet spectrophotometry, and adjusting the concentration of the sample DNA to be consistent;

(3) detection of SSR molecular markers: carrying out PCR amplification of gene SSR markers on the extracted DNA;

(4) and (3) electrophoresis detection: mixing the product obtained by PCR amplification with formamide sample adding buffer solution, denaturing, and detecting on a computer;

(5) GeneMapper data analysis.

The specific process for extracting the genome DNA of the hyphae by the kit method in the step (2) comprises the following steps:

(1) adding liquid nitrogen into the hypha sample, and fully grinding;

(2) adding 360 mu L of Buffer STE and 40 mu L of Buffer SDS into the ground powder quickly, quickly whirling and uniformly mixing, placing the centrifuge tube in a water bath at 65 ℃ for 15min, and reversing the centrifuge tube in the water bath process to mix the sample for a plurality of times;

(3) adding 5 μ L RNase Solution into the lysate, mixing by vortex, and standing at room temperature for 15-30 min;

(4) adding 140 mu LBuffERPS, vortexing and shaking for 30s, and standing on ice for 10 min;

(5) 13000g was centrifuged for 5min at room temperature, and 400. mu.L of the supernatant was carefully transferred to a new centrifuge tube;

(6) add 600 u LBufferPBD (has been diluted with absolute ethanol) to the sample, vortex and mix for 30 s;

(7) loading the DNA binding column in a collecting tube, transferring half of the mixed solution to the column, and centrifuging at 8000g for 1 min;

(8) pouring off the filtrate, putting the column back into the collecting pipe, transferring the residual mixed solution into the column, and centrifuging for 1min at 8000 g;

(9) pouring the filtrate and putting the column back into the collecting tube, adding 600 μ L Buffer GW2 (diluted with absolute ethyl alcohol) into the column, and centrifuging at 8000g for 1 min;

(10) repeating the step 9;

(11) pouring off the filtrate, putting the column back into the collecting pipe, centrifuging for 2min at 10000g to remove the residual ethanol in the column;

(12) transferring the column to a new 1.5ml centrifuge tube, adding 30 μ L of BufferAE preheated to 65 ℃ to the center of the membrane of the column, standing at room temperature for 2min, and centrifuging at 10000g for 1 min;

(13) mu.L of DNA was subjected to 1.2% agarose gel electrophoresis, 2. mu.L of DNA was subjected to NanoDrop spectrophotometry, and the remaining DNA was stored at-20 ℃.

The PCR amplification system in the step (3) is as follows: total volume 10 μ L, including: 10 XPCR buffer 1 uL, 2.5mmol/L dNTP 0.8 uL, 5U/uL HSTaq DNA enzyme 0.1 uL, 5 umol/L SSR mark forward primer and reverse primer total volume 0.6 uL respectively, template DNA extracted with concentration of 20 ng-30 ng/uL 1 uL, ddH₂O 5.9μL；

And (3) PCR reaction conditions: 5min at 95 ℃; 30second at 95 ℃, 30second at 59 ℃, 30second at 72 ℃, 35 cycles; 30min at 60 ℃.

The sample adding buffer solution in the step (4) is 9 mu L of mixed solution of molecular weight internal standard and formamide (0.5: 8.5); the amount of the PCR amplification product added was 1. mu.L.

The specific process of denaturation in the step (4) is to denature at 95 ℃ for 3min, and then to cool in an ice-water mixture for 3 min.

The electrophoresis in the step (4) has the following technological parameters: the modified polyacrylamide gel is commercial POP7 gel, the electrophoresis buffer solution is 3730buffer EDTA, the injection voltage is 2000V, the running voltage is 15000V, the sample injection time is 10s, the temperature is 60 ℃, the capillary length is 50cm, the power is 200W, the electrophoresis is performed for 20min, and the current and the power are dynamic.

The data analysis in the step (5) is specifically as follows: and (3) importing the detected original data file into analysis software genemapper ID3.2, and performing group structure analysis, clustering and heterozygosity analysis and core germplasm resource calculation analysis by using POPGENE, NTSYS and other software. Allele factors (Na, Ne), Nei's genetic diversity index (He), shannon's diversity information index (I) and gene observation heterozygosity (Ho) were analyzed.

TABLE 2 summary of allelic fragment information from SSR primer amplification

The invention discloses an application of an SSR marker fingerprint of a flammulina velutipes strain J4137, which is characterized in that 6 pairs of SSR primers developed by simple repetitive sequence segments of flammulina velutipes genomes are utilized, a large number of SSR primers are screened, the number of allelic segments amplified by the 6 pairs of SSR primers in each flammulina velutipes cultivar is determined and numbered (table 2) by performing banding amplification on the SSR primers of 105 collected main flammulina velutipes cultivars, and the strain J4137 can be effectively identified in 105 collected main flammulina velutipes cultivars by the number combination of different SSR allelic sites. The relative molecular weight of the allelic locus amplified by each SSR primer can be determined by analyzing capillary electrophoresis combined with software, the strain with the specific SSR allelic fragment combination of the strain J4137 is the strain of the flammulina velutipes J4137, and the numbering combination of the strain is as follows: (6+11)/(3+4)/8/2/7/(2+3).

The invention has the beneficial effects that: compared with conventional morphological detection, antagonism test and fruiting test, the method has the advantages of short detection time, high accuracy and good repeatability. The operation time required for detection is within 24h (including genome DNA extraction, PCR amplification, electrophoresis analysis and data analysis), while the time required for a conventional antagonism test is at least two weeks, and the time required for a fruiting test is at least 3 months; the method has the specificity of 'J4137' strains in 105 collected main culture strains of the commercially available flammulina velutipes (including Fv-DY, Fv-FY, Fv-SB, Fv-CYS, Fv-YH, Fv-KL, Fv-MG, Fv-MH, Fv-MI, Fv-MJ, Fv-SY, Fv-FM, Fv-WC, Fv-GF, Fv-BY, Fv-HL, Fv-RYJ, Fv-XH, Fv-HTC, Fv-GR and the like), and has good application prospect.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:

FIG. 1 is a diagram of allelic locus relative molecular weight peaks obtained by sequential detection of primers FfSSR2 in selected needle mushroom cultivation material J4137 and several main cultivation commercial varieties respectively;

FIG. 2 is a diagram of allelic locus relative molecular weight peaks obtained by sequential detection of primers FfSSR4 in selected Flammulina velutipes cultivation material J4137 and several main cultivation commercial varieties respectively;

FIG. 3 is a diagram of the allelic locus relative molecular weight peaks obtained by sequential detection of the primers FfSSR8 in selected Flammulina velutipes cultivation material J4137 and several main cultivation commercial varieties respectively;

FIG. 4 is a diagram of the allelic locus relative molecular weight peaks obtained by sequential detection of the primers FfSSR10 in selected Flammulina velutipes cultivation material J4137 and several main cultivation commercial varieties respectively;

FIG. 5 is a diagram of the allelic locus relative molecular weight peaks obtained by sequential detection of the primers FfSSR12 in selected Flammulina velutipes cultivation material J4137 and several main cultivation commercial varieties respectively;

FIG. 6 is a diagram of the peak of the relative molecular weight of the allelic locus obtained by the primer FfSSR17 detected in turn in the selected needle mushroom cultivation material J4137 and several main cultivation commercial varieties respectively.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with examples are described in detail below.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.

Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.

Example 1:

(1) hypha culture: inoculating needle mushroom strain to potato glucose agar solid culture medium (PDA), culturing at 25 deg.C for 7d, and collecting mycelium;

(2) extraction of genomic DNA: extracting the genome DNA of the hyphae by using a TaqHotStart amplification kit of TAKARA, detecting the concentration and purity of the total genome DNA by an ultraviolet spectrophotometry, and adjusting the concentration of the sample DNA to be consistent;

the CTAB method for extracting genome DNA of hyphae comprises the following steps:

adding a hypha sample into liquid nitrogen for fully grinding;

quickly adding 360 mu LBuffersT and 40 mu LBuffer SDS into the ground powder, quickly whirling and uniformly mixing, placing the centrifugal tube in a water bath at 65 ℃ for 15min, and reversing the centrifugal tube in the water bath process to mix the sample for a plurality of times;

③ adding 5 mu LRNase Solution into the lysate, uniformly mixing by vortex, and standing for 15-30min at room temperature;

adding 140 mu LBuffERPS, whirling and shaking for 30s, and standing on ice for 10 min;

at room temperature, 13000g is centrifuged for 5min, and 400 mu L of supernatant is carefully transferred to a new centrifuge tube;

sixthly, adding 600 mu LBufferPBD (diluted by absolute ethyl alcohol) into the sample, and evenly mixing by vortex for 30 s;

seventhly, the DNA binding column is arranged in a collecting pipe, half of the mixed solution is transferred into the column, and 8000g of the mixed solution is centrifuged for 1 min;

eighthly, pouring the filtrate, putting the column back into a collecting pipe, transferring the residual mixed liquid into the column, and centrifuging for 1min at 8000 g;

ninthly, pouring the filtrate, putting the column back to the collecting pipe, adding 600 mu LBufferGW2 (diluted by absolute ethyl alcohol) into the column, and centrifuging for 1min at 8000 g;

r repeats step 9;

pouring off the filtrate, putting the column back into the collecting pipe, centrifuging for 2min at 10000g to remove the residual ethanol in the column;

transferring the column to a new 1.5ml centrifuge tube, adding 30 μ L of BufferAE preheated to 65 ℃ to the center of the membrane of the column, standing at room temperature for 2min, and centrifuging at 10000g for 1 min;

mu.L of DNA was subjected to 1.2% agarose gel electrophoresis, 2. mu.L of DNA was subjected to NanoDrop spectrophotometry, and the remaining DNA was stored at-20 ℃.

(3) Detection of SSR molecular markers: carrying out PCR amplification of gene SSR markers on the extracted DNA;

the PCR amplification system is as follows: total volume 10 μ L, including: 10 XPCR buffer 1 uL, 2.5mmol/L dNTP 0.8 uL, 5U/uL HSTaq DNA enzyme 0.1 uL, 5 umol/L SSR mark forward primer and reverse primer total volume 0.6 uL respectively, template DNA extracted with concentration of 20 ng-30 ng/uL 1 uL, ddH₂O 5.9μL；

And (3) PCR reaction conditions: 5min at 95 ℃; 30second at 95 ℃, 30second at 59 ℃, 30second at 72 ℃, 35 cycles; 30min at 60 ℃.

(4) And (3) electrophoresis detection: mixing the product obtained by PCR amplification with 1 μ L sample buffer solution, denaturing at 95 deg.C for 3min, and cooling in ice water mixture for 3 min; 3 mu L of sample is applied to a modified polyacrylamide gel for electrophoresis, the modified polyacrylamide gel is commercial POP7 gel, the electrophoresis buffer solution is 3730buffer EDTA, the injection voltage is 2000V, the operation voltage is 15000V, the sample injection time is 10s, the temperature is 60 ℃, the length of a capillary is 50cm, the power is 200W, the electrophoresis is 20min, the current and the power are dynamic,

(5) analysis of results

Performing PCR amplification and capillary electrophoresis on needle mushroom strains by adopting 6 pairs of SSR primers, and finding a matched code combination by analyzing allele factors (Na, Ne), Nei's genetic diversity index (He), shannon's diversity information index (I) and gene observation heterozygosity (Ho) in combination with a relative molecular weight peak diagram of an allele: FfSSR2, FfSSR4, FfSSR8, FfSSR10, FfSSR12 and FfSSR17, and the corresponding band types are: the strain of (6+11)/(3+4)/8/2/7/(2+3) can be determined to be the strain of flammulina velutipes J4137. To ensure the accuracy of the identification, three replicates were recommended.

Taking several main-cultivated commercial varieties as examples, the peak diagrams of the relative molecular weights of the allelic loci obtained by 6 pairs of primers through sequential detection are given, as shown in FIGS. 1-6 (sequentially

). The fingerprint spectrum of the invention refers to the combination of the primer and the band type thereof.

It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.

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Claims (9)

1. An identification method of an SSR marker fingerprint of a flammulina velutipes J4137 strain is characterized by comprising the following steps: the fingerprint comprises 6 pairs of SSR markers, and the specific sequence is as follows:

2. the method for constructing an SSR marker fingerprint of flammulina velutipes J4137 strain according to claim 1, which is characterized in that: comprises the steps of (a) preparing a mixture of a plurality of raw materials,

(1) hypha culture: inoculating needle mushroom strain to potato glucose agar solid culture medium, culturing at 25 deg.C for 7d, and collecting mycelium;

(2) extraction of genomic DNA: extracting the genome DNA of the hyphae by using a TaqHotStart amplification kit of TAKARA, detecting the concentration and purity of the total genome DNA by an ultraviolet spectrophotometry, and adjusting the concentration of the sample DNA to be consistent;

(3) detection of SSR molecular markers: carrying out PCR amplification of gene SSR markers on the extracted DNA;

(4) and (3) electrophoresis detection: mixing the product obtained by PCR amplification with formamide sample adding buffer solution, denaturing, and detecting on a computer;

(5) GeneMapper data analysis.

3. The method for constructing an SSR marker fingerprint of flammulina velutipes J4137 strain according to claim 2, wherein the method comprises the following steps: the step (2) of extracting the genome DNA comprises the following specific steps:

(1) adding liquid nitrogen into the hypha sample, and fully grinding;

(2) adding 360 mu L of Buffer STE and 40 mu L of Buffer SDS into the ground powder quickly, quickly whirling and uniformly mixing, placing the centrifuge tube in a water bath at 65 ℃ for 15min, and reversing the centrifuge tube in the water bath process to mix the sample for a plurality of times;

(3) adding 5 μ L RNase Solution into the lysate, mixing by vortex, and standing at room temperature for 15-30 min;

(4) adding 140 mu LBuffERPS, vortexing and shaking for 30s, and standing on ice for 10 min;

(5) 13000g was centrifuged for 5min at room temperature, and 400. mu.L of the supernatant was carefully transferred to a new centrifuge tube;

(6) add 600 u LBufferPBD to the sample, vortex and mix for 30 s;

(7) loading the DNA binding column in a collecting tube, transferring half of the mixed solution to the column, and centrifuging at 8000g for 1 min;

(8) pouring off the filtrate, putting the column back into the collecting pipe, transferring the residual mixed solution into the column, and centrifuging for 1min at 8000 g;

(9) pouring the filtrate and putting the column back into the collecting tube, adding 600 mu LBuffer GW2 into the column, and centrifuging for 1min at 8000 g;

(10) repeating the step 9;

(11) pouring off the filtrate, putting the column back into the collecting pipe, centrifuging for 2min at 10000g to remove the residual ethanol in the column;

(12) transferring the column to a new 1.5ml centrifuge tube, adding 30 μ L of Buffer AE preheated to 65 deg.C to the center of the membrane of the column, standing at room temperature for 2min, centrifuging at 10000g for 1 min;

(13) mu.L of DNA was subjected to 1.2% agarose gel electrophoresis, 2. mu.L of DNA was subjected to NanoDrop spectrophotometry, and the remaining DNA was stored at-20 ℃.

4. The method for constructing an SSR marker fingerprint of flammulina velutipes J4137 strain according to claim 2, wherein the method comprises the following steps: in the step (3), the PCR amplification is carried out by using an amplification system: total volume 10 μ L, including: 10 XPCR buffer 1 uL, 2.5mmol/L dNTP 0.8 uL, 5U/uL HSTaq DNA enzyme 0.1 uL, 5 umol/L SSR mark forward primer and reverse primer total volume 0.6 uL respectively, template DNA extracted with concentration of 20 ng-30 ng/uL 1 uL, ddH₂O 5.9μL；

And (3) PCR reaction conditions: 5min at 95 ℃; 30second at 95 ℃, 30second at 59 ℃, 30second at 72 ℃, 35 cycles; 30min at 60 ℃.

5. The method for constructing an SSR marker fingerprint of flammulina velutipes J4137 strain according to claim 2, wherein the method comprises the following steps: in the step (4), the product obtained by the PCR amplification is uniformly mixed with a formamide sample adding buffer solution, wherein the volume ratio of the molecular weight internal standard of the sample adding buffer solution to formamide is 0.5: 8.5, the volume of the molecular weight internal standard of the sample adding buffer solution and formamide is totally 9 mu L; the amount of the PCR-amplified product added was 1. mu.L.

6. The method for constructing an SSR marker fingerprint of flammulina velutipes J4137 strain according to claim 2, wherein the method comprises the following steps: in the step (4), the denaturation is carried out for 3min at 95 ℃, and then the mixture is placed in an ice-water mixture for cooling for 3 min.

7. The method for constructing an SSR marker fingerprint of flammulina velutipes J4137 strain according to claim 2, wherein the method comprises the following steps: in the step (4), the modified polyacrylamide gel of the electrophoresis is commercial POP7 gel, the electrophoresis buffer solution is 3730bufferEDTA, the injection voltage is 2000V, the operation voltage is 15000V, the sample injection time is 10s, the temperature is 60 ℃, the length of the capillary is 50cm, the power is 200W, the electrophoresis is 20min, and the current and the power are dynamic.

8. The method for constructing an SSR marker fingerprint of flammulina velutipes J4137 strain according to claim 2, wherein the method comprises the following steps: in the step (5), the data analysis is to import the original data file into analysis software genemapperID3.2, perform group structure analysis, clustering and heterozygosity analysis by using POPGENE and NTSYS software, and perform core germplasm resource calculation analysis; allele factors (Na, Ne), Nei's genetic diversity index (He), shannon's diversity information index (I) and gene observation heterozygosity (Ho) were analyzed.

9. The application of the SSR labeled fingerprint spectrum of flammulina velutipes J4137 strain in claim 1 is characterized in that: the SSR marker fingerprint of the flammulina velutipes J4137 strain is used for identifying the specific allelic variation of the flammulina velutipes J4137 strain and/or identifying the specificity of the flammulina velutipes J4137 strain.

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