CN112813193B - Identification method of microsatellite DNA marker fingerprint of flammulina velutipes X995 strain and construction method and application thereof - Google Patents
Identification method of microsatellite DNA marker fingerprint of flammulina velutipes X995 strain and construction method and application thereof Download PDFInfo
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Abstract
The invention discloses a method for identifying a microsatellite DNA marker fingerprint of a needle mushroom X995 strain and a construction method and application thereof, wherein the fingerprint consists of 6 pairs of microsatellite DNA markers. The construction method comprises the following steps: (1) hypha culture; (2) extracting genome DNA; (3) detecting microsatellite DNA molecular markers; and (4) detecting by capillary electrophoresis. The application comprises the following steps: performing microsatellite DNA marker amplification on the flammulina velutipes strain, comparing the obtained banding pattern with the fingerprint spectrum, and obtaining the flammulina velutipes X995 strain if the banding pattern is 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 X995 strain in 105 collected main culture strains for flammulina velutipes cultivation at home and abroad.
Description
Technical Field
The invention belongs to the technical field of detection of flammulina velutipes strains, and particularly relates to an identification method of a microsatellite DNA marker fingerprint of a flammulina velutipes X995 strain, a construction method and application thereof.
Background
Flammulina velutipes (Flammulina filiformis) 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 improved year by year, and the strain is a variety with the fastest development speed and the largest scale in industrial 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. Compared with the current dominant industrialized strains, china has rich wild and natural needle mushroom cultivation resources, the high-quality resources are efficiently utilized, the genetic basis of the strains is expanded, and the breeding of high-yield, high-quality and characteristic needle mushroom strains in China is facilitated. In order to establish a new edible fungus species registration system to really protect the species property rights of China, a mature species identification technology must be established at first to lay a foundation for new species registration. The requirements of industrial cultivation modes and strain degeneration phenomena on the quality of needle mushroom cultivation strains are higher and higher, and a simple, convenient, rapid and accurate strain identification technology needs to be developed to ensure that each batch of strains is high-quality and accurate.
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
The invention aims to solve the technical problem of providing a microsatellite DNA marker fingerprint of flammulina velutipes 'X995' strain and a construction method and application thereof.
Flammulina velutipes (Flammulina filiformis) X995 was collected at Guangdong province culture collection center on 8.2.20.2021, and was collected at the institute of microbiology, guangdong province, guangzhou, minglie Zhonglu No. 100, lou 5, and Guangdong province, with the collection number of GDMCC No. 61521.
The invention relates to a micro-satellite DNA marker fingerprint of flammulina velutipes 'X995' strain, which consists of 6 pairs of micro-satellite DNA markers, is an SSR primer developed based on simple repetitive sequence fragments of flammulina velutipes genome, has good amplification band type and high repeatability, and has detailed marking information as shown in Table 1:
TABLE 1 microsatellite DNA marker details List
The invention relates to a method for constructing a microsatellite DNA marker fingerprint of an X995 strain of flammulina velutipes, 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 microsatellite DNA molecular markers: carrying out PCR amplification of gene microsatellite DNA 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 15-30min;
(4) Adding 140 mu L of Buffer PS, vortexing and shaking for 30s, and standing on ice for 10min;
(5) 13000g was centrifuged for 5min at room temperature, 400. Mu.L of the supernatant was carefully transferred to a new centrifuge tube;
(6) Add 600. Mu.L Buffer PBD (diluted with absolute ethanol) to the sample, vortex and mix for 30s;
(7) Loading the DNA binding column in a collecting tube, transferring half of the mixed solution to the column, and centrifuging at 8000g for 1min;
(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 pipe, adding 600 μ L Buffer GW2 (diluted with absolute ethyl alcohol) into the column, and centrifuging at 8000g for 1min;
(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, and centrifuging at 10000g for 1min;
(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 buffer1 uL, 2.5mmol/L dNTP0.8 uL, 5U/uL HSTaq DNA enzyme 0.1 uL, 5 umol/L microsatellite DNA 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, ddH 2 O 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 a molecular weight internal standard and formamide mixed solution (0.5; 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 3min.
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 operation 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, performing group structure analysis, clustering and heterozygosity analysis by using POPGENE, NTSYS and other software, and performing 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.
TABLE 2 summary of allelic fragment information from SSR primer amplification
The invention discloses an application of a microsatellite DNA marker fingerprint of flammulina velutipes 'X995' strains, which is characterized in that 6 pairs of SSR primers developed by simple repetitive sequence fragments of flammulina velutipes genomes are utilized, a large number of SSR primers are screened, the number of allelic fragments amplified by the 6 pairs of SSR primers in each flammulina velutipes cultivar is determined and numbered (table 2) by performing banding amplification on 105 collected SSR primers of main flammulina velutipes cultivars, and the 'X995' strains can be effectively identified in 105 collected main flammulina velutipes cultivars by combining the numbers of different SSR allelic sites. The relative molecular weight of the allelic sites amplified by each SSR primer can be determined by analyzing through capillary electrophoresis combined with software, the strain with the strain specific SSR allelic fragment combination of 'X995' is the flammulina velutipes 'X995' strain, and the number combination of the strain is as follows: 8/(2+12)/(3+6)/(4+6+10)/(1+2)/(3+6).
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 'X995' 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.
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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 sequentially detecting a primer FfSSR1 in a selected needle mushroom cultivation material X995 and several main cultivation commercial varieties respectively;
FIG. 2 is a diagram of allelic site relative molecular weight peaks obtained by sequential detection of primers FfSSR2 in selected needle mushroom cultivation material X995 and several main cultivation commercial varieties respectively;
FIG. 3 is a diagram showing the relative molecular weight peaks of the allelic sites obtained by sequentially detecting the primers FfSSR4 in the selected Flammulina velutipes cultivation material X995 and several main cultivation commercial varieties;
FIG. 4 is a diagram of the peak of the relative molecular weight of the allelic site obtained by the primer FfSSR13 respectively and sequentially detected in the selected needle mushroom cultivation material X995 and several main cultivation commercial varieties;
FIG. 5 is a diagram showing the relative molecular weight peaks of the allelic sites obtained by sequentially detecting the primers FfSSR15 in the selected Flammulina velutipes cultivation material X995 and several main cultivation commercial varieties;
FIG. 6 is a diagram of the peak of the relative molecular weight of the allelic site obtained by the primer FfSSR17 in the selected needle mushroom cultivation material X995 and several main cultivation commercial varieties in turn.
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, the references herein to "one embodiment" or "an embodiment" refer to a particular feature, structure, or characteristic that may be included in at least one implementation of the present 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:
(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 15-30min;
(4) adding 140 μ L Buffer PS, vortexing and shaking for 30s, and standing on ice for 10min;
(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. Mu.L Buffer PBD (diluted with absolute ethanol) to the sample, vortex and mix for 30s;
(7) loading the DNA binding column in a collecting tube, transferring half of the mixed solution to the column, and centrifuging at 8000g for 1min;
(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 pipe, adding 600 μ L Buffer GW2 (diluted with absolute ethyl alcohol) into the column, and centrifuging at 8000g for 1min;
r repeats step 9;
(3) Detection of microsatellite DNA molecular markers: carrying out PCR amplification of gene microsatellite DNA markers on the extracted DNA;
PCR amplificationThe system is as follows: total volume 10 μ Ι _, comprising: 10 XPCR buffer1 uL, 2.5mmol/L dNTP0.8 uL, 5U/uL HSTaq DNA enzyme 0.1 uL, 5 umol/L microsatellite DNA mark forward primer and reverse primer total volume each 0.6 uL, template DNA 1 uL, ddH extracted with the concentration of 20 ng-30 ng/uL 2 O 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 3min; 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
PCR amplification and capillary electrophoresis are carried out on needle mushroom strains by adopting 6 pairs of SSR primers, and by analyzing allele factors (Na, ne), nei's genetic diversity index (He), shannon's diversity information index (I) and gene observation heterozygosity (Ho) and combining a relative molecular weight peak diagram of an allele site, a combination of Fu Gebian is found out: ffSSR1, ffSSR2, ffSSR4, ffSSR13, ffSSR15, ffSSR17, the corresponding banding pattern is: 8/(2 + 12)/(3+6)/(4 +6+ 10)/(1+2)/(3+6), and the strain is determined to be the flammulina velutipes 'X995' strain. To ensure the accuracy of the identification, three replicates were recommended.
BY taking several main commercial varieties as examples, peak maps of the relative molecular weights of the allelic sites obtained BY sequential detection of 6 pairs of primers are shown in FIGS. 1 to 6 (sequential (1 X995 (2))
Fv-GR)。
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.
Sequence listing
<110> Shanghai city academy of agricultural sciences
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Claims (2)
1. A method for identifying a microsatellite DNA marker fingerprint of flammulina velutipes X995 strain is characterized by comprising the following steps: the fingerprint consists of 6 pairs of microsatellite DNA markers, and the specific sequences of 6 pairs of corresponding primers are as follows:
FfSSR1 forward primer: TCTGAATGTCCCGGAGCGT;
reverse primer: GATACGAGCAGCACTCGCG;
FfSSR2 forward primer: TCTTCTTGGGTGGAAGACG;
reverse primer: CTGAGCTAGGTTCCTCTAC;
FfSSR4 forward primer: GAAGGTGTGTTCGCTGTTC;
reverse primer: CATTGGAGTGGGTAAAGAG;
FfSSR13 forward primer: ATTTCTTCGGATGCTTTGGA;
reverse primer: TTCTCTTTGCACACGTCGAA;
FfSSR15 forward primer: AGTCGTCGTTCAAGGTGTCG;
reverse primer: CGGTTGTTTGTTCCACTTTT;
FfSSR17 forward primer: CCCAGATGATGCTGCAATGC;
reverse primer: CGCTTTGTGGCACTATCTGC;
the corresponding combination of the belt type numbers is as follows: 8/(2 + 12)/(3+6)/(4 +6+ 10)/(1+2)/(3+6);
wherein the band type number corresponding to the primer FfSSR1 is 8, and the band size of the corresponding allele segment is 285 to 285.99bp; the band type number corresponding to the primer FfSSR2 is 2+12, wherein the size of the strip of the allele segment corresponding to the number 2 is 377-377.99bp, and the size of the strip of the allele segment corresponding to the number 12 is 401-401.99bp; the belt type number corresponding to the primer FfSSR4 is 3+6, wherein the size of the belt of the allele segment corresponding to the number 3 is 311 to 311.99bp, and the size of the belt of the allele segment corresponding to the number 6 is 319 to 319.99bp; the belt type number corresponding to the primer FfSSR13 is 4+6+10, wherein the belt size of the allele segment corresponding to the number 4 is 199 to 199.99bp, the belt size of the allele segment corresponding to the number 6 is 211 to 211.99bp, and the belt size of the allele segment corresponding to the number 10 is 229 to 229.99bp; the belt type number corresponding to the primer FfSSR15 is 1+2, wherein the size of the belt of the allele segment corresponding to the number 1 is 186 to 186.99bp, and the size of the belt of the allele segment corresponding to the number 2 is 195 to 195.99bp; the belt type number corresponding to the primer FfSSR17 is 3+6, wherein the size of the belt of the allele segment corresponding to the number 3 is 291 to 291.99bp, and the size of the belt of the allele segment corresponding to the number 6 is 294 to 294.99bp;
the preservation number of the flammulina velutipes X995 strain is GDMCC No. 61521.
2. The use of the microsatellite DNA marker fingerprint of flammulina velutipes X995 strain according to claim 1 wherein: the microsatellite DNA marker fingerprint of the flammulina velutipes X995 strain is used for identifying the specific allelic variation of the flammulina velutipes X995 strain and/or identifying the specificity of the flammulina velutipes X995 strain.
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| CN104131086A (en) * | 2014-07-04 | 2014-11-05 | 上海市农业科学院 | SSR labeled fingerprint of flammulina velutipes F3 strain and applications thereof |
| CN104152547A (en) * | 2014-07-04 | 2014-11-19 | 上海市农业科学院 | SSR marker fingerprint spectrum of needle mushroom Qinghe strain and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104131086A (en) * | 2014-07-04 | 2014-11-05 | 上海市农业科学院 | SSR labeled fingerprint of flammulina velutipes F3 strain and applications thereof |
| CN104152547A (en) * | 2014-07-04 | 2014-11-19 | 上海市农业科学院 | SSR marker fingerprint spectrum of needle mushroom Qinghe strain and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| Development and characterization of simple sequence repeat (SSR) markers for the mushroom Flammulina velutipes;Ruiying Zhang等;《Journal of Bioscience and Bioengineering》;20101231;第110卷(第3期);第274页右栏第2-3段-第275页左栏第1-2段、表1-2、图1 * |
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