CN113005220B - Identification method of microsatellite DNA marker fingerprint of flammulina velutipes J1011 strain, construction method and application thereof - Google Patents
Identification method of microsatellite DNA marker fingerprint of flammulina velutipes J1011 strain, construction method and application thereof Download PDFInfo
- Publication number
- CN113005220B CN113005220B CN202110387632.8A CN202110387632A CN113005220B CN 113005220 B CN113005220 B CN 113005220B CN 202110387632 A CN202110387632 A CN 202110387632A CN 113005220 B CN113005220 B CN 113005220B
- Authority
- CN
- China
- Prior art keywords
- strain
- primer
- flammulina velutipes
- microsatellite dna
- band
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 240000006499 Flammulina velutipes Species 0.000 title claims abstract description 38
- 235000016640 Flammulina velutipes Nutrition 0.000 title claims abstract description 38
- 108091092878 Microsatellite Proteins 0.000 title claims abstract description 24
- 238000000034 method Methods 0.000 title claims abstract description 15
- 239000003550 marker Substances 0.000 title claims abstract description 13
- 238000010276 construction Methods 0.000 title abstract description 8
- 235000001674 Agaricus brunnescens Nutrition 0.000 claims abstract description 11
- 108700028369 Alleles Proteins 0.000 claims description 19
- 239000012634 fragment Substances 0.000 claims description 13
- 238000004321 preservation Methods 0.000 claims 1
- 108020004414 DNA Proteins 0.000 abstract description 36
- 102000053602 DNA Human genes 0.000 abstract description 36
- 238000001514 detection method Methods 0.000 abstract description 13
- 230000003321 amplification Effects 0.000 abstract description 4
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 4
- 230000008485 antagonism Effects 0.000 abstract description 3
- 238000005251 capillar electrophoresis Methods 0.000 abstract description 3
- 238000000605 extraction Methods 0.000 abstract description 3
- 238000001228 spectrum Methods 0.000 abstract description 3
- 230000000877 morphologic effect Effects 0.000 abstract description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 10
- 239000000523 sample Substances 0.000 description 10
- 238000001962 electrophoresis Methods 0.000 description 9
- 238000012408 PCR amplification Methods 0.000 description 7
- 238000011049 filling Methods 0.000 description 6
- 239000000706 filtrate Substances 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 238000011084 recovery Methods 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 239000000872 buffer Substances 0.000 description 5
- 239000000499 gel Substances 0.000 description 5
- 239000011259 mixed solution Substances 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 4
- 241000233866 Fungi Species 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 238000007405 data analysis Methods 0.000 description 3
- 238000004925 denaturation Methods 0.000 description 3
- 230000036425 denaturation Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 229920002401 polyacrylamide Polymers 0.000 description 3
- 239000001965 potato dextrose agar Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 230000004568 DNA-binding Effects 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 241001537207 Flammulina Species 0.000 description 2
- 101001122597 Homo sapiens Ribonuclease P protein subunit p20 Proteins 0.000 description 2
- 108091081062 Repeated sequence (DNA) Proteins 0.000 description 2
- 102100028674 Ribonuclease P protein subunit p20 Human genes 0.000 description 2
- 102000006382 Ribonucleases Human genes 0.000 description 2
- 108010083644 Ribonucleases Proteins 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 238000012197 amplification kit Methods 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 235000019441 ethanol Nutrition 0.000 description 2
- 238000000227 grinding Methods 0.000 description 2
- 239000005457 ice water Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000002798 spectrophotometry method Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000002155 anti-virotic effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000012364 cultivation method Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
Images
Classifications
-
- 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
-
- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Botany (AREA)
- Mycology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses an identification method of microsatellite DNA (deoxyribonucleic acid) marker fingerprint of flammulina velutipes J1011 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) extraction of genomic DNA; (3) detecting microsatellite DNA molecular markers; (4) capillary electrophoresis detection. The application comprises the following steps: and (3) performing microsatellite DNA mark amplification on the flammulina velutipes strain, and comparing the obtained banding pattern with the fingerprint spectrum, wherein the banding pattern is consistent with the group of fingerprint spectrum to obtain the flammulina velutipes J1011 strain. Compared with the 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 needle mushroom J1011 strain in 105 needle mushroom cultivation main cultivated strains collected 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 microsatellite DNA marker fingerprint of flammulina velutipes J1011 strains, and a construction method and application thereof.
Background
Needle mushrooms (Flammulina filiformis) are a commonly cultivated edible fungus, and are generally classified into white and yellow varieties. The cultivation history is long, the total production amount is improved year by year, and the cultivation method is a variety with the fastest development speed and maximum standard in industrial edible fungi enterprises in China. The daily yield of the industrial cultivation of the flammulina velutipes in China is about 47.12 percent of the industrial total yield of the edible fungi in China. The flammulina velutipes has rich nutrition, delicious taste and medicinal value, is rich in various nutritional ingredients such as protein, mineral substances, vitamins and the like, has various medicinal health care effects such as anti-tumor, immunity-enhancing regulation, antivirus, blood fat reduction, fatigue resistance, liver protection and the like, and is deeply favored by consumers.
The contribution rate of the high-quality strain in single yield and quality of flammulina velutipes is important. The industrial strain of golden mushroom in China is mainly white strain with high first tide yield, short growth period and long storage life, which is bred abroad. Compared with foreign countries, the domestic needle mushroom breeding work is relatively lagged, and the method is also a cause of small market share of the current domestic strains. However, compared with the existing dominant industrial strain, china has rich wild and natural cultivation resources of flammulina velutipes, high-quality resources are effectively utilized, the genetic basis of the strain is expanded, and the method is favorable for breeding of high-yield, high-quality and special flammulina velutipes strains in China. In 1999, china signed International New plant species protection law, which not only requires us to respect the species intellectual property rights of other countries, but also strengthens and protects our own species intellectual property rights of our country. In order to establish a new edible fungus variety registration system to truly protect the variety property rights of China, a mature variety identification technology must be established first, and a foundation is laid for new variety registration. In China, the phenomenon of product homogeneity caused by low diversity of flammulina velutipes culture strains not only brings economic loss to production enterprises, but also greatly influences the rapid development of the China flammulina velutipes industry; on the other hand, the industrial cultivation mode and strain degradation phenomenon have higher and higher requirements on the quality of flammulina velutipes cultivated strains, and a strain identification technology which is simpler, more convenient, faster and more accurate needs to be developed so as to ensure that high-quality and accurate strains are used in each batch.
Aiming at the current development situation of the flammulina velutipes industry, the development of a precise and effective flammulina velutipes strain identification system by utilizing the modern molecular biology technology is an extremely important work.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a microsatellite DNA marker fingerprint of a needle mushroom J1011 strain, and a construction method and application thereof.
Needle mushroom (Flammulina filiformis) J1011, deposited at the microorganism seed collection of Guangdong province at 2 months 8 of 2021, was assigned the address of Building 5, no. 59, of the university of Mitsui 100, guangzhou City, and was deposited at the microorganism institute (Building 59,No.100 Central Xian Lie Road,Guangzhou,China) of Guangdong province under the accession number GDMCC No:61517.
the microsatellite DNA marker fingerprint of the flammulina velutipes 'J1011' strain consists of 6 pairs of microsatellite DNA markers, is an SSR primer developed based on flammulina velutipes genome simple repeated sequence segments, has good amplification band and high repeatability, and the marker detailed information is shown in table 1:
TABLE 1 microsatellite DNA marker detailed information list
The invention relates to a construction method of microsatellite DNA marker fingerprint of flammulina velutipes 'J1011' strain, which comprises the following steps:
(1) Hypha culture: transferring flammulina velutipes strains to a potato dextrose agar solid medium (PDA), culturing at 25 ℃ for 5-10 d, and collecting hyphae;
(2) Extraction of genomic DNA: extracting genome DNA of the mycelium by using TaqHotStart amplification kit of TAKARA company, detecting the total genome DNA concentration and purity by ultraviolet spectrophotometry, and adjusting the concentration of 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 PCR amplified product with formamide sampling buffer, denaturing and detecting;
(5) GeneMapper data analysis.
The specific process for extracting the genome DNA of the mycelium by the kit method in the step (2) comprises the following steps:
(1) Adding the mycelium sample into liquid nitrogen for fully grinding;
(2) Adding 360 mu L of Buffer STE and 40 mu L of Buffer SDS into the ground powder rapidly, vortex and mix uniformly rapidly, placing the centrifuge tube in a water bath at 65 ℃ for 15min, and inverting the centrifuge tube to mix the samples for several times during the water bath;
(3) Adding 5 mu L of RNase Solution into the lysate, mixing by vortex, and standing at room temperature for 15-30min;
(4) Adding 140 mu L Buffer PS, vortex shaking for 30s, and standing on ice for 10min;
(5) At room temperature, 13000g was centrifuged for 5min and 400 μl of supernatant was carefully transferred to a new centrifuge tube;
(6) 600 μl Buffer PBD (diluted with absolute ethanol) was added to the sample, and vortexed for 30s;
(7) Loading the DNA binding column in a collecting pipe, transferring half of the mixed solution into the column, centrifuging at 8000g for 1min;
(8) Pouring out the filtrate, filling the column into a recovery header, transferring the residual mixed solution into the column, and centrifuging for 1min at 8000 g;
(9) Pouring out the filtrate, filling the column into a recovery header, adding 600 mu L Buffer GW2 (diluted by absolute ethyl alcohol) into the column, centrifuging 8000g for 1min;
(10) Repeating the step 9;
(11) Pouring out the filtrate, filling the column into a recovery header, centrifuging 10000g for 2min, and removing residual ethanol in the column;
(12) Transferring the column to a new 1.5ml centrifuge tube, adding 30. Mu.L Buffer AE preheated to 65 ℃ to the center of the membrane of the column, standing for 2min at room temperature, and centrifuging for 1min with 10000 g;
(13) mu.L of DNA was used for detection by 1.2% agarose gel electrophoresis, 2. Mu.L of DNA was used for concentration measurement by a NanoDrop spectrophotometer, and the remaining DNA was stored at-20 ℃.
The PCR amplification system in the step (3) is as follows: a total volume of 10 μl, comprising: 10 XPCR buffer 1. Mu.L, 2.5mm0.8 mu L of ol/L dNTP, 0.1 mu L of 5U/mu L HSTaq DNase, 0.6 mu L of 5 mu mol/L microsatellite DNA labeling forward primer and reverse primer respectively, and 1 mu L of extracted template DNA with the concentration of 20 ng-30 ng/mu L and ddH 2 O 5.9μL；
PCR reaction conditions: 95 ℃ for 5min;95℃30second,60℃30second,72℃30second,35 cycles; 30min at 60 ℃.
The sample adding buffer solution in the step (4) is 9 mu L of a mixed solution (0.5:8.5) of an internal molecular weight standard and formamide; the PCR amplification product was added in an amount of 1. Mu.L.
The specific process of denaturation in the step (4) is that denaturation is carried out for 3min at 95 ℃, and the mixture is placed in an ice-water mixture for cooling for 3min after the denaturation is finished.
The electrophoresis in the step (4) has the following technological parameters: the modified polyacrylamide gel is commercial POP7 gel, the electrophoresis buffer 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 20min, and the current and the power are all dynamic.
The data analysis in the step (5) specifically includes: and (3) importing the detected original data file into analysis software genemoper ID3.2, and performing analysis of group structure, clustering and heterozygosity by using POPGENE, NTSYS and other software, wherein the analysis is performed on core germplasm resources. Allele factors (Na, ne), nei's genetic diversity index (He), shannon's diversity information index (I), and gene observed heterozygosity (Ho) were analyzed.
TABLE 2 allelic fragment information summary of SSR primer amplifications
The invention discloses an application of microsatellite DNA (deoxyribonucleic acid) marker finger-print of flammulina velutipes 'J1011' strain, which is to utilize 6 pairs of SSR primers developed by flammulina velutipes genome simple repeated sequence segments, screen a large number of SSR primers, and determine the number and numbering (Table 2) of allelic fragments amplified by the 6 pairs of SSR primers in each flammulina velutipes cultivar through carrying out band-type amplification on the SSR primers of 105 main cultivars collected, so that the 'J1011' strain can be effectively identified in 105 main cultivars collected through the numbering combination of different SSR allelic sites. The relative molecular weight of the allelic site amplified by each SSR primer can be determined by capillary electrophoresis combined software analysis, the strain with the combination of the specific SSR allelic fragments of the strain 'J1011' is the strain 'J1011' of flammulina velutipes, and the serial number combination of the strains is as follows: 6/5/(2+5)/(2+8)/(5+11)/(2+8).
The invention has the beneficial effects that: compared with the conventional morphological detection, antagonism test and fruiting test, the invention has the advantages of short detection time, high accuracy and good repeatability. The operation time required for detection is within 24 hours (including genomic DNA extraction, PCR amplification, electrophoresis analysis and data analysis), while the time required for the conventional antagonism test is at least two weeks, and the fruiting test is at least 3 months; the method of the invention has specificity of 'J1011' strains in 105 commercial flammulina velutipes main cultivation strains (comprising 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 that are needed in the description of the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art. Wherein:
FIG. 1 is a graph showing the relative molecular weight peaks of allele sites detected by a primer FfSSR2 in sequence in a selected flammulina velutipes cultivation material J1011 and several main cultivated commercial varieties, respectively;
FIG. 2 is a graph showing the relative molecular weight peaks of the allele sites detected by the primer FfSSR4 in sequence in the selected flammulina velutipes cultivation material "J1011" and several main cultivated commercial varieties, respectively;
FIG. 3 is a graph showing the relative molecular weight peaks of the allele sites detected by the primer FfSSR7 in sequence in the selected flammulina velutipes cultivation material "J1011" and several main cultivated commercial varieties, respectively;
FIG. 4 is a graph showing the relative molecular weight peaks of the allele sites detected by the primer FfSSR12 in sequence in the selected flammulina velutipes cultivation material "J1011" and several main cultivated commercial varieties, respectively;
FIG. 5 is a graph showing the relative molecular weight peaks of the allele sites detected by the primer FfSSR15 in sequence in the selected flammulina velutipes cultivation material "J1011" and several main cultivated commercial varieties, respectively;
FIG. 6 is a graph showing the relative molecular weight peaks of the allele of the primer FfSSR19 detected sequentially in the selected flammulina velutipes cultivation material "J1011" and several main cultivated commercial varieties, respectively.
Detailed Description
In order that the above-recited objects, features and advantages of the present invention will become more apparent, a more particular description of the invention will be rendered by reference to specific embodiments thereof.
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 other than those described herein, and persons skilled in the art will readily appreciate that the present invention is not limited to the specific embodiments disclosed below.
Further, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic can be 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: transferring needle mushroom strain onto potato glucose agar solid medium (PDA), culturing at 25deg.C for 7d, and collecting mycelium;
(2) Extraction of genomic DNA: extracting genome DNA of the mycelium by using TaqHotStart amplification kit of TAKARA company, detecting the total genome DNA concentration and purity by ultraviolet spectrophotometry, and adjusting the concentration of sample DNA to be consistent;
the process for extracting the genome DNA of the mycelium by the CTAB method comprises the following steps:
(1) adding the mycelium sample into liquid nitrogen for fully grinding;
(2) adding 360 mu L of Buffer STE and 40 mu L of Buffer SDS into the ground powder rapidly, vortex and mix uniformly rapidly, placing the centrifuge tube in a water bath at 65 ℃ for 15min, and inverting the centrifuge tube to mix the samples for several times during the water bath;
(3) adding 5 mu L of RNase Solution into the lysate, mixing by vortex, and standing at room temperature for 15-30min;
(4) adding 140 mu L Buffer PS, vortex shaking for 30s, and standing on ice for 10min;
(5) at room temperature, 13000g was centrifuged for 5min and 400 μl of supernatant was carefully transferred to a new centrifuge tube;
(6) 600 μl Buffer PBD (diluted with absolute ethanol) was added to the sample, and vortexed for 30s;
(7) loading the DNA binding column in a collecting pipe, transferring half of the mixed solution into the column, centrifuging at 8000g for 1min;
(8) pouring out the filtrate, filling the column into a recovery header, transferring the residual mixed solution into the column, and centrifuging for 1min at 8000 g;
(9) pouring out the filtrate, filling the column into a recovery header, adding 600 mu L Buffer GW2 (diluted by absolute ethyl alcohol) into the column, centrifuging 8000g for 1min;
repeating step 9;
(3) Detection of microsatellite DNA molecular markers: carrying out PCR amplification of gene microsatellite DNA markers on the extracted DNA;
the PCR amplification system is as follows: a total volume of 10 μl, comprising: 10 XPCR buffer 1. Mu.L, 2.5mmol/L dNTP 0.8. Mu.L, 5U/mu.L HSTaq DNA enzyme 0.1. Mu.L, 5. Mu.mol/L microsatellite DNA labeled forward and reverse primers 0.6. Mu.L each, template DNA 1. Mu.L extracted at a concentration of 20 ng-30 ng/. Mu.L, ddH 2 O 5.9μL；
PCR reaction conditions: 95 ℃ for 5min;95℃30second,60℃30second,72℃30second,35 cycles; 30min at 60 ℃.
(4) And (3) electrophoresis detection: mixing the PCR amplified product with 1 μl of sample buffer, denaturing at 95deg.C for 3min, and cooling in ice-water mixture for 3min; applying 3 μL sample to a denaturing polyacrylamide gel for electrophoresis, wherein the denaturing polyacrylamide gel is commercial POP7 gel, the electrophoresis buffer 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 20min, the current and the power are dynamic, (5) the result analysis is carried out
Performing PCR amplification and capillary electrophoresis on flammulina velutipes strains by adopting 6 pairs of SSR primers, and finding out the combination meeting the serial numbers by analyzing an allele factor (Na, ne), a Nei's genetic diversity index (He), a shannon's diversity information index (I) and a gene observation heterozygosity (Ho) and combining a relative molecular weight peak diagram of an allele site: fSSR2, fSSR4, fSSR7, fSSR12, fSSR15, and fSSR19, with the corresponding bands: the strain of 6/5/(2+5)/(2+8)/(5+11)/(2+8) can be determined to be the needle mushroom J1011 strain. To ensure accuracy of the identification, three replicates were recommended.
BY taking several main cultivated commercial varieties as an example, allele-site relative molecular weight peak diagrams obtained BY sequentially detecting 6 pairs of primers are given as shown in FIGS. 1 to 6 (which are sequentially shown as (1J 1011. Mu.m.2-SY (3J. Mu.m). It 4-Fv-WC (5V-GF (6V-BY (7V-HL) (8V-RYJ) (9V-XH-HTC))
Fv-GR)。
The fingerprint spectrum of the invention refers to the primer and the band type combination thereof.
It should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, 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 the technical solution of the present invention may be modified or substituted without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered in the scope of the claims of the present invention.
Sequence listing
<110> Shanghai national academy of sciences of agriculture
<120> identification method of microsatellite DNA marker fingerprint of needle mushroom J1011 strain, construction method and application thereof
<130> 2021040901-zf-wjn
<141> 2021-04-09
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
tcttcttggg tggaagacg 19
<210> 2
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
ctgagctagg ttcctctac 19
<210> 3
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
gaaggtgtgt tcgctgttc 19
<210> 4
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
cattggagtg ggtaaagag 19
<210> 5
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
tcttcctgtc accactgttt 20
<210> 6
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
cgtctcgacc atcgttgga 19
<210> 7
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
atgcggcggt ttgtcagaac 20
<210> 8
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
aaccacaacc ttccttcccc 20
<210> 9
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
agtcgtcgtt caaggtgtcg 20
<210> 10
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
cggttgtttg ttccactttt 20
<210> 11
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
ggtacgctaa tccgcttgtt 20
<210> 12
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
cgtcactcgt gtaagtcaat 20
Claims (2)
1. A microsatellite DNA mark fingerprint identification method of flammulina velutipes J1011 strain is characterized in that: the fingerprint consists of 6 pairs of microsatellite DNA markers, and the specific sequences of the corresponding 6 pairs of primers are as follows:
FfSSR2 forward primer: TCTTCTTGGGTGGAAGACG;
reverse primer: CTGAGCTAGGTTCCTCTAC;
FfSSR4 forward primer: GAAGGTGTGTTCGCTGTTC;
reverse primer: CATTGGAGTGGGTAAAGAG;
FfSSR7 forward primer: TCTTCCTGTCACCACTGTTT;
reverse primer: CGTCTCGACCATCGTTGGA;
FfSSR12 forward primer: ATGCGGCGGTTTGTCAGAAC;
reverse primer: AACCACAACCTTCCTTCCCC;
FfSSR15 forward primer: AGTCGTCGTTCAAGGTGTCG;
reverse primer: CGGTTGTTTGTTCCACTTTT;
FfSSR19 forward primer: GGTACGCTAATCCGCTTGTT;
reverse primer: CGTCACTCGTGTAAGTCAAT;
the corresponding band number combinations are: 6/5/(2+5)/(2+8)/(5+11)/(2+8);
the primer FfSSR2 corresponds to a band type number of 6, and the corresponding allele fragment has a band size of 384-384.99 bp; the corresponding band type number of the primer FfSSR4 is 5, and the band size of the corresponding allele fragment is 318-318.99 bp; the band type corresponding to the primer FfSSR7 is numbered 2+5, wherein the band size of the allele fragment corresponding to the number 2 is 139-139.99 bp, and the band size of the allele fragment corresponding to the number 5 is 145-145.99 bp; the band type number corresponding to the primer FfSSR12 is 2+8, wherein the band size of the allele fragment corresponding to the number 2 is 245-245.99 bp, and the band size of the allele fragment corresponding to the number 8 is 251-251.99 bp; the band type corresponding to the primer FfSSR15 is numbered 5+11, wherein the band size of the allele fragment corresponding to the number 5 is 245-245.99 bp, and the band size of the allele fragment corresponding to the number 11 is 264-264.99 bp; the band type number corresponding to the primer FfSSR19 is 2+8, wherein the band size of the allele fragment corresponding to the number 2 is 246-246.99 bp, and the band size of the allele fragment corresponding to the number 8 is 261-261.99 bp;
the preservation number of the flammulina velutipes J1011 strain is GDMCCNo:61517.
2. the application of the microsatellite DNA marker fingerprint of the needle mushroom J1011 strain as claimed in claim 1, which is characterized in that: the microsatellite DNA marker fingerprint of the flammulina velutipes J1011 strain is used for identifying the specific allelic variation of the flammulina velutipes J1011 strain and/or identifying the specificity of the flammulina velutipes J1011 strain.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110387632.8A CN113005220B (en) | 2021-04-09 | 2021-04-09 | Identification method of microsatellite DNA marker fingerprint of flammulina velutipes J1011 strain, construction method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110387632.8A CN113005220B (en) | 2021-04-09 | 2021-04-09 | Identification method of microsatellite DNA marker fingerprint of flammulina velutipes J1011 strain, construction method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113005220A CN113005220A (en) | 2021-06-22 |
| CN113005220B true CN113005220B (en) | 2023-06-06 |
Family
ID=76388304
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110387632.8A Active CN113005220B (en) | 2021-04-09 | 2021-04-09 | Identification method of microsatellite DNA marker fingerprint of flammulina velutipes J1011 strain, construction method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113005220B (en) |
-
2021
- 2021-04-09 CN CN202110387632.8A patent/CN113005220B/en active Active
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274页右栏第2-3段-第275页左栏第1-2段、表1-2、图1） * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113005220A (en) | 2021-06-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113186327B (en) | Identification method of microsatellite DNA marker fingerprint of flammulina velutipes FC89 strain, construction method and application thereof | |
| CN112941224A (en) | Identification method of SSR (simple sequence repeat) marker fingerprint of golden 6046 strain of flammulina velutipes, construction method and application thereof | |
| CN113005220B (en) | Identification method of microsatellite DNA marker fingerprint of flammulina velutipes J1011 strain, construction method and application thereof | |
| CN112795690A (en) | Identification method of SSR (simple sequence repeat) marker fingerprint of flammulina velutipes J3931 strain as well as construction method and application of identification method | |
| CN112941223A (en) | Golden needle mushroom 6168 strain and identification method and construction method and application of SSR marker fingerprint spectrum thereof | |
| CN112725522A (en) | Golden needle mushroom 1767 strain and identification method and construction method and application of SSR marker fingerprint spectrum thereof | |
| CN113151547B (en) | Identification method of microsatellite DNA marker fingerprint of flammulina velutipes FL1980 strain, construction method and application thereof | |
| CN112980992B (en) | Identification method of microsatellite DNA marker fingerprint of flammulina velutipes FW178 strain, construction method and application thereof | |
| CN113186328B (en) | Identification method of microsatellite DNA marker fingerprint of flammulina velutipes xujin 18 strain and construction method and application thereof | |
| CN112980995B (en) | Identification method of microsatellite DNA marker fingerprint of golden needle mushroom 1754 strain and construction method and application thereof | |
| CN112961933B (en) | Identification method of microsatellite DNA marker fingerprint spectrum of flammulina velutipes FL159 strain and construction method and application thereof | |
| CN113151548B (en) | Identification method of microsatellite DNA marker fingerprint of flammulina velutipes FV1923 strain and construction method and application thereof | |
| CN112813193B (en) | Identification method of microsatellite DNA marker fingerprint of flammulina velutipes X995 strain and construction method and application thereof | |
| CN112961934B (en) | Identification method of microsatellite DNA marker fingerprint of flammulina velutipes SCY12 strain and construction method and application thereof | |
| CN112980994B (en) | Identification method of SSR marker fingerprint of needle mushroom strain and construction method and application thereof | |
| CN112746128B (en) | SSR (simple sequence repeat) marker fingerprint of hypsizigus marmoreus HM3 strain, and construction method and application thereof | |
| CN112695131B (en) | SSR (simple sequence repeat) marker fingerprint of Shanghai Zhen 29 strain of hypsizigus marmoreus, construction method and application thereof | |
| CN112760407B (en) | SSR (simple sequence repeat) marker fingerprint of hypsizigus marmoreus HM37 strain, and construction method and application thereof | |
| CN112795682B (en) | SSR (simple sequence repeat) marker fingerprint of Shanghai Zhen 6 strain of hypsizigus marmoreus, construction method and application thereof | |
| CN112795685B (en) | SSR (simple sequence repeat) marker fingerprint of Shanghai Zhen 11 strain of hypsizigus marmoreus, construction method and application thereof | |
| CN112980991B (en) | Identification method of SSR (simple sequence repeat) marker fingerprint of Shanghai Zhen 30 strain of hypsizigus marmoreus, construction method and application thereof | |
| CN112795687B (en) | SSR (simple sequence repeat) marker fingerprint spectrum of hypsizigus marmoreus HM21 strain as well as construction method and application thereof | |
| CN112760404B (en) | SSR (simple sequence repeat) marker fingerprint of hypsizigus marmoreus Finc-B-5 strain, and construction method and application thereof | |
| CN112877458B (en) | SSR (simple sequence repeat) marker fingerprint spectrum of hypsizigus marmoreus HM36 strain as well as construction method and application thereof | |
| CN112795680B (en) | SSR marker fingerprint of Hypsizigus marmoreus Finc-N-11 strain and construction method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |