CN105755116A - Microsatellite-labeled primer linked with precocious puberty or no precocious puberty of eriocheir sinensis and application thereof - Google Patents
Microsatellite-labeled primer linked with precocious puberty or no precocious puberty of eriocheir sinensis and application thereof Download PDFInfo
- Publication number
- CN105755116A CN105755116A CN201610067789.1A CN201610067789A CN105755116A CN 105755116 A CN105755116 A CN 105755116A CN 201610067789 A CN201610067789 A CN 201610067789A CN 105755116 A CN105755116 A CN 105755116A
- Authority
- CN
- China
- Prior art keywords
- eriocheir sinensis
- primer
- individual
- microsatellite
- precocious puberty
- 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.)
- Granted
Links
- 241000371997 Eriocheir sinensis Species 0.000 title claims abstract description 74
- 108091092878 Microsatellite Proteins 0.000 title claims abstract description 28
- 208000006155 precocious puberty Diseases 0.000 title abstract 9
- 108020004414 DNA Proteins 0.000 claims abstract description 28
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 claims abstract description 22
- 229910052709 silver Inorganic materials 0.000 claims abstract description 22
- 239000004332 silver Substances 0.000 claims abstract description 22
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 claims abstract description 3
- 230000001568 sexual effect Effects 0.000 claims description 29
- 230000003321 amplification Effects 0.000 claims description 9
- 239000003550 marker Substances 0.000 claims description 9
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 9
- 238000012408 PCR amplification Methods 0.000 claims description 6
- 238000002372 labelling Methods 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 4
- 238000001962 electrophoresis Methods 0.000 claims description 4
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 4
- 239000012498 ultrapure water Substances 0.000 claims description 4
- 102000004190 Enzymes Human genes 0.000 claims description 3
- 108090000790 Enzymes Proteins 0.000 claims description 3
- 238000000137 annealing Methods 0.000 claims description 3
- 230000004087 circulation Effects 0.000 claims description 3
- 230000007850 degeneration Effects 0.000 claims description 3
- 238000004925 denaturation Methods 0.000 claims description 3
- 230000036425 denaturation Effects 0.000 claims description 3
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L magnesium chloride Substances [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 3
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 3
- 229920002401 polyacrylamide Polymers 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 3
- 238000004153 renaturation Methods 0.000 claims description 3
- 238000012360 testing method Methods 0.000 claims description 3
- 238000011144 upstream manufacturing Methods 0.000 claims description 3
- 208000033240 Progressive symmetric erythrokeratodermia Diseases 0.000 claims description 2
- 238000000034 method Methods 0.000 abstract description 12
- 238000011161 development Methods 0.000 abstract description 4
- 230000003416 augmentation Effects 0.000 abstract 2
- 238000003752 polymerase chain reaction Methods 0.000 abstract 2
- 238000004043 dyeing Methods 0.000 abstract 1
- 108090000623 proteins and genes Proteins 0.000 abstract 1
- 238000010186 staining Methods 0.000 abstract 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 6
- 238000009395 breeding Methods 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 230000001488 breeding effect Effects 0.000 description 4
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 4
- 239000011435 rock Substances 0.000 description 4
- 229960004756 ethanol Drugs 0.000 description 3
- 210000003205 muscle Anatomy 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 238000007476 Maximum Likelihood Methods 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 235000009827 Prunus armeniaca Nutrition 0.000 description 1
- 244000018633 Prunus armeniaca Species 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 229960000935 dehydrated alcohol Drugs 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000013505 freshwater Substances 0.000 description 1
- 210000002149 gonad Anatomy 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000003147 molecular marker Substances 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
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
-
- 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
- C12Q1/686—Polymerase chain reaction [PCR]
-
- 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
- C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
-
- 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
-
- 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
- C12Q2600/158—Expression markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a microsatellite-labeled primer linked with precocious puberty or no precocious puberty of eriocheir sinensis and application thereof. The microsatellite-labeled primer is characterized in that 5 pairs of microsatellite primers are provided, wherein 2 microsatellite sites are linked with the precocious puberty character of the eriocheir sinensis; 3 microsatellite sites are linked with the normal character of the eriocheir sinensis; the primer can be used for identifying whether the eriocheir sinensis has the precocious puberty. The specific identification method comprises the following steps of: taking DNA of a gene group of the eriocheir sinensis to be identified, then carrying out PCR (Polymerase Chain Reaction) augmentation by 5 pairs of microsatellite primers, respectively carrying out polyacrylamide gel electrophoresis on augmentation products, then carrying out silver dyeing staining, and judging the condition of the precocious puberty of the eriocheir sinensis to be identified by the size of strips at different positions. The microsatellite-labeled primer and the application disclosed by the invention have the advantages that normal individuals without precocious puberty can be selected for culture in a targeted manner, so that the normal individuals can be prevented from mixing the young eriocheir sinensis of precocious puberty in the process of early culture, and the development of the eriocheir sinensis in China can be effectively improved.
Description
Technical field
The present invention relates to the primer of a kind of microsatellite marker whether mutually chain with Eriocheir sinensis sexual precosity and application thereof, belong to animal molecular genetics field.
Background technology
Eriocheir sinensis (Eriocheirsinensis), is the important economy aquatic animals of China, is also called crab, is distributed widely in lake, various places on both sides of the Changjiang River in China.Precocious Eriocheir sinensis refers to that the Eriocheir sinensis kind cultivated then is individual bigger and body weight is in that more than 20g, gonad is reached maturity, the Eriocheir sinensis kind for sexual precosity of the Eriocheir sinensis kind nearly 20% that pond is cultivated then, if cultivating commodity crab with precocious Eriocheir sinensis, mortality rate can reach 60%-90%, brings the loss of heaviness to vast Eriocheir sinensis agricultures.The existing method distinguishing precocious Eriocheir sinensis generally sees outward appearance exactly, precocious Eriocheir sinensis and normal Eriocheir sinensis have slight difference in appearance really, but contrast inconspicuous, and to just look at outward appearance method must be that Eriocheir sinensis grows up and just can be seen that to a certain extent, thus waste substantial amounts of manpower and materials.If can not well distinguish precocious Eriocheir sinensis will the strong influence development prospect of China's Eriocheir sinensis, so this area is in the urgent need to developing a kind of identification technology effectively distinguishing precocious Eriocheir sinensis.
Summary of the invention
It is an object of the invention to provide to overcome above the deficiencies in the prior art micro-satellite primers and the application thereof of the microsatellite marker whether mutually chain with Eriocheir sinensis sexual precosity, apply whether this primer can identify Eriocheir sinensis sexual precosity.
Technical scheme is as follows:
The micro-satellite primers of the microsatellite marker whether mutually chain with Eriocheir sinensis sexual precosity, with there being 5 pairs of micro-satellite primers, specific as follows:
A kind of molecule labelling method whether differentiating Eriocheir sinensis sexual precosity, comprises the steps:
(1) Eriocheir sinensis individual specimen to be identified is taken；
(2) genomic DNA of sample is extracted；
(3) utilizing above-described 5 pairs of micro-satellite primers respectively the genomic DNA of sample to be carried out pcr amplification, the product after amplification is in the polyacrylamide gel electrophoresis of non denatured, and then silver contaminates；
(4) contaminate result according to silver in conjunction with stripe size, the judgement that whether precocious Eriocheir sinensis individual specimen Progressive symmetric erythrokeratodermia is is as follows: for the interpretation tested with primer 1, occur that at 144bp the individual specimen of band is that sexual precosity is individual；For the interpretation carrying out testing with primer 2, occur that at 138bp the individual specimen of band is that sexual precosity is individual；To the interpretation tested with primer 3, occur that at 108bp the individual specimen of band is that non-sexual precosity is individual；To the interpretation tested with primer 4, occur that at 150bp and 153bp the individual specimen of band is that non-sexual precosity is individual；To the interpretation tested with primer 5, occur that at 138bp and 144bp the individual specimen of band is that non-sexual precosity is individual.
Further, in step (3), pcr amplification reaction system is 15ul:10 × PCRBuffer1.5ul, 2.5mmol/LdNTP0.5ul, MgCl21.5ul, each 0.5ul of upstream and downstream primer, Taq enzyme 0.2ul, DNA profiling 2ul, ultra-pure water 8.3ul.
Further, in step (3), pcr amplification reaction program is: 95 DEG C of denaturation 2min, 95 DEG C of degeneration 30s, annealing temperature renaturation 30s, and 72 DEG C extend 30s, 35 circulations；72 DEG C extend 10min；4 DEG C of preservations.
Further, the product after the amplification described in step (3) carries out electrophoresis with the non-denaturing polyacrylamide gel of 12%.
Compared with prior art, the present invention has following beneficial effect:
1. SSR primer sets provided by the invention and method more can distinguish Eriocheir sinensis precocity Eriocheir sinensis effectively accurately than traditional outward appearance method of seeing.
2. the present invention just can distinguish precocious Eriocheir sinensis when Eriocheir sinensis is very immature.
3. select, containing normal growth colony linked marker, to remove the individuality containing precocious colony linked marker and carry out resistance precocity crab breeding strain breeding as parent, it will greatly speed up selection-breeding progress.
4., in actual production, it is possible to select the normal individual of non-sexual precosity to cultivate targetedly, can completely avoid in early days breeding process being mixed into sexual precosity germling, improve the development of the Eriocheir sinensis of China greatly.
Accompanying drawing explanation
Fig. 1 is the product silver dye result figure after using primer 1 amplification in the embodiment of the present invention 1；Wherein (1) is normal Eriocheir sinensis silver dye figure, and (2) are precocious crab silver dye figure, and wherein BB is sized to 144bp；
Fig. 2 is the product silver dye result figure after using primer 2 amplification in the embodiment of the present invention 1；Wherein (1) is normal Eriocheir sinensis silver dye figure, and (2) are precocious crab silver dye figure, and wherein II is sized to 138bp；
Fig. 3 is the product silver dye result figure after using primer 3 amplification in the embodiment of the present invention 1；Wherein (1) is normal Eriocheir sinensis silver dye figure, and (2) are precocious crab silver dye figure, and wherein FF is sized to 108bp；
Fig. 4 is the product silver dye result figure after using primer 4 amplification in the embodiment of the present invention 1；Wherein (1) is normal Eriocheir sinensis silver dye figure, and (2) are precocious crab silver dye figure, and wherein CD is sized to 150bp and 153bp；
Fig. 5 is the product silver dye result figure after using primer 5 amplification in the embodiment of the present invention 1；Wherein (1) is normal Eriocheir sinensis silver dye figure, and (2) are precocious crab silver dye figure, and wherein DE is sized to 138bp and 144bp.
Detailed description of the invention:
Embodiment 1
(1) extraction of Eriocheir sinensis DNA:
1. take 24 normal Eriocheir sinensiss of Eriocheir sinensis and 24 precocious Eriocheir sinensiss, take 0.2g muscle from Eriocheir sinensis leg muscle, put in 1.5ml centrifuge tube, add 470ulSET.Muscle grinding rod is smashed to pieces.
2. add 25ul20%SDS (final concentration of 0.5%), be subsequently adding 5ul20mg/ml E.C. 3.4.21.64 (final concentration 200ug/ml).
3. put into 55 DEG C water bath water-baths and see that solution is clarified in 5 hours.Period needs rock for each hour.
4. add 1uRNAse37 C water bath one hour.
5. adding the saturated phenol of 500 milliliters, turn upside down half an hour, action must be slow, it is impossible to acutely rocks, it is prevented that destroys DNA.
Centrifugal 10 minutes of 6.10000rpm.
7. with rifle head sucking-off supernatant, putting into another clean centrifuge tube, the tip of rifle head to be reduced, it is prevented that destroys DNA.Note the troubled liquor not being drawn onto lower floor.
8. repeat 5-7 step.
9. adding phenol: chloroform: isoamyl alcohol (25:24:1), turn upside down half an hour, action is necessarily slow, it is impossible to acutely rock, it is prevented that destroy DNA.Centrifugal 10 minutes of 10000rpm on centrifuge.With rifle head sucking-off supernatant, putting into another clean centrifuge tube, the tip of rifle head to be reduced, it is prevented that destroys DNA.Note the troubled liquor not being drawn onto lower floor.
10. addition chloroform: isoamyl alcohol (24:1), turns upside down half an hour, and action is necessarily slow, it is impossible to acutely rock, it is prevented that destroy DNA.Centrifugal 10 minutes of 10000rpm on centrifuge.With rifle head sucking-off supernatant, putting into another clean centrifuge tube, the tip of rifle head to be reduced, it is prevented that destroys DNA.Note the troubled liquor not being drawn onto lower floor.
11. add the dehydrated alcohol of the pre-cooling of 1ml in supernatant, firmly quickly rotate, until precipitating out white precipitate, or in subzero 20 degrees Celsius of refrigerator overnight.
12. rotate 3 minutes at centrifuge 10000rpm, abandoning supernatant, remaining solid, bar precipitation washed by the ethanol of the 70% of addition 1ml.
13. 10000rpm rotates three minutes on centrifuge, discarding supernatant, now DNA precipitation adheres to loosely with wall, it is easy to skid off with ethanol.Reclaim DNA 70% ethanol and wash away the salt in DNA precipitation.
14.40-50 DEG C drying, add appropriate ultra-pure water.
(2) Eriocheir sinensis PCR primer amplified reaction and electrophoresis detection
5 pairs of micro-satellite primers and condition that this experiment is selected are as shown in table 1, and PCR reaction system is 15ul:10 × PCRBuffer1.5ul, 2.5mmol/LdNTP0.5ul, MgCl21.5ul, each 0.5ul of upstream and downstream primer, Taq enzyme 0.2ul, DNA profiling 2ul, ultra-pure water 8.3ul.PCR response procedures is: 95 DEG C of denaturation 2min；95 DEG C of degeneration 30s, annealing temperature renaturation 30s, 72 DEG C extend 30s, 35 circulations；72 DEG C extend 10min；4 DEG C of preservations.
PCR primer is separated by electrophoresis with 12% non-denaturing polyacrylamide gel, takes pictures after silver dye.Take pictures result as Figure 1-Figure 5.
Table 1 Eriocheir sinensis 5 essential information to micro-satellite primers
(3) data process:
Result is contaminated according to silver, stripe size is analyzed, spss software is utilized to carry out the linear regression analysis of genotype and phenotypic character, adopt maximum likelihood ratio method, using the P=0.05 linkage relationship as the single molecular marker of threshold test Yu character, two microsatellite locus (site 1 and site 2) and Eriocheir sinensis Early mature apricot close linkage detected, three microsatellite locus (site 3, site 4 and site 5) and the normal character close linkage of Eriocheir sinensis.The P value result less than 0.05 is as shown in table 2.
Analysis result and hereditary effect that table 2 labelling returns are estimated
(4) experimental result:
The experimental result tested with primer 1 is analyzed, and the frequency that the homozygote of 144bp band occurs in non-precocious Eriocheir sinensis is 0, and the frequency occurred in precocious Eriocheir sinensis is 16.67%.The experimental result tested with primer 2 is analyzed, and the frequency that the homozygote of 138bp band occurs in non-precocious Eriocheir sinensis is 0, and the frequency occurred in precocious Eriocheir sinensis is 25%.The experimental result tested with primer 3 is analyzed, and the frequency that the homozygote of 108bp band occurs in non-precocious Eriocheir sinensis is 21%, and the frequency occurred in precocious Eriocheir sinensis is 0.The experimental result tested with primer 4 is analyzed, and the frequency that the heterozygote of 150bp band and 153bp band occurs in non-precocious Eriocheir sinensis is 29%, and the frequency occurred in precocious Eriocheir sinensis is 0.The experimental result tested with primer 5 is analyzed, and the frequency that the heterozygote of 138bp band and 144bp band occurs in non-precocious Eriocheir sinensis is 25%, and the frequency occurred in precocious Eriocheir sinensis is 0.
The micro-satellite primers of the microsatellite marker whether mutually chain with Eriocheir sinensis sexual precosity provided by the invention and apply it and identify Eriocheir sinensis sexual precosity whether method, can effectively to whether Eriocheir sinensis sexual precosity be identified, its bigger meaning is in that to identify out by the non-precocious crab in Eriocheir sinensis colony simultaneously, and form the cultivation of scale, completely avoid sexual precosity individuality proportion in current large-scale groups breeding process bigger, and during germling the problem of bad qualification, fundamentally eliminate the adverse effect that potentiality precocity is individual, the development of current Eriocheir sinensis industry can be greatly enhanced.
Sequence table
<110>China Aquatic Science Research Academy Fresh Water Fishery Research Center
<120>micro-satellite primers of the microsatellite marker whether mutually chain with Eriocheir sinensis sexual precosity and application thereof
<130>1
<160>10
<170>PatentInversion3.3
<210>1
<211>24
<212>DNA
<213>artificial sequence
<400>1
tgaatgaaagatgaaaggaggagg24
<210>2
<211>24
<212>DNA
<213>artificial sequence
<400>2
gccaacagatcccaaaacaataac24
<210>3
<211>24
<212>DNA
<213>artificial sequence
<400>3
tttcaacttttctccgggttgtta24
<210>4
<211>24
<212>DNA
<213>artificial sequence
<400>4
cggtgatcctaattacattctggg24
<210>5
<211>24
<212>DNA
<213>artificial sequence
<400>5
ggagagctttaagatgatgccaaa24
<210>6
<211>24
<212>DNA
<213>artificial sequence
<400>6
ttggaggcaagaaagttagtggag24
<210>7
<211>24
<212>DNA
<213>artificial sequence
<400>7
tcctcgtcatcctcatctcttttc24
<210>8
<211>24
<212>DNA
<213>artificial sequence
<400>8
tcgtggctgattctccactacata24
<210>9
<211>24
<212>DNA
<213>artificial sequence
<400>9
ggcaaacaaagagagaagggagac24
<210>10
<211>26
<212>DNA
<213>artificial sequence
<400>10
gaagaattgaaagacagacacaagca26
Claims (5)
1. the micro-satellite primers of the microsatellite marker whether mutually chain with Eriocheir sinensis sexual precosity, it is characterised in that have 5 pairs of micro-satellite primers, specific as follows:
2. the molecule labelling method whether differentiating Eriocheir sinensis sexual precosity, it is characterised in that comprise the steps:
(1) Eriocheir sinensis individual specimen to be identified is taken；
(2) genomic DNA of sample is extracted；
(3) utilizing 5 described in claim 1 pair micro-satellite primers respectively the genomic DNA of sample to be carried out pcr amplification, the product after amplification is in the polyacrylamide gel electrophoresis of non denatured, and then silver contaminates；
(4) contaminate result according to silver in conjunction with stripe size, the judgement that whether precocious Eriocheir sinensis individual specimen Progressive symmetric erythrokeratodermia is is as follows: for the interpretation tested with primer 1, occur that at 144bp the individual specimen of band is that sexual precosity is individual；For the interpretation carrying out testing with primer 2, occur that at 138bp the individual specimen of band is that sexual precosity is individual；To the interpretation tested with primer 3, occur that at 108bp the individual specimen of band is that non-sexual precosity is individual；To the interpretation tested with primer 4, occur that at 150bp and 153bp the individual specimen of band is that non-sexual precosity is individual；To the interpretation tested with primer 5, occur that at 138bp and 144bp the individual specimen of band is that non-sexual precosity is individual.
3. the molecule labelling method whether differentiating Eriocheir sinensis sexual precosity according to claim 2, it is characterised in that in step (3), pcr amplification reaction system is 15ul:10 × PCRBuffer1.5ul, 2.5mmol/LdNTP0.5ul, MgCl21.5ul, each 0.5ul of upstream and downstream primer, Taq enzyme 0.2ul, DNA profiling 2ul, ultra-pure water 8.3ul.
4. the molecule labelling method whether differentiating Eriocheir sinensis sexual precosity according to claim 2, it is characterised in that in step (3), pcr amplification reaction program is: 95 DEG C of denaturation 2min, 95 DEG C of degeneration 30s, annealing temperature renaturation 30s, 72 DEG C extend 30s, 35 circulations；72 DEG C extend 10min；4 DEG C of preservations.
5. the molecule labelling method whether differentiating Eriocheir sinensis sexual precosity according to claim 2, it is characterised in that the product after the amplification described in step (3) carries out electrophoresis with the non-denaturing polyacrylamide gel of 12%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610067789.1A CN105755116B (en) | 2016-01-31 | 2016-01-31 | Primer and its application with microsatellite marker mutually chain whether Eriocheir sinensis sex premature |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610067789.1A CN105755116B (en) | 2016-01-31 | 2016-01-31 | Primer and its application with microsatellite marker mutually chain whether Eriocheir sinensis sex premature |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105755116A true CN105755116A (en) | 2016-07-13 |
| CN105755116B CN105755116B (en) | 2019-04-12 |
Family
ID=56342896
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610067789.1A Expired - Fee Related CN105755116B (en) | 2016-01-31 | 2016-01-31 | Primer and its application with microsatellite marker mutually chain whether Eriocheir sinensis sex premature |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105755116B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107841565A (en) * | 2017-11-22 | 2018-03-27 | 镇江华大检测有限公司 | Differentiate the molecular specificity labeled primers and method of Eriocheir sinensis |
| CN108753995A (en) * | 2018-07-19 | 2018-11-06 | 江苏省淡水水产研究所 | A kind of and Eriocheir sinensis sex premature character significantly correlated SNP site and application |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103555727A (en) * | 2013-10-14 | 2014-02-05 | 中国水产科学研究院淡水渔业研究中心 | Living molecular detection method for precocious puberty individual of Eriocheir sinensis |
| CN103667272A (en) * | 2013-12-04 | 2014-03-26 | 天津市水生动物疫病预防控制中心 | Eriocheir-sinensis-growth-trait-related EST (expressed sequence tag)-SSR (simple sequence repeat) molecular marker and application thereof |
| CN104004750A (en) * | 2014-03-27 | 2014-08-27 | 江苏省淡水水产研究所 | Eriocheir sinensis sexual precocity property related SNP marker and its application |
| CN105385767A (en) * | 2015-12-17 | 2016-03-09 | 中国水产科学研究院淡水渔业研究中心 | Primers and method for identifying different families of eriocheir sinensis |
-
2016
- 2016-01-31 CN CN201610067789.1A patent/CN105755116B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103555727A (en) * | 2013-10-14 | 2014-02-05 | 中国水产科学研究院淡水渔业研究中心 | Living molecular detection method for precocious puberty individual of Eriocheir sinensis |
| CN103667272A (en) * | 2013-12-04 | 2014-03-26 | 天津市水生动物疫病预防控制中心 | Eriocheir-sinensis-growth-trait-related EST (expressed sequence tag)-SSR (simple sequence repeat) molecular marker and application thereof |
| CN104004750A (en) * | 2014-03-27 | 2014-08-27 | 江苏省淡水水产研究所 | Eriocheir sinensis sexual precocity property related SNP marker and its application |
| CN105385767A (en) * | 2015-12-17 | 2016-03-09 | 中国水产科学研究院淡水渔业研究中心 | Primers and method for identifying different families of eriocheir sinensis |
Non-Patent Citations (3)
| Title |
|---|
| QIU G F等: ""A first generation microsatellite-based linkage map of the Chinese mitten crab Eriocheir sinensis, and its application in quantitative trait loci (QTL) detection"", 《AQUACULTURE》 * |
| XIONG L W等: ""Large-Scale Isolation of Microsatellites from Chinese Mitten CrabEriocheir sinensisvia a Solexa Genomic Survey"", 《INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES》 * |
| 吴滟等: ""中华绒螯蟹微卫星标记与生长性状相关性的初步分析"", 《水生生物学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107841565A (en) * | 2017-11-22 | 2018-03-27 | 镇江华大检测有限公司 | Differentiate the molecular specificity labeled primers and method of Eriocheir sinensis |
| CN108753995A (en) * | 2018-07-19 | 2018-11-06 | 江苏省淡水水产研究所 | A kind of and Eriocheir sinensis sex premature character significantly correlated SNP site and application |
| CN108753995B (en) * | 2018-07-19 | 2021-07-02 | 江苏省淡水水产研究所 | SNP (Single nucleotide polymorphism) site obviously related to sexual precocity traits of Eriocheir sinensis and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105755116B (en) | 2019-04-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105154550B (en) | A kind of method using 1217 purity of EST-SSR molecular labeling Rapid identification variety of watermelon treasure sweet tea | |
| CN102134593A (en) | Gender-specific microsatellite marker for Cynoglossus semilaevis and application of same in identification of superfemale Cynoglossus semilaevis | |
| CN104419706B (en) | Snp marker and application thereof | |
| CN109694906B (en) | Specific molecular marker for identifying sex of eriocheir sinensis | |
| CN106520939B (en) | A kind of method and its application of madai Germplasm Identification | |
| CN105176989B (en) | It is a kind of to differentiate fugu obscurus and the primer and method of Fugu Xanthopterus (Temminck et Schlegel) fry | |
| CN104328119B (en) | The related microsatellite molecular marker of megalobrama amblycephala growth traits and application | |
| CN105755143B (en) | A kind of kit and detection method for being used to detect bacterium | |
| CN104419705B (en) | Snp marker and application thereof | |
| CN105755116A (en) | Microsatellite-labeled primer linked with precocious puberty or no precocious puberty of eriocheir sinensis and application thereof | |
| CN105385767A (en) | Primers and method for identifying different families of eriocheir sinensis | |
| CN111321231B (en) | Method for molecular identification of rainbow trout gynogenesis offspring | |
| CN103667272B (en) | The EST-SSR molecule marker relevant to mitten crab growth traits and application | |
| CN102876777B (en) | The special primer of brown croaker EST microsatellite marker and screening method | |
| CN106591489A (en) | Rice grain length gene GW7 molecular marker and special primer sequences thereof | |
| CN111057771B (en) | SNP molecular marker for distinguishing 'Zhongyang No. 1' from common fugu obscurus and application thereof | |
| CN109554483A (en) | A method of common ox and zebu are quickly detected using Y chromosome molecular labeling | |
| CN103866038B (en) | For detecting tobacco to the N gene-specific primer of TMV resistance to, detection method and test kit | |
| CN115679004B (en) | Primer, method and kit for identifying pseudobagrus vachelli, leiocassis longirostris and hybrid species | |
| CN105602945A (en) | Detection primer group, detection kit, and detection method used for simultaneous detection of three pathogenic bacteria of marine fishes | |
| CN103196980B (en) | Rapid pulse field gel electrophoresis typing method for streptococcus fish | |
| CN106282379B (en) | Hybridize the CH of Pelteobagrus fulvidraco4Digestion identification method | |
| CN106868198A (en) | It is a kind of at the same detect SILURIFORMES four kinds of pathogenic bacteria of fish multiple PCR primer group and detection method | |
| CN106868134B (en) | Real-time quantitative PCR detection method for spores/gametes of ulva aquatica, ulva scleoides and hemerocallis fulva in sea cucumber culture water | |
| CN102758021B (en) | Recombination activating gene (RAG) 2 molecular marker restriction fragment length polymorphism (RFLP) identifying method for aquaculture of catfish hybrids |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20190412 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |