CN103866043B - A kind ofly identify silver carp, flathead hybridization and the microsatellite marker of Introgression In Hatchery Stocks individuality and Auele Specific Primer and application - Google Patents
A kind ofly identify silver carp, flathead hybridization and the microsatellite marker of Introgression In Hatchery Stocks individuality and Auele Specific Primer and application Download PDFInfo
- Publication number
- CN103866043B CN103866043B CN201410146788.7A CN201410146788A CN103866043B CN 103866043 B CN103866043 B CN 103866043B CN 201410146788 A CN201410146788 A CN 201410146788A CN 103866043 B CN103866043 B CN 103866043B
- Authority
- CN
- China
- Prior art keywords
- flathead
- silver carp
- individuality
- introgression
- hybridization
- 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
- 241000252234 Hypophthalmichthys nobilis Species 0.000 title claims abstract description 126
- 241000755266 Kathetostoma giganteum Species 0.000 title claims abstract description 123
- 108091092878 Microsatellite Proteins 0.000 title claims abstract description 76
- 238000009396 hybridization Methods 0.000 title claims abstract description 36
- 239000003550 marker Substances 0.000 title claims abstract description 36
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 15
- 238000012408 PCR amplification Methods 0.000 claims abstract description 11
- 238000012360 testing method Methods 0.000 claims abstract description 10
- 239000002773 nucleotide Substances 0.000 claims abstract description 5
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 5
- 108020004414 DNA Proteins 0.000 claims description 24
- 238000001962 electrophoresis Methods 0.000 claims description 18
- 238000001514 detection method Methods 0.000 claims description 16
- 238000006243 chemical reaction Methods 0.000 claims description 15
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 claims description 12
- 229960005542 ethidium bromide Drugs 0.000 claims description 12
- 238000004519 manufacturing process Methods 0.000 claims description 11
- 238000012797 qualification Methods 0.000 claims description 8
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 7
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 7
- 238000000137 annealing Methods 0.000 claims description 7
- 229920002401 polyacrylamide Polymers 0.000 claims description 7
- 230000004087 circulation Effects 0.000 claims description 6
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 6
- 239000012498 ultrapure water Substances 0.000 claims description 6
- 230000001186 cumulative effect Effects 0.000 claims description 5
- 238000013461 design Methods 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 238000004321 preservation Methods 0.000 claims description 5
- 230000001954 sterilising effect Effects 0.000 claims description 5
- 238000004925 denaturation Methods 0.000 claims description 3
- 230000036425 denaturation Effects 0.000 claims description 3
- 238000000605 extraction Methods 0.000 claims description 3
- 238000003556 assay Methods 0.000 claims description 2
- 238000001426 native polyacrylamide gel electrophoresis Methods 0.000 claims description 2
- 238000007447 staining method Methods 0.000 claims description 2
- 239000003643 water by type Substances 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 26
- 238000009360 aquaculture Methods 0.000 abstract description 4
- 244000144974 aquaculture Species 0.000 abstract description 4
- 238000005516 engineering process Methods 0.000 abstract description 3
- 238000009395 breeding Methods 0.000 abstract description 2
- 230000001488 breeding effect Effects 0.000 abstract description 2
- 238000012544 monitoring process Methods 0.000 abstract description 2
- 230000004304 visual acuity Effects 0.000 abstract description 2
- 239000008239 natural water Substances 0.000 abstract 1
- 239000000499 gel Substances 0.000 description 18
- 230000000877 morphologic effect Effects 0.000 description 8
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 239000011541 reaction mixture Substances 0.000 description 6
- 241000252210 Cyprinidae Species 0.000 description 5
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 5
- 230000002068 genetic effect Effects 0.000 description 4
- 238000003384 imaging method Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 3
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 239000012160 loading buffer Substances 0.000 description 3
- 210000003205 muscle Anatomy 0.000 description 3
- 238000012549 training Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 108700028369 Alleles Proteins 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241001417935 Platycephalidae Species 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 238000005336 cracking Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 230000009182 swimming Effects 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 239000004160 Ammonium persulphate Substances 0.000 description 1
- 241000252233 Cyprinus carpio Species 0.000 description 1
- 241001269238 Data Species 0.000 description 1
- 241000252232 Hypophthalmichthys Species 0.000 description 1
- 241000720946 Hypophthalmichthys molitrix Species 0.000 description 1
- 108010044467 Isoenzymes Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- ROOXNKNUYICQNP-UHFFFAOYSA-N ammonium persulfate Chemical compound [NH4+].[NH4+].[O-]S(=O)(=O)OOS([O-])(=O)=O ROOXNKNUYICQNP-UHFFFAOYSA-N 0.000 description 1
- 235000019395 ammonium persulphate Nutrition 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 231100000290 environmental risk assessment Toxicity 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 229910052736 halogen Inorganic materials 0.000 description 1
- 150000002367 halogens Chemical class 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- -1 polypropylene acrylamide Polymers 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000009666 routine test Methods 0.000 description 1
- 238000005204 segregation Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention belongs to colony's authenticate technology field in aquaculture, specifically a kind of identify silver carp and flathead microsatellite marker and Auele Specific Primer and hybridize and application in Introgression In Hatchery Stocks Individual identification silver carp, flathead.Namely specificity microsatellite locus between 18 silver carps, flathead kind is provided, its nucleotides sequence is classified as any one in SEQ ID NO:1-18, the present invention also provides the primer designed for above-mentioned microsatellite locus, pcr amplification result has specificity between kind, can identify silver carp, flathead and hybridization thereof and Introgression In Hatchery Stocks individuality accurately and efficiently, and can be used to carry out Real-Time Monitoring to natural water area and breeding farm parental population gene pool purity.The invention also discloses for above-mentioned silver carp, flathead and hybridization thereof and the individual test kit carrying out identifying of Introgression In Hatchery Stocks.Authentication method of the present invention has that accuracy is high, resolving power is high and fireballing advantage.
Description
Technical field
The invention belongs to colony's authenticate technology field in aquaculture, specifically a kind of identify silver carp and flathead microsatellite marker and Auele Specific Primer and hybridize and application in Introgression In Hatchery Stocks Individual identification silver carp, flathead.
Background technology
Micro-satellite (microsatellites) or title simple sequence repeats (simple sequence repeats, SSR) refer to the simple tandem sequence repeats DNA sequence dna be made up of 1 ~ 6 Nucleotide.All found its existence in all biological species studied so far, and distribution density is very large.Because micro-satellite is distributed widely in genome, have that density is large, rich polymorphism, height heterozygosis and good stability, follow Mendel's law of segregation codominant inheritance, be easy to the features such as pcr amplification, become the most noticeable Novel DNA mark in recent years, and be widely used in many research fields such as the certification of family pedigree, gene linkage analysis, genetic map construction, Idioplasm identification, population genetic diversity of Biological resources.
Silver carp, flathead have the cultivation history of more than one thousand years in China, critical role is occupied in China's culture fishery, because the cultured output of these two kinds of fish is high, cost is low, disease is few, management is simple, they have been introduced into multiple countries and regions as human food source or regulation and control water quality.But, because the habitat in state's external environment and their country of origin is variant, silver carp, flathead intermolecular hybrid and Introgression In Hatchery Stocks have report (Kolar CS et al.Asian Carps of the Genus Hypophthalmichthys (Pisces in a lot of countries and regions, Cyprinidae) – A Biological Synopsis and Environmental Risk Assessment.2005, Report to U.S.Fish and Wildlife Service per Interagency Agreement94400-3-0128; T á trai I et al.Studies on the biological role and effects of Asian carps in Lake Balaton.A Balaton kutat á s á nak, 2006, é vi eredm é nyei Magyar Tudom á nyos Akad é miaBudapest ISSN1419-1075; Verigin BV et al.A case of natural hybridization betweenHypophthalmichthys molitrix and Aristichthys nobilis (Pisces, Cyprinidae) .Zoologicheskii Zhurnal, 1979,58:190 – 196.).In addition, silver carp, flathead hybrid individual was also once found that there is in China, but to be only limitted in artificial propagation field (Gong Luojun etc. there is flathead silver carp hybridization phenomenon in Diao Cha Hu plant. hubei agricultural science, 1991,12:41.).
Silver carp, the flathead hybrid individual speed of growth are all slow than silver carp, flathead, other side do not show yet advantage (Gong Luojun etc. there is flathead silver carp hybridization phenomenon in Diao Cha Hu plant. hubei agricultural science, 1991,12:41.).Therefore, the hybridization between silver carp, flathead and Introgression In Hatchery Stocks will certainly cause the degeneration of both economic characters, and this is concerning the aquaculture of China " four large Chinese carps " being a huge potential threat.Therefore effective method must be taked individual to identify silver carp, flathead hybridization and Introgression In Hatchery Stocks, and these are individually removed, with the gene pool keeping China silver carp, flathead excellent.But, only rely on morphological method accurately can not judge silver carp, the purebred individuality of flathead and hybridization thereof and Introgression In Hatchery Stocks individuality, because a lot of silver carp, flathead hybridization and Introgression In Hatchery Stocks individuality do not show the intermediate character of both, but show tend to both one of phenotype, the individuality how for Introgression In Hatchery Stocks is likely difficult to precise Identification in morphological specificity.Therefore, need to set up one method fast and accurately, silver carp, flathead hybridization and Introgression In Hatchery Stocks individuality are effectively identified.Mia etc. once adopted hybrid individual (the Mia MY detected without total allelic microsatellite marker between 3 silver carps, flathead in plant of Bangladesh, et al.Detection ofhybridization between Chinese carp species (Hypophthalmichthys molitrix andAristichthys nobilis) in hatchery broodstock in Bangladesh, using DNA microsatelliteloci.Aquaculture, 2005,247:267 – 273.).But, the method Shortcomings part, because be only difficult to accurately judge that test individual belongs to species hybridization or Introgression In Hatchery Stocks is individual with 3 sites, even cannot determine purebred silver carp, flathead, need multiple marker site to combine to be analyzed, could effectively distinguish, marker site detects more, and identification will be more accurate.Therefore this invention exploits specificity microsatellite marker between 18 silver carps, flathead kind, by the comprehensive analysis in these sites, more accurately can differentiate that silver carp, flathead hybridization and Introgression In Hatchery Stocks are individual.
Summary of the invention
The object of this invention is to provide a kind of microsatellite marker and the Auele Specific Primer of identifying silver carp and flathead, namely 18 are provided to the microsatellite marker of silver carp, flathead species specificity and corresponding polymorphism primer, for qualification silver carp, flathead hybridization and Introgression In Hatchery Stocks individuality provide effective instrument.
For achieving the above object, the present invention adopts following technical scheme to realize:
One aspect of the present invention provides the microsatellite marker to silver carp, flathead species specificity, and its nucleotide sequence is any one in SEQ IDNO:1-18.
The present invention also provides the primer pair for above-mentioned 18 microsatellite markers design on the other hand, and its sequence is SEQ ID NO:19-54.
Micro-satellite primers of the present invention, to for the identification of silver carp, flathead hybridization and the detection method of Introgression In Hatchery Stocks individuality, comprises the steps:
1) extraction of genomic dna: extract target individual genomic dna;
2) microsatellite PCR amplification: the primer adopting the present invention's design, target individual genomic dna is that template carries out pcr amplification, obtains the micro-satellite amplified production of target individual;
3) electrophoresis detection amplified production: adopt native polyacrylamide gel electrophoresis and ethidium bromide staining method to detect micro-satellite amplified production;
4) gene type assay: according to the molecular size range determination genotype of the micro-satellite amplified production of each individuality, if a certain individuality all shows as silver carp allelotrope at all 18 microsatellite locus, then this individuality is purebred silver carp; If a certain individuality all shows as flathead allelotrope in all 18 sites, then this individuality is purebred flathead; If a certain individuality manifests silver carp, flathead allelotrope in all 18 sites all simultaneously, then this individuality be silver carp,
Flathead hybrid individual; If at least 1 site of a certain individuality in 18 sites manifests silver carp, flathead etc. simultaneously
Position gene, then this individuality is silver carp, flathead Introgression In Hatchery Stocks individuality.
Improvement to technique scheme: extract target individual genomic dna, is diluted for 20-50ng/ μ L, and add 1 μ L in each PCR reaction, reaction cumulative volume is 12.5 μ L.
Further improvement to technique scheme: the application of sample parameter of described pcr amplification is: each PCR reaction system cumulative volume 12.5 μ L, comprise the template DNA of 20-50ng, 0.4U Taq archaeal dna polymerase, 1.25 μ L10 × PCR Buffer, 0.4 μ L dNTP (2.5mM), the forward and reverse mix primer of 0.4 μ L (each 2.5 μMs), finally adds appropriate sterilizing ultrapure water to 12.5 μ L; The program parameter that PCR instrument is set during this primer is used to be: 94 DEG C of denaturations 5 minutes, (94 DEG C of sex change, 35 seconds, 50 ~ 60 DEG C annealing 35 seconds, 72 DEG C extend 40 seconds) 35 circulations; Last 72 DEG C extend 8 minutes eventually, and PCR primer is in 4 DEG C of preservations.
Further improvement to technique scheme: by PCR primer 10% non-denaturing polyacrylamide gel, 30W invariable power electrophoresis 2h is separated; By offset plate by ethidium bromide (EB) the solution colour developing 5-10 minute of 1%, the gene type of target individual at 18 locus can be obtained.
Present invention also offers the above-mentioned test kit identified silver carp, flathead and hybridization thereof and Introgression In Hatchery Stocks individuality, this test kit comprises the above-mentioned micro-satellite primers carrying out identifying for silver carp, flathead and hybridization thereof and Introgression In Hatchery Stocks individuality.According to practical situation, described test kit comprises Taq archaeal dna polymerase, 10 × PCR Buffer, dNTPs, sterilizing ultrapure water, MgCl further
2.
When the extraction of the synthesis of primer, genomic dna, dNTPs and Taq archaeal dna polymerase reagent are from different batches, PCR reaction conditions, comprises annealing temperature etc. and all certain change can occur.So before carrying out PCR in enormous quantities at every turn, a small amount of individuality all to be selected to carry out thermograde, primer concentration grads PCR, carry out 2% agarose gel electrophoresis, detect expanding effect, to determine best PCR reaction conditions.In addition, after determining the annealing temperature (Tm) of single marking primer PCR, according to the size of PCR primer, suitably part primer can be combined, then grope the condition of multi-PRC reaction.The target product fragment of answering due to used primer pair is no more than 300bp, so the extension time in circulation all controls at about 45 seconds.
Apply above-mentioned primer and detection technique and efficiently can identify that silver carp, flathead hybridization and Introgression In Hatchery Stocks are individual, Real-Time Monitoring can be carried out to the gene pool purity of silver carp, flathead natural population and breeding parent, and the isozyme always continued to use in other species and microsateilite site etc. can be substituted in carry out hybridizing and the detection method of Introgression In Hatchery Stocks.
The present invention compared with prior art has the following advantages:
1. simple to operate, accuracy of judgement;
2. detection speed is fast, because allelotrope number is few, easily distinguishing, can judge without the need to carrying out complicated calculations;
3. resolving power is high.
Method according to the present invention develops corresponding detection kit, for the identification of silver carp, flathead hybridization and Introgression In Hatchery Stocks Individual identification, this test kit has easy, quick, the sensitive advantage of using method, do not need special instrument, be applicable to the needs that Routine Test Lab detects, and detected result is reliable, stable, accurately, for efficiently identifying silver carp, flathead hybridizes and Introgression In Hatchery Stocks individuality provides convenient.
Accompanying drawing explanation
Fig. 1 is the polyacrylamide gel electrophoresis banding pattern figure of the individual specificity microsatellite locus between 3 silver carps, flathead kind of purebred silver carp individuality (left side) and purebred flathead (right side).
Fig. 2 is silver carp, the polyacrylamide gel electrophoresis banding pattern figure of the individual specificity microsatellite locus between 2 silver carps, flathead kind of flathead artificial hybridization family (the 1st swimming lane is DNA Marker, the 2nd, 3 swimming lanes are respectively silver carp, flathead parent.）。Fig. 3 is that (figure A is that Introgression In Hatchery Stocks is individual, and figure B is hybrid individual, and both sides are PBR322DNA Marker for the polyacrylamide gel electrophoresis banding pattern figure of the individual and hybrid individual specificity microsatellite locus between 10 silver carps, flathead kind of random selecting of Introgression In Hatchery Stocks.）。
Fig. 4 is that to the column diagram of the silver carp that morphology judges, flathead and hybrid individual qualification result thereof, (in figure, white represents the distinctive allelotrope of flathead according to specificity microsatellite marker between 18 silver carps, flathead kind, black represents the peculiar allelotrope of silver carp, every bar vertical line represents body one by one, and vertical line is entirely for same color represents that this individuality is for purebred silver carp or flathead; If a certain individual white is equal with black length, then this individuality is hybrid individual, if length does not wait, is that Introgression In Hatchery Stocks is individual.）。
Fig. 5 is that to the column diagram of the silver carp that morphology judges, flathead and hybrid individual qualification result thereof, (in figure, white represents the allelotrope that can detect in flathead according to 8 polymorphic micro-satellite markers, black represents the allelotrope that can detect in silver carp, and every bar vertical line represents body one by one.）。
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.These embodiments are only not used in restriction the scope of protection of present invention for illustration of the present invention; unreceipted specific experiment condition and method in the following example; usually conveniently condition as chief editors such as J. Pehanorm Brookers; Science Press; 2002; Molecular Cloning: A Laboratory guide (third edition), or according to the condition that manufacturer advises.
The screening of [embodiment 1] microsatellite locus
Between 18 silver carps in the present invention, flathead kind, specificity microsatellite marker is strictly screened by three times obtained.Construct silver carp, flathead enriched microsatellite library by magnesphere in the research that this laboratory is previous, and obtained the sequence repeated containing micro-satellite by cloning and sequencing.From the sequence containing micro-satellite, choose core and repeat more than 5 times and the sequence meeting design of primers requirement, adopt Primer Premier5.0 to carry out design of primers.Significant parameter is set to: primer length 20-25bp, PCR primer fragment length scope 120-350bp, the suitableeest annealing temperature 50-60 DEG C.GC content, generally between 40%-60%, avoids secondary structure to occur as far as possible.Finally have successfully been obtained the silver carp of 227 Absorbable organic halogens amplifications, flathead microsatellite marker.
By these 227 microsatellite markers silver carp, flathead each 5 from the individuality of different groups in increase, if the total allelotrope of a certain microsatellite marker nothing between silver carp, flathead, then this site is preserved for next round screening, if there is total allelotrope, directly eliminates.After being screened by the first round, again the microsatellite marker filtered out is detected in each 8 individualities of 14 Ge Lian colonies and 15 Ge Yong colonies, if a certain microsatellite marker is the total allelotrope (Fig. 1) of nothing between silver carp, flathead still, then this site is retained for subsequent use, and in the event of total allelotrope, then eliminated.After taking turns screening by second, the microsatellite marker filtered out is screened in 2 silver carps, each 40 individualities of flathead positive and negative friendship artificial hybridization family, be marked at if a certain in all individualities and show silver carp, flathead unique allele all simultaneously, then this mark can be used as specificity microsatellite marker between silver carp, flathead kind, and can be applicable to the Molecular Identification (Fig. 2) of silver carp, flathead; And if in some individuality, only show the allelotrope of silver carp or flathead, then this site may contain Null allele, if for silver carp, flathead qualification may cause error, therefore this microsatellite marker is also excluded.Finishing screen selects the microsatellite marker that 18 can be used for carrying out silver carp, flathead and hybridization thereof and Introgression In Hatchery Stocks individuality Molecular Identification, SEQ ID NO:1-19, and primer (SEQID NO:19-54) information of each mark is as shown in the table:
Between [embodiment 2] silver carp, flathead kind, specificity microsatellite marker identifies composition and the preparation of PCR system and electrophoresis detection amplified production system
A) .PCR system composition:
DNTPs(10mM) be the raw work Products in Shanghai; Taq archaeal dna polymerase (5U/ μ L), 10 × PCRBuffer are Dalian TaKaRa Products;
B) .10 × PCR Buffer forms:
Tris-HCl(pH8.3)100mM、KCl500mM、MgCl215mM；
C) .PCR reaction system preparation:
Cumulative volume 12.5 μ L, comprises the template DNA of 20-50ng, 0.4U Taq archaeal dna polymerase, 1.25 μ L10 × PCR Buffer, the forward and reverse mix primer of 0.4 μ L dNTP (2.5mM), 0.4 μ L (each 2.5 μMs), finally adds appropriate sterilizing ultrapure water to 12.5 μ L;
D). polyacrylamide gel is prepared:
30% acrylamide 8.5mL, 5 × TBE5mL, 10% ammonium persulphate 0.5mL, TEMED5 μ L, distilled water 11.5mL.
The silver carp between [embodiment 3] silver carp, flathead kind, specificity microsatellite marker qualification morphology judged, flathead and hybrid individual thereof
A). get the three kinds of individualities to be identified being judged as silver carp, flathead and cross-fertilize seed according to morphological specificity, utilize the genomic dna of each individuality of phenol/chloroform method extracting, and DNA is diluted to the concentration of 20-50ng/ μ L.
B). with A) DNA that step is carried is template, according to embodiment 2C) the system preparation PCR reaction mixture of step.The PCR reaction mixture that need are prepared in addition to be accredited as silver carp, the purebred individual DNA of flathead is template in contrast.
C). increase in PCR instrument, 94 DEG C of denaturations, after 5 minutes, carry out 35 PCR reaction cycle, and under each circulation comprises 94 DEG C of sex change 35 seconds, suitable annealing temperature, annealing 35 seconds, 72 DEG C extends 40 seconds; 72 DEG C extend 8 minutes eventually.
D) .PCR product is with according to embodiment 2D) polyacrylamide gel electrophoresis of step system preparation is separated, with 1% ethidium bromide (EB) solution, the gel completing electrophoresis being dyeed, and clear with the training of JS-A380(Shanghai) gel imaging instrument is to often opening gel image scanning and preservation of taking pictures.
E). after between 18 kinds of the present invention, specificity microsatellite marker carries out pcr amplification in target group, by PCR primer electrophoresis in non-denaturing polyacrylamide gel, put after PCR primer being added appropriate sample-loading buffer and carry out electrophoresis in gel, electrophoresis power is 30W, voltage control, at 230V, can manifest the amplification banding pattern of each individuality in colony by ethidium bromide (EB) on ultraviolet gel imaging instrument after being dyeed.Carry out silver carp, flathead species hybridization and Introgression In Hatchery Stocks individuality by the following method to differentiate: if a certain individuality all shows as silver carp allelotrope at all 18 marker sites, then this individuality is purebred silver carp; If a certain individuality all shows as flathead allelotrope in all 18 sites, then this individuality is purebred flathead; If a certain individuality manifests silver carp, flathead allelotrope in all 18 sites all simultaneously, then this individuality is silver carp, flathead hybrid individual; If at least 1 site of a certain individuality in 18 sites manifests silver carp, flathead allelotrope simultaneously, then this individuality is silver carp, flathead Introgression In Hatchery Stocks individuality.In order to the discriminating of more directviewing description Introgression In Hatchery Stocks and hybrid individual, random selecting 10 microsatellite markers carry out gene type in Introgression In Hatchery Stocks and each individuality of hybridization, as shown in Figure 3: in figure, A, B are respectively the polyacrylamide gel electrophoresis banding pattern figure of an Introgression In Hatchery Stocks individuality and hybrid individual specificity microsatellite locus between 10 silver carps, flathead kind, both sides are PBR322DNA Marker, shown in figure A, Introgression In Hatchery Stocks is individual only shows as heterozygosis banding pattern at moiety site, and shown in figure B, silver carp, flathead hybrid individual then all show as heterozygosis banding pattern at all sites.
The comparison of specificity microsatellite marker method and microsateilite method determination rates and accuracy rate between [embodiment 4] silver carp, flathead kind
1, specificity microsatellite marker method between silver carp, flathead kind:
Acquire 167 tail silver carps, flathead sample altogether from U.S.'s Mississippi and korneforos, Illinois, being identified by morphological feature wherein has 60 tail silver carps, 83 tail flatheads and 24 tail hybrid individual.Every tail sample is got appropriate muscle tissue and is pulverized in liquid nitrogen, and drying is placed in centrifuge tube, and is kept in 4 DEG C of refrigerators for subsequent use.When carrying out extracting genome DNA, the muscle tissue of getting each sample is about 10mg, after cracking, digestion, utilizes phenol/chloroform method to extract genomic dna.After extracting genome DNA completes, be diluted to 20-50ng/ μ l with aseptic ultrapure water for subsequent use.According to embodiment 2C) system of step preparation PCR reaction mixture, reaction the primer for announce in the present invention 18 pairs of silver carps, specificity micro-satellite primers between flathead kind, its sequence is SEQ ID NO:19-54.Carry out amplified reaction by the Veriti96Well ThermalCycler PCR amplification instrument being placed in ABI company containing the PCR pipe of reaction mixture prepared, PCR circulation program thereby is according to embodiment 3C) step arranges.According to embodiment 2D) the system configurations polyacrylamide gel of step, put after PCR primer being added appropriate sample-loading buffer and carry out electrophoresis in gel, electrophoresis power is 30W, and voltage control, at 230V, controls electrophoresis time at 2-3h according to the different sizes of each microsatellite marker amplified production.After electrophoresis completes, dye gel in the EB solution of 1% 5-10min, and rear JS-A380(Shanghai training of having dyeed is clear) gel imaging instrument scans and preservation of taking pictures often opening gel.
The genotype of each individuality specificity microsatellite locus between 18 silver carps, flathead kind in statistics electrophoretic image, and according to embodiment 3E) authentication method of step identify each individuality belong to silver carp, flathead, hybridization and Introgression In Hatchery Stocks which kind of.The gene type statistic datas input Structure2.2 software marked these 18 in addition carries out genetic construction calculating, to obtain the degree of silver carp by identification by morphological characters, flathead Introgression In Hatchery Stocks.Utilize specificity microsatellite marker between 18 silver carps, flathead kind to 167 tail samples identify rear discovery morphology is identified silver carp, to there is a large amount of Introgression In Hatchery Stocks in flathead individual, as shown in Figure 4: in figure, white represents the distinctive allelotrope of flathead, black represents the distinctive allelotrope of silver carp, every bar vertical line represents body one by one, and vertical line is entirely for same color represents that this individuality is for purebred silver carp or flathead; If a certain individual white is equal with black length, then this individuality is hybrid individual, if length does not wait, is that Introgression In Hatchery Stocks is individual.This figure can reflect hybridization in silver carp, flathead colony and Introgression In Hatchery Stocks phenomenon accurately, intuitively.In addition, by the analysis of the banding pattern that increases in specificity microsatellite marker between 18 kinds to each individuality, following detailed appraising datum can be obtained:
This qualification result shows, and in U.S.'s water body, has the individuality of considerable part to be accredited as Introgression In Hatchery Stocks individuality in the silver carp of being identified by morphological method, flathead, and by the hybrid individual of identification of morphology in fact major part be also that Introgression In Hatchery Stocks is individual.Therefore, along with the lasting generation of silver carp, flathead species hybridization and Introgression In Hatchery Stocks, the result only relying on morphological method to carry out Identification of Species to silver carp, flathead is very unreliable.
2. microsateilite method:
Acquire 167 tail silver carps, flathead sample altogether from U.S.'s Mississippi and korneforos, Illinois, being identified by morphological feature wherein has 60 tail silver carps, 83 tail flatheads and 24 tail hybrid individual.The muscle tissue of getting this kept dry of various kinds is about 10mg, after cracking, digestion, utilizes phenol/chloroform method to extract genomic dna.After extracting genome DNA completes, be diluted to 20-50ng/ μ L with aseptic ultrapure water for subsequent use.According to embodiment 2C) system of step preparation PCR reaction mixture, reaction the primer is second take turns 8 pairs of silver carps that screening obtains, flathead polymorphic micro-satellite primer, SEQ ID NO:55-70 in the embodiment of the present invention 1, as shown in the table:
Carry out amplified reaction by the Veriti96Well ThermalCycler PCR amplification instrument being placed in ABI company containing the PCR pipe of reaction mixture prepared, PCR circulation program thereby settles embodiment 3C) step arranges.
According to embodiment 2D) the system configurations polyacrylamide gel of step, put after PCR primer being added appropriate sample-loading buffer and carry out electrophoresis in gel, electrophoresis power is 30W, and voltage control, at 230V, controls electrophoresis time at 2-3h according to the different sizes of each microsatellite marker amplified production.After electrophoresis completes, dye gel in the EB solution of 1% 5-10min, and rear JS-A380(Shanghai training of having dyeed is clear) gel imaging instrument scans and preservation of taking pictures often opening gel.
The genotype of each individuality in 8 silver carps, flathead polymorphic micro-satellite site in statistics electrophoretic image, and the gene type statistic data input Structure2.2 software marked these 8 carries out genetic construction calculating, to assess the degree of silver carp, flathead Introgression In Hatchery Stocks.As shown in Figure 5, occur in some individuality although can observe to represent the allelic white that can detect in flathead and represent in silver carp the allelic black that can detect simultaneously, but due to allelic existence total between silver carp, flathead, cannot judge that the color mixed is by total allelotrope or is caused by Introgression In Hatchery Stocks by this figure.Therefore, although adopt polymorphic micro-satellite markers also can find to there occurs hybridization or Introgression In Hatchery Stocks between silver carp, flathead, but because polymorphic site has phase isoallele between silver carp, flathead, therefore accurately can not distinguish hybridization and Introgression In Hatchery Stocks individual, also cannot obtain purebred, hybridize and the concrete number of Introgression In Hatchery Stocks individuality and ratio.In addition, owing to often there is multiple allelotrope after polymorphic micro-satellite markers somatotype, therefore genotypic statistics need consume the plenty of time and carries out, between the allelotrope in especially some site, clip size difference is little, only be difficult to distinguish this kind of allelotrope with plain polypropylene acrylamide gel electrophoretic method, i.e. enable somatotype, also easily causes error in statistics.To carry out accurate statistics and record to the allelotrope in each site, need to carry out fluorescent mark to often pair of primer, adopt sequenator to carry out gene type, this considerably increases research cost undoubtedly.
And 18 silver carps disclosed in the present invention, the allelotrope number of specificity microsatellite marker is few between flathead kind, and be perfectly clear in silver carp, flathead allelic scope, therefore, adopt present method without the need to by complicated gene type and data statistics and method of calculation, can identify accurately and rapidly research object.Low, consuming time short, simple to operate, the accuracy of judgement of present method cost, is worth promoting in silver carp, flathead qualification work.
Claims (9)
1. a specific microsatellite marker between silver carp and flathead kind, is characterized in that, without total allelotrope between silver carp and flathead, can be used for qualification silver carp and flathead, its nucleotide sequence is SEQ ID NO:1-18.
2. micro-satellite primers pair, described primer pair is microsatellite locus design according to claim 1, and it is characterized in that, described micro-satellite primers comprises following primer pair, and nucleotide sequence is SEQ ID NO:19-54:
3. micro-satellite primers according to claim 2 is to for the identification of silver carp, flathead hybridization and the detection method of Introgression In Hatchery Stocks individuality, it is characterized in that comprising the steps:
1) extraction of genomic dna: extract target individual genomic dna;
2) microsatellite PCR amplification: the primer adopting claim 2, target individual genomic dna is that template carries out pcr amplification, obtains the micro-satellite amplified production of target individual;
3) electrophoresis detection amplified production: adopt native polyacrylamide gel electrophoresis and ethidium bromide staining method to detect micro-satellite amplified production;
4) gene type assay: according to the molecular size range determination genotype of the micro-satellite amplified production of each individuality, if a certain individuality all shows as silver carp allelotrope at all 18 microsatellite locus, then this individuality is purebred silver carp; If a certain individuality all shows as flathead allelotrope in all 18 sites, then this individuality is purebred flathead; If a certain individuality manifests silver carp, flathead allelotrope in all 18 sites all simultaneously, then this individuality is silver carp, flathead hybrid individual; If at least 1 site of a certain individuality in 18 sites manifests silver carp, flathead allelotrope simultaneously, then this individuality is silver carp, flathead Introgression In Hatchery Stocks individuality.
4. micro-satellite primers according to claim 3 is to for the identification of silver carp, flathead hybridization and the detection method of Introgression In Hatchery Stocks individuality, it is characterized in that, extract target individual genomic dna, diluted for 20-50ng/ μ L, add 1 μ L in each PCR reaction, reaction cumulative volume is 12.5 μ L.
5. the micro-satellite primers according to claim 3 or 4 is to for the identification of silver carp, flathead hybridization and the detection method of Introgression In Hatchery Stocks individuality, it is characterized in that, the application of sample parameter of described pcr amplification is: each PCR reaction system cumulative volume 12.5 μ L, comprise the template DNA of 20-50ng, 0.4U Taq archaeal dna polymerase, 1.25 μ L10 × PCR Buffer, 2.5mM dNTP 0.4 μ L, containing the mix primer 0.4 μ L of each 2.5 μMs of forward and reverse primer, 9.4 μ L sterilizing ultrapure waters; The program parameter that PCR instrument is set during this primer is used to be: 94 DEG C of denaturations 5 minutes; 94 DEG C of sex change, 35 seconds, 50 ~ 60 DEG C annealing 35 seconds, 72 DEG C extend 40 seconds, 35 circulations of increasing; Last 72 DEG C extend 8 minutes eventually, and PCR primer is in 4 DEG C of preservations.
6. the micro-satellite primers according to claim 3 or 4 is to for the identification of silver carp, flathead hybridization and the detection method of Introgression In Hatchery Stocks individuality, it is characterized in that, the detection to PCR primer: by PCR primer 10% non-denaturing polyacrylamide gel 30W invariable power electrophoresis 2h be separated; By offset plate by the ethidium bromide solution colour developing 5-10 minute of 1%, the gene type of target individual at 18 locus can be obtained.
7. micro-satellite primers according to claim 5 is to for the identification of silver carp, flathead hybridization and the detection method of Introgression In Hatchery Stocks individuality, it is characterized in that, detection to PCR primer: by PCR primer 10% non-denaturing polyacrylamide gel, 30W invariable power electrophoresis 2h is separated; By offset plate by the ethidium bromide solution colour developing 5-10 minute of 1%, the gene type of target individual at 18 locus can be obtained.
8. carry out the test kit identified for silver carp, flathead and hybridization thereof and Introgression In Hatchery Stocks individuality, it is characterized in that, this test kit comprises micro-satellite primers pair according to claim 2.
9. test kit according to claim 8, is characterized in that, described test kit comprises Taq archaeal dna polymerase, 10 × PCR Buffer, dNTPs, sterilizing ultrapure water, MgCl further
2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410146788.7A CN103866043B (en) | 2014-04-14 | 2014-04-14 | A kind ofly identify silver carp, flathead hybridization and the microsatellite marker of Introgression In Hatchery Stocks individuality and Auele Specific Primer and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410146788.7A CN103866043B (en) | 2014-04-14 | 2014-04-14 | A kind ofly identify silver carp, flathead hybridization and the microsatellite marker of Introgression In Hatchery Stocks individuality and Auele Specific Primer and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103866043A CN103866043A (en) | 2014-06-18 |
| CN103866043B true CN103866043B (en) | 2015-08-19 |
Family
ID=50905043
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410146788.7A Active CN103866043B (en) | 2014-04-14 | 2014-04-14 | A kind ofly identify silver carp, flathead hybridization and the microsatellite marker of Introgression In Hatchery Stocks individuality and Auele Specific Primer and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103866043B (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107058572B (en) * | 2017-05-26 | 2020-03-31 | 浙江省淡水水产研究所 | Method for identifying hybrid individuals of culter alburnus and megalobrama amblycephala |
| CN108753987A (en) * | 2018-06-01 | 2018-11-06 | 中国水产科学研究院淡水渔业研究中心 | A kind of method of the general microsatellite Multiplex fluorescent PCR of silver carp flathead |
| CN112553347B (en) * | 2020-12-28 | 2022-04-15 | 中国科学院水生生物研究所 | Development method of bighead carp sex identification molecular marker by taking Gapdh gene as reference |
| CN113215272B (en) * | 2021-05-07 | 2022-06-17 | 西安理工大学 | Silver carp mark release and release effect evaluation method |
| CN113481302B (en) * | 2021-05-07 | 2022-06-17 | 西安理工大学 | Catfish marking releasing effect evaluation method |
| CN113621711B (en) * | 2021-07-13 | 2023-09-22 | 武汉中科瑞华生态科技股份有限公司 | Dual PCR microsatellite primer for bighead genetic diversity analysis and application |
| CN113621710B (en) * | 2021-07-13 | 2023-11-03 | 武汉中科瑞华生态科技股份有限公司 | Bighead microsatellite marked primer and bighead mark releasing effect evaluation method |
| CN116949135A (en) * | 2023-06-20 | 2023-10-27 | 中国水产科学研究院长江水产研究所 | Identification method of Changfeng silver carp introgression gene and application thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2375276A1 (en) * | 2001-03-09 | 2002-09-09 | Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Agriculture And Agri-Food Canada | Methods to isolate gene coding and flanking dna |
| CN101748190B (en) * | 2008-12-22 | 2012-08-01 | 上海基康生物技术有限公司 | Method for detecting related loci of type-2 diabetes |
| CN102912013A (en) * | 2012-09-19 | 2013-02-06 | 长沙三济生物科技有限公司 | Sequencing primer for qualitative detection of cytochrome oxidase CYP2C19 genetic typing and kit of sequencing primer |
-
2014
- 2014-04-14 CN CN201410146788.7A patent/CN103866043B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN103866043A (en) | 2014-06-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103866043B (en) | A kind ofly identify silver carp, flathead hybridization and the microsatellite marker of Introgression In Hatchery Stocks individuality and Auele Specific Primer and application | |
| CN104357553B (en) | A kind of Pelteobagrus fulvidraco microsatellite Parentage determination method | |
| CN107326077A (en) | A kind of molecular labeling for differentiating spotted maigre genetic sex and its application | |
| Ostroverkhova et al. | Genetic diversity of the locus COI-COII of mitochondrial DNA in honeybee populations (Apis mellifera L.) from the Tomsk region | |
| CN105603098A (en) | Microsatellite marker primers used for penaeus monodon microsatellite family identification, identification method and application | |
| Ma et al. | Parentage assignment of the mud crab (Scylla paramamosain) based on microsatellite markers | |
| CN106811540B (en) | Microsatellite marker and specific primer for identifying male and female individuals of Pseudobagrus ussuriensis and application of microsatellite marker and specific primer | |
| CN104561355A (en) | Multiplex-PCR method for parentage assignment of scapharca broughtonii | |
| CN110331217B (en) | Microsatellite marker paternity test primer suitable for Nile tilapia, Oreochromis aureus and hybrid thereof, method and application | |
| Bian et al. | Development of a multiplex PCR assay for parentage assignment of the redclaw crayfish (Cherax quadricarinatus) | |
| CN105441576A (en) | Microsatellite multiplex-PCR (Polymerase Chain Reaction) method for carrying out paternity testing on crassostrea gigas | |
| CN108034729A (en) | A kind of and the relevant EST-STR label Ls v-F36a of Growth Trait in White Shrimp Litopenaeus vannamei and its amplimer and application | |
| CN102321752B (en) | Fluorescence labeled detection kit for simultaneously analyzing 17 gene loci of canine genomic DNA, detection method and application thereof | |
| CN103484459B (en) | Primer group, marking method and application of EST-SSR (Expressed Sequence Tag-Simple Sequence Repeats) molecular marker of macrobrachium nipponense | |
| Gross et al. | Development and characterization of novel tetranucleotide microsatellite markers in the noble crayfish (Astacus astacus) suitable for highly multiplexing and for detecting hybrids between the noble crayfish and narrow-clawed crayfish (A. leptodactylus) | |
| Feng et al. | Characterization of 40 single nucleotide polymorphism (SNP) via T m-shift assay in the mud crab (Scylla paramamosain) | |
| CN104946787A (en) | Molecular marker for identifying carp varieties and method for identifying by utilizing molecular marker | |
| CN104450697A (en) | SNP marker associated with oyster antiviral properties and application thereof | |
| CN116769891A (en) | SNP (Single nucleotide polymorphism) marker for rapidly identifying genetic sex of Chinese soft-shelled turtles as well as primer and application thereof | |
| CN103605913B (en) | Method applied to identification of pacific oyster family | |
| CN105603092A (en) | SNP marker related to sheep multi-horn character and application thereof | |
| CN107849566A (en) | For detecting the method and kit of powdery mildew | |
| Ma et al. | Isolation and characterization of 54 polymorphic microsatellite markers in Scylla paramamosain by FIASCO approach | |
| CN106434974B (en) | A kind of micro-satellite primers and application thereof for Macrobrachium nipponensis diversity analysis | |
| CN103525933A (en) | PCR-RFLP method for distinguishing ctenopharyngodon idellus from mylopharyngodon piceus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C53 | Correction of patent of invention or patent application | ||
| CB03 | Change of inventor or designer information |
Inventor after: Tong Jingou Inventor after: Zhu Chuankun Inventor after: Yu Xiaomu Inventor before: Tong Jingou Inventor before: Zhu Chuankun Inventor before: Yu Xiaomu Inventor before: Zhang Wang |
|
| COR | Change of bibliographic data |
Free format text: CORRECT: INVENTOR; FROM: TONG JINGOU ZHU CHUANKUN YU XIAOMU ZHANG WANG TO: TONG JINGOU ZHU CHUANKUN YU XIAOMU |
|
| EE01 | Entry into force of recordation of patent licensing contract |
Application publication date: 20140618 Assignee: Honghu Yingmeng Ecological Agriculture Co.,Ltd. Assignor: Institute of Hydrobiology, Chinese Academy of Sciences Contract record no.: X2023980053452 Denomination of invention: A microsatellite marker and specific primers for identifying hybrid and genetic introgression individuals of silver carp and bighead carp and their application Granted publication date: 20150819 License type: Common License Record date: 20231222 |
|
| EE01 | Entry into force of recordation of patent licensing contract |