CN1814793A - Cynoglossus semilaevis gunther specific molecular label and genetic sex identifying method - Google Patents
Cynoglossus semilaevis gunther specific molecular label and genetic sex identifying method Download PDFInfo
- Publication number
- CN1814793A CN1814793A CN 200510045554 CN200510045554A CN1814793A CN 1814793 A CN1814793 A CN 1814793A CN 200510045554 CN200510045554 CN 200510045554 CN 200510045554 A CN200510045554 A CN 200510045554A CN 1814793 A CN1814793 A CN 1814793A
- Authority
- CN
- China
- Prior art keywords
- dna
- cynoglossus semilaevis
- sex
- female
- primers
- 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.)
- Pending
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
This invention relates to specific molecule labeling of the sex of semi-sliding tongue soles and its genetic sex test method including the pick-up of the blood DNA, the AFLP analysis of DNA in the gene set, selection of the AFLP molecular labeling and a method for genetic sex check up of semi-sliding tongue sole, which selects 4 from 64 primer combinations to generate female specific DNA segments and selects 7 female specific AFLP molecular labels of the sole to set up the molecule labeling technology of its genetic sex authentication.
Description
Affiliated technical field:
The invention belongs to fish genetic sex identification and sex control technology in the aquatic living things technical field, is a kind of fish sex specific molecular marker and the genetic sex identification method that can use in the breeding of fish unisexuality.
Background technology:
There is abundant fish stock in China, wherein many fish exist gender difference on growth velocity, for example, tilapia is female fast more about more than 40% than male growth, fish such as carp, lefteye flounder and Cynoglossus semilaevis, female individuals is than the male fast 30%-100% that grows, therefore, carry out the existing important scientific meaning of research of these fish sex genes involveds and sex specific molecular marker, great application potential and promotion prospect are arranged again.
Cynoglossus semilaevis (Cynoglossus semiliaevis) is the distinctive a kind of famous and precious economic seawater fish of China, belongs to coastal waters warm water demersal fish, and China is coastal all distribution, is many with the Huanghai Sea, the Bohai Sea.Cynoglossus semilaevis is because its delicious flavour, and fine and tender taste is nutritious, welcome by consumers in general, and its marketable value is high, and the breed prospect is boundless.Although over nearly 2 years, the Cynoglossus semilaevis artificial breeding technique obtains to break through, produce nearly 1,000,000 tails of semi-smooth tongue sole offspring breed per year, but, studies show that there are huge difference in Cynoglossus semilaevis female individuals and male on growth velocity, its female individuals is than the big 2-3 of male times (Huanghai Sea aquatic products institute, Meng Tianxiang etc., 1988).Because male was grown slow, reduced the cultured output of Cynoglossus semilaevis, increased aquaculture cost, thereby the popularization of semi-smooth tongue sole offspring breed and the formation of aquaculture industry have been had a strong impact on, owing to be difficult to identify the genetic sex of Cynoglossus semilaevis, had a strong impact on the carrying out of cynoglossus semilaevis gynogenesis and sex control research.
The research of relevant fish sex related molecular marker was just carried out on a few fish at present.The main method that adopts comprises RAPD, SSR and AFLP etc.The Canada breadboard Devlin of West Vancouver (1994) has found the special dna fragmentation of chinook Y chromosome; African catfish male and female gene pool is scanned with RAPD in (2000) such as Kovacs of Hungary Agricultural Biotechnology Ct, find the relevant RAPD mark of two male sexs, one (CgaY1) be 2.6kb approximately, another (CgaY2) 458bp, this is to find the special dna marker of sex first from catfish.The linked dna marker of tilapia phenotypic sex is sought with separating group's analytical method in (2004) such as Lee of U.S. New Hampshire university, finds 10 microsatellite markers chain with the tilapia phenotypic sex.These marks can be directly used in the research of different Y chromosome allelotrope functions and have the evaluation of the parent population of one or several Y chromosome copies.(2004) such as Ezaz of Britain Stirling university AFLP technology to analyze bolti genome, find the AFLP mark of three Y linkages (OniY425, OniY382, OniY227) and an X-linkage (OniX420), wherein OniX420 and OniY425 prove allelotrope, yet these marks but can not be differentiated the sex of the individuality that does not have sibship, do not reach the level of identifying the tilapia genetic sex.Because exist very big-difference between the different fish, the sex specific molecular marker of a kind of fish can not be used, therefore must set up the sex specific molecular marker of various fish oneself on another kind of fish.Relevant Cynoglossus semilaevis sex specific molecular marker and genetic sex detection technique there is no report at present both at home and abroad.
Summary of the invention:
The objective of the invention is in order to filter out Cynoglossus semilaevis sex specific molecular marker, set up the molecular biology method that genetic sex is identified, for Cynoglossus semilaevis sex control and complete female breeding research provide molecule marker and technological method.
Its technology contents of the present invention is as follows: one, Cynoglossus semilaevis sex specific molecular marker comprises: 1, the extraction of Cynoglossus semilaevis blood DNA; 2, the aflp analysis of genomic dna; 3, the screening of sex specific molecular marker; Two, Cynoglossus semilaevis genetic sex authentication method.
One, Cynoglossus semilaevis sex specific molecular marker comprises: 1, the extraction of Cynoglossus semilaevis blood DNA; 2, the aflp analysis of genomic dna; 3, the screening of sex specific molecular marker:
1, the extraction of Cynoglossus semilaevis blood DNA:
Extract Cynoglossus semilaevis fish blood, leave standstill the centrifugal removal upper plasma in back on ice, add 4-6 times of volume dissolved in distilled water hemocyte precipitation, shake up, leave standstill, centrifugal again, remove supernatant; Add 4-6 times of volume STE damping fluid, washing and precipitating, with the STE damping fluid that contains proteolytic enzyme-K, digestion is spent the night, till the throw out dissolving; Add the saturated phenol of equal-volume, extracting once; With with the identical phenol of above-mentioned STE damping fluid volume: chloroform: primary isoamyl alcohol (25: 24: 1) mixed solution extracting once, use then and the identical chloroform of above-mentioned STE damping fluid volume: primary isoamyl alcohol (24: 1) extracting is once; At last with the dehydrated alcohol deposit D NA of 2-3 times of volume.In-20 ℃ centrifugal in 12000rpm after placing 20-30 minute, remove supernatant, collect the DNA precipitation, after 70% washing with alcohol and seasoning, with the TE dissolving, DNA is kept at-20 ℃ standby.
The concentration of described genomic dna should be not less than 20ng/ μ l, the purity requirement OD of genomic dna
260/ OD
280Value is 1.76-1.80.
2, the aflp analysis of genomic dna:
The aflp analysis of genomic dna comprises the steps: that mainly the first step carries out enzyme to dna profiling and cut, and second step was that special joint is connected on the endonuclease bamhi, and the 3rd step was to increase in advance, and the 4th step was to carry out selective amplification, and the 5th step was electrophoretic analysis.
Describedly dna profiling is carried out enzyme cut, its method is: digested the 80-110ng template DNA 4-6 hour with EcoRI/MseI enzyme mixture (1.25U/ μ l) earlier, whether detect enzyme with 1% agarose gel electrophoresis and cut fully, the dna solution that abundant enzyme is cut places 70 ℃ to hatch 15 minutes deactivation restriction enzymes.
Described special joint is connected on the endonuclease bamhi, and its method is: add 12.0 μ l joint mixtures and 0.5 μ l T4 dna ligase in the endonuclease reaction mixed solution upward, hatched 12-20 hour for 20 ℃.
Described pre-amplification, its method is: with 10 times of above-mentioned connection product dilutions, as the template of the pre-amplification of PCR; The PCR primer is the primer mixture that 3 ' end has a selectivity base (A or C), carries out PCR and increases in advance.
Described selective amplification: its method is: pre-expansion is increased production thing dilution 30-50 doubly, as the template of selective amplification.Carry out the selectivity pcr amplification with fluorescently-labeled MseI primer and EcoRI primer, these two kinds of primer 3 ' ends have three selectivity bases.In pcr amplification product, add an amount of sample solution, place stand-by after 3 minutes immediately on ice in 94 ℃ of sex change.Used 64 combination of primers to carry out selective amplification altogether, the PCR product of acquisition carries out electrophoretic separation with 6.5% polyacrylamide gel after 94 ℃ of sex change.
Described electrophoretic analysis, its method is: the amplified production that sex change is good carries out electrophoresis with the polyacrylamide gel of 5.5-6.5%.Deposition condition: voltage 1500V, power 40W, electric current 40mA, 45 ℃ of temperature, 3.5 hours time.Then the DNA band that produces behind the electrophoresis is observed, taken a picture and analysis.
3, the screening of sex specific molecular marker:
With 64 combination of primers the genomic dna of 15 Cynoglossus semilaevis female individuals and 13 Cynoglossus semilaevis males has been carried out the AFLP-PCR amplification altogether, filter out 4 combination of primers by the SAGA software analysis and produced 7 female special dna fragmentations, these fragments do not exist in the male genomic dna: wherein combination of primers M1-E1 amplifies three female special dna fragmentations, and length is respectively 378--382bp, 570-575bp and 780-783bp; Combination of primers M2-E2 amplifies the female specific DNA fragment of a 460-464bp; Combination of primers M3-E3 amplifies two female special dna fragmentations, and length is respectively 132-136bp and 614-618bp; Combination of primers M4-E4 amplifies the female specific DNA fragment of a 300-305bp.To only in female individuals, occur, and absent variable fragment promptly can be used for genetic sex and identifies as female specific DNA mark in male.
Two, genetic sex identification method
Extract the blood of Cynoglossus semilaevis fish to be measured, extract genomic dna, carry out aflp analysis with the AFLP primer that produces female specific DNA fragment, if produced these specific fragments, can think that these fishes are the female individuals in the heredity, not have these segmental individualities then to be considered to male in the heredity.
The present invention and prior art contrast are characterized in: the present invention adopts molecular marking technique, is that material screens the sex specific DNA molecular mark first with the Cynoglossus semilaevis, has set up the Protocols in Molecular Biology that China's cultured fishes genetic sex is identified first.Advantages such as this technology has accurately, sensitive, reliable have been opened up new technological approaches for fish sex control and unisexuality breeding, suit to apply in all cultured fishes, to the significant and using value of cultured fishes unisexuality breeding.
Embodiment:
Adopt modern molecular biology technique, filter out the special molecule marker of sex, set up the molecular engineering that Cynoglossus semilaevis genetic sex is identified, this is for cultivating the complete female seed of Cynoglossus semilaevis, carrying out complete female seed cultures, improve the cultured output of Cynoglossus semilaevis, improve the economic benefit of culturing, really Cynoglossus semilaevis is developed as a good breed variety of being accepted by numerous raisers, promote the development of cynoglossus semilaevis cultivation industry and the update of marine fish culture kind, have important practical significance and huge economic benefit.
Below by the screening of the female specific AFLP molecule marker of Cynoglossus semilaevis and the foundation of genetic sex identification method, technology contents of the present invention is elaborated:
One, Cynoglossus semilaevis sex specific molecular marker comprises: 1, the extraction of Cynoglossus semilaevis blood DNA, 2, the aflp analysis of genomic dna, 3, the screening of sex specific molecular marker.
1, the extraction of Cynoglossus semilaevis blood DNA:
Extract Cynoglossus semilaevis fish blood 200-400 μ l, get 100-150 μ l blood centrifugal 15 minutes in 3500rpm, remove upper plasma after, add 5 times of volume distilled water (sterilization) dissolving hemocyte precipitation, shake up, left standstill 5-10 minute, centrifugal 15 minutes again, remove supernatant with 3500rpm; Add 5 times of volume STE (0.1mol/L NaCl, 10mmol/L TrisCl (pH8.0), 1mmol/LEDTA (pH8.0)), washing and precipitating 2 times; At every turn at the centrifugal 7-8 of 12000rpm minute; Go to add 0.5ml STE behind the supernatant, 10%SDS 25-30 μ l, proteolytic enzyme-K is to final concentration 100 μ g/ml, and 37 ℃ of digestion are spent the night, till the throw out dissolving; Add the saturated phenol of equal-volume, reversing shakes up 10 minutes, and centrifugal 5 minutes of 12000rpm sucts a layer stillness of night and goes into another 1.5ml centrifuge tube; Add equal-volume phenol: chloroform: primary isoamyl alcohol (25: 24: 1), reversing mixing after 10 minutes centrifugal 5 minutes in 12000rpm, sucting a layer stillness of night goes into another 1.5ml centrifuge tube, in supernatant liquor, add the equal-volume chloroform: primary isoamyl alcohol (24: 1), reversing mixing 10 minutes, in 12000rpm centrifugal 5 minutes, get supernatant and go into another pipe; Add 1/12 volume 3M sodium-acetate, add 4 ℃ of dehydrated alcohols of 3 times of volumes behind the mixing again, soft jolting; Can be observed white floss and (DNA) occur, in-20 ℃ place 20-30 minute after in centrifugal 10 minutes of 12000rpm low temperature (4 ℃), remove supernatant, collect the DNA precipitation, add the 1ml70% washing with alcohol, centrifugal 10 minutes of 12000rpm low temperature (4 ℃), remove supernatant, repeat 1 time; After the seasoning, with pH8.0TE dissolving, be kept at-20 ℃ standby.The concentration of genomic dna should be not less than 20ng/ μ l, the purity requirement OD of genomic dna
260/ OD
280Value is about 1.8.
2, the aflp analysis of genomic dna: mainly be divided into following 5 steps:
(1), enzyme is cut:
Digested the 80-110ng template DNA 4-6 hour with 1.0 μ l EcoRI/MseI enzyme mixtures (1.25U/ μ l), whether detect enzyme with 1% agarose gel electrophoresis again and cut fully, the dna solution that abundant enzyme is cut is hatched 15 minutes deactivation restriction enzymes in 70 ℃ then.
(2), connect:
Add 12.0 μ l joint mixtures and 0.5 μ l T4DNA ligase enzyme in the endonuclease reaction mixed solution upward, hatched 12-20 hour for 20 ℃, guarantee that joint fully connects.
(3), pre-amplification:
To connect 10 times of product dilutions, as the template of pre-amplification; Pre-amplification primer is the primer mixture that 3 ' end has a selectivity base (A or C).The PCR reaction conditions is: 92-94 ℃, and 30 seconds; 54-56 ℃, 1 minute; 70-72 ℃, 1 minute.After 20 circulations, 4 ℃ stand-by.
(4), selective amplification:
Pre-expansion is increased production thing dilution 30-50 doubly, as the template of selective amplification.Increase with primer that contains restriction enzyme site MseI and the primer that contains restriction enzyme site EcoRI, these two kinds of primer 3 ' ends have three selectivity bases.The PCR reaction was divided into for three steps: the first step is carried out 1 circulation: 92-94 ℃, 30 seconds; 63-65 ℃, 30 seconds; 70-72 ℃, 1 minute; Second step was carried out 12 circulation: 92-94 ℃, 30 seconds; 65-54 ℃, 30 seconds; 70-72 ℃, 1 minute; The 3rd step was carried out 23 circulations--and 92-94 ℃, 30 seconds; 54-56 ℃, 30 seconds; 70-72 ℃, 1 minute.Amplified production adds an amount of sample solution, and 94 ℃ of sex change 3 minutes place on ice immediately.
(5), electrophoretic analysis:
The amplified production that sex change is good carries out electrophoresis with the polyacrylamide gel of 5.5-6.5%.Deposition condition: voltage 1500V, power 40W, electric current 40mA, 45 ℃ of temperature, 3.5 hours time.Used 64 combination of primers that the genomic dna of 15 Cynoglossus semilaevis female individuals and 13 Cynoglossus semilaevis males has been carried out aflp analysis altogether, behind SAGA software analysis electrophoretogram, filter out 4 of combination of primers that produce specific fragment, these 4 combination of primers common properties have been given birth to 7 female special dna fragmentations.
3, the screening of sex specific molecular marker:
With 64 combination of primers the genomic dna of 15 Cynoglossus semilaevis female individuals and 13 Cynoglossus semilaevis males has been carried out the AFLP-PCR amplification altogether, filter out 4 combination of primers by the SAGA software analysis and produced 7 female special dna fragmentations, these fragments do not exist in the male genomic dna: wherein combination of primers M1-E1 amplifies three female special dna fragmentations, and length is respectively 378--382bp, 570-575bp and 780-783bp; Combination of primers M2-E2 amplifies the female specific DNA fragment of a 460-464bp; Combination of primers M3-E3 amplifies two female special dna fragmentations, and length is respectively 132-136bp and 614-618bp; Combination of primers M4-E4 amplifies the female specific DNA fragment of a 300-305bp.To only in female individuals, occur and in male absent variable fragment promptly can be used for genetic sex and identify as female specific DNA mark.
Two, genetic sex identification method
The Cynoglossus semilaevis fish blood DNA extractive technique that application is set up above extracts blood, isolates Cynoglossus semilaevis fish genomic dna, and this method need not be killed fish, is non-invasive technique, is suitable for applying; And then genomic dna is carried out aflp analysis with the AFLP primer that can produce the female specific DNA fragment of Cynoglossus semilaevis that screens above, electrophoresis confirms having or not of DNA band, if produced the special dna fragmentation of sex in the genomic dna of certain bar fish, can think that these fishes are the female individuals in the heredity, not have these segmental individualities then can think male in the heredity.Just can identify the genetic sex of Cynoglossus semilaevis by this method, for Cynoglossus semilaevis sex control and complete female breeding research provide technique means.This method accurately, reliably has significant application value in Cynoglossus semilaevis sex control and unisexuality breeding.
Claims (2)
1), the extraction of Cynoglossus semilaevis blood DNA 1, a kind of Cynoglossus semilaevis sex specific molecular marker is characterized in that its technology contents comprises following three aspects:; 2), the aflp analysis of genomic dna; 3), the screening of sex specific molecular marker;
1), the extraction of Cynoglossus semilaevis blood DNA:
Extract Cynoglossus semilaevis fish blood, leave standstill the centrifugal removal upper plasma in back on ice, add 4-6 times of volume dissolved in distilled water hemocyte precipitation, shake up, leave standstill, centrifugal again, remove supernatant; Add 4-6 times of volume STE damping fluid, washing and precipitating, with the STE damping fluid that contains proteolytic enzyme-K, digestion is spent the night, till the throw out dissolving; Add the saturated phenol of equal-volume, extracting once; With phenol, chloroform, the extracting of primary isoamyl alcohol mixed solution once, use chloroform, the extracting of primary isoamyl alcohol mixed solution then once; At last with the dehydrated alcohol deposit D NA of 2-3 times of volume; In-20 ℃ centrifugal in 12000rpm after placing 20-30 minute, remove supernatant, collect the DNA precipitation, after 70% washing with alcohol and seasoning, with the TE dissolving, DNA is kept at-20 ℃ standby;
The concentration of described genomic dna should be not less than 20ng/ μ l, the purity requirement OD of genomic dna
260/ OD
280Value is 1.76-1.80;
2), the aflp analysis of genomic dna:
The aflp analysis of genomic dna comprises the steps: that mainly the first step carries out enzyme to dna profiling and cut, and second step was that special joint is connected on the endonuclease bamhi, and the 3rd step was to increase in advance, and the 4th step was to carry out selective amplification, and the 5th step was electrophoretic analysis;
Describedly dna profiling is carried out enzyme cut, its method is: digested the 80-110ng template DNA 4-6 hour with 1.25U/ μ l EcoRI/MseI enzyme mixture earlier, whether detect enzyme with 1% agarose gel electrophoresis and cut fully, the dna solution that abundant enzyme is cut places 70 ℃ to hatch 15 minutes deactivation restriction enzymes;
Described special joint is connected on the endonuclease bamhi, and its method is: add 12.0 μ l joint mixtures and 0.5 μ l T4 dna ligase in the endonuclease reaction mixed solution upward, hatched 12-20 hour for 20 ℃;
Described pre-amplification, its method is: with 10 times of above-mentioned connection product dilutions, as the template of the pre-amplification of PCR; The PCR primer is the primer mixture that 3 ' end has the selectivity base of an A or C, carries out PCR and increases in advance;
Described selective amplification: its method is: pre-expansion is increased production thing dilution 30-50 doubly, as the template of selective amplification; Carry out the selectivity pcr amplification with fluorescently-labeled MseI primer and EcoRI primer, these two kinds of primer 3 ' ends have three selectivity bases; In pcr amplification product, add an amount of sample solution, place stand-by after 3 minutes immediately on ice in 94 ℃ of sex change; Used 64 combination of primers to carry out selective amplification altogether, the PCR product of acquisition carries out electrophoretic separation with 6.5% polyacrylamide gel after 94 ℃ of sex change;
Described electrophoretic analysis, its method is: the amplified production that sex change is good carries out electrophoresis with the polyacrylamide gel of 5.5-6.5%; Deposition condition: voltage 1500V, power 40W, electric current 40mA, 45 ℃ of temperature, 3.5 hours time; Then the DNA band that produces behind the electrophoresis is observed, taken a picture and analysis;
3), the screening of sex specific molecular marker:
With 64 combination of primers the genomic dna of 15 Cynoglossus semilaevis female individuals and 13 Cynoglossus semilaevis males has been carried out the AFLP-PCR amplification altogether, filter out 4 combination of primers by the SAGA software analysis and produced 7 female special dna fragmentations, these fragments do not exist in the male genomic dna: wherein combination of primers M1-E1 amplifies three female special dna fragmentations, and length is respectively 378--382bp, 570-575bp and 780-783bp; Combination of primers M2-E2 amplifies the female specific DNA fragment of a 460-464bp; Combination of primers M3-E3 amplifies two female special dna fragmentations, and length is respectively 132-136bp and 614-618bp; Combination of primers M4-E4 amplifies the female specific DNA fragment of a 300-305bp; To only in female individuals, occur, and absent variable fragment promptly can be used for genetic sex and identifies as female specific DNA mark in male.
2, a kind of Cynoglossus semilaevis genetic sex authentication method, the method that it is characterized in that it is: the blood that extracts Cynoglossus semilaevis fish to be measured, extract genomic dna, carry out aflp analysis with the AFLP primer that produces female specific DNA fragment, if produced these specific fragments, can think that these fishes are the female individuals in the heredity, not have these segmental individualities then to be considered to male in the heredity.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510045554 CN1814793A (en) | 2005-12-15 | 2005-12-15 | Cynoglossus semilaevis gunther specific molecular label and genetic sex identifying method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510045554 CN1814793A (en) | 2005-12-15 | 2005-12-15 | Cynoglossus semilaevis gunther specific molecular label and genetic sex identifying method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1814793A true CN1814793A (en) | 2006-08-09 |
Family
ID=36907149
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200510045554 Pending CN1814793A (en) | 2005-12-15 | 2005-12-15 | Cynoglossus semilaevis gunther specific molecular label and genetic sex identifying method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1814793A (en) |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100469896C (en) * | 2007-12-01 | 2009-03-18 | 中国水产科学研究院黄海水产研究所 | Tongue sole molecular marker auxiliary sex control method |
| CN101613750B (en) * | 2009-02-27 | 2011-08-24 | 河南师范大学 | Molecular biology method for identifying gender of chub fry |
| CN101381769B (en) * | 2008-10-24 | 2011-09-07 | 长沙学院 | Molecular identification method for siniperca kneri garman and siniperca scherzeri and kit |
| CN102181554A (en) * | 2011-04-21 | 2011-09-14 | 中国海洋大学 | Specific genomic DNA fragment of female tongue sole and application thereof |
| WO2012083681A1 (en) * | 2010-12-23 | 2012-06-28 | 中国水产科学研究院黄海水产研究所 | Cynoglossus semilaevis gender-specific microsatellite marker and application thereof in super-female fish identification |
| CN101805785B (en) * | 2009-04-23 | 2012-07-04 | 河南师范大学 | Molecular biological method for fast identifying sex of bighead seedlings |
| CN101240348B (en) * | 2008-01-23 | 2012-07-11 | 中国水产科学研究院黄海水产研究所 | Rapid identification method for half-smooth tongue-sole genetic sex |
| CN103320517A (en) * | 2013-07-04 | 2013-09-25 | 中国水产科学研究院黄海水产研究所 | Primer and method for quickly detecting gender difference of juvenile fishes of fugu rubripes |
| CN106947827A (en) * | 2017-05-09 | 2017-07-14 | 中国科学院水生生物研究所 | One kind obtains flathead sex specific molecular marker and its screening technique and application |
| CN109055520A (en) * | 2018-09-03 | 2018-12-21 | 天津渤海水产研究所 | A kind of Cynoglossus semilaevis excretion body Sexual differentially expressed label and kit |
| CN109825565A (en) * | 2019-01-22 | 2019-05-31 | 天津渤海水产研究所 | A kind of Cynoglossus semilaevis true and false milter discriminating method based on fluorescent molecule label system |
-
2005
- 2005-12-15 CN CN 200510045554 patent/CN1814793A/en active Pending
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100469896C (en) * | 2007-12-01 | 2009-03-18 | 中国水产科学研究院黄海水产研究所 | Tongue sole molecular marker auxiliary sex control method |
| CN101240348B (en) * | 2008-01-23 | 2012-07-11 | 中国水产科学研究院黄海水产研究所 | Rapid identification method for half-smooth tongue-sole genetic sex |
| CN101381769B (en) * | 2008-10-24 | 2011-09-07 | 长沙学院 | Molecular identification method for siniperca kneri garman and siniperca scherzeri and kit |
| CN101613750B (en) * | 2009-02-27 | 2011-08-24 | 河南师范大学 | Molecular biology method for identifying gender of chub fry |
| CN101805785B (en) * | 2009-04-23 | 2012-07-04 | 河南师范大学 | Molecular biological method for fast identifying sex of bighead seedlings |
| WO2012083681A1 (en) * | 2010-12-23 | 2012-06-28 | 中国水产科学研究院黄海水产研究所 | Cynoglossus semilaevis gender-specific microsatellite marker and application thereof in super-female fish identification |
| CN102181554A (en) * | 2011-04-21 | 2011-09-14 | 中国海洋大学 | Specific genomic DNA fragment of female tongue sole and application thereof |
| CN102181554B (en) * | 2011-04-21 | 2016-01-20 | 中国海洋大学 | The female special genomic DNA fragment of Cynoglossus semilaevis and application thereof |
| CN103320517A (en) * | 2013-07-04 | 2013-09-25 | 中国水产科学研究院黄海水产研究所 | Primer and method for quickly detecting gender difference of juvenile fishes of fugu rubripes |
| CN103320517B (en) * | 2013-07-04 | 2014-12-03 | 中国水产科学研究院黄海水产研究所 | Primer and method for quickly detecting gender difference of juvenile fishes of fugu rubripes |
| CN106947827A (en) * | 2017-05-09 | 2017-07-14 | 中国科学院水生生物研究所 | One kind obtains flathead sex specific molecular marker and its screening technique and application |
| CN109055520A (en) * | 2018-09-03 | 2018-12-21 | 天津渤海水产研究所 | A kind of Cynoglossus semilaevis excretion body Sexual differentially expressed label and kit |
| CN109825565A (en) * | 2019-01-22 | 2019-05-31 | 天津渤海水产研究所 | A kind of Cynoglossus semilaevis true and false milter discriminating method based on fluorescent molecule label system |
| CN109825565B (en) * | 2019-01-22 | 2022-04-15 | 天津渤海水产研究所 | Cynoglossus semilaevis true and false male fish screening method based on fluorescent molecular marker system |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1814793A (en) | Cynoglossus semilaevis gunther specific molecular label and genetic sex identifying method | |
| CN101270389A (en) | Cynoglossus semilaevis special numerator mark and uses thereof | |
| CN101240348B (en) | Rapid identification method for half-smooth tongue-sole genetic sex | |
| CN102358902B (en) | Silkworm fibroin heavy-chain gene mutation sequence and mutation method and application | |
| Yarra et al. | Characterization of the mantle transcriptome in bivalves: Pecten maximus, Mytilus edulis and Crassostrea gigas | |
| CN101429557B (en) | Special molecular marker for sex of verasper variegates, and genetic sex identification method | |
| CN104059963B (en) | Detection method of Chinese simmental cattle carcass and meat quality trait genetic markers | |
| CN111394445B (en) | Indel marker for sex identification of channa maculata and application thereof | |
| CN109576275B (en) | Cynoglossus semilaevis antibacterial disease related gene and application method thereof | |
| CN106947827A (en) | One kind obtains flathead sex specific molecular marker and its screening technique and application | |
| CN102134593A (en) | Gender-specific microsatellite marker for Cynoglossus semilaevis and application of same in identification of superfemale Cynoglossus semilaevis | |
| CN112048014B (en) | Penaeus monodon PmGLUT2 gene and application thereof | |
| CN109161603A (en) | The microsatellite marker primer and genetic sex identification method of channel catfish gunther sex-linked | |
| CN110684776B (en) | Penaeus monodon Na+/K+/2Cl-Cotransporter NKCC gene and application thereof | |
| CN1880478A (en) | Female-specific AFLP fragment of Cynoglossus semilaevis Gunther and PCR method for identification of genetic sex | |
| CN105349691B (en) | It is a kind of identify Crustin genetic sex DNA sequence tag and its application | |
| CN113930525B (en) | Specific sequence for snakehead sex identification and application | |
| CN108084261A (en) | A kind of Cynoglossus semilaevis anti pathologic immunity related gene and its application | |
| CN111560401A (en) | Molecular breeding method for thickening interpuscular spurs of erythroculter ilishaeformis and megalobrama amblycephala | |
| CN107937395B (en) | Microsatellite molecular marker for polymorphism of high-sea swimming crabs, and identification method and application thereof | |
| CN100415884C (en) | DNA molecular marking method for researching fish genetic relation | |
| CN111363834B (en) | SNP molecular marker related to resistance of white spot syndrome of procambarus clarkii | |
| CN110564867B (en) | SNP molecular marker of Qinchuan cattle CFL1 gene and detection method thereof | |
| Shifat et al. | Use of RAPD fingerprinting for discriminating two populations of Hilsa shad (Tenualosa ilisha Ham.) from inland rivers of Bangladesh | |
| CN108588238B (en) | Method for rapidly identifying sex specific molecular marker and genetic sex of Charybdis feriatus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |