CN102972322A - Simplified releasing method capable of accurately tracing released Fenneropenaeus chinensis - Google Patents
Simplified releasing method capable of accurately tracing released Fenneropenaeus chinensis Download PDFInfo
- Publication number
- CN102972322A CN102972322A CN2012105595936A CN201210559593A CN102972322A CN 102972322 A CN102972322 A CN 102972322A CN 2012105595936 A CN2012105595936 A CN 2012105595936A CN 201210559593 A CN201210559593 A CN 201210559593A CN 102972322 A CN102972322 A CN 102972322A
- Authority
- CN
- China
- Prior art keywords
- releasing
- pcr
- quadruple
- primer
- 1min
- 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
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
A simplified releasing method capable of accurately tracing released Fenneropenaeus chinensis comprises the following steps: in the releasing season each year, capturing wild mated female shrimps and tagging eyestalks of the wild mated female shrimps; then culturing seeds; preserving the female shrimps having production finished in a refrigerator at an ultralow temperature of -75 DEG C; in the autumn, capturing and sampling released individuals at each releasing migration point; detecting the released individuals and the number of the released individuals through two groups of Fenneropenaeus chinensis fluorescent labeled micro-satellite quadruple PCR (Polymerase Chain Reaction) individual identification technologies after samples are captured; and finally determining the releasing effect of the Fenneropenaeus chinensis. According to the invention, the released Fenneropenaeus chinensis can be tracked and identified as long as samples are collected from Fenneropenaeus chinensis parents producing the released seeds and parent-child traces of the collected released samples are analyzed by using the two groups of fluorescent labeled micro-satellite quadruple PCRs. Complicated operations of preparing Fenneropenaeus chinensis full-sib families on a large scale, culturing parents of the overwintering Fenneropenaeus chinensis and identifying male parent shrimps in the prior art are avoided.
Description
Technical field
The invention belongs to the seawater animal field of releasing, relate to particularly a kind of ground simple and easy to operate and can realize the method for releasing that the Chinese prawn individuality of releasing is accurately reviewed.
Background technology
Chinese prawn (Fenneropenaeus chinensis) is the distinctive annual large-scale economic shrimps of China, mainly is distributed in the coastal zone along the Huanghai Sea and the Bohai Sea sea area, remarkable in economical benefits.Since the 1980s, because the continuing to increase of catching intensity, Ecological Changes, water pollution, disease takes place frequently and propagate industry artificially to the impact of the composite factors such as blindness demand of wild Chinese prawn, the rapid atrophy of Chinese prawn wild resource amount.Be protection, recover Chinese prawn population and fishery resources, China carries out Chinese prawn in beginning in 1984 and moves and grow/enhancement releasing the young shrimp of about 3,000,000,000 tails of releasing every year in specific sea area.Release through extensive seedling for years, Penaeus Chinensis Resources has measured certain recovery.But owing to can't accurately distinguish release in the recapture colony individual and wild individuality and quantity, thereby the additional level of colony to wild resource of can't Scientific evaluation releasing, analyze the dynamic change of wild resource and the planning of releasing of formulating science, this is the Chinese prawn significant problem in the urgent need to address of releasing.Weigh and release one of important indicator of effect of assessment is the rate of recapture estimation of releasing, this is the planning of releasing of formulation science, effectively promotes the important indicator of wild Chinese prawn Stock resoures recovery.But existing detection method can't provide the accurate rate of recapture owing to there are various defectives, mainly has following reason: 1) can't accurately distinguish in the recapture sample and release and the Wild prawn individuality; 2) release individual physical markings process operation loaded down with trivial details, can't carry out on a large scale, generally only the part individuality of releasing is carried out mark; 3) the individual death rate is large behind physical markings, the mark harsh to individual specification requirement, affects the rate of recapture and accurately estimates.Therefore, in the urgent need to a kind of technology the colony of releasing is indicated in enormous quantities at present that releasing as Chinese prawn provides accurate rate of recapture data.And this technology should possess following characteristics: the individuality of 1) releasing carries distinctive tag system, and this system can accurately distinguish wild and artificial releasing's individuality; 2) individual all one's life can be cultivated and maintain to the individuality that carries this tag system in enormous quantities, can not have any inherence or physical injury to identifying individuality; 3) this sign individuality can be differentiated rapidly and be made a distinction with other individuality; 4) the sign individuality is released and can not be destroyed or affect wild Chinese prawn colony resource structures.
Summary of the invention
The technical problem to be solved in the present invention provides and a kind ofly can realize the method for releasing accurately review to the Chinese prawn of releasing, the precise evaluation Chinese prawn effect of releasing, the precise evaluation rate of recapture of releasing, calculate the purpose of wild population stock number, formulate the planning of releasing of science, effectively promote wild Chinese prawn Stock resoures to recover.
The effect evaluation method of releasing of the present invention meets to release and detects 5 conditions that will possess: 1) have definite genetic background, do not carry the extensive many familys seed rearing of Chinese prawn of specific pathogen; 2) the efficient accurately identification of Chinese prawn fluorescence labeling microsatellite Quadruple-PCR individuality/family; 3) carry the specific dna molecular finger-print individual tagging of releasing; 4) carry individual the detecting on a large scale of releasing of specific dna molecular finger-print; 5) release and the accurate Calculation of wild resource and the rate of recapture.
The present invention is achieved through the following technical solutions:
A kind ofly can realize may further comprise the steps the method for releasing accurately review to the Chinese prawn of releasing:
Release season every year, and the female shrimp of wild mating is caught in the sea, and the optic stalk mark, then carries out seed rearing, and the female shrimp-75 ℃ ultra low temperature freezer after laying eggs saves backup; Autumn, in the migration point of respectively the releasing individual sampling of fishing for of releasing, pass through two groups of Chinese prawn fluorescence labeling microsatellite Quadruple-PCR individual identification technology for detection recapture samples after the sample recapture, and determine the individual quantity of releasing, finally assess the effect of releasing of Chinese prawn;
Wherein said Chinese prawn fluorescence labeling microsatellite Quadruple-PCR individual identification technology, first group of little satellite Quadruple-PCR combination of primers and sequence are as follows:
EN0033 forward sequence is: 5 '-6-FAM-CCTTGACACGGCATTGATTGG-3 ', reverse sequence is: 5 '-TACGTTGTGCAAACGCCA AGC-3 ';
RS0622 forward sequence is: 5 '-VIC-TCAGTCCGTAGTTCATACTTGG-3 ', reverse sequence is: 5 '-CACATGCCTTTGTGTGAAAACG-3 ';
FCKR002 forward sequence is: 5 '-NED-CTCAACCCTCACCTCAGGAACA-3 ', reverse sequence is: 5 '-AATTGTGGAGGCGACTAAGTTC-3 ';
FCKR013 forward sequence is: 5 '-PET-GCACATATAAGCACAAACGCTC-3 ', reverse sequence is: 5 '-CTCTCTCGCAATCTCTCCAACT-3 ';
Further, the reaction system of described first group of Quadruple-PCR primer is: 25 μ l reaction systems, concentration comprising 2.5 μ l10 * PCR buffer solution, four kinds of fluorescent dye primers is 10 μ M, every of the positive anti-primer of EN0033 is 0.25 μ l, every of the positive anti-primer of RS0622 is 0.5 μ l, every of the positive anti-primer of FCKR002 is 0.5 μ l, and every of the positive anti-primer of FCKR013 is 0.5 μ l; 100ng genomic DNA, 250 μ mol/LdNTPs, 2.5 μ l, 2.0mmol/L Mg
2+2.0 μ l, 1 Taq of unit archaeal dna polymerase;
Further, the pcr amplification program of described first group of Quadruple-PCR primer is: the 1:94 ℃ of sex change 40s that circulate behind 94 ℃ of sex change 4min, 70 ℃ of each cycle annealing temperature of annealing 1min(reduce by 1 ℃), 72 ℃ are extended 1min, circulate altogether 5 times; The 2:94 ℃ of sex change 40s that circulate subsequently, 65 ℃ of annealing 1min, 72 ℃ are extended 1min, circulate altogether 8 times; The 3:94 ℃ of sex change 40s that circulate again, 64 ℃ of each cycle annealing temperature of annealing 1min(reduce by 1 ℃), 72 ℃ are extended 1min, circulate altogether 4 times; The 4:94 ℃ of sex change 40s that circulate, 61 ℃ of annealing 1min, 72 ℃ are extended 1min, circulate altogether 12 times; Last 72 ℃ are extended 5min, preserve and termination routine for 4 ℃.
Wherein said Chinese prawn fluorescence labeling microsatellite Quadruple-PCR individual identification technology, second group of little satellite Quadruple-PCR combination of primers and sequence are as follows:
RS1101 forward sequence is: 5 ' 6-FAM--CGAGTGGCAGCGAGTCCT-3 ', reverse sequence is: 5 '-TATTCCCACGCTCTTGTC-3 '
FCKR005 forward sequence is: 5 ' VIC--CATCGAATCTAAGAGCTGGAAT-3 ', reverse sequence is: 5 '-TTTGTTTGTGAATAATGTGTGT-3 '
FCKR007 forward sequence is: 5 ' NED--CGAAATAAGTTAAATGAAAAAA-3 ', reverse sequence is: 5 '-CAACATAAGACTCACGAGACAG-3 '
FCKR009 forward sequence is: 5 ' PET--GCACGAAAACACATTAGTAGGA-3 ', reverse sequence is: 5 '-ATATCTGGAATGGCAAAGAGTC-3 '.
Further, the reaction system of described second group of Quadruple-PCR primer is: 25 μ l reaction systems, concentration comprising 2.5 μ l10 * PCR buffer solution, four kinds of fluorescent dye primers is 10 μ M, every of the positive anti-primer of EN0033 is 0.25 μ l, every of the positive anti-primer of RS0622 is 0.5 μ l, every of the positive anti-primer of FCKR002 is 0.5 μ l, and every of the positive anti-primer of FCKR013 is 0.5 μ l; 100ng genomic DNA, 250 μ mol/LdNTPs, 2.5 μ l, 2.0mmol/L Mg
2+2.0 μ l, 1 Taq of unit archaeal dna polymerase;
Further, the pcr amplification program of described second group of Quadruple-PCR primer is: the 1:94 ℃ of sex change 40s that circulate behind 94 ℃ of sex change 4min, and 52 ℃ of annealing 1min, 72min extends 1min, circulates altogether 10 times; The 2:94 ℃ of sex change 40s that circulate subsequently, 51 ℃ of each cycle annealing temperature of annealing 1min(reduce by 0.5 ℃), 72 ℃ are extended 1min, circulate altogether 4 times; The 3:94 ℃ of sex change 40s that circulate again, 49 ℃ of annealing 1min, 72 ℃ are extended 1min, circulate altogether 10 times; Last 72 ℃ are extended 5min, preserve and termination routine for 4 ℃.
Further, release effect determine be calculated as follows: the rate of recapture of the releasing=individual amount/total number of samples of releasing.
The present invention carries out individual principle of accurately reviewing: utilize the interior individuality of the same family of Chinese prawn to possess the same molecular finger-print, in the known situation of single female parent, the characteristics that same family individuality can accurately be identified are grown seedlings in conjunction with the extensive many familys of Chinese prawn, produce the colony of releasing.During the autumn flood recapture, utilize accurately efficient molecular fingerprint atlas analysis identification, the identification individuality of releasing calculates release individual amount and wild individual amount from the recapture sample, thereby reaches the precise evaluation rate of recapture of releasing.
The present invention's beneficial effect compared with prior art:
The present invention realizes the maternal accurately selection of all colonies of releasing to a region, and accurately reviewing of rear recapture individuality laid a good foundation in order to release.Advantage compared with prior art mainly is to have simplified the preparation of the colony of releasing, that is: the present invention only needs the release Chinese prawn female parent of seed of production is carried out sample collection, and utilize two groups of fluorescence labeling microsatellite Quadruple-PCRs that the sample of releasing that gathers is carried out parent-offspring's Source Tracing, can realize the Tracking Recognition to the Chinese prawn of releasing.Avoided prior art to need the troublesome operation of preparation Chinese prawn family full-sibs in enormous quantities, the Chinese prawn parents culture of surviving the winter, male shrimp parent identification.
The detection method that the present invention is used for the recapture individuality is by two groups of Chinese prawn fluorescence labeling microsatellite Quadruple-PCR individual identification technology, utilize the molecular fingerprint atlas analysis, from the recapture sample, identify the individuality of releasing, this is labeled as the individual inheritance material, can both play mark action for any specification Chinese prawn individuality of releasing, and can maintain all one's life, can not cause any inherence or physical damnification to tagging.And in the whole process of releasing, the individuality of releasing possesses life ecological habit capable, the identical migration route identical with other individuality, can not possess more obvious existence/dead advantage than other individuality; Releasing, sign is individual can be differentiated rapidly and be made a distinction with other individuality by little satellite Quadruple-PCR technology, can be used for to release in the recapture sample, wild population quantity and the rate of recapture carry out accurate Calculation; And the sign individuality is released and can not be destroyed or affect wild Chinese prawn colony resource structures.Therefore the effect of releasing that method of the present invention can the accurate evaluation Chinese prawn.
Embodiment
Embodiment 1: the release establishment of population and method of mark
One, has reviewing of specific molecular finger-print Chinese prawn family individuality
The offspring that the every couple of father and mother breed has consistent dna molecular fingerprint, and this molecular fingerprint is different from other family and individual and maintain all one's life for individuality/family is peculiar.Utilize this species specific molecular fingerprint collection of illustrative plates, the reference parent genotype can accurately identify specific individuality/family from population mixture.The little satellite of Chinese prawn is widely distributed in genome, and the genetic variation level is abundant, and neutral characteristics are selected in tool heredity.Microsatellite molecular marker different genotype combination and the molecular fingerprint collection of illustrative plates that forms is the ideal tools of carrying out individuality/parentage identification.Take the Chinese prawn microsatellite locus of laboratory independent development as example, the allelomorph quantity in most of sites does not wait at 7-13.In the known situation of single parent, utilize eight mutual not chain microsatellite molecular markers, the allelomorph number in each site is 10, and then the molecular fingerprint spectrum recognition system that forms of these four little satellites can reach the level of family/individual elimination factor 99.9999% in theory.That is to say that its molecular fingerprint collection of illustrative plates is consistent between the individuality in the same family, and is different from other individual molecular fingerprint collection of illustrative plates.
Two, many familys acquisition of population of releasing
The seed of releasing annual spring produces season, and it is some that the female shrimp individuality of selecting mating in the prawn is caught in the sea, carries out optic stalk mark (namely putting plastic hoop with label information at the prawn optic stalk).Carry out seed rearing after the fortification, carry out subsequently the Chinese prawn seed and release.
Three, recapture and the recruitment evaluation of releasing
In autumn, in the migration point of respectively releasing individual the fishing for of releasing, each place number of samples of releasing is 5000 tails.Each sample is got the swimming appendage and is adopted conventional phenol/chloroform method for extracting to carry out the DNA extraction.Then carry out the fluorescence labeling microsatellite Quadruple-PCR and detect analysis.At first carrying out first group of fluorescence labeling microsatellite Quadruple-PCR detects.The PCR reaction system is: 25 μ l reaction systems, concentration comprising 2.5 μ l10 * PCR buffer solution, four kinds of fluorescent dye primers (combination of primers and sequence see Table 1) is 10 μ M, every of the positive anti-primer of EN0033 is 0.25 μ l, every of the positive anti-primer of RS0622 is 0.5 μ l, every of the positive anti-primer of FCKR002 is 0.5 μ l, and every of the positive anti-primer of FCKR013 is 0.5 μ l; 100ng genomic DNA, 250 μ mol/L dNTPs2.5 μ l, 2.0mmol/L Mg
2+2.0 μ l, 1 Taq of unit archaeal dna polymerase; Amplification program is: the 1:94 ℃ of sex change 40s that circulate behind 94 ℃ of sex change 4min, 70 ℃ of each cycle annealing temperature of annealing 1min(reduce by 1 ℃), 72 ℃ are extended 1min, circulate altogether 5 times; The 2:94 ℃ of sex change 40s that circulate subsequently, 65 ℃ of annealing 1min, 72 ℃ are extended 1min, circulate altogether 8 times; The 3:94 ℃ of sex change 40s that circulate again, 64 ℃ of each cycle annealing temperature of annealing 1min(reduce by 1 ℃), 72 ℃ are extended 1min, circulate altogether 4 times; Then the 4:94 ℃ of sex change 40s that circulate, 61 ℃ of annealing 1min, 72 ℃ are extended 1min, circulate altogether 12 times; Last 72 ℃ are extended 5min, preserve and termination routine for 4 ℃.The PCR product detects and carries out at ABI3130 sequenator (U.S. Applied Biosystems company), utilizes GeneMapper software to carry out peak value and transforms and the interpretation of microsatellite locus allelomorph.Adopt same method female parent to be carried out the genotype detection of four microsatellite locus.Family parent and sample data are inputted respectively and are carried out parent and offspring individual identification in Cervus 3.0 softwares, the individuality individual and that parent's allelomorph does not conform to of releasing in first group of detection excludes, the individuality that conforms to parent's allelomorph utilizes second group of fluorescence labeling microsatellite Quadruple-PCR system to detect once again, and its data and maternal data are inputted respectively and carried out female parent and offspring individual identification in Cervus 3.0 softwares.Infer release in the test sample individuality and quantity thereof with this.The rate of recapture=the individual amount of the releasing/sample total amount (%) of releasing.
The used micro-satellite primers combination of table 1. Chinese prawn individuality/parentage identification and sequence
Embodiment 2: small-scale detection
2010, defend Experimental Base at the Huanghai Sea aquatic products Ao Shan of research institute and carried out the simulated experiment that utilizes the inventive method to release.Embodiment is: spring, the female shrimp of mating was caught also in random acquisition 10 tail seas, by the optic stalk mark individuality is carried out the female shrimp of physical markings mating and cultivate separately family, then May, when young shrimp individuality reached the 3cm left and right sides, each random choose 100 tail carried out the physics fluorescence labeling from 3 familys respectively.300 tail individualities of the young shrimp of random acquisition 2000 tail same specifications and above 3 familys are raised together with.Then mid-August, after all individual collections, adopt the described first group of Chinese prawn fluorescence labeling microsatellite Quadruple-PCR reaction system of table 1, PCR reaction composition and amplification program are with embodiment 1.Use this Quadruple-PCR reaction system, in conjunction with family female genotype data, raise together with the family individuality to 3 and carried out the molecular labeling Source Tracing.Then utilize second group of Chinese prawn fluorescence labeling microsatellite Quadruple-PCR reaction system, PCR reaction composition and amplification program are with embodiment 1.The result shows that through the individual fluorescence labeling checking, all family individualities all can obtain the affirmation of tracing to the source through two groups of Chinese prawn fluorescence labeling microsatellite Quadruple-PCR reaction systems.
Technical indicator
Under the condition of known single female parent, the individuality of two groups of Chinese prawn fluorescence labeling microsatellite Quadruple-PCR technology/parentage identification rate is more than 99.9993%; This technology can be carried out tracing to annual 6000 Chinese prawn family full-sibs parents and filial generation thereof, namely can satisfy the need that the annual 3000000000 tail Released Penaeus Orientalis of China are followed the tracks of and be used for carrying out the accurate estimation of the rate of recapture; The tracking of not only can releasing in the single place of releasing of this technology, can also release simultaneously in many places a volume tracing and the rate of recapture are calculated.
Range of application
This technology is mainly used in the Chinese prawn accurate differentiation individual and wild individuality of releasing in the practice of releasing, and then realizes the precise evaluation to the effect of releasing.
Claims (5)
1. method of releasing that can realize accurately reviewing to the Chinese prawn of releasing, it is characterized in that may further comprise the steps: release season every year, and the female shrimp of wild mating is caught in the sea, and the optic stalk mark, then carry out seed rearing, produce female shrimp-75 a ℃ ultra low temperature freezer of finishing and save backup; In autumn, in the migration point of respectively the releasing individual sampling of fishing for of releasing, by two groups of Chinese prawn fluorescence labeling microsatellite Quadruple-PCR individual identification technology for detection release individuality and quantity, finally determine the effect of releasing of Chinese prawn after the sample recapture;
Described two groups of Chinese prawn fluorescence labeling microsatellite Quadruple-PCR individual identification technology, first group of little satellite Quadruple-PCR combination of primers and sequence are as follows:
EN0033 forward sequence is: 5 '-6-FAM-CCTTGACACGGCATTGATTGG-3 ', reverse sequence is: 5 '-TACGTTGTGCAAACGCCA AGC-3 ';
RS0622 forward sequence is: 5 '-VIC-TCAGTCCGTAGTTCATACTTGG-3 ', reverse sequence is: 5 '-CACATGCCTTTGTGTGAAAACG-3 ';
FCKR002 forward sequence is: 5 '-NED-CTCAACCCTCACCTCAGGAACA-3 ', reverse sequence is: 5 '-AATTGTGGAGGCGACTAAGTTC-3 ';
FCKR013 forward sequence is: 5 '-PET-GCACATATAAGCACAAACGCTC-3 ', reverse sequence is: 5 '-CTCTCTCGCAATCTCTCCAACT-3 ';
Described two groups of Chinese prawn fluorescence labeling microsatellite Quadruple-PCR individual identification technology, second group of little satellite Quadruple-PCR combination of primers and sequence are as follows:
RS1101 forward sequence is: 5 '-6-FAM-CGAGTGGCAGCGAGTCCT-3 ', reverse sequence is: 5 '-TATTCCCACGCTCTTGTC-3 '
FCKR005 forward sequence is: 5 '-VIC-CATCGAATCTAAGAGCTGGAAT-3 ', reverse sequence is: 5 '-TTTGTTTGTGAATAATGTGTGT-3 '
FCKR007 forward sequence is: 5 '-NED-CGAAATAAGTTAAATGAAAAAA-3 ', reverse sequence is: 5 '-CAACATAAGACTCACGAGACAG-3 '
FCKR009 forward sequence is: 5 '-PET-GCACGAAAACACATTAGTAGGA-3 ', reverse sequence is: 5 '-ATATCTGGAATGGCAAAGAGTC-3 '.
2. according to claim 1ly a kind ofly can realize the method for releasing accurately review to the Chinese prawn of releasing, its spy is that the reaction system of described first group of little satellite Quadruple-PCR primer is: 25 μ l reaction systems, concentration comprising 2.5 μ l10 * PCR buffer solution, four kinds of fluorescent dye primers is 10 μ M, every of the positive anti-primer of EN0033 is 0.25 μ l, every of the positive anti-primer of RS0622 is 0.5 μ l, every of the positive anti-primer of FCKR002 is 0.5 μ l, and every of the positive anti-primer of FCKR013 is 0.5 μ l; 100ng genomic DNA, 250 μ mol/LdNTPs, 2.5 μ l, 2.0mmol/L Mg
2+2.0 μ l, 1 Taq of unit archaeal dna polymerase, surplus is supplied with distilled water.
3. according to claim 1 and 2ly a kind ofly can realize the method for releasing accurately review to the Chinese prawn of releasing, its spy is that the pcr amplification program of described first group of little satellite Quadruple-PCR primer is: 1:94 ℃ of sex change 40s circulates behind 94 ℃ of sex change 4min, 70 ℃ of annealing 1min, each cycle annealing temperature reduces by 1 ℃, 72 ℃ are extended 1min, circulate altogether 5 times; The 2:94 ℃ of sex change 40s that circulate subsequently, 65 ℃ of annealing 1min, 72 ℃ are extended 1min, circulate altogether 8 times; The 3:94 ℃ of sex change 40s that circulate again, 64 ℃ of annealing 1min, each cycle annealing temperature reduces by 1 ℃, and 72 ℃ are extended 1min, circulate altogether 4 times; The 4:94 ℃ of sex change 40s that circulate, 61 ℃ of annealing 1min, 72 ℃ are extended 1min, circulate altogether 12 times; Last 72 ℃ are extended 5min, preserve and termination routine for 4 ℃.
4. according to claim 1ly a kind ofly can realize the method for releasing accurately review to the Chinese prawn of releasing, its spy is that the reaction system of described second group of little satellite Quadruple-PCR primer is: 25 μ l reaction systems, concentration comprising 2.5 μ l10 * PCR buffer solution, four kinds of fluorescent dye primers is 10 μ M, EN 0033 every of positive anti-primer is 0.25 μ l, every of the positive anti-primer of RS0622 is 0.5 μ l, every of the positive anti-primer of FCKR002 is 0.5 μ l, and every of the positive anti-primer of FCKR013 is 0.5 μ l; 100ng genomic DNA, 250 μ mol/LdNTPs, 2.5 μ l, 2.0mmol/L Mg
2+2.0 μ l, 1 Taq of unit archaeal dna polymerase, surplus is supplied with distilled water.
5. according to claim 1 or 4 describedly a kind ofly can realize the method for releasing accurately review to the Chinese prawn of releasing, its spy is that the pcr amplification program of described second group of little satellite Quadruple-PCR primer is: 1:94 ℃ of sex change 40s circulates behind 94 ℃ of sex change 4min, 52 ℃ of annealing 1min, 72min extends 1min, circulates altogether 10 times; The 2:94 ℃ of sex change 40s that circulate subsequently, 51 ℃ of annealing 1min, each cycle annealing temperature reduces by 0.5 ℃, and 72 ℃ are extended 1min, circulate altogether 4 times; The 3:94 ℃ of sex change 40s that circulate again, 49 ℃ of annealing 1min, 72 ℃ are extended 1min, circulate altogether 10 times; Last 72 ℃ are extended 5min, preserve and termination routine for 4 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012105595936A CN102972322A (en) | 2012-12-20 | 2012-12-20 | Simplified releasing method capable of accurately tracing released Fenneropenaeus chinensis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012105595936A CN102972322A (en) | 2012-12-20 | 2012-12-20 | Simplified releasing method capable of accurately tracing released Fenneropenaeus chinensis |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102972322A true CN102972322A (en) | 2013-03-20 |
Family
ID=47846898
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2012105595936A Pending CN102972322A (en) | 2012-12-20 | 2012-12-20 | Simplified releasing method capable of accurately tracing released Fenneropenaeus chinensis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102972322A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103155892A (en) * | 2013-04-03 | 2013-06-19 | 中国水产科学研究院黄海水产研究所 | Chinese prawn releasing effect assessment method |
| CN104099412A (en) * | 2014-06-05 | 2014-10-15 | 中国水产科学研究院东海水产研究所 | Evaluation method for releasing effect of Marsupenaeus Japonicus |
| CN106577425A (en) * | 2016-12-22 | 2017-04-26 | 中国科学院南海海洋研究所 | Panmixis parent tracing method for litopenaeus vannamei |
| CN106757379A (en) * | 2016-12-20 | 2017-05-31 | 上海赛安生物医药科技有限公司 | Lung cancer polygenic variation library constructing method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1488765A (en) * | 2002-10-09 | 2004-04-14 | 中国水产科学研究院黄海水产研究所 | Chinese prawn S33 microsatellite label detecting technique |
| CN102687692A (en) * | 2012-06-04 | 2012-09-26 | 中国水产科学研究院黄海水产研究所 | Release method capable of accurately tracing released Chinese prawns |
-
2012
- 2012-12-20 CN CN2012105595936A patent/CN102972322A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1488765A (en) * | 2002-10-09 | 2004-04-14 | 中国水产科学研究院黄海水产研究所 | Chinese prawn S33 microsatellite label detecting technique |
| CN102687692A (en) * | 2012-06-04 | 2012-09-26 | 中国水产科学研究院黄海水产研究所 | Release method capable of accurately tracing released Chinese prawns |
Non-Patent Citations (6)
| Title |
|---|
| 于飞等: "微卫星标记在大菱鲆(Scophthalmus maximus L.)家系系谱印证中的应用研究", 《海洋学报》 * |
| 孔杰等: "微卫星三重PCR基因扫描技术在中国明对虾家系标识中的应用", 《中国水产科学》 * |
| 张建勇等: "四引物扩增受阻突变体系PCR技术在中国明对虾SNP基因分型中的研究", 《中国水产科学》 * |
| 李伟亚等: "中国对虾微卫星四重PCR技术的建立及其在模拟放流效果评估方面的应用", 《海洋学报》 * |
| 田燚等: "中国对虾遗传连锁图谱的构建", 《科学通报》 * |
| 高焕等: "中国对虾(Fenneropenaeus chinensis)基因组微卫星特征分析", 《海洋与湖沼》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103155892A (en) * | 2013-04-03 | 2013-06-19 | 中国水产科学研究院黄海水产研究所 | Chinese prawn releasing effect assessment method |
| CN104099412A (en) * | 2014-06-05 | 2014-10-15 | 中国水产科学研究院东海水产研究所 | Evaluation method for releasing effect of Marsupenaeus Japonicus |
| CN104099412B (en) * | 2014-06-05 | 2016-05-11 | 中国水产科学研究院东海水产研究所 | The release appraisal procedure of effect of a kind of Marsupenaeus japonicus |
| CN106757379A (en) * | 2016-12-20 | 2017-05-31 | 上海赛安生物医药科技有限公司 | Lung cancer polygenic variation library constructing method |
| CN106757379B (en) * | 2016-12-20 | 2018-08-28 | 上海赛安生物医药科技股份有限公司 | Lung cancer polygenic variation library constructing method |
| CN106577425A (en) * | 2016-12-22 | 2017-04-26 | 中国科学院南海海洋研究所 | Panmixis parent tracing method for litopenaeus vannamei |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102687692B (en) | Release method capable of accurately tracing released Chinese prawns | |
| CN101911918B (en) | Selective breeding method for disease-resistant superior strains of paralichthys olivaceus | |
| CN102134593B (en) | Gender-specific microsatellite marker for Cynoglossus semilaevis and application of same in identification of superfemale Cynoglossus semilaevis | |
| CN104498613B (en) | SSR fluorescent dye primer and application for mandarin sturgeon paternity test | |
| CN105349645A (en) | Roe and fry quick classification and determination methods based on CoI DNA bar codes | |
| CN104357553A (en) | Pelteobagrus fulvidraco microsatellite family identification method | |
| CN102972322A (en) | Simplified releasing method capable of accurately tracing released Fenneropenaeus chinensis | |
| Chakrabarty et al. | Is sexual selection driving diversification of the bioluminescent ponyfishes (Teleostei: Leiognathidae)? | |
| CN103993081B (en) | A kind of method differentiating gynogenetic grass carp and common grass carp | |
| CN103525814B (en) | A kind of special SCAR mark of Cynoglossus semilaevis sex and application method | |
| CN109122438A (en) | A kind of purple sea hybrid scallop bottom sowing aquaculture method | |
| CN104450694A (en) | q SRBSDV6 (Southern rice black-streaked dwarf virus 6) and molecular marker method thereof | |
| CN105349543A (en) | DNA (deoxyribonucleic acid) sequence used for identifying genetic sex of fenneropenaeus chinensis as well as obtaining method and application thereof | |
| Barwick et al. | Redeye bass (Micropterus coosae) and Alabama spotted bass (M. punctulatus henshalli) hybridization in Keowee Reservoir | |
| CN103484459B (en) | Primer group, marking method and application of EST-SSR (Expressed Sequence Tag-Simple Sequence Repeats) molecular marker of macrobrachium nipponense | |
| CN105349691A (en) | DNA (deoxyribonucleic acid) sequence tag for identifying genetic sex of fenneropenaeus chinensis and application thereof | |
| Ashton et al. | Evaluating microsatellite markers for parentage-based tagging of hatchery burbot | |
| Zhang et al. | Application of a fluorescence in situ hybridization (FISH) method to study green tides in the Yellow Sea | |
| Hidayania et al. | The morphometric character and mitochondrial 16S rRNA sequence of Portunus pelagicus | |
| Wang et al. | Integration genetic linkage map construction and several potential QTLs mapping of Chinese shrimp (Fenneropenaeus chinensis) based on three types of molecular markers | |
| CN103276089A (en) | Paternity test method and kit of common suckers | |
| CN106916884A (en) | A kind of bolti salt tolerant related molecular marker SSR450 and its application | |
| CN103911444B (en) | Primer for discriminating anguilla marmorata and anguilla bicolor pacifica fry and method thereof | |
| CN106529780A (en) | Portunus trituberculatus release effect evaluation method | |
| Çakmak et al. | Triploid Black Sea Trout (Salmo labrax Pallas, 1814) induced by heat shock and evaluation of triploidy with different techniques |
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 |
Application publication date: 20130320 |