CN102687692B - Release method capable of accurately tracing released Chinese prawns - Google Patents
Release method capable of accurately tracing released Chinese prawns Download PDFInfo
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- CN102687692B CN102687692B CN201210179604.8A CN201210179604A CN102687692B CN 102687692 B CN102687692 B CN 102687692B CN 201210179604 A CN201210179604 A CN 201210179604A CN 102687692 B CN102687692 B CN 102687692B
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- 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
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Abstract
The invention discloses a release method capable of accurately tracing the released Chinese prawns, belonging to the field of proliferation release of seawater animals. The method comprises the following steps of: selecting multiple parents of the Chinese prawns; performing one-to-one directional copulation on the male and female parents; sleeving a mark ring on the eyestalks of the female prawns after successful copulation, and performing overwintering cultivation; marking the eyestalks of the male prawns and placing at -70 DEG C for standby; performing offspring seed cultivation of copulated female prawns next year; releasing cultivated seedlings of the Chinese prawns in the release season; in autumn, fishing and sampling at migration points; after recapturing the samples, detecting released individuals and numbers by using a Chinese prawn fluorescence labeling microsatellite quadruple PCR individual recognition technology; and finally determining the release effect of the Chinese prawns. According to the method, the detection method of the recaptured individuals adopts the Chinese prawn fluorescence labeling microsatellite quadruple PCR individual recognition technology; the released individuals are recognized from the recaptured samples by using molecular fingerprint spectrum analysis; each mark is an individual genetic material and does not bring any internal or physical injury to the individual; and the release effect of the Chinese prawns can be accurately evaluated.
Description
Technical field
The invention belongs to seawater animal enhancement releasing field, relate to particularly a kind of method of releasing that the Chinese prawn individuality of releasing is accurately reviewed that realizes.
Background technology
Chinese prawn (Fenneropenaeus chinensis) is the distinctive annual large-scale economic shrimps of China, is mainly distributed in coastal zone along the Huanghai Sea and the Bohai Sea sea area, remarkable in economical benefits.Since the 1980s, due to the continuing to increase of catching intensity, Ecological Changes, water pollution, disease takes place frequently and propagate the impact of industry on the composite factors such as blindness demand of wild Chinese prawn, the sharply atrophy of Chinese prawn wild resource amount artificially.For protection, recovery Chinese prawn population and fishery resources, China started to carry out Chinese prawn in specific sea area in 1984 and moves grow/enhancement releasing, the young shrimps of approximately 3,000,000,000 tails of releasing every year.Through extensive seedling for years, release, Penaeus Chinensis Resources has measured certain recovery.But owing to cannot accurately distinguishing release in recapture colony individual and wild individuality and quantity, thereby the supplementary level of colony to wild resource of cannot Scientific evaluation releasing, analyze the dynamic change of wild resource the planning of releasing of formulating science, this is the Chinese prawn significant problem in the urgent need to address of releasing.One of important indicator of weighing and assessing the effect of releasing is the rate of recapture estimation of releasing, and this is the planning of releasing of formulation science, the important indicator that effectively promotes the recovery of wild Chinese prawn Stock resoures.But existing detection method cannot provide the accurate rate of recapture owing to there are various defects, mainly has following reason: 1) in recapture sample, cannot accurately distinguish and release and Wild prawn individuality; 2) to release physical markings process operation individual loaded down with trivial details, cannot carry out on a large scale, generally only the part individuality of releasing carried out to mark; 3) after physical markings, mark harsh to individual specification requirement, individual death rate is large, 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, as Chinese prawn, release accurate rate of recapture data are provided.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 individual; 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) 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 is to provide a kind of method of releasing that can accurately review the Chinese prawn realization of releasing, precise evaluation Chinese prawn release effect and the rate of recapture of releasing, calculate 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 listed releasing in background technology 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:
Can realize the method for releasing of accurately reviewing to the Chinese prawn of releasing, comprise the following steps:
Select to determine genetic background, a plurality of parents of Chinese prawn that do not carry specific pathogen, by the directed mating one to one of female male parent, the successful female shrimp of mating overwintering culture after optic stalk puts Marking ring, after male shrimp optic stalk mark, be placed on-70 ℃ standby, the female shrimp of mating carries out seed rearing next year, and release the season of releasing to cultivated Chinese prawn seedling; In autumn, in the migration point of respectively the releasing individual sampling of fishing for of releasing, after sample recapture, by 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;
Wherein said Chinese prawn fluorescence labeling microsatellite Quadruple-PCR individual identification technology, micro-satellite primers combination and sequence are as follows:
EN0033 forward sequence is: 5 '-CCTTGACACGGCATTGATTGG-3 ', and reverse sequence is: 5 '-TACGTTGTGCAAACGCCA AGC-3 ';
RS0622 forward sequence is: 5 '-TCAGTCCGTAGTTCATACTTGG-3 ', and reverse sequence is: 5 '-CACATGCCTTTGTGTGAAAACG-3 ';
FCKR002 forward sequence is: 5 '-CTCAACCCTCACCTCAGGAACA-3 ', and reverse sequence is: 5 '-AATTGTGGAGGCGACTAAGTTC-3 ';
FCKR013 forward sequence is: 5 '-GCACATATAAGCACAAACGCTC-3 ', and reverse sequence is: 5 '-CTCTCTCGCAATCTCTCCAACT-3 ';
PCR reaction system is: 25 μ l reaction systems, concentration comprising 2.5 μ l 10 * PCR buffer solutions, 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/L dNTPs, 2.0mmol/L Mg
2+, 1 Taq of unit archaeal dna polymerase;
Amplification program is: the 1:94 ℃ of sex change 40s that circulate after 94 ℃ of sex change 4min, and 70 ℃ of annealing 1min, each cycle annealing temperature reduces by 1 ℃ afterwards, and 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 ℃ afterwards, 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 ℃.
Further, the definite of effect that release is 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 individuality in the same family of Chinese prawn to possess same molecular finger-print, in the situation that Parent is known, the feature that same family individuality can accurately be identified, grows seedlings in conjunction with the extensive many familys of Chinese prawn, produces the colony of releasing.During autumn flood recapture, utilize accurately efficient molecular fingerprint atlas analysis identification, from recapture sample, the identification individuality of releasing, calculates release individual amount and wild individual amount, thereby reaches the precise evaluation rate of recapture of releasing.
The present invention's beneficial effect compared with prior art:
The present invention selects meticulously to the parent of colony that releases, directed mating one to one, and mark is also kept the parent of colony that releases, accurately reviewing and laying a good foundation for recapture individuality after releasing.
The present invention is Chinese prawn fluorescence labeling microsatellite Quadruple-PCR individual identification technology for the detection method of recapture individuality, utilize molecular fingerprint atlas analysis, from recapture sample, identify the individuality of releasing, this is labeled as individual inheritance material, for any specification Chinese prawn individuality of releasing, can play mark action, and can maintain all one's life, can not cause any inherence or physical damnification to tagging.And whole, release in process, the individuality of releasing possesses the migration route capable, identical with other individual identical life ecological habit, 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 micro-satellite Quadruple-PCR technology, can be used for to releasing in recapture sample, wild population quantity and the rate of recapture carry out accurate Calculation; And 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 accurate evaluation Chinese prawn.
Embodiment
Embodiment 1: the release establishment of population and method of mark
One, there is 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 peculiar other family and individual and maintain all one's life that is different from for individual/family resembles.Utilize this species specific molecular fingerprint collection of illustrative plates, reference parent genotype can accurately identify specific individuality/family from population mixture.The micro-satellite of Chinese prawn is widely distributed in genome, and genetic variation level is abundant, and neutral feature is 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.The Chinese prawn microsatellite locus of laboratory independent development of take is example, and the allelomorph quantity in most of sites is not at 7-13 etc.In the situation that parents are known, utilize four mutual not chain microsatellite molecular markers, the allelomorph number in each site is 10, and the molecular fingerprint spectrum recognition system that these four micro-satellites form in theory can reach the level of family/individual elimination factor 99.9999%.That is to say, between the individuality in same family, its molecular fingerprint collection of illustrative plates is consistent, and is different from other individual molecular fingerprint collection of illustrative plates.
Two, many familys acquisition of population of releasing
From sea, catch that prawn, to select male shrimp and the female shrimp individuality of not mating some autumn in the first year, be placed on one to one 3m
3directed mating in Glass fibre reinforced plastic tub.(putting the plastic hoop with label information on prawn optic stalk) overwintering culture after the successful female shrimp optic stalk mark of mating, after male shrimp eye mark, be placed on-70 ℃ standby.The female shrimp of mating carries out seed rearing next year, and release the season of releasing to cultivated Chinese prawn seedling.
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 swimming appendage and is adopted conventional phenol/chloroform method for extracting to carry out DNA extraction.Then carry out fluorescence labeling microsatellite Quadruple-PCR and detect analysis.PCR reaction system is: 25 μ l reaction systems, concentration comprising 2.5 μ l 10 * PCR buffer solutions, four kinds of fluorescent dye primers (combination of primers and sequence are in 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 dNTPs, 2.0mmol/L Mg
2+, 1 Taq of unit archaeal dna polymerase; Amplification program is: the 1:94 ℃ of sex change 40s that circulate after 94 ℃ of sex change 4min, and 70 ℃ of annealing 1min, each cycle annealing temperature reduces by 1 ℃ afterwards, and 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 ℃ afterwards, 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 ℃.PCR product detects and carries out on ABI3130 sequenator (U.S. Applied Biosystems company), utilizes GeneMapper software to carry out peak value conversion and the interpretation of microsatellite locus allelomorph.Adopt same method many familys Parent to be carried out to the genotype detection of four microsatellite locus.Family parent and sample data are inputted respectively and in Cervus 3.0 softwares, are carried out parent and offspring individual identification, detect the individual amount of releasing in sample.The rate of recapture=individual amount/sample the total amount (%) of releasing of releasing.
The micro-satellite primers combination used of table 1. Chinese prawn individuality/parentage identification and sequence
Embodiment 2: small-scale detection
2010, at the Huanghai Sea aquatic products Ao Shan of research institute, defend Experimental Base and carried out the simulated experiment that utilizes the inventive method to release.Embodiment is: random acquisition in autumn in the first year 100 tail seas are caught prawn and by optic stalk mark, individuality carried out to physical markings, therefrom selects female shrimp and each 6 tails of male shrimp of not mating, according to proportion control mating in 1: 1, completes the male shrimp-76 ℃ preservation of mating.Backup group is set simultaneously.Remain female shrimp and the random mating of male shrimp, mating completes female shrimp and mixes and survive the winter with the female shrimp of control mating.Spring in 2010,3 tails are controlled the female shrimp of mating and are cultivated separately family, and then May, when young shrimp individuality reaches 3cm left and right, from 3 familys, each random choose 100 tails carry out physics fluorescence labeling respectively.All the other female shrimp synchronized mixes are cultivated young shrimp 30000 tails, and therefrom random choose 1988 tail same specification prawns are individual raises together with 300 tail individualities of above 3 familys.Mid-August then, after all individual collections, adopts 4 pairs of Chinese prawn SSR primers described in table 1 to form Chinese prawn fluorescence labeling microsatellite Quadruple-PCR reaction systems, and PCR reaction composition and amplification program are with embodiment 1.Apply this micro-satellite Quadruple-PCR technology, in conjunction with family parent genotype data, to 3, raise together with family individuality and carried out molecular labeling Source Tracing.Result shows, through individual fluorescence labeling checking, all family individualities all can utilize the confirmation that obtains tracing to the source of this micro-satellite Quadruple-PCR reaction system.
Technical indicator
Individuality/parentage identification rate of Chinese prawn fluorescence labeling microsatellite Quadruple-PCR technology is more than 99.9995% (Parent is known); This technology can be carried out tracing to annual 6000 Chinese prawn family full-sibs parents and filial generation thereof, namely can meet need that the annual 3000000000 tail Released Penaeus Orientalis of China are followed the tracks of and 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 in many places a volume tracing and the rate of recapture are calculated simultaneously.
Range of application
This technology is mainly used in the Chinese prawn accurate differentiation individual and wild individuality of releasing in practice of releasing, and then realizes the precise evaluation to the effect of releasing.
Claims (1)
1. can realize the method for releasing of accurately reviewing to the Chinese prawn of releasing, it is characterized in that comprising the following steps: select to determine genetic background, a plurality of parents of Chinese prawn that do not carry specific pathogen, female male parent is placed on to 3m one to one
3directed mating in Glass fibre reinforced plastic tub, the successful female shrimp of mating overwintering culture after optic stalk puts Marking ring, after male shrimp optic stalk mark, be placed on-70 ℃ standby, the female shrimp of mating carries out seed rearing next year, release the season of releasing to cultivated Chinese prawn seedling; In autumn, in the migration point of respectively the releasing individual sampling of fishing for of releasing, after sample recapture, by 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;
Described Chinese prawn fluorescence labeling microsatellite Quadruple-PCR individual identification technology, micro-satellite primers combination and sequence are as follows:
EN0033 forward sequence is: 5 '-CCTTGACACGGCATTGATTGG-3 ', and reverse sequence is: 5 '-TACGTTGTGCAAACGCCA AGC-3 ';
RS0622 forward sequence is: 5 '-TCAGTCCGTAGTTCATACTTGG-3 ', and reverse sequence is: 5 '-CACATGCCTTTGTGTGAAAACG-3 ';
FCKR002 forward sequence is: 5 '-CTCAACCCTCACCTCAGGAACA-3 ', and reverse sequence is: 5 '-AATTGTGGAGGCGACTAAGTTC-3 ';
FCKR013 forward sequence is: 5 '-GCACATATAAGCACAAACGCTC-3 ', and reverse sequence is: 5 '-CTCTCTCGCAATCTCTCCAACT-3 ';
PCR reaction system is: 25 μ l reaction systems, concentration comprising 2.5 μ l10 * PCR buffer solutions, 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/L dNTPs, 2.0mmol/L Mg
2+, 1 Taq of unit archaeal dna polymerase;
Amplification program is: the 1:94 ℃ of sex change 40s that circulate after 94 ℃ of sex change 4min, and 70 ℃ of annealing 1min, each cycle annealing temperature reduces by 1 ℃ afterwards, and 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 ℃ afterwards, 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 ℃;
Described the definite of the effect of releasing is calculated as follows: the rate of recapture of the releasing=individual amount/total number of samples of releasing.
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Families Citing this family (8)
Publication number | Priority date | Publication date | Assignee | Title |
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CN102919172A (en) * | 2012-11-03 | 2013-02-13 | 中国水产科学研究院淡水渔业研究中心 | High-efficiency using method of freshwater shrimp eyestalk label |
CN102919171A (en) * | 2012-11-03 | 2013-02-13 | 中国水产科学研究院淡水渔业研究中心 | Method for distinguishing parent freshwater shrimps from autumn breeding seedlings |
CN102972322A (en) * | 2012-12-20 | 2013-03-20 | 中国水产科学研究院黄海水产研究所 | Simplified releasing method capable of accurately tracing released Fenneropenaeus chinensis |
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 |
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 |
CN110710477B (en) * | 2019-10-10 | 2024-06-25 | 云南大学 | Monitoring device for evaluating fish proliferation and releasing effects |
CN114375888A (en) * | 2022-01-12 | 2022-04-22 | 山东交通学院 | Recapture rate analysis method for evaluating marine fishery organism proliferation effect |
CN114375889B (en) * | 2022-01-27 | 2023-08-29 | 华能西藏雅鲁藏布江水电开发投资有限公司 | Labeling reagent and labeling method for released fries of phyllostachys praecox |
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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 |
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