CN103004653A - Preparation method for triploid grouper - Google Patents
Preparation method for triploid grouper Download PDFInfo
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- CN103004653A CN103004653A CN2012104801245A CN201210480124A CN103004653A CN 103004653 A CN103004653 A CN 103004653A CN 2012104801245 A CN2012104801245 A CN 2012104801245A CN 201210480124 A CN201210480124 A CN 201210480124A CN 103004653 A CN103004653 A CN 103004653A
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Abstract
The invention discloses a preparation method for a triploid grouper, which comprises the following steps: selecting different types of triploid groupers with the genetic relationship of 0.140 to 0.145 genetic distances as parent fishes; adopting a dry method to perform artificial fertilization and distant hybridization; transferring germ cells into seawater after fertilization to perform cold shock treatment; and catching and domesticating for culture of seedlings and identifying the triploid groupers. The preparation method has the advantages of short period for breeding, fast growth speed and no influence on the marine ecological environment.
Description
Technical field
The invention belongs to fish genetic breeding field, be specifically related to the preparation method of a kind of triploid grouper.
Background technology
Ablen is subordinate to Perciformes (Perciformes) ， Sushi section (Serranidae), Epinephelinae (Epinephelinae), world-famous famous and precious fish, it is nutritious, be a kind ofly be low in fat content, the first-class food fish of high protein, liked by consumers in general.Become the main object of mariculture ground groupers such as coastal areas of southern China Guangdong, Hainan, Fujian, Guangxi, economic worth is huge.
Triploid refers to have the genomic individuality of three covers, and normal individuality only has two cover chromosome sets.Triploid individual reproduction archaeocyte normal joint conference in the reduction division process, can not produce normal gamete, in the triploid ontogenetic process, the undesired growth of sexual gland has been saved the reproduction energy and this part energy is used for growing, add the triploid individuality than dliploid Duoed one the cover chromosome set, gene expression is more active has accelerated the process of growing, so that triploid has very significantly advantage aspect growth rate, has huge using value.
At present, the breeding method breeding work year limit for length of traditional grouper, breeding work is made slow progress and grouper crossbreeding pattern in hybridization dliploid escape easily, thereby the marine eco-environment is caused unpredictable serious consequence.
Summary of the invention
Technical problem to be solved by this invention provides the preparation method of a kind of triploid grouper, this preparation method's breeding cycle weak point, fast growth, on the marine eco-environment without impact.
Above-mentioned technical problem of the present invention is achieved by the following technical solution: the preparation method of a kind of triploid grouper, may further comprise the steps: select genetic distance and be the grouper not of the same race of 0.140-0.145 affiliation as parent population, adopt the dry method artificial insemination to carry out distant hybridization, transfer to behind the ovum fertilization and carry out cold shock treatment in the seawater, grow seedlings through harvesting, identify the triploid grouper after raising and train and get final product.
The present invention cooperates physical stimulation to produce the triploid grouper by distant hybridization, has greatly accelerated the process of grouper polyploid breeding work.
The preferred epinephelus lanceolatus fish of grouper of the present invention (E.lanceolatus) and Epinephelus coioides (E.coioides).
Temperature when the present invention preferably adopts the dry method artificial insemination to carry out distant hybridization is 25 ~ 28 ℃.
The present invention transfers to behind ovum fertilization and carries out cold shock treatment in the seawater, and the temperature during cold shock treatment is preferably 4 ℃, and the cold shock treatment time is preferably 20 ~ 25min.
The present invention grows seedlings through harvesting, raise and train to grow to and identify the triploid grouper after 8 ~ 9 monthly ages and get final product.
The present invention grows seedlings through harvesting, adopt the ploidy analyser to detect the haemocyte ploidy after raising and train, thereby and carries out chromosome karyotype analysis and size of blood cells and measure and identify the triploid grouper.
Can adopt the haemocyte smear method to measure size of blood cells.
Compared with prior art, the present invention has following advantage:
(1) the present invention select genetic distance be the grouper not of the same race of 0.140-0.145 affiliation as parent population, after hybridization, carry out cold treatment, can obtain the triploid grouper.The present invention is simple to operate, and the triploid grouper fast growth of preparing is irreproducible, on the marine eco-environment without impact;
(2) the present invention has accelerated the process of ablen genetic breeding research, for later seawater fish polyploid genetic breeding research provides important evidence; Aspect production application, has huge economic worth; All has great reference value aspect ablen genetic breeding, genetic evolution and the research of endocrine regulation scheduling theory.
Description of drawings
Fig. 1-2 is that the chromosome map among the embodiment 1 carries out as a result figure of karyotyping.
Embodiment
Below the reagent that adopts among each embodiment, if no special instructions, be commercially available.
Embodiment 1
Go out 5 kinds of grouper mitochondrial cytochrome bs of CHINESE OFFSHORE grouper (Cyt b) gene by pcr amplification, utilize MEGA3.1 software and calculate genetic distance according to the Kimura two-parameter model.The concrete kind of grouper is as follows: epinephelus akaara (E.akaara), brown some grouper (E.fuscoguttatus), epinephelus lanceolatus fish (E.lanceolatus), Epinephelus coioides (E.coioides) and leopard line gill sour jujube perch (P.leopardus).Select healthy grouper as the hybridization parent population.
Table 1PCR amplification grouper mitochondrial cytochrome b (Cyt b) gene primer sequence is as follows
Genetic distance between 13 kinds of ablen kinds of table 2
Front 1 ~ 2 month of breeding period, select lovely luster, body surface not damaged, the gill filament scarlet without parasite, healthy female Epinephelus coioides and male epinephelus lanceolatus fish as parent population, genetic distance is 0.14419, specially the pond meticulous bait of throwing something and feeding provides good nutrition for parent population, strengthen the physique of parent population in order to obtain the measured gamete of matter, keep a close eye on during this time temperature and the salinity of water in the parent fish pond, note parent population ingest and animation in order to keep parent population to enter breeding period at the state of the best.
Adopt the dry method artificial insemination that female Epinephelus coioides and male epinephelus lanceolatus fish are carried out distant hybridization, after entering breeding period, carrying the previous day hastens parturition to the female Epinephelus coioides injection human chorionic gonadtropin 500IU/kg that belly obviously swells, near the scrub raun cloacal aperture seawater is pushed the belly of fish and is caught ovum with clean beaker; Near the scrub milter cloacal aperture seawater is pushed the belly of fish and is caught the milky seminal fluid with clean test tube, notes avoiding the urine of fish as far as possible.
Be 25 ℃ seawater with adding temperature after ovum and the seminal fluid mixing and stirring, start the fertilization of grouper ovum, behind the ovum fertilization 3 minutes, be transferred to cold shock 20min in 4 ℃ the seawater, grouper fertilized egg after processing is changed in the normal water temperature seawater, leave standstill a moment, get the ovum that floats on the upper strata and put into the little inflation hatching of tank interior.
Change the fry that hatches over to outdoor large tracts of land sea pond and carry out ecological breeding, according to the situations such as transparency of sunlight, temperature, water colour splash around in the pond different amounts by fermentation peanut press pulp, fry grows to 3cm and changes Xiao Chi after above over to again and carry out pellet and raise and train.
Fry physique was large when pellet was raised and train fry to 8 ~ 9 monthly age, adaptability is stronger, was difficult for because of the manual operation unexpected death, and the ablen hatching has the habit of cannibalism, noted regular larger separately raising of fry that size is differed; Afterbody is noted guaranteeing that with the anticoagulant dilute blood of vast scale thereby the blood adhesion does not affect testing result after getting blood, uses the ploidy analyser to detect the haemocyte ploidy, identifies hybridization triploid Green Dragon spot.
In the present embodiment, measure sample take the fish haemocyte as dna content, use the ploidy analyser (instrument and reagent are all provided by German Partec company) to measure; At first, fish blood takes a morsel, be that the haemocyte dilution is made in 0.75% liquaemin anticoagulant dilution more than 10 times with the quality percentage composition, stir in the DAPI of 0.8mL dye liquor (being provided by German Partec company) with sticking a little the haemocyte dilution of getting of 1mL blue electron gun head, leave standstill moments later and detect.After testing, the dna content value between Epinephelus coioides, epinephelus lanceolatus fish and the hybridization dliploid Green Dragon spot is more or less the same, and is respectively 1:1,1:1,1.03:1 with the ratio of Epinephelus coioides; The ratio of hybridization triploid Green Dragon spot dna content value and Epinephelus coioides is 1.34:1, obviously greater than the dliploid grouper, thereby identifies triploid Green Dragon spot.
On the basis of identifying, get head-kidney behind injection phytolectin (PHA) and the colchicine, the preparation chromosome map carries out karyotyping, and the result proves that the chromosome set of Epinephelus coioides, epinephelus lanceolatus fish, hybridization dliploid Green Dragon spot is 2n=48; The chromosome set of hybridization triploid Green Dragon spot is 3n=72.
On the basis of identifying, preparation haemocyte smear, with the size of Photoshop software measurement haemocyte, further the Analysis and Identification result is as shown in table 2, and all the every value than dliploid grouper is larger for the every numerical value of haemocyte of hybridization triploid Green Dragon spot.
In the present embodiment, the method for chromosome preparation operation is as follows: 1) injection: test fish belly chamber injection PHA(10ug/g), the PHA of injection same amount injects colchicine (3ug/g) after 22 hours after three hours; 2) get kidney: the injection colchicine is cut the tail bloodletting after 4 hours, bloodletting 30 minutes, head-kidney is got in dissection, the head-kidney tissue is put into physiological saline to be cleaned 2-3 time, the head-kidney tissue is put into a little physiological saline culture dish to be shredded, filter immigration 15ml centrifuge tube with 400 mesh sieve thin,tough silk, 1000rpm, 8min is centrifugal; 3) hypotonic: centrifugal rear removal supernatant adds 0.075mol/LKCl liquid, in 37 ℃ hypotonic 30 minutes; 4) fixing: with the hypotonic centrifugal 8min of cell 1000rpm that handles well, abandon supernatant, add 6ml Ka Nuoshi fixer, with suction pipe piping and druming evenly, leave standstill 20min, the centrifugal supernatant of abandoning repeats twice; 5) drip sheet: add the 1-2ml fixer, even every the slide of piping and druming drips 1-2 and drips air drying; 6) dyeing: with 10%Giemsa dye liquor dyeing 30min, the running water flushing is dried; 7) microscopy: choose finely dispersed metacinesis and take pictures mutually, statistics chromosome number.
In the present embodiment, the operating procedure of haemocyte smear is as follows: 1) get blood: use the 1ml syringe with 0.75% liquaemin anticoagulant, get the about 0.1ml of blood at afterbody; 2) smear: bleed at slide one end, with cover glass be close to slide obliquely 45 ° at the uniform velocity push the other end to, naturally dry; 3) fixing: the haemocyte slide that will dry places methanol solution fixedly to dry behind the 5min; 4) dyeing: after slide dries, be coated with the part of cell, 30min after washing with the 10%Gimsa dye liquor; 5) microscopy: naturally dry rear microscopy and take pictures.(table 3)
Table 3 haemocyte measurement data
Annotate: above data are got by standard error analysis by the measuring and calculating of Photoshop software.
In the present embodiment, when 8 ~ 9 monthly age, the average weight of hybridization triploid Green Dragon spot is 174.60g, the average weight of hybridization dliploid Green Dragon spot is 82.33g, the growth rate of triploid Green Dragon spot is 2.12 times of dliploid Green Dragon spot, growth rate is with the obvious advantage, can provide important material and foundation for the fish genetic breeding work, and potential economic worth and scientific research value are huge.
Equally, select female brown healthy grouper and male epinephelus lanceolatus fish as parent population, genetic distance is 0.12033; Female epinephelus akaara and male Epinephelus coioides are as parent population, and genetic distance is 0.15040; Female leopard line gill sour jujube perch and male epinephelus lanceolatus fish are as parent population, and genetic distance is 0.22347; After 25 ℃ of artificial inseminations 3 minutes, be transferred to cold shock 20min in 4 ℃ the seawater.Grouper gynogenesis ovum after processing is changed in the normal water temperature seawater, by common lithosporic fry management, raise and train fry to 8 ~ detect after 9 monthly ages, all do not find the triploid grouper.The applicant finds to only have when genetic distance is 0.140-0.145 through lot of experiments, adopts the preparation method among the present invention could obtain the triploid grouper.
Above-described embodiment is the better embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under Spirit Essence of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included in protection scope of the present invention.
Claims (7)
1. the preparation method of a triploid grouper, it is characterized in that may further comprise the steps: select genetic distance and be the grouper not of the same race of 0.140-0.145 affiliation as parent population, adopt the dry method artificial insemination to carry out distant hybridization, transfer to behind the ovum fertilization and carry out cold shock treatment in the seawater, grow seedlings through harvesting, identify the triploid grouper after raising and train and get final product.
2. the preparation method of triploid grouper according to claim 1 is characterized in that: adopting molecular labeling method to select genetic distance is that the grouper not of the same race of 0.140-0.145 affiliation is as parent population.
3. the preparation method of triploid grouper according to claim 1, it is characterized in that: described grouper comprises epinephelus lanceolatus fish and Epinephelus coioides.
4. the preparation method of triploid grouper according to claim 1 is characterized in that: the temperature when adopting artificial dry method fertilization to carry out distant hybridization is 25 ~ 28 ℃.
5. the preparation method of triploid grouper according to claim 1, it is characterized in that: transfer to behind the ovum fertilization and carry out cold shock treatment in the seawater, the temperature during cold shock treatment is 4 ℃, and the cold shock treatment time is 20 ~ 25min.
6. the preparation method of triploid grouper according to claim 1 is characterized in that: grow seedlings through harvesting, raise and train and identify that obtaining the triploid grouper gets final product after growing to 8 ~ 9 monthly ages.
7. the preparation method of triploid grouper according to claim 1, it is characterized in that: grow seedlings through harvesting, adopt the ploidy analyser to detect the haemocyte ploidy after raising and train, thereby and carry out chromosome karyotype analysis and size of blood cells and measure and identify the triploid grouper.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105941332A (en) * | 2016-06-30 | 2016-09-21 | 湖南文理学院 | Method for increasing breeding rate of Channa argus triploid |
| CN106135082A (en) * | 2016-06-27 | 2016-11-23 | 海南晨海水产有限公司 | A kind of artificial fecundation method of black speck cabrilla |
| CN110055213A (en) * | 2019-04-23 | 2019-07-26 | 中国海洋大学 | A kind of separation method of dwarf clam egg membrane |
| CN113197132A (en) * | 2021-06-22 | 2021-08-03 | 海南大学 | Method for artificially inducing triploid of grouper |
| CN113711955A (en) * | 2021-06-30 | 2021-11-30 | 广东省海洋渔业试验中心 | Method for improving green-red hybrid spot germplasm |
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2012
- 2012-11-22 CN CN201210480124.5A patent/CN103004653B/en active Active
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106135082A (en) * | 2016-06-27 | 2016-11-23 | 海南晨海水产有限公司 | A kind of artificial fecundation method of black speck cabrilla |
| CN105941332A (en) * | 2016-06-30 | 2016-09-21 | 湖南文理学院 | Method for increasing breeding rate of Channa argus triploid |
| CN110055213A (en) * | 2019-04-23 | 2019-07-26 | 中国海洋大学 | A kind of separation method of dwarf clam egg membrane |
| CN113197132A (en) * | 2021-06-22 | 2021-08-03 | 海南大学 | Method for artificially inducing triploid of grouper |
| CN113711955A (en) * | 2021-06-30 | 2021-11-30 | 广东省海洋渔业试验中心 | Method for improving green-red hybrid spot germplasm |
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