CN103004653B - Preparation method for triploid grouper - Google Patents
Preparation method for triploid grouper Download PDFInfo
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- CN103004653B CN103004653B CN201210480124.5A CN201210480124A CN103004653B CN 103004653 B CN103004653 B CN 103004653B CN 201210480124 A CN201210480124 A CN 201210480124A CN 103004653 B CN103004653 B CN 103004653B
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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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- 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
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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 genetics-breeding in fish field, be specifically related to a kind of preparation method 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 to be low in fat content, the first-class food fish of high protein, deeply like 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 can not joint conference normally in reduction division process, normal gamete can not be produced, in triploid ontogenetic process, the abnormal growth of sexual gland saves reproduction energy and is used for growing by this part energy, add individual more than the dliploid a set of chromosome sets of triploid, gene expression is more active accelerates the process of growing, and makes triploid in growth rate, have obviously advantage, has huge using value.
At present, the breeding method breeding work year limit for length of traditional grouper, breeding work are made slow progress and are hybridized dliploid in grouper crossbreeding pattern and easily escape, thus cause unpredictable serious consequence to the marine eco-environment.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of preparation method of triploid grouper, this preparation method's breeding cycle is short, fast growth, on the marine eco-environment without impact.
Above-mentioned technical problem of the present invention is achieved by the following technical solution: a kind of preparation method of triploid grouper, comprise the following steps: selecting genetic distance is that the grouper not of the same race of 0.140-0.145 affiliation is as parent population, dry method artificial insemination is adopted to carry out distant hybridization, transfer to after ovum fertilization in seawater and carry out cold shock treatment, nursery through harvesting, raise and train after identify triploid grouper.
The present invention coordinates physical stimulation to produce triploid grouper by distant hybridization, greatly accelerates the process of grouper polyploid breeding work.
The preferred epinephelus lanceolatus fish (E.lanceolatus) of grouper of the present invention and Epinephelus coioides (E.coioides).
Temperature when the present invention preferably adopts dry method artificial insemination to carry out distant hybridization is 25 ~ 28 DEG C.
The present invention transfers in seawater and carries out cold shock treatment after ovum fertilization, and temperature during cold shock treatment is preferably 4 DEG C, and the cold shock treatment time is preferably 20 ~ 25min.
Nursery of the present invention through harvesting, raise and train grow to 8 ~ 9 monthly ages after identify triploid grouper.
Nursery of the present invention through harvesting, raise and train after adopt the ploidy analyser to detect haemocyte ploidy, and carry out chromosome karyotype analysis and size of blood cells is measured thus identifies triploid grouper.
Haemocyte smear method can be adopted to measure size of blood cells.
Compared with prior art, tool of the present invention has the following advantages:
(1) the present invention select genetic distance be the grouper not of the same race of 0.140-0.145 affiliation as parent population, carry out cold treatment after hybridization, can triploid grouper be obtained.The present invention is simple to operate, the triploid grouper fast growth prepared, irreproducible, on the marine eco-environment without impact;
(2) the present invention accelerates the process of ablen genetic breeding research, for later seawater fish polyploid genetic breeding research provides important evidence; There is huge economic worth in production application; In ablen genetic breeding, genetic evolution and the research of endocrine regulation scheduling theory, all there is great reference value.
Accompanying drawing explanation
Fig. 1-2 is that the chromosome map in embodiment 1 carries out karyotyping result figure.
Embodiment
The reagent adopted in following embodiment, if no special instructions, is commercially available.
Embodiment 1
Go out CHINESE OFFSHORE grouper 5 kinds of grouper mitochondrial cytochrome bs (Cyt b) gene by pcr amplification, utilize MEGA3.1 software and calculate genetic distance according to 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 hybridization parent population.
Table 1PCR amplification grouper mitochondrial cytochrome b (Cyt b) gene primer sequence is as follows
Genetic distance between table 2 13 kinds of ablen kinds
Before breeding period 1 ~ 2 month, 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, special pond meticulous bait of throwing something and feeding provides excellent nutrition for parent population, strengthen the physique of parent population to obtain the measured gamete of matter, period keeps a close eye on temperature and the salinity of water in parent fish pond, note parent population ingest and animation to keep parent population to enter breeding period in the state of the best.
Dry method artificial insemination is adopted to carry out distant hybridization to female Epinephelus coioides and male epinephelus lanceolatus fish, after entering breeding period, carry the previous day to hasten parturition to the female Epinephelus coioides injection human chorionic gonadtropin 500IU/kg that belly obviously swells, seawater near scrub raun cloacal aperture, the belly pushing fish catches ovum with clean beaker; Seawater near scrub milter cloacal aperture, the belly pushing fish catches milky seminal fluid with clean test tube, notes the urine as far as possible avoiding fish.
The seawater that temperature is 25 DEG C is added by after ovum and seminal fluid mixing and stirring, start the fertilization of grouper ovum, after ovum fertilization 3 minutes, be transferred to cold shock 20min in the seawater of 4 DEG C, grouper fertilized egg after process is proceeded in normal water temperature seawater, leave standstill a moment, get the ovum floated on upper strata and put into the micro-inflation hatching of tank interior.
The fry hatched is proceeded to outdoor large area sea pond and carry out ecological breeding, to splash around pond different amount peanut press pulp by fermentation according to situations such as the transparencies of sunlight, temperature, water colour, fry proceeds to little Chi again and carries out pellet and raise and train after growing to more than 3cm.
When fry to 8 ~ 9 monthly age raised and train by pellet, fry physique is comparatively large, adaptability is comparatively strong, and not easily because of manual operation unexpected death, ablen hatching has the habit of cannibalism, notes regular separately being raised by fry larger for size difference; After afterbody gets blood, the anticoagulant dilute blood of attention vast scale is guaranteed blood adhesion thus does not affect testing result, uses the ploidy analyser to detect haemocyte ploidy, identifies hybridization triploid Green Dragon spot.
In the present embodiment, with fish haemocyte for DNA content measures sample, the ploidy analyser (instrument and reagent are all provided by German Partec company) is used to measure; First, take a morsel fish blood, be that 0.75% sodium heparin anticoagulant dilutes more than 10 times and makes haemocyte dilution with mass percentage, glue with 1mL blue electron gun head and get a little haemocyte dilution in the DAPI dye liquor (being provided by German Partec company) of 0.8mL and stir, leave standstill and detect in a moment.After testing, Epinephelus coioides, epinephelus lanceolatus fish and the DNA content value of hybridizing between dliploid Green Dragon spot are more or less the same, and are 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, is obviously greater than dliploid grouper, thus identifies triploid Green Dragon spot.
On the basis of qualification, get head-kidney after injection phytolectin (PHA) and colchicine, prepare chromosome map and carry out karyotyping, result proves, 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 qualification, prepare haemocyte smear, by the size of Photoshop software measurement haemocyte, further Analysis and Identification result, as shown in table 2, the every numerical value of haemocyte of hybridization triploid Green Dragon spot is all larger than each entry value of dliploid grouper.
In the present embodiment, method of chromosome preparation operation is as follows: 1) inject: test fish lumbar injection PHA(10ug/g), the PHA of the identical amount of 22 hr later injection, three hr later injection colchicines (3ug/g); 2) kidney is got: injection colchicine cut tail bloodletting after 4 hours, bloodletting 30 minutes, solution takes head-kidney, head-kidney tissue is put into physiological saline and cleans 2-3 time, head-kidney tissue is put into a little physiological saline culture dish to shred, move into 15ml centrifuge tube with 400 order silk cover filterings, 1000rpm, 8min are centrifugal; 3) hypotonic: centrifugal rear removal supernatant, adds 0.075mol/LKCl liquid, in 37 DEG C hypotonic 30 minutes; 4) fixing: by the centrifugal 8min of cell 1000rpm good for Hypotonic treatment, to abandon supernatant, add 6ml Ka Nuoshi fixer, with suction pipe piping and druming evenly, leave standstill 20min, centrifugally abandon supernatant, repeat twice; 5) drip sheet: add 1-2ml fixer, piping and druming is evenly often opened slide and is dripped 1-2 and drip, air drying; 6) dye: with 10%Giemsa dye liquor dyeing 30min, tap water, dries; 7) microscopy: choose finely dispersed division phases and take pictures, statistics chromosome number.
In the present embodiment, the operating procedure of haemocyte smear is as follows: 1) get blood: with the 1ml syringe with 0.75% sodium heparin anticoagulant, get blood be about 0.1ml at afterbody; 2) smear: bleed in 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 dried to be placed in after methanol solution fixes 5min and to dry; 4) dye: after slide dries, be coated with the part of cell with 10%Gimsa dye liquor, 30min after washing; 5) microscopy: naturally dry rear microscopy and take pictures.(table 3)
Table 3 haemocyte measurement data
Note: above data obtain through standard error analysis after being calculated by 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 genetics-breeding in fish work, potential economic worth and scientific research value 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 DEG C of artificial inseminations 3 minutes, be transferred to cold shock 20min in the seawater of 4 DEG C.Grouper gynogenesis ovum after process is proceeded in normal water temperature seawater, by common lithosporic fry management, detects after raising and train fry to 8 ~ 9 monthly age, all do not find triploid grouper.Applicant finds through lot of experiments, only has when genetic distance is 0.140-0.145, adopts the preparation method in the present invention to obtain triploid grouper.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from Spirit Essence of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included in protection scope of the present invention.
Claims (5)
1. the preparation method of a triploid grouper, it is characterized in that comprising the following steps: selecting genetic distance is that the grouper not of the same race of 0.140-0.145 affiliation is as parent population, dry method artificial insemination is adopted to carry out distant hybridization, transfer to after ovum fertilization in seawater and carry out cold shock treatment, nursery through harvesting, raise and train after identify triploid grouper;
The temperature adopting artificial dry method to be fertilized when carrying out distant hybridization is 25 ~ 28 DEG C;
Transfer to after ovum fertilization in seawater and carry out cold shock treatment, temperature during cold shock treatment is 4 DEG C, and the cold shock treatment time is 20 ~ 25min.
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, 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: nursery through harvesting, raise and train grow to 8 ~ 9 monthly ages after qualification obtain triploid grouper.
5. the preparation method of triploid grouper according to claim 1, it is characterized in that: nursery through harvesting, raise and train after adopt the ploidy analyser to detect haemocyte ploidy, and carry out chromosome karyotype analysis and size of blood cells is measured thus identifies triploid grouper.
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| CN106135082B (en) * | 2016-06-27 | 2018-10-23 | 海南晨海水产有限公司 | A kind of artificial fecundation method of blackspot grouper |
| 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 |
| CN113197132B (en) * | 2021-06-22 | 2022-12-27 | 海南大学 | Method for artificially inducing triploid of grouper |
| CN113711955B (en) * | 2021-06-30 | 2022-09-30 | 广东省农业技术推广中心 | Method for improving green-red hybrid spot germplasm |
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