CN104585091A - Subfamily distant hybridization for German mirror carp and megalobrama amblycephala and application of tetraploid hybrid fishes - Google Patents
Subfamily distant hybridization for German mirror carp and megalobrama amblycephala and application of tetraploid hybrid fishes Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
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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
- 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
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- Marine Sciences & Fisheries (AREA)
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- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Farming Of Fish And Shellfish (AREA)
Abstract
The invention discloses a subfamily distant hybridization for German mirror carp and megalobrama amblycephala. The method comprises the following steps of artificial dry-process fertilization on the ova of the pubescent German mirror carp and sperms of the megalobrama amblycephala by taking the German mirror carp as a female parent and the megalobrama amblycephala as a male parent, hatching fertilized ova at the water temperature of 19-20 DEG C, and breeding fries in a pond; then, detecting the DNA (deoxyribonucleic acid) content, the chromosome number and the blood cell sizes of the fries by a flow cytometry DNA content determination method, a chromosome ploidy detection method of peripheral blood cell culture and a blood smear method, screening, and then preparing to obtain triploid hybrid fishes and tetraploid hybrid fishes. A tetraploid fish strain which is genetically stable and has four sets of crucian carp chromosomes can be formed by subsequent self-cross selective breeding of the tetraploid hybrid fishes, and the tetraploid fish strain can be used as a resource for breeding the infertile triploid fishes. The method disclosed by the invention has the advantages that not only are the characters of hybrid offspring good but also a good foundation can be laid for variety breeding, and a precious resource is provided for subsequent breeding of new polyploid fishes.
Description
Technical field
The present invention relates to fish hybridization method, be specifically related to a kind of inter-subfamily distant hybridization method of crucian and megalobrama amblycephala.
Background technology
Hybridization technique is the method that improvement fish proterties is commonly used, and is that it is one of most basic fish breeding method, is also the breeding technique be widely adopted by hybridizing the breeding method integrated by the part merit of two or more kinds.Affiliation plant between or plant between more than hybridization be commonly referred to as distant hybridization.Distant hybridization is as a kind of important genetics-breeding in fish and character improvement method, it can integrate a whole set of foreign gene, thus change the expression regulation of filial generation gene, make filial generation may show hybrid vigour in growth rate, resistance, disease resistance and meat etc.The filial generation that inter-genera distant hybridization as red crucian carp and Xiangjiang wild carps is formed has fast growth, Fresh & Tender in Texture, premunition strong, survival rate advantages of higher; And in the filial generation of red crucian carp and megalobrama amblycephala, find that there is triploid (3n=124) and tetraploid (4n=148) filial generation.Carry out although Japanese white crucian in the past and the hybridization of megalobrama amblycephala also have, in its offspring, only found triploid hydrid offspring individuals, and tetraploid do not detected.
How to utilize and to improve existing biology techniques and method, in existing fish products kind, select suitable parent fish carry out distant hybridization, thus it is carried out that genetic improvement is more excellent with acquired character, kind is more abundant and to the more significant novel Polyploid fish of genetic research, just becomes the problem that those skilled in the art face for a long time and need solution.
Summary of the invention
Technical problem to be solved by this invention is, overcome the deficiency and defect mentioned in above background technology, there is provided a kind of not only filial generation proterties excellent but also good basis can be established for breed breeding, provide the Japanese white crucian of precious resources and the method for megalobrame amblycephala subfamily distant hydridization for the seed selection of follow-up novel Polyploid fish, and the application of Tetraploid fish that the method obtains.
For solving the problems of the technologies described above, the technical scheme that the present invention proposes is a kind of method of Japanese white crucian and megalobrame amblycephala subfamily distant hydridization, it is characterized in that: described method take Japanese white crucian as female parent, take megalobrama amblycephala as male parent, the sperm of the ovum of sexually matured Japanese white crucian and megalobrama amblycephala is carried out artificial dry method fertilization, fertilized egg is hatched under 20 DEG C ~ 21 DEG C water temperatures, and fry is raised in pond; The ploidy detection method that recycling fluidic cell DNA content determination method, peripheral blood cells are cultivated and blood film method carry out the detection of DNA content, chromosome number and size of blood cells to fry, prepare triploid hydrid fish and Tetraploid fish after screening.
In the technical scheme of the invention described above, Japanese white crucian (Carassius auratus cuvieri Temminck et Schlegel) is under the jurisdiction of Cypriniformes, Cyprinidae, liploid fish (2n=100) during carp subfamily crucian carp belongs to, Japanese white crucian strong adaptability, cold-resistant, high temperature resistant, early sexual maturity, reproduction rate are high, aquaculture cost is low, are a kind of high-quality fish that are fast, high financial profit that grow up; Megalobrama amblycephala (Megalobrama amblycephala Yih) is under the jurisdiction of Cypriniformes, Cyprinidae, Culter subfamily, the liploid fish (2n=48) during triangular bream belongs to, height is flat-sided, caudal peduncle is high and short, and build is graceful, phytophagy, fast growth, strong stress resistance, fine and tender taste, fat are beautiful, and fat is abundant.The logical Japanese white crucian of selecting of the present invention is that male parent carries out distant hybridization for maternal, megalobrama amblycephala, obtain possess multiple heterosis, hybrid vigor novel triploid, Tetraploid fish.Filial generation incorporates the merits such as father, maternal bodily form grace, fast growth, strong stress resistance, fine and tender taste, nutritive value height, has good market prospects.Further, the tetraploid offspring obtained in filial generation, is expected to large-scale application in the production of triploid fish, significant in aquaculture.
Above-mentioned Japanese white crucian and the method for megalobrame amblycephala subfamily distant hydridization, preferably, described method specifically comprises the following steps:
(1) selection of parent population and cultivation: before breeding period 3 ~ 4 months, select sexual maturity feature is obvious, sign is good female Japanese white crucian and male megalobrama amblycephala and carry out special pond cultivation (the good parent population of sign generally show as build is good, body colour is bright-coloured, physique is healthy and strong, anosis without wound) respectively as the maternal parent population of distant hybridization and male parent parent population, keep water quality good; Raise with smart bait about the breeding period previous moon, every 2 ~ 3 days stimulation by running water once, to promote parent population gonadal maturation;
(2) artificial induced spawning: in mid-May then to early June, when water temperature stability is more than 20 DEG C, for parent population injection Luteinizing hormone releasing hormone analog (LRH-A) is hastened parturition with the ocyodinic that mixes of human chorionic gonadtropin (HCG), the consumption of described Luteinizing hormone releasing hormone analog is 10 μ g/kg, and the consumption of human chorionic gonadtropin is 600IU/kg; First inject maternal parent population, inject male parent parent population again after 4 ~ 5h, the injected dose of male parent parent population reduces by half; (water surface area is 80m by the number ratio of 1: 6 ~ 8, mother, male parent parent population to be put into same spawning pond after injection
2left and right), then wash by water in pond in time depending on regimen; 3 ~ 4h before laying eggs, after injecting certain water yield, stops water filling, allows fish hasten parturition in hydrostatic, until it starts to lay eggs and produce essence smoothly;
(3) insemination hatching: select the maternal parent population smoothly that lays eggs, then measure large male parent parent population with product essence and carry out artificial dry method insemination, fertilized egg is placed in the culture dish Lentic hatching that water temperature is 19 DEG C ~ 20 DEG C;
(4) raise: after fry all hatches, continue cultivation 2 ~ 3 days in circular hatching channal, occur being proceeded to by fry after waist point in the pond of fostering and apply fertilizer in advance until fry and raise, soya-bean milk of splashing after 1 ~ 2 day, makes every effort to thin, even by 2 ~ 3 times/day; The ploidy detection method that recycling fluidic cell DNA content determination method, peripheral blood cells are cultivated and blood film method carry out the detection of DNA content, chromosome number and size of blood cells to fry, obtain triploid hydrid fish and Tetraploid fish that Japanese white crucian and megalobrama amblycephala hybridize after detecting screening.
Above-mentioned Japanese white crucian and the method for megalobrame amblycephala subfamily distant hydridization, preferably, adopt pectoral fin base portion without abdominal cavity one, squama place pin injection, to improve the survival rate of parent population during injection in described step (2).
Above-mentioned Japanese white crucian and the method for megalobrame amblycephala subfamily distant hydridization, preferably, fix maternal parent population with canvas stretcher with male parent parent population carries out artificial dry method insemination during insemination hatching in described step (3), so that operation and the damage of minimizing to maternal parent population.
Above-mentioned Japanese white crucian and the method for megalobrame amblycephala subfamily distant hydridization, preferably, described fluidic cell DNA content determination method comprises following operating procedure: with the disposable syringe soaked through heparin from fish tail vein blood vessel blood sampling 0.2mL, inject the Eppendorf pipe that 0.8% physiological saline is housed, 1mL nucleus extraction liquid DAPI-A is added, processing time 10 ~ 15min in blood and mixed liquor of normal saline; Sample filters through 20 μm of NFs; DNA dyeing liquor is statically placed in dark place stained specimens 5 ~ 10min, then goes up machine testing.
Above-mentioned Japanese white crucian and the method for megalobrame amblycephala subfamily distant hydridization, preferably, the ploidy detection method that described peripheral blood cells is cultivated comprises following operating procedure: 1) in superclean bench, prepare medium, 100ml medium is containing following composition: 84ml RPMI-1640,15ml calf serum, 2 PHA, 1ml 0.1% liquaemin, with 7.5%NaHCO
3or 1NHCl adjusts pH to 7.2 ~ 7.4; 2) sterilizing heparin sodium aqua is drawn with syringe, in the experiment fish tail portion venous blood sampling after iodine disinfection; 3) by adding the anticoagulant standard of 0.2ml in every 10ml culture fluid, anticoagulation is added in culture fluid, in 24 DEG C, cultivate 68 ~ 72 hours in the incubator of 5% gas concentration lwevel, between culture period, regular jog is even, fully contacts medium to make cell; 4) stop cultivating first 24 hours, in culture fluid, be added dropwise to the colchicin of 10 μ g/ml with 1ml syringe, make final concentration be 0.05 ~ 0.07 μ g/ml.
Above-mentioned Japanese white crucian and the method for megalobrame amblycephala subfamily distant hydridization, preferably, the operating procedure of described blood film method comprises: 1) with iodine cotton swab wiping fish tail intravenous regional, appropriate blood is got with disposable syringe, touch one in clean slide one end, make drop of blood invest on slide face; 2) with the two ends of this slide hand-held, with another hand-held live one end of another slide, contact drop of blood dripping to have on the slide of blood after by forward direction, make two slides angle about at 45 °, moving gently, make drop of blood in line, is then a uniform thin slice by forward direction pusher; 3) smear in atmosphere drying be placed on staining rack, drip Wright's staining liquid, make smear be colored liquid and cover; 4), after dyeing 5 ~ 10 minutes, in the slide back side, distilled water evenly rinses, and after seeing smear pinkiness, naturally dries, last microscopy.
As a total technical conceive, the application of the Tetraploid fish that the present invention also provides a kind of method of above-mentioned Japanese white crucian and megalobrame amblycephala subfamily distant hydridization to obtain, by described Tetraploid fish by follow-up selfed breeding, what form an inheritance stability has the chromosomal tetraploid fish strain of the white crucian carp of four covers, and by this tetraploid fish strain as the resource of breeding sterile triploid fish.
In former red crucian carp and the hybrid experiment of megalobrama amblycephala, allotetraploid crucian carp triangular bream (4n=148) colony having both sexes to educate in crucian carp triangular bream first familiar generation.In former female Japanese white crucian and male megalobrama amblycephala hybrid experiment, we only obtain triploid (3n=124) crucian carp triangular bream filial generation, and find no allotetraploid individuality.In R&D process of the present invention, we are by refinement distant hybridization operation and strict selective mechanisms, in female Japanese white crucian and male megalobrama amblycephala filial generation, we not only obtain the crucian carp triangular bream offspring of triploid (3n=124), have also obtained allotrtraploid fish (4n=148) crucian carp triangular bream offspring, and this allotrtraploid fish (4n=148) has significant difference with female red crucian carp in shape with the allotrtraploid fish (4n=148) in the first familiar generation of male megalobrama amblycephala, the most obvious characteristic be the former without mouth must, and the latter has mouth palpus.In reciprocal cross experiment, namely carry out distant hybridization experiment as maternal, Japanese white crucian as male parent using megalobrama amblycephala, there is no the filial generation of survival.
The filial generation F1 generation allotetraploid crucian carp triangular bream of red crucian carp and megalobrama amblycephala has the special reproductive characteristic that simultaneously can produce subtrahend and unreduced gamete, and they define the autotetraploid fish (4n=200) with four red chromatography crucian carps by selfing.In the present invention, by in the filial generation F1 of female Japanese white crucian and male megalobrama amblycephala, on the basis obtaining novel tetraploid crucian carp triangular bream (4n=148), by follow-up selfed breeding, by what be expected to a formation inheritance stability, there is the white crucian carp of four covers chromosomal tetraploid fish (4n=200) new lines.And once the formation of this tetraploid fish new lines, the large-scale production for sterile triploid fish is provided valuable tetraploid fish resource, great society, economy and ecological benefits will be produced.Therefore, the present invention has meaning of crucial importance in genetics-breeding in fish and fish Study on Evolution.
Visible, the cultivation of Japanese white crucian and megalobrama amblycephala filial generation, good basis has been established in the seed selection not being only two improved seeds, and provides precious resources for the seed selection of follow-up novel Polyploid fish, in organic evolution and genetics-breeding in fish, have important biological significance.
Accompanying drawing explanation
In order to be illustrated more clearly in the embodiment of the present invention or technical scheme of the prior art, be briefly described to the accompanying drawing used required in embodiment or description of the prior art below, apparently, accompanying drawing in the following describes is some embodiments of the present invention, for those of ordinary skill in the art, under the prerequisite not paying creative work, other accompanying drawing can also be obtained according to these accompanying drawings.
Fig. 1 is the profile photo of triploid hydrid fish in Japanese white crucian (♀) in the embodiment of the present invention × megalobrama amblycephala (♂) offspring.
Fig. 2 is the profile photo of Tetraploid fish in Japanese white crucian (♀) in the embodiment of the present invention × megalobrama amblycephala (♂) offspring.
Fig. 3 is the colored graph (3n=124) of triploid hydrid fish in Japanese white crucian (♀) in the embodiment of the present invention × megalobrama amblycephala (♂) offspring..
Fig. 4 is the chromosome map (4n=148) of Tetraploid fish in Japanese white crucian (♀) in the embodiment of the present invention × megalobrama amblycephala (♂) offspring.
Fig. 5 is the DNA content figure of Japanese white crucian in the embodiment of the present invention.
Fig. 6 is the DNA content figure of three times of crossbred fishes in Japanese white crucian (♀) in the embodiment of the present invention × megalobrama amblycephala (♂) offspring.
Fig. 7 be in Japanese white crucian (♀) in the embodiment of the present invention × megalobrama amblycephala (♂) offspring four times of crossbred fishes DNA content figure.
Fig. 8 is the blood film figure (100 ×) of triploid hydrid fish in Japanese white crucian (♀) in the embodiment of the present invention × megalobrama amblycephala (♂) offspring.
Fig. 9 is the blood film figure (100 ×) of Tetraploid fish in Japanese white crucian (♀) in the embodiment of the present invention × megalobrama amblycephala (♂) offspring.
Embodiment
For the ease of understanding the present invention, hereafter will do to describe more comprehensively, meticulously to the present invention in conjunction with Figure of description and preferred embodiment, but protection scope of the present invention is not limited to following specific embodiment.
It should be noted that, when a certain element is described on " be fixed on, be fixed in, be connected to or be communicated in " another element, it can be directly fixing, affixed, connect or be communicated with on another element, also can be by other intermediate connectors indirectly fixing, affixed, connection or connection on another element.
Unless otherwise defined, hereinafter used all technical terms are identical with the implication that those skilled in the art understand usually.The object of technical term used herein just in order to describe specific embodiment is not be intended to limit the scope of the invention.
Unless otherwise specified, the various raw material, reagent, instrument and equipment etc. used in the present invention are all bought by market and are obtained or prepare by existing method.
Embodiment:
A kind of method of Japanese white crucian of the present invention and megalobrame amblycephala subfamily distant hydridization, the method take Japanese white crucian as female parent, take megalobrama amblycephala as male parent, the sperm of the ovum of sexually matured Japanese white crucian and megalobrama amblycephala is carried out artificial dry method fertilization, fertilized egg is hatched under certain water temperature, and fry is raised in pond; The ploidy detection method that recycling fluidic cell DNA content determination method, peripheral blood cells are cultivated and blood film method carry out the detection of DNA content, chromosome number and size of blood cells to fry, prepare triploid hydrid fish and Tetraploid fish after screening.
The Japanese white crucian of above-mentioned the present embodiment and the method for megalobrame amblycephala subfamily distant hydridization, specifically comprise the following steps:
(1) selection of parent population and cultivation: before breeding period 3 ~ 4 months, select sexual maturity feature is obvious, sign is good female Japanese white crucian and male megalobrama amblycephala and carry out special pond cultivation (the good parent population of sign generally show as build is good, body colour is bright-coloured, physique is healthy and strong, anosis without wound) respectively as the maternal parent population of distant hybridization and male parent parent population, keep water quality good; Raise with smart bait about the breeding period previous moon, every 2 ~ 3 days stimulation by running water once, to promote parent population gonadal maturation;
(2) artificial induced spawning: in mid-May then to early June, when water temperature stability is more than 20 DEG C, for parent population injection Luteinizing hormone releasing hormone analog (LRH-A) is hastened parturition with the ocyodinic that mixes of human chorionic gonadtropin (HCG), the consumption of Luteinizing hormone releasing hormone analog is 10 μ g/kg, and the consumption of human chorionic gonadtropin is 600IU/kg; First inject maternal parent population, inject male parent parent population again after 4 ~ 5h, the injected dose of male parent parent population reduces by half; By the number ratio of 1: 6 ~ 8, mother, male parent parent population being put into same water surface area after injection is 80m
2in the spawning pond of left and right, then wash by water in pond in time depending on regimen; 3 ~ 4h before laying eggs, after injecting certain water yield, stops water filling, allows fish hasten parturition in hydrostatic, until it starts to lay eggs and produce essence smoothly; Adopt pectoral fin base portion without abdominal cavity one, squama place pin injection during injection, to improve the survival rate of parent population;
(3) insemination hatching: after discovery parent population mutually chases in spawning pond, start to draw in the net to salvage, parent population is detected and (notes using soft net, operation wants light, detect in water), good for spawning effect, lay eggs female Japanese white crucian and male megalobrama amblycephala are smoothly carried out artificial dry method insemination, fixes maternal parent population with canvas stretcher with male parent parent population carries out artificial dry method insemination, so that operate and the damage of minimizing to maternal parent population; Smart ovum is clamp-oned in clean porcelain basin, ceaselessly stirs 2 ~ 3min rapidly with clean agitator, then rapid culture dish of fertilized egg being poured into gently carries out Lentic hatching, and water temperature controls between 20 DEG C ~ 21 DEG C.
(4) raise: after fry all hatches, continue cultivation 2 ~ 3 days in circular hatching channal, occur being proceeded to by fry after waist point in the pond of fostering and apply fertilizer in advance until fry and raise, soya-bean milk of splashing after 1 ~ 2 day, makes every effort to thin, even by 2 ~ 3 times/day; The ploidy detection method that recycling fluidic cell DNA content determination method, peripheral blood cells are cultivated and blood film method carry out the detection of DNA content, chromosome number and size of blood cells to fry.
The ploidy detection method that fry grows up to the cultivation of rear employing peripheral blood cells detects, operating procedure is: 1) in superclean bench, prepare medium, 100ml medium is containing following composition: 84ml RPMI-1640,15ml calf serum, 2 PHA, 1ml 0.1% liquaemin, with 7.5%NaHCO
3(aseptic) or 1N HCl adjust pH to 7.2 ~ 7.4; 2) a small amount of sterilizing heparin sodium aqua is drawn with syringe, in the experiment fish tail portion venous blood sampling after iodine disinfection; 3) by adding the anticoagulant standard of about 0.2ml in every 10ml culture fluid, anticoagulation is added in culture fluid, in 24 DEG C, cultivate 68 ~ 72 hours in the incubator of 5% gas concentration lwevel, between culture period, regular jog is even, fully contacts medium to make cell; 4) stop cultivating first 24 hours, in culture fluid, be added dropwise to the colchicin of 10 μ g/ml with 1ml syringe, make final concentration be 0.05 ~ 0.07 μ g/ml.Above step all needs sterile working.
Chromosome preparation process is: 1) all proceeded to by culture in clean 10ml centrifuge tube, with 1000rpm centrifugal 5 minutes, abandons supernatant; 2) in centrifuge tube, hypotonic medium 9ml is added, mixing, hypotonic 25 ~ 30 minutes; 3) add 1ml fixer, centrifugal 5 minutes of the rear 1000rpm of mixing, abandons supernatant gently; 4) add 5ml fixer, mix gently, leave standstill centrifugal 5 minutes of 1000rpm after 20 minutes, abandon supernatant; 5) step 4 is repeated) once; 6) how much add after appropriate fixer makes cell suspension (being generally 1 ~ 2ml) depending on last cell quantity, draw cell suspension and drip sheet from 20 ~ 30cm eminence, featheriness is fallen apart, natural seasoning in air; 7) Giemsa dye liquor dyeing 20 ~ 40 minutes, thin current rinse the slide back side and remove dye liquor, and gas is done rear microscopy, taken pictures.
Find after screening after testing, in the Japanese white crucian that the present embodiment obtains and megalobrama amblycephala filial generation, contain triploid hydrid fish and Tetraploid fish (see Fig. 3 and Fig. 4).
Observed by profile and find, the profile photo of the triploid hydrid fish that the present embodiment method obtains and Tetraploid fish as shown in Figure 1 and Figure 2, they have the properties and characteristics of Japanese white crucian and megalobrama amblycephala concurrently, such as their head is partial to megalobrama amblycephala, and body part is partial to Japanese white crucian, its triploid hydrid offspring is partial to Japanese white crucian in body colour, and body colour is whiter, Tetraploid offspring body colour is then partial to megalobrama amblycephala, and color is darker; Tail fin aspect, triploid hydrid offspring individuals caudal peduncle is longer, and closer to Japanese white crucian, and Tetraploid offspring caudal peduncle is short and high, with megalobrama amblycephala closer to.Following table 1, table 2 be can measure in triploid hydrid fish and Tetraploid fish morphological feature in Japanese white crucian (♀) × megalobrama amblycephala (♂) offspring, denumerable proterties.
Table 1: metric characters in triploid hydrid fish and Tetraploid fish morphological feature
Table 2: denumerable proterties in triploid hydrid fish and Tetraploid fish morphological feature
Through obtaining Japanese white crucian and Japanese white crucian and megalobrama amblycephala filial generation blood DNA content with flow cytomery, flow cytometry assay step is: to take a blood sample about 0.2mL from fish tail vein blood vessel with through the wetting disposable syringe of heparin, inject the Eppendorf pipe that 0.8% physiological saline is housed, 1mL nucleus extraction liquid DAPI-A (nuclei extraction solution is added in blood and mixed liquor of normal saline, Germany Partec Gmbh provides), processing time 10 ~ 15min; Sample filters through 20 μm of NFs (German Partec GmbH provides); DNA dyeing liquor (DAPI-B, Germany Partec GmbH provides) be statically placed in dark place stained specimens about 5 ~ 10min, then go up machine testing, the testing result of Japanese white crucian and triploid hydrid fish and Tetraploid fish is respectively as shown in Fig. 5, Fig. 6 and Fig. 7.Table 3 is the comparing of triploid hydrid fish and Tetraploid fish DNA content and Japanese white crucian DNA content in Japanese white crucian × megalobrama amblycephala offspring.
Table 3: triploid hydrid fish and Tetraploid fish DNA content compare with Japanese white crucian DNA content
Blood film method step is: 1) with iodine cotton swab wiping fish tail intravenous regional, get appropriate blood, touch one in clean slide one end, make drop of blood invest on slide face with disposable syringe; 2) with the two ends of this slide hand-held, rapid another hand-held live one end of another slide, contact drop of blood dripping to have on the slide of blood after by forward direction, make two slides angle about at 45 °, moving gently, make drop of blood in line, is then a uniform thin slice by forward direction pusher; 3) smear in atmosphere drying be placed on staining rack, drip Wright's staining liquid, make smear be colored liquid and cover; 4), after dyeing 5 ~ 10 minutes, in the slide back side, distilled water evenly rinses, and after seeing smear pinkiness, naturally dries, last microscopy.
In Japanese white crucian (♀) × megalobrama amblycephala (♂) offspring, the blood film figure of triploid hydrid fish and Tetraploid fish respectively as shown in Figure 8, Figure 9, observe under an optical microscope, find in the red blood cell of triploid hydrid fish and Tetraploid fish, all have some red blood cells and there is dumbbell shaped cell nucleus, account for 18% of Erythrocytes, 26% respectively.Observe under an electron microscope, the cell nucleus of dumbbell shaped shows as two flexural deformation, and nuclear major part shows as the heterochromatin that aggegation degree is higher, dyeing is darker, and small part is the euchromatin that aggegation degree is lower, dyeing is more shallow.Erythrocytic cytoplasm is homogeneous shape, and the organelle in it is not obvious.Under light microscope and electron microscope, we also observe the separating phenomenon that being divided into two appears in a small amount of erythrocyte nucleus, and just cytoplasm is still interconnected and does not separate completely.In Japanese white crucian × megalobrama amblycephala offspring, red blood cell (core) measured value of triploid hydrid fish and Tetraploid fish is as shown in table 4 below.
Table 4 is triploid fish in Japanese white crucian × megalobrama amblycephala offspring, tetraploid fish red blood cell (core) measured value.
Therefore, the inventive method is for utilizing distant hybridization to produce novel triploid and tetraploid fish open new way, by further seed selection, can select novel tetraploid fish and the triploid fish of the white crucian carp of collection and megalobrama amblycephala advantage, this has important biological significance in organic evolution research and genetics-breeding in fish.
Claims (8)
1. the method for a Japanese white crucian and megalobrame amblycephala subfamily distant hydridization, it is characterized in that: described method take Japanese white crucian as female parent, take megalobrama amblycephala as male parent, the sperm of the ovum of sexually matured Japanese white crucian and megalobrama amblycephala is carried out artificial dry method fertilization, fertilized egg is hatched under 19 DEG C ~ 20 DEG C water temperatures, and fry is raised in pond; The ploidy detection method that recycling fluidic cell DNA content determination method, peripheral blood cells are cultivated and blood film method carry out the detection of DNA content, chromosome number and size of blood cells to fry, prepare triploid hydrid fish and Tetraploid fish after screening.
2. the method for Japanese white crucian according to claim 1 and megalobrame amblycephala subfamily distant hydridization, is characterized in that, described method specifically comprises the following steps:
(1) selection of parent population and cultivation: before breeding period 3 ~ 4 months, select sexual maturity feature is obvious, sign is good female Japanese white crucian and male megalobrama amblycephala and carry out the cultivation of special pond respectively as the maternal parent population of distant hybridization and male parent parent population, keep water quality good; Raise with smart bait about the breeding period previous moon, every 2 ~ 3 days stimulation by running water once, to promote parent population gonadal maturation;
(2) artificial induced spawning: in mid-May then to early June, when water temperature stability is more than 20 DEG C, for parent population injection Luteinizing hormone releasing hormone analog is hastened parturition with the ocyodinic that mixes of human chorionic gonadtropin, the consumption of described Luteinizing hormone releasing hormone analog is 10 μ g/kg, and the consumption of human chorionic gonadtropin is 600IU/kg; First inject maternal parent population, inject male parent parent population again after 4 ~ 5h, the injected dose of male parent parent population reduces by half; By the number ratio of 1: 6 ~ 8, mother, male parent parent population are put into same spawning pond after injection, then wash by water in pond in time depending on regimen; 3 ~ 4h before laying eggs, after injecting certain water yield, stops water filling, allows fish hasten parturition in hydrostatic, until it starts to lay eggs and produce essence smoothly;
(3) insemination hatching: select the maternal parent population smoothly that lays eggs, then measure large male parent parent population with product essence and carry out artificial dry method insemination, fertilized egg is placed in the culture dish Lentic hatching that water temperature is 19 DEG C ~ 20 DEG C;
(4) raise: after fry all hatches, in circular hatching channal, continue cultivation 2 ~ 3 days, occur after waist point, fry being proceeded in the pond of fostering and apply fertilizer in advance until fry and raise, soya-bean milk of splashing after 1 ~ 2 day, 2 ~ 3 times/day; The ploidy detection method that recycling fluidic cell DNA content determination method, peripheral blood cells are cultivated and blood film method carry out the detection of DNA content, chromosome number and size of blood cells to fry, prepare triploid hydrid fish and Tetraploid fish after screening.
3. the method for Japanese white crucian according to claim 2 and megalobrame amblycephala subfamily distant hydridization, is characterized in that, adopts pectoral fin base portion without abdominal cavity one, squama place pin injection in described step (2) during injection.
4. the method for Japanese white crucian according to claim 2 and megalobrame amblycephala subfamily distant hydridization, is characterized in that, in described step (3) during insemination hatching is to fix maternal parent population with canvas stretcher and male parent parent population carries out artificial dry method insemination.
5. the Japanese white crucian according to any one of Claims 1 to 4 and the method for megalobrame amblycephala subfamily distant hydridization, it is characterized in that, described fluidic cell DNA content determination method comprises following operating procedure: with the disposable syringe soaked through heparin from fish tail vein blood vessel blood sampling 0.2mL, inject the Eppendorf pipe that 0.8% physiological saline is housed, 1mL nucleus extraction liquid DAPI-A is added, processing time 10 ~ 15min in blood and mixed liquor of normal saline; Sample filters through 20 μm of NFs; DNA dyeing liquor is statically placed in dark place stained specimens 5 ~ 10min, then goes up machine testing.
6. the Japanese white crucian according to any one of Claims 1 to 4 and the method for megalobrame amblycephala subfamily distant hydridization, it is characterized in that, the ploidy detection method that described peripheral blood cells is cultivated comprises following operating procedure: 1) in superclean bench, prepare medium, 100ml medium is containing following composition: 84ml RPMI-1640,15ml calf serum, 2 PHA, 1ml0.1% liquaemins, with 7.5%NaHCO
3or 1N HCl adjusts pH to 7.2 ~ 7.4; 2) sterilizing heparin sodium aqua is drawn with syringe, in the experiment fish tail portion venous blood sampling after iodine disinfection; 3) by adding the anticoagulant standard of 0.2ml in every 10ml culture fluid, anticoagulation is added in culture fluid, in 24 DEG C, cultivate 68 ~ 72 hours in the incubator of 5% gas concentration lwevel, between culture period, regular jog is even, fully contacts medium to make cell; 4) stop cultivating first 24 hours, in culture fluid, be added dropwise to the colchicin of 10 μ g/ml with 1ml syringe, make final concentration be 0.05 ~ 0.07 μ g/ml.
7. the Japanese white crucian according to any one of Claims 1 to 4 and the method for megalobrame amblycephala subfamily distant hydridization, it is characterized in that, the operating procedure of described blood film method comprises: 1) with iodine cotton swab wiping fish tail intravenous regional, appropriate blood is got with disposable syringe, touch one in clean slide one end, make drop of blood invest on slide face; 2) with the two ends of this slide hand-held, with another hand-held live one end of another slide, contact drop of blood dripping to have on the slide of blood after by forward direction, make two slides angle about at 45 °, moving gently, make drop of blood in line, is then a uniform thin slice by forward direction pusher; 3) smear in atmosphere drying be placed on staining rack, drip Wright's staining liquid, make smear be colored liquid and cover; 4), after dyeing 5 ~ 10 minutes, in the slide back side, distilled water evenly rinses, and after seeing smear pinkiness, naturally dries, last microscopy.
8. the application of Tetraploid fish that obtains of the method for a Japanese white crucian according to any one of claim 1 ~ 7 and megalobrame amblycephala subfamily distant hydridization, it is characterized in that, by described Tetraploid fish by follow-up selfed breeding, what form an inheritance stability has the chromosomal tetraploid fish strain of the white crucian carp of four covers, and by this tetraploid fish strain as the resource of breeding sterile triploid fish.
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Effective date of registration: 20210601 Address after: Room 201, real estate management office building, Hunan Normal University, 36 Lushan Road, Yuelu District, Changsha City, Hunan Province, 410000 Patentee after: Hunan Normal University Asset Management Co.,Ltd. Address before: 410081 School of life sciences, Hunan Normal University, Changsha 36, Yuelu District, Hunan Patentee before: HUNAN NORMAL University Effective date of registration: 20210601 Address after: No. A3-3, taozihu cultural and Creative Industrial Park, Hunan Normal University, Yuelu District, Changsha City, Hunan Province, 410000 Patentee after: Hunan Yuelu Shanshui Breeding Technology Co.,Ltd. Address before: Room 201, real estate management office building, Hunan Normal University, 36 Lushan Road, Yuelu District, Changsha City, Hunan Province, 410000 Patentee before: Hunan Normal University Asset Management Co.,Ltd. |
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