CN104585083B - Method for distant hybridization between subfamilies of cyprinus carpio and megalobrama amblycephala - Google Patents
Method for distant hybridization between subfamilies of cyprinus carpio and megalobrama amblycephala Download PDFInfo
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- CN104585083B CN104585083B CN201410771279.3A CN201410771279A CN104585083B CN 104585083 B CN104585083 B CN 104585083B CN 201410771279 A CN201410771279 A CN 201410771279A CN 104585083 B CN104585083 B CN 104585083B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; 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
Abstract
A method for distant hybridization between subfamilies of cyprinus carpio and megalobrama amblycephala includes the following steps that firstly, female cyprinus carpio and male megalobrama amblycephala which have obvious sexual maturity characteristics and good somatic features are selected as parent fishes for special pool breeding, then, artificial spawning induction is conducted on the parent finishes in a breeding season, the parent fishes with a good spawning induction effect are selected to be subjected to artificial dry-method fertilization, running water incubation is conducted on fertilized embryos in a pure water incubation device, fish fry are raised after being incubated, the raised fish fry are detected and screened, hybrid offspring of the cyprinus carpio and the megalobrama amblycephala are obtained through flow cytometry DNA content determination and renal tissue chromosome ploidy detection, and the hybrid offspring comprise diploid natural gynogenesis cyprinus carpio, diploid natural gynogenesis mirror carps, diploid fish like the cyprinus carpio and tetraploid hybrid fish. The method has the advantages that the rate of fertilization is high, the incubating rate is high, the offspring are excellent in character, and the method has great significance in genetic breeding.
Description
Technical field
The present invention relates to fish hybridization method, more particularly to the distant hybridization method in cyprinid fish between subfamily.
Background technology
Hybridization of the assay affiliation more than inter-species or inter-species is referred to as distant hybridization, is that fish character improvement is commonly used
Method.Used as a kind of important genetics-breeding in fish and character improvement method, it can integrate a whole set of external source base for distant hybridization
Cause, so as to change the expression regulation of filial generation gene so that filial generation may the speed of growth, resistance, disease resistance with
And the aspect such as meat shows hybrid vigour.Distant hybridization can be formed in phenotype and genotype according to different parental combinations
On all different different types of fish.Such as F in the filial generation that red crucian carp is formed with the inter-genera distant hybridization of Xiangjiang wild carpses1-F2For two
Times body hybridization fish (2n=100), F3-F24For allotrtraploid fish (4n=200)；Using allotrtraploid fish and relevant dliploid
Fish mates, and the triploid fish (3n=150) for having the advantages that fast growth, Fresh ＆ Tender in Texture, premunition are strong is defined on a large scale.
The F of the inter-subfamily distant hybridization of red crucian carp and megalobrama amblycephala1In define the red crucian carp of dliploid Natural Gynogenesiss (2n=100), heterologous
Tetraploid crucian carp triangular bream (4n=148) and allotriploid crucian carp triangular bream (3n=124)；Wherein allotetraploid crucian carp triangular bream is defined by selfing
Autotetraploid fish products system (4n=200).
Carp (Cyprinus carpio carpio) belongs to the liploid fish (2n=100) in Cyprinidae, carp subfamily, rolls into a ball head
Triangular bream (Megalobrama amblycephala) belongs to the liploid fish (2n=48) in Cyprinidae, Culter subfamilies；Heredity, classification and
, there is larger difference in the biological properties such as bodily form aspect, especially body cell dyeing between them between carp and megalobrama amblycephala
Body number is widely different, and the chromosome number of carp is 100, and the chromosome number of megalobrama amblycephala is 48, and not into ploidy pass
System.So far also successfully report with megalobrame amblycephala subfamily distant hydridization without carp both at home and abroad, exploring one kind makes carp with group's head
Hybridizing method between the triangular bream and biological characteristics to its offspring is researched and analysed with important using value.
The content of the invention
The technical problem to be solved is to overcome the shortcomings of to be mentioned and defect in background above technology, there is provided one
Plant remote between the carp and megalobrama amblycephala subfamily that rate of fertilization is high, incubation rate is high, characters of progenies is excellent, significant to genetic breeding
The method of edge hybridization.
To solve above-mentioned technical problem, technical scheme proposed by the present invention is that remote edge is miscellaneous between a kind of carp and megalobrama amblycephala subfamily
The method of friendship, comprises the following steps：Select that sexal maturity feature is obvious first, the female carp that sign is good and male megalobrama amblycephala make
Cultivate for the special pond of parent population, then artificial induced spawning is carried out to the parent population in mating period, select the good parent population of spawning effect and enter
Pedestrian's work dry method is inseminated, and the embryo after the completion of insemination carries out Lotic hatching in water purification hatching apparatus, after the completion for the treatment of fry hatching
Raised, then the fry after raising is detected, sieved, by fluidic cell DNA content measure, nephridial tissue chromosome times
Property detection obtain carp and megalobrama amblycephala filial generation, filial generation includes that dliploid Natural Gynogenesiss carp, dliploid are natural
Gynogenesis mirror carp, the liploid fish of class crucian and Tetraploid fish.Four types obtained using preceding solution
The advantages of hybridization fish is respectively provided with bodily form grace, fast growth, strong stress resistance, educates in new excellent fish strain development and heredity
There is important value in terms of kind.
The method of above-mentioned carp and megalobrame amblycephala subfamily distant hydridization, it is preferred that methods described specifically includes following step
Suddenly：
The selection and cultivation of (a) parent population：3～4 months before breeding period, select that sexal maturity feature is obvious, sign is good
Female carp and male megalobrama amblycephala carry out special pond cultivation respectively as the maternal parent population and male parent parent population of distant hybridization, keep water quality
Well；Or so breeding period previous moon with smart bait raising, every 2～3 days stimulation by running water once, promote parent population gonad development into
It is ripe；The good parent population of sign normally behaves as that build is good, body colour is bright-coloured, physique healthy and strong, disease-free without wound；
(b) artificial induced spawning：It is the parent when water temperature stability is more than 20 DEG C in mid-May then to early June
Fish injection Luteinizing hormone releasing hormone analog (LRH-A) is hastened parturition with the mixing ocyodinic of human chorionic gonadtropin (HCG),
The consumption of the Luteinizing hormone releasing hormone analog is 6～10 μ g/kg (most preferably 10 μ g/kg), human chorionic gonadtropin
Consumption is 500～800IU/kg (most preferably 600IU/kg)；Maternal parent population is first injected, male parent parent population, male parent is injected after 4～5h again
The injection dosage of parent population halves；Maternal parent population, male parent parent population are put into same spawning by injection after finishing by 1: 6～8 quantity ratio
Chi Zhong, then washes by water in time regarding regimen in pond, and 3～4h especially before spawning after injecting certain water yield, stops water filling, allows fish
Hasten parturition in hydrostatic, until it starts smoothly spawning and produces essence；Preferably noted without the pin of abdominal cavity at squama one using pectoral fin base portion during injection
Method is penetrated, to improve the survival rate of parent population；
(c) insemination hatching：Spawning smoothly maternal parent population is selected, is fixed with canvas stretcher, in order to operating and reducing right
The damage of maternal parent population；Then the male parent parent population big with essence amount is produced carries out artificial dry method insemination, and embryonated egg is placed in water temperature for 20 DEG C
Lotic hatching is carried out in～21 DEG C of water purification hatching apparatus；
D () raises：After fry all hatches, continue to cultivate 2～3 days in incubator, by fish after waist point occurs in fry
Seedling is proceeded in the pond fostered and apply fertilizer in advance and raised, and soya-bean milk of splashing after 1～2 day, makes every effort to thin, even by 2～3 times/day；Fry is led to after growing up to
Overflow-type cell DNA content is determined, the detection of nephridial tissue ploidy obtains filial generation.The dyeing of four type filial generations
Body figure is respectively as shown in Fig. 4, Fig. 6, Fig. 8 and Figure 10.
Fluidic cell DNA content determination method step is in the present invention：With the disposable syringe of Jing heparin wetting from fish tail
Vein blood vessel blood sampling about 0.2mL, in Eppendorf pipes of the injection equipped with 0.8% physiological saline, mixes in blood and physiological saline
1mL nucleus extraction liquid DAPI-A are added in liquid, and (nuclei extraction solution, German Partec Gmbh are carried
For), 10～15min of process time；Sample is filtered through 20 μm of NFs (German Partec GmbH are provided)；DNA dyeing liquors
(DAPI-B, German Partec GmbH are provided) is statically placed in dark place stained specimens about 5～10min, then goes up machine testing.By stream
Formula cell DNA content determine can clearly know dliploid Natural Gynogenesiss carp in carp and megalobrama amblycephala filial generation,
The DNA of dliploid Natural Gynogenesiss mirror carp, the liploid fish of class crucian and Tetraploid fish these four type filial generations
Average content and the ratio with the DNA average contents of the common red crucian carp of object of reference.
The detection method of nephridial tissue ploidy includes in the present invention：The cultural aim fish 1 under 18 DEG C～22 DEG C water temperatures
After～3 days, to testing fish injection PHA 1～3 time, each dosage is 2～8 μ g/g body weight, and interval time is 12～24 hours,
Dissection draw materials before 2～6 hours, inject colchicine, dosage be 2～4 μ g/g body weight.Nephridial tissue is taken out, is cut under physiological saline
Broken nephridial tissue, 0.075mol/L KCL Hypotonic treatments, hypotonic 40～60 minutes at a temperature of 20 DEG C；With glacial acetic acid: methyl alcohol (1: 3)
Fixed nephridial tissue 1～3 time；Piece is dripped on frost slide；Giemsa dye liquors are dyeed 45～60 minutes, and thin current rinse slide
The back side removes dye liquor, air-dries rear microscopy, takes pictures.Can also clearly know carp with group's head by the detection of nephridial tissue ploidy
Dliploid Natural Gynogenesiss carp in triangular bream filial generation, dliploid Natural Gynogenesiss mirror carp, the liploid fish of class crucian and
The chromosome number and its ploidy of Tetraploid fish these four types.
Compared with prior art, it is an advantage of the current invention that：In the present invention, the inter-subfamily distant hybridization of carp and megalobrama amblycephala
The F of formation1In define dliploid Natural Gynogenesiss carp (2n=100), dliploid Natural Gynogenesiss mirror carp (2n=
100) and class crucian liploid fish (2n=100) and Tetraploid fish (4n=148), these filial generations have the bodily form excellent
The advantages of U.S., fast growth, strong stress resistance, while the distinguishing feature of bio-diversity is presented, in new excellent fish strain
Cultivate and genetic breeding aspect has important value.Filial generation in the present invention obviously with the parent of existing Distant crossing combination
Offspring being produced with hybridization and there is marked difference, the method for the present invention has rate of fertilization is high, incubation rate is high, characters of progenies is excellent etc.
Advantage, dliploid Natural Gynogenesiss carp, dliploid Natural Gynogenesiss mirror carp, class that Distant crossing combination of the present invention is obtained
The liploid fish of crucian and Tetraploid fish are expected to cultivate the natural thelykaryon of dliploid by hereditary and selection in generation after hybridization
Development carp strain, dliploid Natural Gynogenesiss mirror carp strain, the liploid fish strain of class crucian and Tetraploid fish products
System, wherein by continuing seed selection to Tetraploid fish products system, being expected to develop new autotetraploid fish products system.The present invention exists
Biological evolution and genetics-breeding in fish aspect have important biological significance.
Description of the drawings
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
The accompanying drawing to be used needed for having technology description is briefly described, it should be apparent that, drawings in the following description are the present invention
Some embodiments, for those of ordinary skill in the art, on the premise of not paying creative work, can be with basis
These accompanying drawings obtain other accompanying drawings.
Fig. 1 is the profile photo of carp (♀) in carp (♀) × megalobrama amblycephala (♂) parent in the embodiment of the present invention.
Fig. 2 is the profile photo of megalobrama amblycephala (♂) in carp (♀) × megalobrama amblycephala (♂) parent in the embodiment of the present invention.
Fig. 3 is dliploid Natural Gynogenesiss carp in carp (♀) × megalobrama amblycephala (♂) offspring in the embodiment of the present invention
Profile photo.
Fig. 4 is dliploid Natural Gynogenesiss carp in carp (♀) × megalobrama amblycephala (♂) offspring in the embodiment of the present invention
Chromosome map (2n=100).
Fig. 5 is dliploid Natural Gynogenesiss mirror carp in carp (♀) × megalobrama amblycephala (♂) offspring in the embodiment of the present invention
Profile photo.
Fig. 6 is dliploid Natural Gynogenesiss mirror carp in carp (♀) × megalobrama amblycephala (♂) offspring in the embodiment of the present invention
Chromosome map (2n=100).
Fig. 7 is that the profile of the liploid fish of class crucian in carp (♀) × megalobrama amblycephala (♂) offspring in the embodiment of the present invention is shone
Piece.
Fig. 8 is the chromosome of the liploid fish of class crucian in carp (♀) × megalobrama amblycephala (♂) offspring in the embodiment of the present invention
Figure (2n=100).
Fig. 9 is the profile photo of Tetraploid fish in carp (♀) × megalobrama amblycephala (♂) offspring in the embodiment of the present invention.
Figure 10 is the chromosome map of Tetraploid fish in carp (♀) × megalobrama amblycephala (♂) offspring in the embodiment of the present invention
(4n=148).
Figure 11 is the DNA content figure of the common red crucian carp of object of reference in the embodiment of the present invention.
Figure 12 is dliploid Natural Gynogenesiss carp in carp (♀) × megalobrama amblycephala (♂) offspring in the embodiment of the present invention
DNA content figure.
Figure 13 is dliploid Natural Gynogenesiss mirror carp in carp (♀) × megalobrama amblycephala (♂) offspring in the embodiment of the present invention
DNA content figure.
Figure 14 is that the DNA of the liploid fish of class crucian in carp (♀) × megalobrama amblycephala (♂) offspring in the embodiment of the present invention contains
Spirogram.
Figure 15 is the DNA content figure of Tetraploid fish in carp (♀) × megalobrama amblycephala (♂) offspring in the embodiment of the present invention.
Specific embodiment
For the ease of understanding the present invention, more complete is made to the present invention below in conjunction with Figure of description and preferred embodiment
Face, meticulously describe, but protection scope of the present invention is not limited to embodiment in detail below.
Unless otherwise defined, the implication that all technical terms used hereinafter are generally understood that with those skilled in the art
It is identical.Technical term used herein is intended merely to describe the purpose of specific embodiment, is not intended to limit the present invention
Protection domain.
Unless otherwise specified, various raw material, reagent, instrument and equipment used in the present invention etc. can pass through city
Field is commercially available or can be prepared by existing method.
Embodiment：
A kind of carp of the present invention and the method for megalobrame amblycephala subfamily distant hydridization, comprise the following steps：The property selected first
The female carp and male megalobrama amblycephala that mature characteristic is obvious, sign is good is cultivated as the special pond of parent population, then in mating period pair
The parent population carries out artificial induced spawning, and selecting the good parent population of spawning effect carries out artificial dry method insemination, the embryo after the completion of insemination
Lotic hatching is carried out in water purification hatching apparatus, treats to be raised after the completion of fry hatching, then the fry after raising is examined
Survey, sieve, carp and megalobrama amblycephala filial generation are obtained by fluidic cell DNA content measure, the detection of nephridial tissue ploidy,
Filial generation include dliploid Natural Gynogenesiss carp, dliploid Natural Gynogenesiss mirror carp, the liploid fish of class crucian and
Tetraploid fish.
The carp of above-mentioned the present embodiment and the method for megalobrame amblycephala subfamily distant hydridization, specifically include following steps：
The selection and cultivation of (a) parent population：The selection of Parent parent population is most important in the present embodiment, and concrete grammar is numerous
Grow 3～4 months before the phase, select that sexal maturity feature is obvious, build is good, body colour is bright-coloured, physique healthy and strong, the disease-free female carp without wound
(referring to Fig. 1) and male megalobrama amblycephala (referring to Fig. 2) carry out special pond training respectively as the maternal parent population and male parent parent population of distant hybridization
Educate, keep water quality good.Particularly feed is rationally fed in or so breeding period previous moon, with the raising of smart bait, every 2～3 days
Stimulation by running water once, to promote parent population gonadal maturation.
(b) artificial induced spawning：It is parent population note when water temperature stability is more than 20 DEG C in mid-May then to early June
Penetrate Luteinizing hormone releasing hormone analog (LRH-A) to be hastened parturition with the mixing ocyodinic of human chorionic gonadtropin (HCG), LRH-
The consumption of A is 10 μ g/kg, and the consumption of HCG is 600IU/kg；Maternal parent population is first injected, male parent parent population, father is injected after 4～5h again
The injection dosage of this parent population halves；Inject after finishing by 1:Maternal parent population, male parent parent population are put into same water by 6～8 quantity ratio
Face area is 80m2In the spawning pond of left and right, then wash by water in pond in time regarding regimen, 3～4h especially before spawning, injection one
After determining the water yield, stop water filling, allow fish to hasten parturition in hydrostatic, until it starts smoothly spawning and produces essence；Pectoral fin base is adopted during injection
Portion without the pin injection of abdominal cavity at squama one, to improve the survival rate of parent population.
(c) insemination hatching：After finding that parent population mutually chases in spawning pond, start to draw in the net to salvage, parent population is examined
Survey, fix by the good female carp wet towel parcel of spawning effect and with canvas stretcher, then enter pedestrian with male megalobrama amblycephala
Work dry method is inseminated, and with clean chicken feather sperm and ovum are sufficiently stirred for.Embryonated egg is placed in water purification hatching apparatus and carries out flowing water and incubate
Change, 20 DEG C～21 DEG C of water temperature, the fry of hatching is raised in pond.
D () raises：After fry all hatches, prior to cultivating 2～3 days in incubator, by fry after there is waist point in fry
Proceed in the pond fostered and apply fertilizer in advance and raise, soya-bean milk of splashing after 1～2 day, makes every effort to thin, even, until fry grows to energy by 2～3 times/day
Normally ingest.During hatching, the fertility rate and hatchability of embryo is counted.The rate of fertilization of carp and megalobrama amblycephala distant hybridization fish and incubate
Rate is respectively 85% and 65%.Fry uses nephridial tissue ploidy detection method to carp (♀) × megalobrama amblycephala after growing up to
(♂) offspring's chromosome number of somatic is detected.Nephridial tissue ploidy detection method be：In 18 DEG C～22 DEG C water
The lower cultural aim fish of temperature is after 1～3 day, and to testing fish injection PHA 1～3 time, each dosage is 2～8 μ g/g body weight, interval time
For 12～24 hours, 2～6 hours before dissection is drawn materials, colchicine is injected, dosage is 2～4 μ g/g body weight.Take out nephridial tissue,
Nephridial tissue, 0.075mol/L KCL Hypotonic treatments, hypotonic 40～60 minutes at a temperature of 20 DEG C are shredded under physiological saline；Use ice
Acetic acid:Methyl alcohol (1:3) fixed nephridial tissue 1～3 time；Piece is dripped on frost slide；Giemsa dye liquors dyeing 45～60 minutes, carefully
Current rinse the slide back side and remove dye liquor, air-dry rear microscopy, take pictures, and the chromosome map of four type filial generations is respectively as schemed
4th, shown in Fig. 6, Fig. 8 and Figure 10.
The present embodiment is finally obtained dliploid Natural Gynogenesiss carp (referring to Fig. 3), dliploid Natural Gynogenesiss
Mirror carp (referring to Fig. 5), the liploid fish (referring to Fig. 7) of class crucian and Tetraploid fish (referring to Fig. 9) these four types.
With common red crucian carp as reference, dliploid Natural Gynogenesiss carp, dliploid are determined with flow cytometer natural female
The DNA average contents of red blood cell in caryogenesis mirror carp, the liploid fish of class crucian and Tetraploid fish these four types, as a result
Respectively as shown in Figure 12, Figure 13, Figure 14, Figure 15, data understand in analysis chart, dliploid Natural Gynogenesiss carp, dliploid
The DNA average contents of Natural Gynogenesiss mirror carp, the liploid fish of class crucian and Tetraploid fish are respectively 104.28,
93.25th, 99.17 and 143.62, dliploid Natural Gynogenesiss carp is 1.025 with the DNA average contents ratio for compareing red crucian carp
(referring to Figure 11), the ratio and estimated 1:Without significant difference between 1 theoretical ratio；Dliploid Natural Gynogenesiss mirror carp with compare
The DNA average contents ratio of red crucian carp is 0.917, the ratio and estimated 1:Without significant difference between 1 theoretical ratio；The two of class crucian
Times body fish is 0.975 with the DNA average contents ratio for compareing red crucian carp, the ratio and estimated 1:Without significance difference between 1 theoretical ratio
It is different；Tetraploid fish is 1.412 with the DNA average contents ratio for compareing red crucian carp, the ratio and estimated 1:1.5 theoretical ratio
Between without significant difference.Dliploid Natural Gynogenesiss carp, dliploid Natural Gynogenesiss mirror carp, the liploid fish of class crucian and
The DNA average contents of Tetraploid fish and it is shown in Table 1 with the DNA average content ratios of common red crucian carp：
Table 1：DNA average contents and the DNA average content ratios with common red crucian carp in four kinds of filial generations
Fluidic cell DNA content determination method step is：With the disposable syringe of Jing heparin wetting from fish tail vein blood vessel
Blood sampling about 0.2mL, in Eppendorf pipes of the injection equipped with 0.8% physiological saline, adds in blood and mixed liquor of normal saline
1mL nucleus extraction liquid DAPI-A (nuclei extraction solution, German Partec Gmbh are provided), during process
Between 10～15min；Sample is filtered through 20 μm of NFs (German Partec GmbH are provided)；DNA dyeing liquors (DAPI-B, moral
State Partec GmbH are provided) dark place stained specimens about 5～10min is statically placed in, then go up machine testing.
Dliploid Natural Gynogenesiss carp, dliploid Natural Gynogenesiss mirror carp and similar crucian that the present invention is obtained
Liploid fish and Tetraploid fish these four types, not only incorporate the multinomial merit of Parent parent population, also, logical
It is excessive for seed selection, be expected in generation after hybridization by hereditary and selection cultivate dliploid Natural Gynogenesiss carp strain, two times
The liploid fish strain and Tetraploid fish products system of body Natural Gynogenesiss mirror carp strain and similar crucian, wherein by right
Tetraploid fish products system continues seed selection, is expected to develop new autotetraploid fish products system.The present invention is in biological evolution and fish
Class genetic breeding aspect has important biological significance.
Claims (1)
1. a kind of carp and the method for megalobrame amblycephala subfamily distant hydridization, comprise the following steps：It is bright that sexal maturity feature is selected first
The good female carp of aobvious, sign and male megalobrama amblycephala are cultivated as the special pond of parent population, then the parent population are entered in mating period
Row artificial induced spawning, selecting the good parent population of spawning effect carries out artificial dry method insemination, and the embryo after the completion of insemination is hatched in water purification
Lotic hatching is carried out in device, treats to be raised after the completion of fry hatching, then the fry after raising is detected, sieved, led to
Overflow-type cell DNA content is determined, the detection of nephridial tissue ploidy obtains carp and megalobrama amblycephala filial generation, filial generation bag
Include dliploid Natural Gynogenesiss carp, dliploid Natural Gynogenesiss mirror carp, the liploid fish of class crucian and Tetraploid
Fish；
Methods described specifically includes following steps：
（a）The selection and cultivation of parent population：3～4 months before breeding period, select that sexal maturity feature is obvious, the female that sign is good
Carp and male megalobrama amblycephala carry out special pond cultivation respectively as the maternal parent population and male parent parent population of distant hybridization, keep water quality good
It is good；Raised with smart bait in the breeding period previous moon, every 2～3 days stimulation by running water once；The female carp（Cyprinus carpio carpio）Belong to the liploid fish in Cyprinidae, carp subfamily, 2n=100；
（b）Artificial induced spawning：It is the parent population note when water temperature stability is more than 20 DEG C in mid-May then to early June
Penetrate Luteinizing hormone releasing hormone analog to be hastened parturition with the mixing ocyodinic of human chorionic gonadtropin, the lutern release swashs
The consumption of plain analog is 6～10 μ g/kg, and the consumption of human chorionic gonadtropin is 500～800 IU/kg；First inject maternal
Parent population, injects again male parent parent population after 4～5h, the injection dosage of male parent parent population halves；Inject the quantity ratio by 1: 6～8 after finishing
Maternal parent population, male parent parent population are put in same spawning pond, are then washed by water in pond in time regarding regimen, 3 especially before spawning～
4h, after injecting certain water yield, stops water filling, allows fish to hasten parturition in hydrostatic, until it starts smoothly spawning and produces essence；
（c）Insemination hatching：Spawning smoothly maternal parent population is selected, is fixed with canvas stretcher, then the male parent parent big with essence amount is produced
Fish carries out artificial dry method insemination, and embryonated egg is placed in the water purification hatching apparatus that water temperature is 20 DEG C～21 DEG C and carries out Lotic hatching；
（d）Raise：After fry all hatches, continue to cultivate 2～3 days in incubator, turn fry after waist point occurs in fry
Enter in the pond fostered and apply fertilizer in advance and raise, soya-bean milk of splashing after 1～2 day, makes every effort to thin, even by 2～3 times/day；Fry passes through stream after growing up to
Formula cell DNA content is determined, the detection of nephridial tissue ploidy obtains filial generation；
What the fluidic cell DNA content was determined concretely comprises the following steps：With the disposable syringe of Jing heparin wetting from fish tail vein
Blood vessel about 0.2 mL of blood sampling, in Eppendorf pipes of the injection equipped with 0.8% physiological saline, in blood and mixed liquor of normal saline
Add 1mL nucleus extraction liquid DAPI-A, the min of process time 10～15；Sample is filtered through 20 m NFs；DNA contaminates
Color liquid is statically placed in the min of dark place stained specimens 5～10, then goes up machine testing；
The concrete steps of the nephridial tissue ploidy detection include：The cultural aim fish 1～3 day under 18 DEG C～22 DEG C water temperatures
Afterwards, to testing fish injection PHA 1～3 time, each dosage is 2～8 μ g/g body weight, and interval time is 12～24 hours, is being dissected
2～6 hours before drawing materials, colchicine is injected, dosage is 2～4 μ g/g body weight；Nephridial tissue is taken out, is shredded under physiological saline
Nephridial tissue, 0.075mol/L KCL Hypotonic treatments, hypotonic 40～60 minutes at a temperature of 20 DEG C；With glacial acetic acid: methyl alcohol fixes kidney
Tissue 1～3 time；Piece is dripped on frost slide；Giemsa dye liquors are dyeed 45～60 minutes, and thin current rinse the slide back side and go
Except dye liquor, air-dry rear microscopy, take pictures.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410771279.3A CN104585083B (en) | 2014-12-15 | 2014-12-15 | Method for distant hybridization between subfamilies of cyprinus carpio and megalobrama amblycephala |
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| CN105145434B (en) * | 2015-09-12 | 2018-02-13 | 上海海洋大学 | The screening and method for group matching that a kind of megalobrama amblycephala advantage forms a complete set of |
| CN106135094A (en) * | 2016-06-30 | 2016-11-23 | 湖南文理学院 | The method being efficiently bred as Monopterus albus (Zuiew) male tetraploid pure lines |
| CN106135092A (en) * | 2016-06-30 | 2016-11-23 | 湖南文理学院 | The method improving silver xenocypris fish male tetraploid incubation rate |
| CN106550909B (en) * | 2016-10-31 | 2019-07-26 | 湖南师范大学 | The method of fancy carp and megalobrame amblycephala subfamily distant hydridization |
| CN108823277A (en) * | 2018-04-18 | 2018-11-16 | 浙江师范大学 | The method of sturgeon Genome Size is quickly measured with Flow Cytometry |
| CN110199918B (en) * | 2019-06-14 | 2021-07-09 | 湖南师范大学 | Method for establishing autotetraploid carp strain and method for breeding autotetraploid carp |
| CN110915727B (en) * | 2019-12-10 | 2021-02-12 | 湖南师范大学 | Method for breeding multiple colorful double-tail goldfish through distant hybridization |
| CN114467808A (en) * | 2021-12-31 | 2022-05-13 | 湖南岳麓山水产育种科技有限公司 | Large-scale preparation method of bighead carp grown from distant hybridization natural gynogenesis |
| CN114698575A (en) * | 2022-05-10 | 2022-07-05 | 湖南文理学院 | Breeding method for variegated carp heterogenous gynogenesis |
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| CN101884314A (en) * | 2010-07-26 | 2010-11-17 | 湖南师范大学 | Method for inter-subfamily distant hybridization of grass carp and megalobrama amblycephala |
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| CN101720698B (en) * | 2009-12-09 | 2011-06-29 | 湖南师范大学 | Method for distant hybridization of megalobrama amblycephala and erythroculter ilishaeformis |
| CN101946738B (en) * | 2010-09-26 | 2012-07-25 | 湖南师范大学 | Method of hybridizing carassius cuvieri and red crucian varieties |
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| CN101884314A (en) * | 2010-07-26 | 2010-11-17 | 湖南师范大学 | Method for inter-subfamily distant hybridization of grass carp and megalobrama amblycephala |
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