CN102893938B - Subfamily distant hybridization method for Xenocypris davidi Bleeker and Erythroculter ilishaeformis Bleeker - Google Patents
Subfamily distant hybridization method for Xenocypris davidi Bleeker and Erythroculter ilishaeformis Bleeker Download PDFInfo
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- CN102893938B CN102893938B CN201210449551.7A CN201210449551A CN102893938B CN 102893938 B CN102893938 B CN 102893938B CN 201210449551 A CN201210449551 A CN 201210449551A CN 102893938 B CN102893938 B CN 102893938B
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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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- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
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Abstract
The invention discloses a subfamily distant hybridization method for Xenocypris davidi Bleeker and Erythroculter ilishaeformis Bleeker. The method comprises the following steps: by using Xenocypris davidi Bleeker as a female parent fish and Erythroculter ilishaeformis Bleeker as a male parent fish, firstly carrying out artificial induced spawning on the female parent fish and the male parent fish, carrying out artificial dry-method insemination on the female parent fish and the male parent fish which have good induced spawning effects, incubating with flowing water in a culture dish after insemination, farming after fries are incubated, and obtaining filial generations of Xenocypris davidi bleeker and Erythroculter ilishaeformis Bleeker by detection after breeding. The subfamily distant hybridization method provided by the invention has the advantages of simple steps, convenient operation, high fertility rate and hatching rate, good characters for later generations and the like.
Description
Technical field
The present invention relates to a kind of method of fish inter-subfamily distant hybridization, relate in particular to the distant hybridization method between the close silver xenocypris of a kind of yellow tail and erythroculter ilishaeformis subfamily.
Background technology
Distant hybridization of fish is two kinds of hybridization between fish not of the same race, can be belong to together fish not fish of the same race hybridization, also can be do not belong to together between, between different subfamily, not equal even with the hybridization between fish individual between order.Hybridization can be integrated parents' advantage, makes filial generation all show hybrid vigour at aspects such as profile, growth rate, survival rate and resistances against diseases, is a kind of important fish genetic breeding and character improvement method.The close silver xenocypris of yellow tail (
xenocypris davidi Bleeker) belong to the liploid fish (2n=48) in Cyprinidae, silver xenocypris subfamily, there is the advantages such as meat is good, feeding habits are wide, erythroculter ilishaeformis (
erythroculter ilishaeformis Bleeker) belong to the liploid fish (2n=48) in Cyprinidae, Culter subfamily, be large-scale economic freshwater fish, have growth rapidly, lower oxygen concentration resistance, adaptability and the advantage such as premunition is extremely strong.There is larger difference aspect the biological propertyes such as classification, the bodily form in these two kinds of fishes, so the distant hybridization of these two exists larger difficulty.The novel hybridization fish that how to utilize and improve existing biology techniques to have merit successfully to obtain has become scientific research person and has faced for a long time and problem demanding prompt solution.
Summary of the invention
The technical problem to be solved in the present invention is to overcome the deficiencies in the prior art, and a kind of step is simple, easy to operate, fertility rate and hatchability is high, characters of progenies the is good close silver xenocypris of yellow tail and the method for erythroculter ilishaeformis inter-subfamily distant hybridization are provided.
For solving the problems of the technologies described above, the technical scheme that the present invention proposes is the method for the close silver xenocypris of a kind of yellow tail and erythroculter ilishaeformis inter-subfamily distant hybridization, comprise the following steps: using the close silver xenocypris of female yellow tail as maternal parent population, using male erythroculter ilishaeformis as male parent parent population, first described maternal parent population and male parent parent population are carried out to artificial induced spawning, the good maternal parent population of spawning effect and male parent parent population are carried out to artificial dry method insemination, after insemination in culture dish Lotic hatching, after fry hatching completes, raise, raise the filial generation that obtains the close silver xenocypris of yellow tail and erythroculter ilishaeformis by detecting.This filial generation is the close silver xenocypris of yellow tail and erythroculter ilishaeformis inter-subfamily distant hybridization F
1in generation, (is called for short silver xenocypris Culter F
1generation), there is higher fertilization rate, incubation rate and survival rate; By further utilizing fluidic cell DNA content detection technique, peripheral blood cells method of chromosome preparation to silver xenocypris Culter F
1in generation, is detected, and confirmation obtains the close silver xenocypris of yellow tail and erythroculter ilishaeformis inter-subfamily distant hybridization dliploid silver xenocypris Culter F
1in generation, this lays a good foundation for the genetic breeding of follow-up silver xenocypris Culter.
The above-mentioned close silver xenocypris of yellow tail and the method for erythroculter ilishaeformis inter-subfamily distant hybridization, the concrete operations of described artificial induced spawning preferably include: annual late June (be the close silver xenocypris of yellow tail with erythroculter ilishaeformis mating season intersect the time), more than choosing for two ages, sexual maturity feature is obvious, the close silver xenocypris of female yellow tail and male erythroculter ilishaeformis that sign is good, first for the close silver xenocypris injection Luteinizing hormone releasing hormone analog of female yellow tail (LRH-A) is hastened parturition with the ocyodinic that mixes of human chorionic gonadtropin (HCG), the consumption of Luteinizing hormone releasing hormone analog is 20 μ g/kg, the consumption of human chorionic gonadtropin is 1500IU/kg, after 4h～5h, more male erythroculter ilishaeformis is injected, the injected dose of male erythroculter ilishaeformis is compared the close silver xenocypris of female yellow tail and is reduced by half, injection is rear by 1: the quantity of (2～3) is put into same spawning pond than by the close silver xenocypris of female yellow tail, male erythroculter ilishaeformis, until hastened parturition.
The above-mentioned close silver xenocypris of yellow tail and the method for erythroculter ilishaeformis inter-subfamily distant hybridization, the concrete operations of described Lotic hatching preferably include: select the maternal parent population that egg laying amount is large, the quality of laying eggs is high and produce the large male parent parent population of essence amount and carry out after artificial dry method insemination, fertilized egg after artificial dry method insemination is for example evenly laid on, in culture dish (glass culture dish that diameter is 22cm) and carries out Lotic hatching, in each culture dish, the control of the ovum grain number of fertilized egg is 1500～2000, and water temperature remains on 22 ℃～26 ℃.
The above-mentioned close silver xenocypris of yellow tail and the method for erythroculter ilishaeformis inter-subfamily distant hybridization, the concrete operations of described raising preferably include: after fry all hatches, prior to cultivating in filtered water tank 1～2 day, regularly change during this time water, until fry, occur waist point, start, after flat trip, fry to be proceeded in the pond of fostering and apply fertilizer in advance and raised, the soya-bean milk of splashing after 3～4 days, 2～3 times/day, along pond, splash, until fry grows up to.
The above-mentioned close silver xenocypris of yellow tail and the method for erythroculter ilishaeformis inter-subfamily distant hybridization, described detection preferably adopts peripheral blood cells to cultivate method of chromosome preparation (peripheral blood cells cultivation), fluidic cell DNA content determination method (flow cytometry assay) is carried out the detection of chromosome number, DNA content to filial generation.
Compared with prior art, the invention has the advantages that: the present invention by repetition test finally select the close silver xenocypris of yellow tail as maternal, select erythroculter ilishaeformis as male parent, carry out the distant hybridization between subfamily, finally successfully obtained and a kind ofly at aspects such as profile, growth rate, survival rate and resistances against diseases, all shown heterotic dliploid silver xenocypris Culter.More similar test, the fertility rate and hatchability of the close silver xenocypris of yellow tail and erythroculter ilishaeformis intermolecular hybrid is all significantly improved, and can stablize and repeat to obtain a large amount of filial generations, hybridization F
1for proterties neat and consistent.Meanwhile, the advantage of the close silver xenocypris of yellow tail and erythroculter ilishaeformis has been integrated in distant hybridization, and under equal conditions, filial generation relatively its Parent all shows hybrid vigour in the aspect such as shape, growth rate, survival rate and resistance against diseases outside.
In addition, take distant hybridization method of the present invention as basis, be also expected to by how obtain tetraploid GuCulter colony from dliploid silver xenocypris Culter for seed selection.Existing research shows, red crucian carp (2n=100, ♀) * Xiangjiang wild carps (2n=100, in filial generation ♂), F
1-F
2for dliploid (2n=100), and at dliploid F
2in individuality, exist and can produce respectively the female, male individual of diplont ovum and diploid sperm, diplont ovum and diploid sperm combination, formed the tetraploid fish F that both sexes can be educated
3(4n=200).Tetraploid crucian carp carp has bred 20 generation (F continuously at present
3-F
22), formed the tetraploid fish colony of a stabilization characteristics of genetics.Accordingly, the acquisition of dliploid silver xenocypris Culter of the present invention is significant aspect fish breeding and organic evolution.
Accompanying drawing explanation
Fig. 1 is the profile photo of the close silver xenocypris of yellow tail (♀) * erythroculter ilishaeformis (♂) offspring in the embodiment of the present invention.
Fig. 2 is the chromosome map (2n=48) of the close silver xenocypris of yellow tail (♀) * erythroculter ilishaeformis (♂) offspring in the embodiment of the present invention.
Fig. 3 is the DNA content figure of the red crucian carp that records in the embodiment of the present invention.
Fig. 4 is the close silver xenocypris of yellow tail (♀) * erythroculter ilishaeformis (♂) offspring's of recording in the embodiment of the present invention DNA content figure.
Embodiment
Below in conjunction with Figure of description, the invention will be further described with concrete preferred embodiment, but protection domain not thereby limiting the invention.
embodiment:
A method for the close silver xenocypris of yellow tail of the present invention and erythroculter ilishaeformis inter-subfamily distant hybridization, specifically comprises the following steps:
1. artificial induced spawning: using the close silver xenocypris of female yellow tail as maternal parent population, using male erythroculter ilishaeformis as male parent parent population, in annual late June, choose the two ages close silver xenocypris of female yellow tail and male erythroculter ilishaeformis above, that sexual maturity feature is obvious, sign is good and hasten parturition.First for maternal parent population injection Luteinizing hormone releasing hormone analog (LRH-A) is hastened parturition with the ocyodinic that mixes of human chorionic gonadtropin (HCG), the injection consumption of LRH-A is 20 μ g/kg, the injection consumption of HCG is 1500IU/kg, adopts pectoral fin base portion depression without 45 ° of thoracic cavities injections of squama place inclination; After 4h～5h, inject male parent parent population, the injected dose of male parent parent population is compared maternal parent population and is reduced by half again, and injecting method is with maternal parent population; Injection is rear by 1: the quantity of (2～3) is put into same spawning pond than by maternal parent population, male parent parent population, and running water stimulates, and before breeding, 3h～4h stops adding water, allows fish hasten parturition in hydrostatic.
2. artificial dry method insemination and hatching: during phenomenon that mutually chasing appears in the parent population in finding spawning pond, start to draw in the net to salvage parent population and detect that (attention will be used soft net, it is light that operation is wanted, in water, detect), select egg laying amount large, the maternal parent population that the quality of laying eggs is high and the large male parent parent population of product essence amount carry out artificial dry method insemination, smart ovum is clamp-oned in clean porcelain basin, with clean feather, ceaselessly stir rapidly, then fertilized egg is evenly laid in the glass culture dish that diameter is 22cm and carries out Lotic hatching, ovum grain number in each culture dish is 1500～2000, water temperature remains on 22 ℃～26 ℃.
3. raise: after fry all hatches, prior to cultivating in filtered water tank 1～2 day, regularly change in the meantime water, until fry, there is waist point, start fry to be proceeded in the pond of fostering and apply fertilizer in advance and raised, the soya-bean milk of splashing after 3～4 days, 2～3 times/day after flat trip, along pond, splash, until fry grows up to.
Between the incubation period, carry out artificial counting, statistics embryo's fertility rate and hatchability.In the present embodiment, the fertility rate and hatchability of the close silver xenocypris of yellow tail and erythroculter ilishaeformis hybridization fish is respectively 85% and 77%, compares with similar cross experiment, and its fertility rate and hatchability is significantly improved.
Choose at random the close silver xenocypris of yellow tail, erythroculter ilishaeformis and the dliploid silver xenocypris Culter (the profile photo of dliploid silver xenocypris Culter is referring to Fig. 1) that cultivated under the same conditions for two ages; External form feature to them detects, and comprises on dorsal fin bar, abdomeinal fin bar, stern fin ray, lateral line scales, side line squama under squama and side line, and testing result is as shown in table 1 below.
Table 1: the denumerable proterties comparison of the close silver xenocypris of yellow tail, erythroculter ilishaeformis and filial generation (unit: sheet or bar)
The related data of above-mentioned denumerable proterties shows: on the side line of dliploid silver xenocypris Culter, squama, between the close silver xenocypris of yellow tail and erythroculter ilishaeformis, shows as intermediate form, embodies the characteristic of hybridization; Although filial generation is more similar to male parent erythroculter ilishaeformis in shape, dliploid silver xenocypris Culter is more partial to the close silver xenocypris of maternal yellow tail on lateral line scales and dorsal fin bar; Under side line, squama is all higher in parent, shows the characteristic of variation.
The ploidy detection method of cultivating by fish peripheral blood cells is again identified the lymphocytic chromosome number of dliploid silver xenocypris Culter.The ploidy detection method operating procedure that peripheral blood cells is cultivated 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) with syringe, draw a small amount of sterilizing heparin sodium aqua, for the experiment fish tail portion venous blood sampling after iodine disinfection; 3) by adding the anticoagulant standard of about 0.2ml that anticoagulation is added in culture fluid in every 10ml culture fluid, in 24 ℃, the incubator of 5% gas concentration lwevel, cultivate 68h～72h, between culture period, regularly jog is even, so that cell fully contacts medium; 4) stop cultivating first 3 hours, add the colchicin of 10 μ g/ml in culture fluid, making final concentration is 0.05 μ g/ml～0.07 μ g/ml, and above step all needs sterile working; 5) culture is all proceeded in 10 clean ml centrifuge tubes, with the centrifugal 5min of 1500rpm, abandon supernatant; 6) in centrifuge tube, add 10ml hypotonic medium, mix, hypotonic 25min～30min; 7) add 1ml fixer, mix gently the centrifugal 5min of rear 1500rpm, abandon supernatant; 8) add 5ml fixer, mix gently, after standing 30min, the centrifugal 5min of 1500rpm, abandons supernatant; 9) repeating step 8 once; 10) depending on last cell quantity, how much add appropriate fixer to make after cell suspension (being generally 1ml～2ml), draw cell suspension and drip sheet from 20cm～30cm eminence, featheriness is loose, natural seasoning in air; 11) Giemsa dye liquor dyeing 50min～60min, thin current rinse the slide back side and remove dye liquor, microscopy after gas is dry, take pictures.The result obtaining with above-mentioned experimental technique as shown in Figure 2, the close silver xenocypris of yellow tail (♀) and erythroculter ilishaeformis (♂) filial generation F
1chromosome number be 48, prove that they are dliploid (2n=48).
Meanwhile, take red crucian carp as reference, with the DNA content in the above-mentioned dliploid silver xenocypris of cells were tested by flow cytometry Culter red blood cell.The general steps of fluidic cell DNA content determination method is: use through the wetting disposable syringe of liquaemin from fish tail vein blood vessel approximately 0.2 mL that takes a blood sample, the Eppendorf pipe of 0.8% physiological saline is equipped with in injection, in blood and mixed liquor of normal saline, add 1mL nucleus extraction liquid DAPI-A(nuclei extraction solution, Germany Partec Gmbh provides), processing times 10～15 min; Sample filters through 20 um NFs (German Partec GmbH provides); DNA dyeing liquor (DAPI-B, German Partec GmbH provides) is statically placed in dark place stained specimens approximately 5～10 min, then goes up machine testing.Measurement result respectively as shown in Figure 3, Figure 4.In analysis chart, data are known, the DNA average content of red crucian carp, dliploid silver xenocypris Culter is respectively 97.35 and 68.40, the DNA average content ratio that contrasts red crucian carp and dliploid silver xenocypris Culter is 1.423, ratio (the red crucian carp: erythroculter ilishaeformis=101.29: 67.40=1.503) without significant difference of this ratio and existing red crucian carp and erythroculter ilishaeformis.Show thus, the result that the ploidy detection method that the result of the test of fluidic cell DNA content determination method is cultivated with peripheral blood cells obtains is consistent.
Claims (3)
1. the method for the close silver xenocypris of yellow tail and erythroculter ilishaeformis inter-subfamily distant hybridization, comprise the following steps: the close silver xenocypris of Yi Huangwei is as maternal parent population, using erythroculter ilishaeformis as male parent parent population, first described maternal parent population and male parent parent population are carried out to artificial induced spawning, the good maternal parent population of spawning effect and male parent parent population are carried out to artificial dry method insemination, after insemination in culture dish Lotic hatching, after fry hatching completes, raise, raise the filial generation that obtains the close silver xenocypris of yellow tail and erythroculter ilishaeformis by detecting;
The concrete operations of described artificial induced spawning comprise: in annual late June, choose sexual maturity feature is obvious, sign the is good close silver xenocypris of female yellow tail and male erythroculter ilishaeformis, first for the close silver xenocypris injection of female yellow tail Luteinizing hormone releasing hormone analog is hastened parturition with the ocyodinic that mixes of human chorionic gonadtropin, the consumption of Luteinizing hormone releasing hormone analog is 20 μ g/kg, and the consumption of human chorionic gonadtropin is 1500IU/kg; After 4h～5h, more male erythroculter ilishaeformis is injected; The injected dose of male erythroculter ilishaeformis is compared the close silver xenocypris of female yellow tail and is reduced by half; Injection is rear by 1: the quantity of (2～3) is put into same spawning pond than by the close silver xenocypris of female yellow tail, male erythroculter ilishaeformis, until hastened parturition;
The concrete operations of described Lotic hatching comprise: the fertilized egg after artificial dry method insemination is evenly laid in culture dish and carries out Lotic hatching, and in each culture dish, the control of the ovum grain number of fertilized egg is 1500～2000, and water temperature remains on 22 ℃～26 ℃.
2. the method for the close silver xenocypris of yellow tail according to claim 1 and erythroculter ilishaeformis inter-subfamily distant hybridization, it is characterized in that, the concrete operations of described raising comprise: after fry all hatches, prior to cultivating in water tank 1～2 day, during regularly change water, until fry, there is waist point, start after flat trip, fry is proceeded in the pond of fostering and apply fertilizer in advance and raised, the soya-bean milk of splashing after 3～4 days, 2～3 times/day, along pond, splash, until fry grows up to.
3. the method for the close silver xenocypris of yellow tail according to claim 1 and 2 and erythroculter ilishaeformis inter-subfamily distant hybridization, it is characterized in that, described detection is to adopt peripheral blood cells cultivation method of chromosome preparation, fluidic cell DNA content determination method filial generation to be carried out to the detection of chromosome number, DNA content.
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| CN105684962B (en) * | 2015-11-17 | 2018-05-11 | 浙江省淡水水产研究所 | One kind sticks up mouth Culter and triangular bream hybrid preparation method |
| 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 |
| CN108184727A (en) * | 2018-03-22 | 2018-06-22 | 广西壮族自治区水产科学研究院 | A kind of artificial fecundation method of the former carp of crow |
| 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 |
| CN115053836B (en) * | 2022-05-20 | 2024-04-19 | 湖南师范大学 | Distant hybridization method between grass carp and xenocypris davidi subfamily and cultivation method of triploid xenocypris davidi |
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