CN102893938A - 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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- CN102893938A CN102893938A CN2012104495517A CN201210449551A CN102893938A CN 102893938 A CN102893938 A CN 102893938A CN 2012104495517 A CN2012104495517 A CN 2012104495517A CN 201210449551 A CN201210449551 A CN 201210449551A CN 102893938 A CN102893938 A CN 102893938A
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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 the 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 the 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, the silver xenocypris subfamily, have 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, the Culter subfamily, be large-scale economic freshwater fish, have growth rapidly, the advantage such as lower oxygen concentration resistance, adaptability and premunition be extremely strong.There is larger difference in these two kinds of fishes aspect the biological propertyes such as classification, the bodily form, so there is larger difficulty in the distant hybridization of these two.How utilizing and improving existing biology techniques has become scientific research person with the novel hybridization fish that successfully obtains to have merit 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, may further comprise the steps: with the close silver xenocypris of female yellow tail as maternal parent population, with male erythroculter ilishaeformis as the male parent parent population, first described maternal parent population and male parent parent population are carried out artificial induced spawning, maternal parent population and the male parent parent population good to spawning effect carry out artificial dry method insemination, insemination is Lotic hatching in culture dish afterwards, after fry hatching is finished, 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, (be called for short silver xenocypris Culter F
1Generation), have 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, detected, and conclusive evidence 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), choose more than two ages, the sexual maturity feature is obvious, the close silver xenocypris of female yellow tail and male erythroculter ilishaeformis that sign is good, at first hasten parturition with the ocyodinic that mixes of human chorionic gonadtropin (HCG) for the close silver xenocypris injection Luteinizing hormone releasing hormone analog of female yellow tail (LRH-A), the consumption of Luteinizing hormone releasing hormone analog is 20 μ g/kg, and the consumption of human chorionic gonadtropin is 1500IU/kg; Again male erythroculter ilishaeformis is injected through behind 4h～5h; 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 with the close silver xenocypris of female yellow tail, male erythroculter ilishaeformis, finishes until hasten 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 after the large male parent parent population of smart amount carries out artificial dry method insemination, fertilized egg after the insemination of artificial dry method evenly is laid in the culture dish (for example diameter is the glass culture dish of 22cm) carries out Lotic hatching, the ovum grain number of fertilized egg is controlled to be 1500～2000 in each culture dish, 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 the filtered water tank 1～2 day, regularly change during this time water, after fry the waist point occurs, begins flat trip, fry changed in the pond of fostering and apply fertilizer in advance raise, the soya-bean milk of splashing after 3～4 days, 2～3 times/days, splash along the pond, 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 adopt 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 a kind ofly all to show heterotic dliploid silver xenocypris Culter at aspects such as profile, growth rate, survival rate and resistances against diseases.More similar test, the fertility rate and hatchability of the close silver xenocypris of yellow tail and erythroculter ilishaeformis intermolecular hybrid all is significantly improved, and can stablize and repeat to obtain a large amount of filial generations, hybridization F
1For the proterties neat and consistent.Simultaneously, 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 aspects such as shape, growth rate, survival rate and resistance against diseases outside.
In addition, take distant hybridization method of the present invention as the basis, also be expected to by how from dliploid silver xenocypris Culter, to obtain tetraploid silver xenocypris Culter colony for seed selection.Existing studies show that, red crucian carp (2n=100, ♀) * Xiangjiang wild carps (2n=100, in filial generation ♂), F
1-F
2Be dliploid (2n=100), and at dliploid F
2Exist in the individuality to produce respectively the female, male individual of diplont ovum and diploid sperm, diplont ovum and diploid sperm combination have 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.
Description of drawings
Fig. 1 is the close silver xenocypris of yellow tail (♀) in the embodiment of the invention * erythroculter ilishaeformis (♂) offspring's profile photo.
Fig. 2 is the close silver xenocypris of yellow tail (♀) in the embodiment of the invention * erythroculter ilishaeformis (♂) offspring's chromosome map (2n=48).
Fig. 3 is the dna content figure of the red crucian carp that records in the embodiment of the invention.
Fig. 4 is the close silver xenocypris of yellow tail (♀) that records in the embodiment of the invention * erythroculter ilishaeformis (♂) offspring's dna content figure.
Embodiment
The invention will be further described with concrete preferred embodiment below in conjunction with Figure of description, but protection domain not thereby limiting the invention.
Embodiment:
The method of a kind of close silver xenocypris of yellow tail of the present invention and erythroculter ilishaeformis inter-subfamily distant hybridization specifically may further comprise the steps:
1. artificial induced spawning: as maternal parent population,,, choose the two ages close silver xenocypris of female yellow tail and male erythroculter ilishaeformis above, that the sexual maturity feature is obvious, sign is good and hasten parturition in annual late June as the male parent parent population with male erythroculter ilishaeformis with the close silver xenocypris of female yellow tail.Hasten parturition with the ocyodinic that mixes of human chorionic gonadtropin (HCG) for maternal parent population injection Luteinizing hormone releasing hormone analog (LRH-A) first, 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; Inject the male parent parent population behind 4h～5h, 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 with maternal parent population, male parent parent population, and running water stimulates, and 3h～4h stops to add water before breeding, 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, begin 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, detect in the water), it is large to select egg laying amount, the large male parent parent population of the smart amount of the maternal parent population that the quality of laying eggs is high and product carries out artificial dry method insemination, smart ovum is clamp-oned in the clean porcelain basin, ceaselessly stir rapidly with clean feather, then fertilized egg evenly is 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, and water temperature remains on 22 ℃～26 ℃.
3. raise: after fry all hatches, prior to cultivating in the filtered water tank 1～2 day, regularly change in the meantime water, the waist point occurs, fry changed in the pond of fostering and apply fertilizer in advance after beginning flat trip and raise the soya-bean milk of splashing after 3～4 days, 2～3 times/days until fry, splash along the pond, until fry grows up to.
Carry out artificial counting between the incubation period, 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 has obtained improving significantly.
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, the side line squama under the squama and side line, and testing result is as shown in table 1 below.
Table 1: the denumerable proterties of the close silver xenocypris of yellow tail, erythroculter ilishaeformis and filial generation is (unit: sheet or bar) relatively
The related data of above-mentioned denumerable proterties shows: squama is between the close silver xenocypris of yellow tail and the erythroculter ilishaeformis on the side line of dliploid silver xenocypris Culter, shows as intermediate form, embodies the characteristic of hybridization; Although filial generation is more similar to the male parent erythroculter ilishaeformis in shape, dliploid silver xenocypris Culter more is partial to the close silver xenocypris of maternal yellow tail on lateral line scales and dorsal fin bar; Squama is then all higher in the parent under the side line, shows the characteristic of variation.
The ploidy detection method of cultivating by the 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) prepare medium in superclean bench, the 100ml medium contains following composition: 84ml RPMI-1640,15ml calf serum, 2 PHA, 1ml 0.1% liquaemin, with 7.5% NaHCO
3(aseptic) or 1N HCl transfer pH to 7.2～7.4; 2) draw a small amount of sterilization heparin sodium aqua with syringe, for the experiment fish tail section venous blood sampling behind the iodine disinfection; 3) by adding the anticoagulant standard of about 0.2ml in every 10ml culture fluid anticoagulation is added in the culture fluid, in 24 ℃, the incubator of 5% gas concentration lwevel, cultivates 68h～72h, between culture period regularly jog spare so that cell fully contacts medium; 4) stop cultivating front 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 changed in the 10 clean ml centrifuge tubes, with the centrifugal 5min of 1500rpm, abandon supernatant; 6) in centrifuge tube, add the 10ml hypotonic medium, mixing, hypotonic 25min～30min; 7) add the 1ml fixer, the centrifugal 5min of 1500rpm behind the mixing abandons supernatant gently; 8) add the 5ml fixer, mixing gently leaves standstill the centrifugal 5min of 1500rpm behind the 30min, abandons supernatant; 9) repeating step 8 once; 10) how much look last cell quantity adds an amount of fixer and makes cell suspension (after being generally 1ml～2ml), drawing cell suspension and drip sheet from 20cm～30cm eminence, featheriness is loose, natural seasoning in the air; 11) Giemsa dye liquor dyeing 50min～60min, dye liquor is removed at the thin current flushing slide back side, microscopy after gas is done, takes pictures.The result who obtains 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).
Simultaneously, take red crucian carp as reference, with the dna content in the above-mentioned dliploid silver xenocypris of the 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 about 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 about 5～10 min of dark place stained specimens, then goes up machine testing.Measurement result respectively as shown in Figure 3, Figure 4.Data as can be known in the analysis chart, 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 that thus the result of the test of fluidic cell dna content determination method is consistent with the result that the ploidy detection method that peripheral blood cells is cultivated obtains.
Claims (5)
1. the method for the close silver xenocypris of yellow tail and erythroculter ilishaeformis inter-subfamily distant hybridization, may further comprise the steps: the close silver xenocypris of Yi Huangwei is as maternal parent population, with erythroculter ilishaeformis as the male parent parent population, first described maternal parent population and male parent parent population are carried out artificial induced spawning, maternal parent population and the male parent parent population good to spawning effect carry out artificial dry method insemination, after the insemination in culture dish Lotic hatching, after fry hatching is finished, raise, raise the filial generation that obtains the close silver xenocypris of yellow tail and erythroculter ilishaeformis by detecting.
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 artificial induced spawning comprise: in annual late June, choose the sexual maturity feature is obvious, sign the is good close silver xenocypris of female yellow tail and male erythroculter ilishaeformis, at first hasten parturition with the ocyodinic that mixes of human chorionic gonadtropin for the close silver xenocypris injection of female yellow tail Luteinizing hormone releasing hormone analog, the consumption of Luteinizing hormone releasing hormone analog is 20 μ g/kg, and the consumption of human chorionic gonadtropin is 1500IU/kg; Again male erythroculter ilishaeformis is injected through behind 4h～5h; 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 with the close silver xenocypris of female yellow tail, male erythroculter ilishaeformis, finishes until hasten parturition.
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, the concrete operations of described Lotic hatching comprise: the fertilized egg after the artificial dry method insemination evenly is laid on carries out Lotic hatching in the culture dish, the ovum grain number of fertilized egg is controlled to be 1500～2000 in each culture dish, and water temperature remains on 22 ℃～26 ℃.
4. 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, the concrete operations of described raising comprise: after fry all hatches, prior to cultivating in the water tank 1～2 day, during regularly change water, after fry the waist point occurs, begins flat trip, fry changed in the pond of fostering and apply fertilizer in advance raise the soya-bean milk of splashing after 3～4 days, 2～3 times/days, splash along the pond, until fry grows up to.
5. 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 the detection of chromosome number, dna content.
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| 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 |
| CN115053836A (en) * | 2022-05-20 | 2022-09-16 | 湖南师范大学 | Method for distant hybridization between subfamilies of grass carp and xenocypris davidi and method for breeding triploid xenocypris davidi hybrid fish |
| 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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