A method for distant hybridization between megaleseus and ereteleus

Abstract

The present application belongs to the field of fish distant hybrid breeding, and discloses a method for distant hybridization between Megalobrama amblycephala and the genus Procyprinus. Megalobrama amblycephala and Procyprinus are selected as parent fish to artificially induce spawning, and are transferred to an ecological breeding net cage to naturally spawn and fertilize, and to flow water natural incubation. For parent fish that fail to successfully spawn after the effect period of the induced spawning drug, artificial dry insemination is adopted, and static water incubation is carried out. After the hatched fry are hatched and all have a waist point, feeding is started, and then the diploid hybrid offspring of Megalobrama amblycephala and Procyprinus is obtained. The hybridization method of the present application is to use Megalobrama amblycephala and Procyprinus for intergeneric distant hybridization, successfully combines the high back, fast growth of Megalobrama amblycephala and the slender body shape, tender meat quality, and good stress resistance of Procyprinus, and creatively breeds a new type of Megalobrama amblycephala and Procyprinus diploid hybrid fish with long body shape, wide back, better muscle quality, and stronger stress resistance, which is a new type of distant hybridization.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of fish distant hybrid breeding, and particularly relates to a method for distant hybridization between Megalobrama amblycephala and Erythroculter dabryi Bleeker. BACKGROUND

[0002] Various fish lines obtained through distant hybridization have important values in genetic breeding and biological evolution. Distant hybridization of fish generally refers to that genetic materials of two parent fish with distant genetic relationship are combined through hybridization, so that the hybrid offspring has significant changes in external phenotypic traits and internal molecular genetics, thereby obtaining a new fish variety meeting the needs of production and life. For example, in the laboratory, female white crucian carp after population selection is used as the female parent, and male red crucian carp is used as the male parent, and the offspring obtained through hybridization has combined excellent traits of the parents, has the advantages of beautiful body shape, fast growth rate, tender meat, high nutritional value, etc., and the hybrid crucian carp has been directly applied to practical production as an excellent fish. For another example, through distant hybridization between Megalobrama amblycephala and Erythroculter dabryi Bleeker, a new hybrid variety, hybrid Erythroculter dabryi Bleeker, is prepared on the basis of the obtained Megalobrama amblycephala and Erythroculter dabryi Bleeker F1, and the female Megalobrama amblycephala F1 is used as the female parent and the male Megalobrama amblycephala is used as the male parent. Compared with the original parent, the hybrid Erythroculter dabryi Bleeker has obvious hybrid advantages, such as tender meat, few muscle spines, strong resistance, low oxygen tolerance, etc.

[0003] The Megalobrama amblycephala (BSB) used in the present application belongs to the family Cyprinidae, the subfamily Cultrinae, and the genus Megalobrama, has high body side, thick back, herbivorous, fast growth rate, good resistance, etc. The Erythroculter dabryi Bleeker (GTC) belongs to the family Cyprinidae, the subfamily Cultrinae, and the genus Erythroculter, has the advantages of tender meat, rich nutrition, etc. However, with the change of natural water environment, the individuals in the Erythroculter dabryi Bleeker population become inbred, causing partial gene loss, natural reproduction begins to degenerate, and the population size begins to decrease, thereby having a significant impact on biological diversity and biological evolution. At the same time, due to the small size of adult Erythroculter dabryi Bleeker, the economic utilization value is relatively low compared with other Cultrinae such as Culter alburnus, so that the aquaculture of Erythroculter dabryi Bleeker is less. Through distant hybridization with Megalobrama amblycephala, the new type of Megalobrama amblycephala and Erythroculter dabryi Bleeker offspring obtained by combining the advantages of the two has a higher growth rate and size than the parent Erythroculter dabryi Bleeker. The hybrid Megalobrama amblycephala and Erythroculter dabryi Bleeker not only improves the economic value of fish individuals, but also promotes the increase of economic benefits of aquaculture; at the same time, it also plays a good protection role for the Erythroculter dabryi Bleeker germplasm resource, and has an important promoting role for fish production practice.

[0004] The far cross is a good technical means for protecting the germplasm resources of species, and thus the new type of gudgeon or gudgeon catfish produced by the far cross between the blue catfish and the blue-red catfish is of great significance to the protection of the germplasm resources of the gudgeon catfish, genetic breeding and biological diversity. SUMMARY

[0005] The technical problem to be solved by the present application is to overcome the deficiencies and defects mentioned in the above background art, and to provide a method for far cross between blue catfish and blue-red catfish, so as to obtain a new type of gudgeon or gudgeon catfish with fast growth, tender meat and high individual economic nutritional value.

[0006] To solve the above technical problem, the technical solution provided by the present application is:

[0007] A method for far cross between blue catfish and blue-red catfish, comprising the following steps:

[0008] Select blue catfish and blue-red catfish as parent fish for special pool breeding, artificially induce spawning, and transfer to the prepared ecological breeding net cage in the hatching tank for natural spawning and fertilization. Then collect the net gauze containing fertilized eggs and put them into another hatching tank for natural flow water hatching. For the parent fish that fail to spawn after the effect period of the induced spawning drug, artificial dry insemination method is used to assist the completion of egg fertilization, and then the fertilized eggs are transferred to the hatching dish for still water hatching. After the hatched fry are hatched and all appear waist points, feeding is started, and then the diploid hybrid offspring of the blue catfish and the blue-red catfish is obtained.

[0009] The present application is a far cross between blue catfish and blue-red catfish, which is a cross between gudgeon and red catfish in the subfamily of Cultrinae. It successfully obtains new type of gudgeon or gudgeon catfish, which is a new type of diploid gudgeon or gudgeon catfish. Through far cross combined with the advantages of the parents, it is found by the present application that the growth rate and size of the hybrid offspring gudgeon catfish are higher than those of the blue-red catfish. At the same time, it also plays a good protective role for the germplasm resources of the blue-red catfish, and has an important promoting effect on fish production practice.

[0010] In the above-mentioned method for far cross between blue catfish and blue-red catfish, preferably, the special pool breeding specifically comprises the following steps: selecting female blue catfish or female blue-red catfish and male blue-red catfish or male blue catfish in good growth state, uniform individual size, no injury and no disease as parent fish and putting them into the special pool for intensive breeding, wherein the sexual maturity age of the blue catfish is 2-3 years old, and the sexual maturity age of the blue-red catfish is 1 year old. The female and male parent fish are stimulated once every 2-3 days by flow water one month before the breeding period to promote the development and maturation of the gonads.

[0011] Preferably, the artificial induced spawning process comprises the following steps: in the middle of May of the breeding season, when the water temperature is stable at 25-27℃, select the sexually mature and active Megalobrama amblycephala and Protosalanx hyalita parent fish in a ratio of 2-3:1, and then inject the mixed induced spawning agent to induce spawning; for the female Megalobrama amblycephala, the dose of luteinizing hormone analogue is 8-10 μg / kg, the dose of chorionic gonadotropin is 800-1000 IU / kg, and the dose of domperidone is 1-2 mg / kg; for the female Protosalanx hyalita, the dose of luteinizing hormone analogue is 8-10 μg / kg, the dose of chorionic gonadotropin is 1600-2000 IU / kg, and the dose of domperidone is 1-2 mg / kg; the dose of induced spawning agent for the male Megalobrama amblycephala and Protosalanx hyalita is half of that for the female, and no domperidone is injected. The mixed solution of the three kinds of induced spawning agents is prepared in a small beaker before injection, and the fish in the parent fish induced spawning tank are fished with soft rope fishnet for injection of the induced spawning agent. The injection site is the slanting abdominal cavity injection from the scaleless part of the base of the pectoral fin. After injection, the parent fish is transferred to the hatching tank with an ecological breeding net cage, and a little gauze is hung on the bamboo pole to observe the spawning of the parent fish. The induced spawning tank is flushed with well water to promote the spawning of the parent fish.

[0012] Preferably, the ecological breeding net cage is a "cuboid" type net frame, and the gauze cloth for collecting eggs is laid in the net frame before induced spawning.

[0013] Preferably, the hatching process of the fertilized eggs obtained by natural spawning and fertilization is as follows: the male and female parent fish are placed in the net cage to naturally chase each other to spawn and fertilize, and the spawning effect time of the parent fish is 9-10 h; after observing that the gauze cloth in the net cage is covered with dense fertilized eggs, the gauze cloth is collected into another hatching tank for natural hatching with flowing water, and the bubbles existing on the gauze cloth are regularly emptied to prevent the fish eggs from dying of oxygen deficiency.

[0014] More preferably, after the fry hatched in the hatching tank emerge from the membrane, the gauze cloth is taken out so that the fry can swim normally in the hatching tank, and part of the gauze cloth is left to provide an attachment site for the fry; when the fry all have a waist point and can swim back and forth in the water, the brine shrimp is started to be fed to the fry for opening of the mouth.

[0015] Preferably, the specific operation of the artificial dry insemination includes the following steps: pressing the body of the Megalobrama amblycephala with hands, taking the fish out of the water, then wiping the fish clean, patting the fish abdomen for several times, and then extruding the eggs into a basin from top to bottom, and then extruding the sperm of the Pseudobrama simoni into the basin by the same method, and water is not allowed to be brought in during the operation, and the sperm and the eggs are fully mixed and fertilized by stirring clockwise with dry feathers for 1-2 minutes.

[0016] Preferably, the hatching process of the fertilized eggs of the artificial dry insemination is as follows: 1 / 3 of the volume of tap water after aeration is added to the hatching culture dish prepared in advance, and then the fertilized eggs are evenly spread in the culture dish with feathers, 800-1200 fertilized eggs per dish, and the fertilized eggs are fully absorbed and expanded after standing for 20-30 minutes; then the water is changed with new clean pond water, and after the fertilized eggs are fully absorbed and expanded, the culture dish is placed in a dripping water system, and the water is opened for 24-36 hours for hatching, and the temperature is kept at 24-26 DEG C and the water is kept clean, and the white mold dead eggs are removed regularly during the period.

[0017] More preferably, after the fish fry in the hatching culture dish is hatched, it is first transferred to a basin, and when the fish fry all have a waist point and can swim back and forth in the water, the fish fry is started to be fed with brine shrimp for opening.

[0018] Preferably, the feeding includes the following steps: after 3-4 days of feeding with brine shrimp, the fish fry is transferred to a special growth pond prepared in advance for feeding, and fresh soy milk is fed once a day.

[0019] Preferably, the new type of Megalobrama amblycephala and Pseudobrama simoni or Pseudobrama simoni and Megalobrama amblycephala hybrid obtained by the step is selected for shape measurement, flow cytometry DNA content determination, and kidney tissue chromosome ploidy number detection after the fish fry grows for a period of time. The shape characteristic data, DNA content, and chromosome ploidy number and morphology of the hybrid offspring are detected and screened, so that the advantage of the hybrid offspring is analyzed.

[0020] Compared with the prior art, the application has the advantages that:

[0021] 1. The hybridization method of the application is to perform intergeneric distant hybridization between Megalobrama amblycephala and Pseudobrama simoni, successfully combines the high back, fast growth of Megalobrama amblycephala and the slender body, tender meat, and good stress resistance of Pseudobrama simoni, and creatively breeds a new type of Megalobrama amblycephala and Pseudobrama simoni diploid hybrid fish which has a long body, a wide back, better muscle quality, and stronger stress resistance, and is a new type of distant hybridization.

[0022] 2、In order to avoid the death of female fish due to difficult delivery or oviposition disorder during the delivery process, and the situation that the ovum is not mature enough to be normally fertilized in the process of simply using artificial ovum squeezing, the present application adopts the method of "ecological breeding net cage + artificial assisted fertilization" for crossbreeding of distant hybrid fish, which can not only improve the utilization rate of parent fish in the simple ecological breeding method and expand the number of offspring seedlings, but also greatly reduce the damage to the parent caused by artificial fertilization.

[0023] 3、In the past hybrid breeding process of Megalobrama amblycephala and Myxocyprinus asiaticus, due to the large size of adult Myxocyprinus asiaticus, the female and male parents need to be chased in a large area of delivery pool after injection of delivery drugs and cannot be stressed, the parent Myxocyprinus asiaticus used in the present application can be delivered in an indoor ordinary small cement tank after injection of delivery drugs, which can save breeding cost.

[0024] 4、In the past hybrid breeding process of Megalobrama amblycephala and Myxocyprinus asiaticus, especially in the process of artificial fertilization operation of female Myxocyprinus asiaticus parent, the parent is prone to die after ovum squeezing due to strong stress, in order to avoid damage to the parent fish in the hybrid breeding process, the present application selects Myxocyprinus asiaticus which has shorter sexual maturity age, smaller adult size and stronger operation tolerance as hybrid parent, which not only makes the breeding operation process more convenient, but also reduces the damage to the parent Myxocyprinus asiaticus and significantly improves the utilization rate of the parent.

[0025] 5、Crossbreeding of Megalobrama amblycephala with sexual maturity age of 2-3 years and Myxocyprinus asiaticus with sexual maturity age of 1 year can shorten the breeding cycle of the parent and promote the continuation of Megalobrama amblycephala and Myxocyprinus asiaticus germplasm, which has an important promoting effect on the formation of Megalobrama amblycephala and Myxocyprinus asiaticus and Myxocyprinus asiaticus. BRIEF DESCRIPTION OF DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the following embodiment or prior art description will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0027] Figure 1 Structure diagram of ecological breeding net cage;

[0028] Figure 2 Natural ovum fertilization and culture dish hatching method diagram;

[0029] Figure 3 Photo of diploid new Megalobrama amblycephala and Myxocyprinus asiaticus offspring of Megalobrama amblycephala (♀) x Myxocyprinus asiaticus (♂);

[0030] Figure 4 A photo of the offspring of the diploid new type of Misgurn-Culter of Misgurn anguillicaudatus (♀) x Culter alburnus (♂) ;

[0031] Figure 5 A chromosome map of the offspring of the diploid new type of Misgurn-Culter of Misgurn anguillicaudatus (♀) x Culter alburnus (♂) ;

[0032] Figure 6 A chromosome map of the offspring of the diploid new type of Misgurn-Culter of Misgurn anguillicaudatus (♀) x Culter alburnus (♂) ;

[0033] Figure 7 A DNA content map of Misgurn anguillicaudatus;

[0034] Figure 8 A DNA content map of Misgurn anguillicaudatus;

[0035] Figure 9 A DNA content map of the offspring of the diploid new type of Misgurn-Culter of Misgurn anguillicaudatus (♀) x Culter alburnus (♂) ;

[0036] Figure 10 A DNA content map of the offspring of the diploid new type of Misgurn-Culter of Misgurn anguillicaudatus (♀) x Culter alburnus (♂). DETAILED DESCRIPTION

[0037] In order to facilitate the understanding of the present application, the following will make a more comprehensive and detailed description of the present application in combination with the drawings of the specification and the preferred embodiments, but the protection scope of the present application is not limited to the following specific embodiments.

[0038] Unless otherwise defined, all the professional terms used in the following have the same meaning as that generally understood by the person skilled in the art. The professional terms used in the present application are only for the purpose of describing the specific embodiments and are not intended to limit the protection scope of the present application.

[0039] Unless otherwise specified, the various raw materials, reagents, instruments and equipment used in the present application can be purchased from the market or can be prepared by the existing method.

[0040] Embodiment:

[0041] A method for the intergeneric distant hybridization of Misgurn anguillicaudatus and Culter alburnus, comprising the following steps:

[0042] 1) Selection and cultivation of parent fish

[0043] The experimental fish of Megalobrama amblycephala were obtained from the Hunan Fish Genetic Breeding Center in Changsha, Hunan Province, and were artificially bred. The wild Protosalanx hyalinus were obtained from the ecological field of the Hunan Fish Genetic Breeding Center, and were temporarily raised in a large pool. In the first winter, the parent fish with better growth and development were selected and placed in a special pool for intensive breeding. In particular, the parent fish were stimulated once every 2-3 days for one month before the breeding period to promote the development and maturation of the gonads of the female and male parent fish.

[0044] 2) Preparation of ecological breeding net cages

[0045] The ecological breeding net cages used in the hybrid breeding process were "cuboid" type net frames made of aluminum square rods according to the size of the hatching pool area. The structure of the net cage is as follows Figure 1 As shown in the figure, the inside of the net frame was filled with gauze cloth for collecting eggs before the induction of labor.

[0046] 3) Artificial induction of labor

[0047] From the middle of May of the breeding period, when the water temperature of the induction pool was stable at 25-27°C, the parent fish of Megalobrama amblycephala and Protosalanx hyalinus with obvious sexual maturity characteristics and active individuals were selected according to the ratio of 2:1 and placed in the parent temporary pool. The male Megalobrama amblycephala or Protosalanx hyalinus were selected after gently squeezing their genital pores, and the individuals with obvious white semen flowing out were selected. Then the parent fish were injected with a mixed labor-inducing agent of luteinizing hormone releasing hormone analogue (LRH-A) and chorionic gonadotropin (HCG) for labor induction; the dose of luteinizing hormone releasing hormone analogue for female Megalobrama amblycephala was 10 μg / kg, the dose of chorionic gonadotropin was 1000 IU / kg, and the dose of domperidone was 2 mg / kg. The mixed labor-inducing agent was injected at a dose of 0.5 mL per fish, and the injection site was the abdominal cavity at an angle from the depressed part of the base of the pectoral fin without scales. After a period of time after injection of female Megalobrama amblycephala parent fish, male Megalobrama amblycephala was injected for tail chasing. The injection dose of male parent fish was halved based on the dose of female drug, and no domperidone was injected. The dose of luteinizing hormone releasing hormone analogue for female Protosalanx hyalinus was 10 μg / kg, the dose of chorionic gonadotropin was 2000 IU / kg, and the dose of domperidone was 2 mg / kg. Similarly, the mixed labor-inducing agent was injected at a dose of 0.5 mL per fish, and the injection method and one-time injection amount per fish were consistent with those of female Megalobrama amblycephala parent fish; the dose of labor-inducing agent for male Protosalanx hyalinus was half of that of female, and no domperidone was injected. After injection, the parent fish were transferred to the hatching tank where the ecological breeding net cages were placed, and a little gauze was hung on the bamboo pole on the pool to observe the spawning of the parent fish. The induction pool was flushed with running water to promote the production of parent fish.

[0048] 4) Fish egg insemination and hatching

[0049] The fish is induced by the ecological breeding net cage and the artificial dry insemination. The male and female parent fish are placed in the net cage to naturally pursue and lay eggs. When the fertilized eggs are observed on the gauze in the net cage, the gauze is collected and put into a new hatching tank. The bubbles on the gauze are emptied at intervals to avoid the death of the fish eggs due to oxygen deficiency. The parent fish which do not successfully lay eggs are inseminated by the artificial dry method. The method is to press the head of the fish with hand, take the fish out of water, wipe the fish, pat the abdomen of the fish, and then squeeze the eggs into a basin. The sperm of the fish is squeezed into the basin by the same method. The water is not brought into the basin during the operation. The dry feather is used to stir the eggs and sperm clockwise for 1-2 minutes to make the eggs and sperm mix well. The 1 / 3 volume of the prepared pond water is added into the 20cm diameter culture dish. The fertilized eggs are evenly spread in the culture dish. The culture dish is placed for 20-30 minutes to make the eggs absorb water and swell. The culture dish is replaced with new pond water. When the eggs absorb water and swell completely, the culture dish is placed in the dripping water system. The water is opened for 24 hours to hatch the eggs. Figure 2 The ecological breeding net cage and the artificial dry insemination are shown in

[0050] 5) Fish fry cultivation

[0051] When the fish eggs in the hatching tank are observed to be hatched, the gauze is taken out to make the fish fry swim normally in the hatching tank. The fish fry in the glass hatching dish is transferred to the white plastic basin. When the fish fry has the waist point and can swim in the water, the artemia is used to feed the fish fry. The artemia is fed for 3-4 days. The fish fry in the hatching tank and the plastic basin is transferred to the fish fry growth pool which is pre-fertilized. The fish fry in the pool is fed with fresh soy milk once a day. Then the fish fry is fed with the feed. The sinking feed and the floating feed are mixed according to the growth of the fish fry.

[0052] In order to further highlight the effect of reducing parent loss and expanding the number of seedlings by using the method of "ecological breeding net cage + artificial insemination", three groups of parallel repeated experiments are designed to verify the control by taking "Megalobrama (♀) x Procydus primoryensis (♂)" as an example in the present application, and the mortality of parents and the number of hybrid offspring are counted. The specific implementation is as follows: 30 3-year-old mature female Megalobrama with uniform size and body weight of about 1 kg are allocated to three parent labor induction tanks of the same size for labor induction, and 15 male Procydus primoryensis and Megalobrama (for chasing) are put into the tank according to the ratio of 2:1. The three hatching tanks are respectively used for fertilized egg hatching by using "ecological net cage natural hatching", "artificial insemination and egg hatching with gauze window", and "ecological net cage natural hatching + residual egg culture dish static water hatching". The number of fish before and after labor induction is counted and recorded. After hatching, the fish in the hatching tank and the plastic basin are fed for 3-4 days (the fish in the culture dish are moved into the hatching tank for feeding), and the number of fish in the hatching tank and the plastic basin is counted by the water cup method. The statistical results are as shown in Table 1.

[0053] Table 1: Comparison of breeding results of different breeding methods

[0054]

[0055] As can be seen from Table 1, the breeding method of "ecological breeding net cage + artificial insemination" has the smallest loss of parents. At the same time, the number of hybrid offspring obtained by using this breeding method is also increased; the number of fish obtained by using the "artificial insemination" breeding method and the "ecological breeding net cage" breeding method is increased by about 28% and 10% respectively.

[0056] The fish fry is counted by the water cup method in the above implementation, and the specific operation steps are as follows: first, the fish fry collected in the hatching tank is removed with a fine mesh bag, then a specific size of measuring cup is used to pour a cup, the total number of fish fry (water spray) in the cup is calculated, and the total number of fish fry in the hatching tank is calculated according to the number of cups poured from the fine mesh bag.

[0057] After the fish fry is fed for six months, the new type of Megalobrama or Procydus primoryensis and the same age parents are randomly sampled for experimental detection, and the biological shape related data are measured for comparison and analysis. At the same time, the DNA content and the number of chromosomes are detected and screened by flow cytometry DNA content determination method and kidney tissue chromosome number detection method, and it is found that the new type of Megalobrama or Procydus primoryensis obtained by distant hybridization of Megalobrama and Procydus primoryensis is a diploid offspring.

[0058] Table 2: Comparison of countable trait data of Megalobrama, Procydus primoryensis, diploid new type of Megalobrama and Procydus primoryensis (unit: piece or strip)

[0059]

[0060] Note: The capital Roman numerals in the above table represent the number of hard spines; the Arabic numerals represent the number of fin rays.

[0061] The countable trait-related data in Table 2 show that the new hybrid Misgurn and Culter offspring basically integrate the characteristics of their parents (Misgurn anguillicaudatus and Culter alburnus) in appearance, such as the lateral line scales of the new hybrid Misgurn being 54-61, which is between the 50-57 of the female parent Misgurn anguillicaudatus and the 62-70 of the Culter alburnus; but the lateral line scales of the new hybrid Culter being 58-69, which is biased towards the 62-70 of the female parent Culter alburnus. Some countable traits are biased towards the female parent, such as the pelvic fin number of the new hybrid Misgurn and Culter being III+25-27 and III+22-26, respectively, which is biased towards the III+25-27 of the female parent Misgurn anguillicaudatus and the III+22-27 of the female parent Culter alburnus, respectively. In addition, the photos of the two kinds of hybrid offspring fish are shown in Figure 1 、 2 From the appearance, the body height of the new hybrid Misgurn is similar to that of the female parent Misgurn anguillicaudatus; but the head is biased towards the male parent Culter alburnus, which is more smooth. The new hybrid Culter is biased towards the head of the male parent Misgurn anguillicaudatus, and the body length is more like the female parent Culter alburnus, which is more slender and elegant. These can all indicate that the new hybrid Misgurn and Culter are the direct hybrid offspring of Misgurn anguillicaudatus and Culter alburnus.

[0062] Table 3: Comparison of the quantifiable trait data of Misgurn anguillicaudatus, Culter alburnus, diploid new hybrid Misgurn and Culter

[0063]

[0064] The comparison and analysis of the quantifiable traits in Table 3 show that the diploid new hybrid Misgurn and Culter both integrate some trait characteristics of their parents Misgurn anguillicaudatus and Culter alburnus in appearance, and the related data is between the parents, showing obvious hybrid characteristics; such as the ratio of body length to body height of the diploid hybrid Misgurn and Culter being 2.98 and 3.42, respectively, which is between the 2.39 of the parent Misgurn anguillicaudatus and the 4.13 of the parent Culter alburnus. But some characteristics are more biased towards the female parent; such as the ratio of body length to head length of the new hybrid Misgurn and Culter being 4.24 and 3.73, respectively, which is biased towards the 4.42 of the female parent Misgurn anguillicaudatus and the 3.74 of the female parent Culter alburnus, respectively. In addition, the photos of the two kinds of direct hybrid offspring fish are shown in Figure 3 、 4 It can be seen that the body back of the new hybrid Misgurn appears higher like Misgurn anguillicaudatus; but the body type of the new hybrid Culter appears more slender like Culter alburnus.

[0065] The kidney tissue chromosome number detection method was used to detect and screen the chromosomes of the direct hybrid offspring of Misgurn anguillicaudatus and Culter alburnus, and it was found that the new hybrid Misgurn (2n = 48) and the new hybrid Culter (2n = 48) are both diploid offspring. The chromosome maps are shown in Figure 5 、 6 .

[0066] At the same time, with Megalobrama amblycephala and Rutilus arcasii as the control, the DNA content in the blood cells of the tail vein of the diploid Megalobrama amblycephala and Rutilus arcasii was determined by flow cytometry, and the results are shown in Figs. 9, 10 and 11. Figure 7 、 8 Fig. 9 shows the DNA content in the blood cells of the tail vein of the diploid Megalobrama amblycephala and Rutilus arcasii; Fig. 10 shows the DNA content in the blood cells of the tail vein of the diploid Megalobrama amblycephala and Rutilus arcasii; and Fig. 11 shows the DNA content in the blood cells of the tail vein of the diploid Megalobrama amblycephala and Rutilus arcasii. The average DNA content of the diploid Megalobrama amblycephala and Rutilus arcasii was 69.48, 69.12, 68.72 and 67.89, respectively. The ratio of the average DNA content of the diploid Megalobrama amblycephala to that of the control Megalobrama amblycephala was 0.99, which was not significantly different from the theoretical ratio of 1:1. The ratio of the average DNA content of the diploid Rutilus arcasii to that of the control Rutilus arcasii was 0.98, which was not significantly different from the theoretical ratio of 1:1. These experimental results were consistent with the results obtained by the method for detecting the chromosome ploidy number of the kidney.

[0067] The specific operation steps for detecting the DNA content in the blood cells of the tail vein in the above embodiment are as follows: 1) 0.2-0.3 mL of 0.2% heparin sodium (ACD) solution was taken by a sterile disposable syringe with a specification of 1 mL, and the needle was soaked, then about 0.5 mL of blood was taken from the tail vein under 4-5 scales of the tail side line of the experimental fish, and was injected into the first set of Eppendorf tubes prepared in advance, and was labeled. 2) 300 μL of DAPI staining solution was taken by a pipette gun according to the dosage, and was added to the second set of Eppendorf tubes prepared in advance, then 0.5-1 μL of blood was taken from the first set of Eppendorf tubes to the second set of Eppendorf tubes, and was added until the solution was reddish, and was closed and dyed for about 15 min. 3) After the sample was filtered through a special nylon filter with a pore size of 20 μm, the sample was detected by a machine, and was recorded and saved.

[0068] The kidney chromosome preparation method used in the above embodiments has the following specific operation steps: 1) select the new hybrid loach, loach, and its parent Megalobrama amblycephala and Hemiculter leucisculus, etc. experimental fish in good growing condition, active individual, and put them into experimental glass tank for oxygenation cultivation. 2) Inject plant hemagglutinin (PHA) three times and colchicine twice in the chest of the experimental fish, generally inject PHA in the left chest fin, and inject colchicine in the right chest fin. The injection time of the first needle of PHA is about 20:30 in the evening of the same day, and the dose is 10 μg / g of body weight. The injection time of the second needle of PHA is about 8:30 in the morning of the next day, and the dose is 15 μg / g of body weight; immediately inject the first needle of colchicine, and the dose is 4 μg / g of body weight. The injection time of the third needle of PHA is about 11:30 in the morning, and the dose is 6 μg / g of body weight, and then inject the last needle of colchicine, and the dose is still 4 μg / g of body weight. 3) About 1 hour after injecting the last needle of colchicine, start to prepare blood, cut the gill with scissors to bleed for 15 minutes, and change the water once in the middle. 4) Kill the fish and take the kidney in a small culture dish, add a small amount of 0.8% physiological saline to cut the kidney, then use a pipette to suck the paste-like kidney tissue liquid into a 15 mL centrifuge tube. After washing the residual tissue liquid in the culture dish with 1-2 mL of 0.8% physiological saline, add 2 mL of physiological saline to the centrifuge tube, and use a dropper to blow for more than 200 times, so as to disperse the tissue cells. 5) Let the cell liquid stand for 10 minutes, then suck the supernatant into a new 15 mL centrifuge tube, add 0.8% physiological saline to 10 mL, centrifuge at 2000 rpm / min for 5 minutes, and discard the supernatant. 6) Low permeate for 2 hours by adding 0.075 mol / L KCl low permeate liquid, and suck the bottom precipitate every half hour during the low permeation period; centrifuge at 1500 rpm / min for 5 minutes, and discard the supernatant. 7) Add 6 mL of Carnoy's fixing solution (methanol: glacial acetic acid = 3:1) to fix the cells, for 30 minutes, then centrifuge at 1500 rpm / min for 5 minutes, and discard the supernatant. Repeat this step for gradient fixation: 4 mL, 30 min; 2 mL, 15 min. 8) Drop on the ice-coated slide, dry the slide with an alcohol lamp, then dye with the prepared Giemsa dye, the time is determined according to the situation, generally 10-15 min. 9) Finally, observe the chromosomes under a microscope, and make a record of the photographs.

[0069] The diploid hybrid fish of Megalobranchius and Culter is a new type of hybrid fish between Megalobrama and Culter, which combines the high body back of Megalobrama, fast growth rate and the slender body shape of Culter, tender meat quality, good resistance and other points, and creatively breeds a new type of hybrid fish of Megalobranchius and Culter with better muscle quality, better resistance and other hybrid advantages. The new type of Megalobranchius or Culter obtained by the method has the characteristics that the method of "ecological breeding net cage + artificial insemination" is used for breeding distant hybrid fish, which can not only improve the utilization rate of parent fish in the simple ecological breeding method, but also expand the number of offspring seedlings. At the same time, it can greatly reduce the damage to the parent caused by artificial insemination. Secondly, the adult individuals of the parent Culter are small, the character is relatively mild, the resistance is strong, the sexual maturity age is short, so the hybrid preparation process is easy to operate, convenient to collect, and can make the parent less damage or death, can improve the utilization rate of germplasm and shorten the breeding cycle and other advantages. It has important significance and economic value for the future research and production application of fish genetic breeding.

Claims

1. A method of outcrossing between Megalobrama amblycephala and the genus Gobiobotia, characterized in that, The method comprises the following steps: selecting Megalobrama amblycephala and Hemibarbus luna as parent fish to be bred in a special pool, artificially inducing spawning, transferring the parent fish to an ecological breeding net cage prepared in advance in a hatching tank to naturally spawn and fertilize, collecting the net gauze containing the fertilized eggs and placing the gauze into another hatching tank to naturally hatch the fertilized eggs in flowing water; for the parent fish that fail to spawn after the effect period of the induced spawning drug, the method of artificial dry insemination is used to complete the fertilization of fish eggs, and then the fertilized eggs are transferred to a hatching dish to be hatched in still water; after the hatched fish fry are hatched and all have hip points, feeding is started, and then the diploid hybrid offspring of Megalobrama amblycephala and Hemibarbus luna is obtained. The special pool breeding specifically comprises the following steps: selecting female Megalobrama amblycephala or female Hemibarbus luna and male Hemibarbus luna or male Megalobrama amblycephala that are in good growth state, have uniform individual size, are free of injury and disease, and are used as parent fish and are placed in a special pool to be intensively bred, wherein the sexual maturation age of Megalobrama amblycephala is 2-3 years, and the sexual maturation age of Hemibarbus luna is 1 year; the female and male parent fish are stimulated once every 2-3 days in flowing water one month before the breeding period to promote the development and maturation of the gonads of the female and male parent fish. The artificial induced spawning specifically comprises the following steps: in the middle of May of the breeding period, when the water temperature is stable at 25-27 DEG C, the female and male Megalobrama amblycephala and Hemibarbus luna parent fish that are obviously in sexual maturation and are active are selected according to the quantity ratio of 2-3:1 and are placed in a parent fish temporary breeding pool, wherein the male Megalobrama amblycephala or Hemibarbus luna that can be seen to have obvious milky white semen flowing out after being gently squeezed at the genital pore is selected, and then the parent fish is injected with a mixed induced spawning agent to induce spawning; for the female Megalobrama amblycephala, the dose of luteinizing hormone analogue is 8-10 μg / kg, the dose of chorionic gonadotropin is 800-1000 IU / kg, and the dose of domperidone is 1-2 mg / kg; for the female Hemibarbus luna, the dose of luteinizing hormone analogue is 8-10 μg / kg, the dose of chorionic gonadotropin is 1600-2000 IU / kg, and the dose of domperidone is 1-2 mg / kg; the dose of the induced spawning drug for the male Megalobrama amblycephala and Hemibarbus luna parent fish is half of that for the female parent fish, and no domperidone is injected. The fertilized egg hatching process of the natural spawning and fertilization is as follows: the female and male parent fish are placed in the net cage to naturally chase and spawn and fertilize, the parent fish spawning effect time is 9-10 h; after the observation of the dense fertilized eggs on the gauze in the net cage, the gauze is collected and placed in another hatching tank to naturally hatch the fertilized eggs in flowing water, and the bubbles existing on the gauze are regularly emptied; after the hatched fish fry are hatched and emerge from the membrane, the gauze is taken out to enable the fish fry to normally swim in the hatching tank, part of the gauze is left to provide an attachment site for the fish fry, and when the fish fry all have hip points and can swim back and forth in water, the brine shrimp is started to be fed for the fish fry to open their mouths. The artificial dry insemination of the fertilized eggs is carried out as follows: 1 / 3 of the volume of the hatching culture dish is filled with the aerated tap water, and then the fertilized eggs are evenly spread in the culture dish by using a feather, 800-1200 fertilized eggs per dish, and the fertilized eggs are allowed to swell by absorbing water for 20-30 minutes; then the water is replaced with new clean pond water, and after the fertilized eggs are fully swollen, the culture dish is placed in a dripping system, and the water is allowed to flow for 24-36 hours for hatching, and the temperature is kept at 24-26℃, and the water is kept clean, and the dead eggs with white mold are removed regularly during the period.

2. The method of outcrossing of Megalobrama amblycephala to a species of the genus Opsariichthys according to claim 1, characterized in that, The ecological breeding net cage is a "cuboid" type net frame, and the net gauze for collecting eggs is fully laid in the net frame before the production is induced.

3. The method of outcrossing of Megalobrama amblycephala to a species of the genus Opsariichthys according to claim 1, characterized in that, The specific operation of the artificial dry insemination includes the following steps: the fish body of the Megalobrama amblycephala is pressed by hand to hold the genital pore, and then the fish is taken out of the water, and then the fish body is wiped clean, and the abdomen is lightly tapped several times, and the eggs are squeezed into the basin from top to bottom, and then the same method is used to squeeze the sperm of the Pseudobrama simoni into the basin, and water is not allowed to be brought in during the operation process, and the dry feather is used to stir clockwise for 1-2 minutes, so that the sperm and eggs are fully mixed and inseminated.

4. The method of outcrossing of Megalobrama amblycephala to Rasborinus genus according to claim 1, characterized in that, After the fry in the hatching culture dish is hatched, it is first transferred to a basin, and when the fry has a waist point and can swim back and forth in the water, the brine shrimp is used to start feeding the fry.

5. The method of outbreeding of Megalobrama, according to any one of claims 1-4, characterized in that, The feeding includes the following steps: after 3-4 days of feeding with brine shrimp, the fry is transferred to a special growth pond that is pre-fertilized, and the fry is fed once a day with fresh soy milk.

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