A method for efficiently inducing and rapidly identifying homologous tetraploid crucian carp male-nuclear development offspring

Abstract

The present application belongs to the technical field of fish breeding, and discloses a method for efficiently inducing and rapidly identifying homologous tetraploid crucian male-nuclear development offspring, comprising the following steps: inducing parturition of female hybrid crucian and male homologous tetraploid crucian, obtaining hybrid crucian ovum, placing the ovum in ovary liquid, inactivating the ovum through ultraviolet lamp irradiation, stirring and uniformly mixing the ovum with sperm of the homologous tetraploid crucian to make the ovum "fertilized", without chromosome doubling treatment, and obtaining survival offspring after hatching and feeding, wherein a pair of oral papillae in the survival offspring is the male-nuclear development offspring. The hybrid crucian ovum selected by the present application contains a heterologous genome itself, has better compatibility, is beneficial to improving the hatching rate, can efficiently induce homologous tetraploid crucian male-nuclear development, and simultaneously utilizes the appearance characteristic of the male-nuclear development individual inheriting a pair of oral papillae from the father to rapidly identify the male-nuclear development offspring, so that the goal of rapidly identifying homologous tetraploid crucian male-nuclear development offspring is achieved, and the present application has important significance in fish genetic breeding.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of fish cultivation, and particularly relates to a method for efficiently inducing and rapidly identifying homologous tetraploid crucian carp male-nucleus development offspring. BACKGROUND

[0002] Male-nucleus development refers to a special sexual reproduction mode in which an ovum develops by means of male pronucleus. Natural male-nucleus development individuals are rarely found in nature, and are occasionally found in fish hybridization, such as the hybridization of common carp (♀) and grass carp (♂), the hybridization of white crucian carp (♀) and bluegill (♂), and the fertilization of common carp ovum by heterozygous gynogenetic common carp sperm. Artificial male-nucleus development refers to the fertilization of haploid sperm and genetically inactivated ovum, the doubling of chromosomes by inhibiting the first cleavage, and the formation of a diploid individual.

[0003] At present, the research object of fish male-nucleus development is mainly diploid, and the operation process of male-nucleus development is complex, especially the influence of chromosome doubling treatment on embryonic development, which leads to low hatching rate, high fry malformation rate and other problems, and it is difficult to achieve the purpose of batch preparation of artificial male-nucleus development fish. If the diploid sperm produced by tetraploid fish is used for male-nucleus development, the diploid sperm itself contains two sets of chromosomes, and the male-nucleus development diploid fish can be obtained without chromosome doubling treatment, which can avoid the damage caused by chromosome doubling treatment and improve the survival rate of male-nucleus development fish. There are few relevant research reports on tetraploid fish male-nucleus development at home and abroad, the main reason is that it is difficult to obtain fertile tetraploid, especially homologous tetraploid. The diploid sperm produced by tetraploid fish itself contains two sets of chromosomes, and the male-nucleus development induction can obtain male-nucleus development diploid fish without chromosome doubling treatment, which can avoid the damage caused by chromosome doubling treatment and improve the survival rate of male-nucleus development fish.

[0004] The patent document with the patent application number CN200610031790.5 discloses a fish male-nucleus development method, which uses diploid sperm produced by heterologous tetraploid crucian carp as material, irradiates the genetic activity of the nuclear DNA of the inactivated goldfish ovum by ultraviolet rays, and then mixes the diploid sperm with the genetically inactivated ovum, to obtain diploid male-nucleus development fish with male and female fertility without chromosome doubling treatment. Although this method can obtain a certain amount of male-nucleus development fish, the hatching rate ((the number of hatched fry / total number of eggs) x 100%) is 4.1%. In the article published by Zhou Rong et al. in 2015, it is mentioned that a small amount of diploid male-nucleus development fish is obtained by mixing inactivated red crucian carp ovum irradiated by ultraviolet rays and diploid sperm produced by homologous tetraploid crucian carp without chromosome doubling treatment, and the hatching rate is 2.10%. The above two kinds of male-nucleus development fish can produce non-reduction diploid gametes, which is obviously different from ordinary diploid fish which produce haploid gametes, and is a high-quality germplasm resource.

[0005] Although the prior art can obtain androgenesis fish, the hatching rate is too low, and it is difficult to achieve the goal of batch and efficient preparation of androgenesis fish. Therefore, by improving the existing androgenesis method, the bottleneck of low fertilization rate is solved, and a large number of androgenetic offspring are prepared, thereby providing more diploid gamete resources for developing improved fish. SUMMARY

[0006] 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 efficiently inducing and rapidly identifying androgenetic offspring of homologous tetraploid crucian carp.

[0007] To solve the above technical problems, the technical solution provided by the present application is:

[0008] A method for efficiently inducing and rapidly identifying androgenetic offspring of homologous tetraploid crucian carp, comprising the following steps:

[0009] (1) Injecting a female hybrid crucian carp and a male homologous tetraploid crucian carp with a labor-inducing agent to induce labor, and after the female hybrid crucian carp is in estrus, the eggs are squeezed out of the fish body and laid on a culture dish containing ovarian fluid in advance;

[0010] (2) Placing the culture dish containing the hybrid crucian carp eggs obtained in step (1) on a shaker with an ice plate, and inactivating under ultraviolet light irradiation;

[0011] (3) Stirring and mixing the hybrid crucian carp eggs inactivated by ultraviolet light irradiation in step (2) with the sperm of the homologous tetraploid crucian carp to make them "fertilized", without chromosome doubling treatment, and dispersing and laying them in a culture dish containing clean water, and incubating at room temperature in still water, and after hatching, the androgenetic fry obtained is transferred to a pond for breeding, and the individuals with a pair of barbels in the surviving offspring are androgenetic offspring.

[0012] The present application has obtained the world's first example of a sexually fertile homologous tetraploid crucian carp, which has been transmitted to the 18th generation.

[0013] The above method, preferably in step (1), selects female hybrid crucian carp with healthy signs and well-developed gonads. The female hybrid crucian carp is derived from fish distant hybrid breeding, and its eggs contain heterologous genomes, which have better compatibility with foreign genomes than the eggs of purebred fish (such as goldfish and red crucian carp), which is beneficial to improve the hatching rate; compared with goldfish and red crucian carp, hybrid crucian carp has a large amount of eggs and a long spawning cycle, which is beneficial to long-term and batch androgenesis experiments, and more androgenetic offspring can be obtained.

[0014] Preferably, in step (1), the dosage of the injection of the labor-inducing agent is a mixture of chorionic gonadotropin 400-600 IU / kg, luteinizing hormone analogue 6-8 ug / kg, and diethylstilbestrol maleate 1 mg / kg.

[0015] Preferably, in step (1), the ovarian fluid is a liquid obtained by precipitating or filtering the egg granules and liquid squeezed from the cloaca of a natural pure female fish after induced estrus.

[0016] Preferably, in step (1), the male homologous tetraploid crucian carp is a 1-year-old fish.

[0017] Preferably, in step (2), the rotation speed of the shaker is set to 100-110 r / min.

[0018] Preferably, in step (2), the ultraviolet lamp is two 15W ultraviolet lamps, the ultraviolet lamp irradiation time is 180s, and the irradiation distance is 10-12 cm.

[0019] The present application uses the external characteristics to quickly identify the androgenetic offspring, and according to the characteristics that the female H. molitrix has no whiskers and the male homologous tetraploid crucian carp has a pair of whiskers, the androgenetic individual inherits a pair of whiskers from the male parent, and the above individual is detected by a flow cytometer to be diploid, which fully proves that the external characteristics can be used as a method for quickly identifying androgenetic offspring.

[0020] Compared with the prior art, the present application has the following beneficial effects:

[0021] 1. The present application uses ultraviolet irradiation to induce genetic inactivation of H. molitrix eggs to induce homologous tetraploid crucian carp to produce diploid sperm for androgenesis, and the H. molitrix eggs themselves contain heterologous genomes, which have better compatibility with foreign genomes than the eggs of purebred fish, which is beneficial to improve the hatching rate and can efficiently induce homologous tetraploid crucian carp androgenesis.

[0022] 2, The present application uses the androgenesis individual to inherit the pair of oral papilla from the male parent, and quickly identifies the androgenesis offspring, so as to quickly identify the androgenesis offspring of the homologous tetraploid crucian carp, and has important significance in fish genetic breeding. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. 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 any creative effort on the basis of these drawings.

[0024] Figure 1 The flow chart for androgenesis and the appearance of androgenetic fish in the embodiments are shown in the figure;

[0025] Figure 2 The figure of blood cell DNA content of the homologous tetraploid crucian carp induced by the genetically inactivated Carassius auratus gibelio eggs in the embodiments is shown in the figure. DETAILED DESCRIPTION

[0026] In order to facilitate the understanding of the present application, the following will combine the drawings in the specification and the preferred embodiments to make a more comprehensive and detailed description of the present application, but the protection scope of the present application is not limited to the following specific embodiments.

[0027] Unless otherwise defined, all the professional terms used in the following have the same meaning as generally understood by those 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.

[0028] Unless otherwise specified, 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.

[0029] Embodiment:

[0030] A method for efficiently inducing and quickly identifying the homologous tetraploid crucian carp androgenetic offspring, comprising the following steps:

[0031] (1) Artificial induction of labor: in the breeding season, select healthy, well-developed gonad Carassius auratus gibelio, male homologous tetraploid crucian carp, inject 400-600IU / kg of chorionic gonadotropin (HCG), 6-8ug / kg of luteinizing hormone releasing hormone analogue (LRH-A) and 1mg / kg of domperidone (DOM) mixed labor induction agent, the injection dose of male individual is half of that of female, after the female Carassius auratus gibelio is in estrus, the eggs are squeezed out from the fish body and laid in the culture dish containing the ovary fluid in advance, and the ovary fluid just covers the eggs;

[0032] (2) Genetic inactivation of the hybrid crucian carp ovum: a shelf is made with fixed supports, wrapped with black cloth to avoid light, and a low-speed shaker is placed at the bottom, with the shaker speed set to 100 r / min. Two 15W ultraviolet lamps are placed 10-12 cm above the shaker, and the culture dishes in step (1) are placed on the shaker with ice plates, and the ultraviolet lamps are irradiated for 180 s;

[0033] (3) Incubation and feeding: the genetically inactivated hybrid crucian carp ovum in step (2) is thoroughly mixed with the sperm of the homologous tetraploid crucian carp, "fertilized", and dispersed and laid flat in a culture dish containing clean water without chromosome doubling treatment, and incubated at room temperature in still water. After hatching, the androgenetic larvae are transferred to a pond for feeding;

[0034] (4) Identification of androgenetic offspring: according to the characteristics that the female hybrid crucian carp has no whiskers and the male homologous tetraploid crucian carp has a pair of whiskers, the individual with a pair of whiskers in the offspring is the androgenetic individual.

[0035] The androgenetic preparation process and the appearance of the androgenetic fish of the present embodiment are shown in Figure 1 , the red box is the position of the whiskers, and the DNA content of the diploid androgenetic offspring is shown in Figure 2 .

[0036] In addition, the comparison of the hatching rate under different ovary liquid immersion conditions is shown in Table 1.

[0037] Table 1 Comparison of hatching rate under different ovary liquid immersion conditions

[0038] Natural ovarian fluid of the present invention Artificial ovarian fluid of the present invention Hatching rate (%) 27.13；28.09；28.27 5.88；5.99；6.21

[0039] The artificial ovary liquid formula is as follows: 4.11 g BSA per liter, 3.8 mmol Na2HPO4, 118.0 mmol NaCl, 12.7 mmol KCl, 0.7 mmol MgCl2·6H2O, 7 mmol CaCl2, 5.5 mmol tyrosine and 5.5 mmol glycine, and the pH value is adjusted to 8.14.

[0040] It can be seen that the hatching rate of the natural ovary liquid obtained by the present application ((the number of hatched fry / total number of eggs) x 100%) can reach up to 28.27%, but the hatching rate of the artificial ovary liquid is only 6.21% at most. In addition, the artificial ovary liquid has strict requirements on the pH value, and it is difficult to control the accuracy of the pH value in the production practice, which will reduce the quality of the ovum; while the natural ovary liquid obtained by the present application by precipitating or filtering the ovum grains and liquid squeezed from the cloaca does not need to be calibrated for the pH value, and the affinity for ovum is retained. Therefore, the ovary liquid shown by the present application has more obvious hatching advantages.

[0041] Overall, the hatching rate of androgenesis induced by the method can reach more than 28.27%, which is much higher than the hatching rate of diploid sperm androgenesis induced by using genetically inactivated goldfish oocytes to induce heterologous tetraploid crucian carp and using genetically inactivated red crucian carp oocytes to induce homologous tetraploid crucian carp (4.1% and 2.1% respectively); and the androgenetic individuals inherit a pair of barb from the father, which is a morphological characteristic, so the androgenetic offspring can be quickly identified, which can achieve the goal of quickly identifying the androgenetic offspring of homologous tetraploid crucian carp; the method of the embodiment overcomes the problem of low hatching rate of tetraploid fish androgenetic offspring in the prior art, and can efficiently induce homologous tetraploid crucian carp androgenesis and quickly identify the androgenetic offspring, thereby achieving the goal of providing more diploid gamete resources for the development of improved fish, and having important significance in fish genetics and breeding.

Claims

1. A method for efficiently inducing and rapidly identifying homologous tetraploid male gynogenetic offspring of Carassius auratus, characterized in that, It comprises the following steps: (1) Injecting the female hybrid crucian carp and male homologous tetraploid crucian carp with a labor-inducing agent, wherein the labor-inducing agent is a mixture of chorionic gonadotropin 400-600 IU / kg, luteinizing hormone analogue 6-8 μg / kg and maleic acid dihydrotestosterone 1 mg / kg; after the female hybrid crucian carp is in estrus, the eggs of the hybrid crucian carp are squeezed out of the fish body and laid in a culture dish containing ovary fluid in advance; the ovary fluid is obtained by precipitating or filtering the ovum and liquid squeezed out of the cloaca of a natural female fish after the female fish is in estrus after labor induction; (2) Placing the culture dish containing the eggs of the hybrid crucian carp obtained in step (1) on a shaker with an ice plate, and inactivating the eggs by ultraviolet light irradiation; the rotation speed of the shaker is set to 100-110 r / min; the ultraviolet light is two 15 W ultraviolet lamps, the irradiation time of the outer lamp is 180 s, and the irradiation distance is 10-12 cm; (3) Stirring and mixing the inactivated eggs of the hybrid crucian carp in step (2) with the sperm of the homologous tetraploid crucian carp to make the eggs "fertilized", without chromosome doubling treatment, and dispersing and laying the eggs in a culture dish containing clean water, and incubating the eggs at room temperature in still water; after hatching, the obtained androgenetic seedlings are transferred to a pond for breeding, and the individuals with a pair of oral papillae in the surviving offspring are androgenetic offspring.

2. The method of claim 1, wherein, In step (1), the female hybrid crucian carp is selected to be healthy in appearance and to have well-developed gonads.

3. The method of claim 1, wherein, In step (1), the male homologous tetraploid crucian carp is a 1-year-old fish.

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