A method for creating a herps virus-resistant double-triploid crucian carp clone with growth advantage

Abstract

The present application belongs to the technical field of fish genetic breeding, and particularly relates to a method for creating a herpes virus-resistant double-triploid crucian carp clone with growth advantage. A female gynogenetic double-triploid gibelion carpio mother is bred with a sexual reproduction double-diploid carassius auratus father to obtain offspring G1, male fish in the offspring G1 is screened and crossed with double-diploid yellow river carp female fish to obtain double-triploid carassius auratus offspring G2; then, female fish in the offspring G2 is used as a mother to naturally gynogenetic with the father Xingguo red carp to obtain offspring G3, and the G3 is a double-triploid carassius auratus clone resistant to herpes virus. The present application establishes a new breeding route for creating a double-triploid carassius auratus clone, and through integration and reconstruction of the genomes of the yellow river carp, the gibelion carpio and the carassius auratus, the double-triploid carassius auratus forms a clear heterosis in the resistance to herpes virus and the growth speed, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of fish genetic breeding technology, specifically relating to a method for creating a double triploid carp-crucian carp clone with growth advantages and resistance to herpesvirus. Background Technology

[0002] Distant hybridization and polyploidization are important breeding techniques. The former can introduce more genes, generate more genetic variation and useful phenotypes, while the latter not only has a buffering effect on hybridization incompatibility, but also may lead to parthenogenesis due to ploidy changes, which will be beneficial for the fixation of heterotic traits. Therefore, integrating the advantages of hybridization breeding and polyploidization breeding technologies, developing new and efficient breeding technology pathways, and creating superior synthetic allopolyploid germplasm remains one of the important directions for the development of modern breeding technology.

[0003] Silver carp ( Carassius gibelio Silver crucian carp is one of the important farmed aquatic fish species in my country, but due to the high-density and monoculture farming methods commonly practiced in major silver crucian carp farming areas, a series of diseases have broken out. In particular, diseases caused by crucian carp herpesvirus (Cercospora spp.) have become more prevalent. Ca Acute gill hemorrhage disease caused by HV infection has caused extremely serious damage to the crucian carp industry. Acute gill hemorrhage disease has a very rapid onset, with all crucian carp dying within a week of infection. Currently, there is no effective treatment, and prevention remains the primary approach in aquaculture, but with limited success.

[0004] The applicant team's previous research found that the gynogenesis of the triploid crucian carp (AAABBB) can be performed, in which the paternal sperm nucleus only triggers the unreduced egg nucleus of the crucian carp to develop into an embryo without fusion with the egg nucleus, and thus the gynogenesis is usually a stable "clonal reproduction". However, the team also found that the crucian carp can occasionally integrate a set of diploid common carp (AABB) sperm to form a restored sexually reproducing tetraploid male (AAAABBBB). Subsequently, the tetraploid male is crossed with the diploid common carp to produce a large number of gynogenetic new triploid (AAABBB). Based on this finding, the applicant has applied for a series of patents, in which the patent CN115486411B discloses a method for creating a new gynogenetic crucian carp strain resistant to herpes virus, which integrates a set of sexually reproducing diploid common carp genome into the gynogenetic heteromict crucian carp to obtain a synthetic tetraploid male fish with increased ploidy and restored sexual reproduction ability, and then uses the sexual reproduction ability to cross with a diploid Japanese white crucian carp to create a new triploid population with restored ploidy and integrated crucian carp, red crucian carp and Japanese white crucian carp genomes. However, only about 4.0% of the female individuals in the G3 offspring produced by the method have a strong disease resistance ability (disease resistance ability improved by more than 50%), and need to be screened and confirmed by anti-virus experiments, so the creation efficiency is low and the process is complicated. In addition, since the growth rate of the diploid Japanese white crucian carp is significantly slower than that of the triploid crucian carp, the disease-resistant clonal line created by using the diploid white crucian carp as the female parent is generally slower than the crucian carp main breeding variety in growth rate, and is difficult to promote.

[0005] Therefore, there is still a lack of new crucian carp varieties with both herpes virus resistance and growth advantages in the current crucian carp breeding industry in China. SUMMARY

[0006] In order to solve the problems in the prior art, the present application provides a method for creating a herpes virus-resistant triploid crucian carp clonal line with growth advantages.

[0007] The present application adopts the following technical solutions:

[0008] A method for creating a herpes virus-resistant triploid crucian carp clonal line with growth advantages, comprising the following steps:

[0009] S1. Creating a tetraploid crucian carp: breeding a female gynogenetic triploid crucian carp with a male sexually reproducing diploid crucian carp to obtain offspring G1, and screening male fish from the offspring G1 to obtain fertile tetraploid male fish;

[0010] S2. Creating a triploid crucian carp population: crossing the male fish in the offspring G1 with a female diploid yellow river carp to obtain triploid crucian carp offspring G2;

[0011] S3. Establishing a diploid triploid carp crucian clone: taking female fish in the offspring G2 as the female parent, stimulating the ovum of the female parent with the sperm of the male parent Xingguo red carp, performing natural gynogenesis to obtain offspring G3, and the G3 is a diploid triploid carp crucian clone with anti-herpes virus.

[0012] Preferably, the gynogenetic diploid triploid crucian carp female parent in step S1 is the artificial crucian carp "Zhongke 3".

[0013] Preferably, in step S1, the specific method of breeding is as follows: in the breeding season of crucian carp, the sperm of the male parent is mixed with the trypsin solution at a volume ratio of 1:50, and the sperm is treated at 25 DEG C for 15 min; the ovum of the female parent is mixed with the treated sperm for fertilization, and the obtained fertilized egg is further hatched; the mass concentration of the trypsin solution is 2%.

[0014] Preferably, in step S1, the method for screening male fish from the offspring G1 is as follows: in the breeding season, the cloaca of the fish is squeezed, and if sperm flows out, the fish is a diploid tetraploid male fish.

[0015] The present application has the following beneficial effects:

[0016] Since the diploid species in the genus Carassius, such as white crucian carp and red crucian carp, can still be infected with the carp pox virus, only the survival rate after infection is different. More importantly, the growth rate of these diploid crucian carp is significantly slower than that of the diploid triploid crucian carp main breeding variety "Zhongke 3", so the diploid crucian carp female is not the best female parent for breeding with the diploid tetraploid. The yellow river carp is the most distant natural diploid in the family Cyprinidae from the genus Carassius, and it has excellent traits such as rapid growth, slender body shape and good body color. More importantly, the inventors have found for the first time that the yellow river carp is not infected with the carp pox virus. Therefore, the present application first uses the diploid yellow river carp as the female parent, and successfully transfers and fixes the excellent traits of the diploid yellow river carp to the diploid triploid carp crucian clone.

[0017] At present, there are some carp crucian hybrids obtained by traditional cross breeding, but these carp crucian hybrids are generally sterile, so further breeding of these hybrid populations cannot be carried out. The diploid triploid carp crucian provided by the present application is not only fertile, but also restores the ability of single female gynogenesis. Therefore, the diploid triploid carp crucian provided by the present application breaks through the traditional cognition of "triploid sterility" and "carp crucian hybrid sterility". At the same time, each female diploid triploid carp crucian obtained by the present application can form a new clone, so that the clone with the most excellent traits can be selected as a candidate new variety.

[0018] The application establishes a breeding route for creating a double-triploid crucian carp clone, which integrates and reconfigures the genomes of double-diploid yellow river carp, double-triploid gibel carp and double-diploid crucian carp, so as to form heterosis of double-triploid crucian carp in growth rate and anti-herpes virus in two important economic traits, and to use the change of ploidy to drive the conversion of reproductive mode, so as to fix the heterosis in the double-triploid crucian carp clone by using the ability of parthenogenetic reproduction. Therefore, the breeding route provided by the application not only integrates the advantages of hybridization and polyploid breeding technology, but also is the first successful case of polyploid genome design by manipulating multi-genome reconfiguration and sexual-parthenogenetic reproductive mode conversion in animal breeding.

[0019] The growth comparison and herpes virus infection experiment shows that the growth rate of the double-triploid crucian carp clone is 1.72 times that of the main breeding variety, gibel carp "Zhongke No.3", and the anti-herpes virus ability is increased by about 98.2% compared with "Zhongke No.3", and the double-triploid crucian carp clone is almost not infected with herpes virus. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The application provides a method for creating a double-triploid crucian carp clone with growth advantage and anti-herpes virus.

[0021] Figure 2 For the comparison of the double-triploid gibel carp, double-diploid yellow river carp and the double-triploid crucian carp clone created in the application, it can be seen that the double-triploid crucian carp is slender in body shape, the scales are dense and uniform, there is no barbel, and the appearance is closer to crucian carp; the sizes of different individuals of the double-triploid crucian carp clone are uniform, and the appearances are consistent.

[0022] Figure 3 The double-triploid crucian carp clone created in the application is resistant to crucian herpes virus Ca The results of the HV experiment.

[0023] Figure 4 The growth experiment results of the double-triploid crucian carp clone created in the application. DETAILED DESCRIPTION

[0024] In order to facilitate understanding, the technical solutions of the application will be described in more detail in combination with specific embodiments:

[0025] As shown in the following Figure 1 A method for creating a double-triploid crucian carp clone with growth advantage and anti-herpes virus, including the following three stages:

[0026] The first stage: creating double-quadruple crucian carp

[0027] Firstly, the natural ability of gynogenesis of the triploid Carassius auratus is used to form the non-reduced triploid eggs (A3n), and then the eggs are fertilized with the reduced sperm (A1n) of the trypsin-treated sexually-reproducing Carassius auratus male, in which part of the sperm nucleus fuses with the egg nucleus of Carassius auratus to form the double tetraploid (A4n) (G1). Then male individuals are selected from the G1, and the male individuals are all double tetraploid fertile offspring.

[0028] In this stage, the female parent meets the condition of the triploid Carassius auratus, and the male parent meets the condition of the diploid Carassius auratus. In the example, the triploid Carassius auratus uses the heterozygous Carassius auratus “Zhongke No. 3”, and the operation steps in this stage are as follows:

[0029] S1. Sperm is treated by trypsin: in the breeding season, sperm is collected by squeezing the cloaca of the diploid Carassius auratus male, and a trypsin solution with a mass concentration of 2.0% is prepared by using a sperm preservation solution. Then, the sperm is mixed with the trypsin solution at a volume ratio of 1:50, and is treated at room temperature for 15 min, during which the sperm membrane is continuously shaken to dissolve the nuclear membrane.

[0030] S2. The trypsin-treated diploid Carassius auratus sperm is fertilized with Carassius auratus eggs: in the breeding season, mature eggs are obtained after artificial induction of the heterozygous Carassius auratus “Zhongke No. 3”, and the sperm treated in step S1 is quickly mixed with the eggs and poured into a porcelain dish with water for fertilization. During the process, the fertilized eggs are evenly attached to the bottom of the porcelain dish by stirring with a feather. After about 1 minute of water fertilization, the water in the porcelain dish is quickly poured out to avoid the residual trypsin continuing to damage the membrane structure of the fertilized eggs. The fertilized eggs are hatched to obtain offspring (G1).

[0031] S3. Male individuals are selected from the synthetic double tetraploid population: in the breeding season, the cloaca is squeezed to determine whether it is a double tetraploid male individual according to the presence or absence of sperm.

[0032] Second stage: creation of a double triploid Carassius auratus population

[0033] The double tetraploid male fish (sexual reproduction) is used as the male parent, and the sexually-reproducing diploid Carassius auratus is used as the female parent to breed the double triploid Carassius auratus offspring G2, in which about 90% of the female individuals regain the ability of gynogenesis. The operation steps in this stage are as follows:

[0034] Fertilization of the ovum of the sexual reproduction diploid yellow river carp with the sperm of the sexual reproduction tetraploid crucian carp: in the breeding season, select the diploid yellow river carp female fish with well-developed gonads for artificial induction of labor, and fertilize the mature ovum of the diploid yellow river carp with the sperm of the tetraploid crucian carp, specifically: simultaneously squeeze the ovum and the sperm into a waterless container, stir uniformly, then pour into a porcelain dish with water for fertilization, during which, stir with a feather to make the fertilized eggs evenly adhere to the bottom of the porcelain dish. The obtained fertilized eggs are hatched to obtain offspring G2, which are all diploid triploid.

[0035] Third stage: creation of diploid triploid carp crucian clone line

[0036] Using the female fish in the diploid triploid carp crucian G2 as the female parent, stimulate it to undergo natural gynogenesis using Xingguo red carp sperm to obtain diploid triploid offspring G3, which is a diploid triploid carp crucian clone line.

[0037] The specific operation steps of this stage are as follows:

[0038] Fertilization of the ovum of the diploid triploid carp crucian with the sperm of the Xingguo red carp: in the breeding season, select female individuals with well-developed gonads from the diploid triploid carp crucian population G2 for artificial induction of labor, then fertilize the ovum with the sperm of the Xingguo red carp male fish, the obtained fertilized eggs are hatched, and the hatched offspring G3 are all diploid triploid carp crucian clone lines.

[0039] Experiment 1

[0040] Crucian herpes virus infection experiment

[0041] 1. Obtaining of diploid triploid carp crucian

[0042] Refer to the steps of stages 1-3 above:

[0043] S1. Fertilize the mature ovum of the hybrid crucian carp "Zhongke No. 3" with the trypsin-treated diploid crucian sperm, a total of about 500 fertilized eggs are obtained; the fish fry obtained by hatching 500 fertilized eggs are raised to sexual maturity, and 406 adult fish are obtained, of which 24 are diploid tetraploid male individuals, accounting for 5.91%.

[0044] S2. Select one diploid yellow river carp female fish and one diploid tetraploid male fish for fertilization to obtain offspring G2; the ovum fertilization rate is 94.8%, and the fertilized egg hatching rate is 92.6%.

[0045] S3. Randomly select 1 female individual from G2 and breed with Xingguo red carp to obtain offspring population G3, which is a diploid triploid carp crucian clone line.

[0046] 2. Crucian herpes virus infection experiment

[0047] The obtained G3 generation double-triploid carp crucian clone line was subjected to herpesvirus of crucian Ca The artificial infection experiment of HV was carried out to verify the anti-virus characteristics, and the experimental process was as follows:

[0048] S1. Preparation of fish to be attacked: the double-triploid carp crucian clone line and the fish fry of the control group of the hybrid crucian carp "Zhongke No. 3" and the yellow river carp were bred to the size of about 20 g under the same environment, and after being taken back to the laboratory, the water temperature was gradually transitioned to 23℃ (±1℃), and the fish were bred for two weeks and observed to confirm that the experimental fish were normal.

[0049] S2. Intraperitoneal injection of virus: the three groups of experimental fish were randomly divided into 3 parallel groups, a total of 9 groups of fish, about 20-30 tails per group, and injection was simultaneously performed. Ca The amount of HV filtrate was 0.1 ml per 10 g of body weight, and the virus amount in the filtrate was 2.9 x 10 8 Individuals of herpesvirus / mL, and the injection was performed from the base of the pectoral fin to the abdominal cavity.

[0050] S3. Evaluation of the anti-disease ability of each clone line: after the injection of the virus, the water temperature was maintained at 23℃ (±1℃), and the water was changed every day to keep the water clean, and the dead individuals were fished out in time. The survival rate of the 9 groups of fish after intraperitoneal injection of the virus was counted every day, and the infection period was 21 days. The average survival rate of the corresponding 3 parallel groups of each experimental group was calculated.

[0051] The results are shown in Figure 3 The average survival rate of the double-triploid carp crucian clone line was 98.2%, which was significantly higher than that of the hybrid crucian carp "Zhongke No. 3" (the average survival rate was 0%). It can be seen that the breeding route provided in the present application can create a new strain of crucian with a significant advantage of resisting herpesvirus of crucian (HV). Ca

[0052] Experiment 2

[0053] Growth comparison experiment

[0054] The obtained G3 generation double-triploid carp crucian clone line and the control group of the hybrid crucian carp "Zhongke No. 3" were mixed and put into a pond of about 1 mu, and after being bred for 6 months, 100 individuals were randomly selected, the two strains were distinguished by shape, and then weighed.

[0055] The results are shown in Figure 4 The average weight of the double-triploid carp crucian clone line was 259.1 g, which was significantly higher than the average weight of the hybrid crucian carp "Zhongke No. 3" (150.6 g), and the double-triploid carp crucian clone line had a significant growth advantage over the hybrid crucian carp "Zhongke No. 3".

[0056] ​The above experiment proves that the breeding route provided by the application can create a new strain of crucian carp with significant growth advantage, anti-sheath virus capacity and also maintaining the ability of gynogenesis, and thus is an important way to cultivate new varieties of silver crucian carp.

[0057] The above embodiments are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that any modifications, equivalent replacements and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A method for creating an anti-herpesvirus diploid carp crucian clonal line with growth advantage, characterized in that, The method comprises the following steps: S1. Creating double tetraploid crucian carp: breeding a female gynogenetic double triploid crucian carp female parent and a sexual reproduction double diploid crucian carp male parent to obtain offspring G1, screening male fish from the offspring G1, and the male fish is a fertile double tetraploid male fish; S2. Creating a double triploid crucian carp group: using the male fish in the offspring G1 as a male parent to cross with a double diploid yellow river carp female fish to obtain double triploid crucian carp offspring G2; S3. Creating a double triploid crucian carp clone line: using a female fish in the offspring G2 as a female parent, stimulating the ovum of the female parent by using a male parent Xingguo red crucian carp sperm, performing natural gynogenesis, obtaining offspring G3, and the G3 is a double triploid crucian carp clone line against herpes virus.

2. A method for the production of an anti-herpesvirus diploid carassius auratus clone line with growth advantage according to claim 1, characterized in that, The gynogenetic double triploid crucian carp female parent in the step S1 is a heterotic crucian carp "Zhongke No. 3".

3. A method for the production of an anti-herpesvirus diploid carassius auratus clone line with growth advantage according to claim 1, characterized in that, In the step S1, the specific method of breeding is as follows: in the breeding season of the crucian carp, the male parent sperm is mixed with a trypsin solution at a volume ratio of 1:50, the sperm is treated at 25 DEG C for 15 min; the ovum of the female parent is mixed with the treated sperm for fertilization, and the obtained fertilized egg is further hatched; the mass concentration of the trypsin solution is 2%.

4. A method for the production of a bi-triploid carassius auratus × cyprinus carpio clonal line with growth advantage against herpes virus according to the method for the production of claim 1, characterized in that, In the step S1, the method for screening the male fish from the offspring G1 is as follows: in the breeding season, the cloaca of the fish is squeezed, and if the sperm flows out, the fish is a double tetraploid male fish.

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