Method for creating anti-herpes virus double-triploid carp and crucian carp cloning line with growth advantage

Abstract

The invention belongs to the technical field of fish genetic breeding, and particularly relates to a creation method of an anti-herpes virus double-triploid carp and crucian carp cloning line with a growth advantage. The method comprises the following steps: breeding a gynogenesis double-triploid prussian carp female parent and a sexual reproduction double-diploid prussian carp male parent to obtain a filial generation G1, screening male fish from the filial generation G1, and hybridizing the male fish with double-diploid Yellow River carp female fish to obtain a double-triploid prussian carp filial generation G2; female fish in the filial generation G2 is used as a female parent, natural gynogenesis is carried out on the female parent and the male parent Xinguo red carp, a filial generation G3 is obtained, and the G3 is an anti-herpes virus double-triploid carp and crucian carp cloning line. A brand-new breeding route for creating the bitriploid carp and crucian carp cloning line is established, genomes of Yellow River carps, prussian carps and crucian carps are integrated and reconstructed, so that the bitriploid carp and crucian carps form obvious heterosis in blister disease virus resistance and growth speed, and the bitriploid carp and crucian carp cloning line has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of fish genetic breeding, and in particular relates to a method for creating a herpes virus-resistant double triploid carp and crucian carp clone system with growth advantage. Technical Background

[0002] Distant hybridization and polyploidization are important breeding methods. The former can introduce more genes, resulting in greater genetic variation and useful phenotypes, while the latter not only has a buffering effect on hybrid incompatibility but also can lead to parthenogenesis through ploidy changes, which is conducive to the fixation of heterotic traits. Therefore, integrating the advantages of hybridization and polyploidization breeding technologies, developing new and efficient breeding pathways, and creating high-quality synthetic allopolyploid germplasm remain important directions for the development of modern breeding technology.

[0003] At present, more than half of the new aquatic species approved in my country are bred through hybridization or polyploidy breeding technology, but there are very few successful cases of integrating distant hybridization and polyploidy technology to create breakthrough varieties with improved multiple traits such as high yield and high resistance.

[0004] The silver crucian carp (Carassius gibelio) is a key aquaculture species in my country. However, due to the high-density, monoculture practices commonly practiced in major aquaculture areas, a series of diseases have emerged. In particular, acute gill hemorrhagic disease (AHD), caused by infection with the Carassius herpes virus (CaHV), has severely damaged the crucian carp industry. AHD develops rapidly, resulting in the death of all carp within a week of infection. Currently, there is no effective treatment, and prevention remains the primary approach in aquaculture, with limited success.

[0005] The applicant's team's preliminary research found that double triploid silver crucian carp (AAABBB) can undergo parthenogenetic gynogenesis. In this model, the paternal sperm nucleus only triggers the embryonic development of the unreduced silver crucian carp egg nucleus, and does not fuse with the silver crucian carp egg nucleus. Therefore, gynogenesis is usually also a stable "clonal reproduction". However, the team also found that silver crucian carp can occasionally integrate a set of sperm from double diploid crucian carp (AABB) to form double tetraploid males (AAAABBBB) that have restored sexual reproduction ability. Subsequently, backcrossing the double tetraploid males with the double diploid crucian carp can produce a large number of new double triploids (AAABBB) with gynogenesis. Based on this discovery, the applicant applied for a series of patents, among which patent CN115486411B discloses a method for creating a new strain of herpes virus-resistant gynogenetic silver crucian carp. By integrating a set of sexually reproductive amphidiploid crucian carp genomes into the gynogenetic albigenetic silver crucian carp, synthetic tetraploid males with increased ploidy and restored sexual reproductive ability were obtained. The males were then backcrossed with amphidiploid Japanese white crucian carp using their sexual reproductive ability, thereby creating a new triploid population with restored ploidy and integrated with the genomes of silver crucian carp, red crucian carp, and Japanese white crucian carp. However, only about 4.0% of the female individuals in the offspring G3 produced by this method can form a clonal line with extremely strong disease resistance (disease resistance increased by more than 50%), and they need to be screened and confirmed through antiviral experiments. Therefore, the creation efficiency is low and the process is cumbersome. In addition, since the growth rate of amphidiploid Japanese white crucian carp is significantly slower than that of triploid silver crucian carp, the disease-resistant clone line created with amphidiploid white crucian carp as the mother plant generally grows slower than the main silver crucian carp species, making it difficult to promote breeding.

[0006] Therefore, my country's crucian carp breeding industry currently still lacks new crucian carp varieties that have both herpes virus resistance and growth advantages. Summary of the Invention

[0007] In order to solve the problems existing in the prior art, the present invention provides a method for creating a herpes virus-resistant double triploid carp and crucian carp clone system with growth advantage.

[0008] The present invention adopts the following technical solutions:

[0009] A method for creating a herpes virus-resistant double triploid carp and crucian carp clone line with growth advantage comprises the following steps:

[0010] S1. Creation of tetraploid crucian carp: Breeding a gynogenetic triploid silver crucian carp female with a sexually reproductive diploid crucian carp male to obtain progeny G1, and selecting male fish from the G1 progeny to obtain fertile tetraploid male fish;

[0011] S2. Create a population of triploid carp and crucian carp: Use the male fish from the G1 progeny as the male parent and cross it with amphidiploid Yellow River carp females to obtain triploid carp and crucian carp progeny G2;

[0012] S3. Create a double triploid carp and crucian carp clone line: take the female fish in the offspring G2 as the mother, use the sperm of the father Xingguo red carp to stimulate the eggs of the mother, perform natural gynogenesis, and obtain the offspring G3, which is a double triploid carp and crucian carp clone line resistant to herpes virus.

[0013] Preferably, the gynogenetic triploid gibel crucian carp mother in step S1 is the allogynogenetic gibel crucian carp "Zhongke No. 3".

[0014] Preferably, in step S1, the specific method of breeding is: in the crucian carp breeding season, the male semen is mixed with the semen: trypsin solution volume ratio of 1:50, and the semen is treated at 25°C for 15 minutes; the maternal eggs are mixed with the treated semen for fertilization, and the obtained fertilized eggs are continued to hatch; the mass concentration of the trypsin solution is 2%.

[0015] Preferably, in step S1, the method for selecting male fish from the offspring G1 is to squeeze the cloaca of the fish body during the breeding season, and if semen flows out, it is a ditetraploid male fish.

[0016] The beneficial effects of the present invention are:

[0017] Since the amphidiploid species in the genus Carassius auratus, such as white carp and red carp, can still be infected with the Carassius auratus virus, the only difference is the survival rate after infection. More importantly, the growth rate of these amphidiploid carps is significantly slower than that of the main breeding variety of ditriploid silver carp "Zhongke No. 3", so these amphidiploid carp females are not the best mothers for breeding with ditetraploids. The Yellow River carp is the natural amphidiploid that is most distantly related to the Carassius auratus among the Cyprinidae fish, and it has excellent traits such as rapid growth, slender body, and good body color. More importantly, the inventors discovered for the first time that the Yellow River carp is not infected with the Carassius auratus virus. Therefore, this application uses amphidiploid Yellow River carp as the mother parent for the first time, and successfully transfers and fixes the excellent traits of the Yellow River carp to the ditriploid carp and Carassius auratus clone system.

[0018] There are currently some carp and crucian carp hybrids obtained through traditional crossbreeding, but these carp and crucian carp hybrids are generally infertile, so it is impossible to further select and breed these hybrid populations. The double triploid carp and crucian carp provided in this application are not only fertile, but also have restored the reproductive capacity of the parthenogenetic gynogenetics. Therefore, the double triploid carp and crucian carp provided in this application have simultaneously broken through the two traditional cognitions of "triploid sterility" and "carp and crucian carp hybrid sterility". At the same time, almost every double triploid carp and crucian carp female obtained in this application can form a new clone line, so that the clone line with the best traits can be further selected as a candidate new variety.

[0019] This application establishes a breeding route for creating a double triploid carp and crucian carp clonal line. By integrating and reconstructing the genomes of amphidiploid Yellow River carp, amphidiploid silver carp, and amphidiploid crucian carp, the double triploid carp and crucian carp develop hybrid vigor in two important economic traits: growth rate and resistance to herpes virus. At the same time, ploidy change is used to drive reproductive mode conversion, thereby utilizing parthenogenetic reproductive capacity to fix heterosis in the double triploid carp and crucian carp clonal line. Therefore, the breeding route provided by this application not only integrates the advantages of hybridization and polyploidization breeding technologies, but also represents the first successful case of polyploid genome design in animal breeding by manipulating multiple genome reconstructions and converting sexual-parthenogenetic reproductive modes.

[0020] Growth comparison and herpes virus infection experiments showed that the growth rate of the double triploid carp and crucian carp clone system was 1.72 times that of the main breed, the heterologous silver crucian carp "Zhongke No. 3", and its resistance to herpes virus was increased by about 98.2% compared with "Zhongke No. 3", and it was almost not infected with the herpes virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The present application provides a route for creating a herpes virus-resistant double triploid carp and crucian carp clone line with growth advantages.

[0022] Figure 2 By comparing the double triploid silver crucian carp and double diploid Yellow River carp used in this application with the double triploid carp and crucian carp clone system created in this application, it can be seen that the double triploid carp and crucian carp have a slender body, dense and neat scales, no barbels and are closer to crucian carp in appearance; different individuals of the double triploid carp and crucian carp clone system are uniform in size and consistent in appearance.

[0023] Figure 3 The experimental results of the double triploid carp and crucian carp clone line created for this application are resistant to crucian carp herpes virus CaHV.

[0024] Figure 4 The growth experimental results of the double triploid carp and crucian carp clone lines created for this application. DETAILED DESCRIPTION

[0025] For ease of understanding, the technical solution of the present invention is described in more detail below with reference to specific embodiments:

[0026] like Figure 1 As shown, a method for creating a herpes virus-resistant double triploid carp and crucian carp clone with growth advantage includes the following three stages:

[0027] Phase I: Creation of tetraploid crucian carp

[0028] First, the natural gynogenetic reproductive capacity of triploid silver crucian carp is utilized to form unreduced triploid eggs (A3n). These eggs are then fertilized with trypsin-treated, reduced sperm (A1n) from sexually reproducing male amphidiploid crucian carp. Some of the sperm nuclei fuse with the egg nuclei of the silver crucian carp to form tetraploids (A4n) (G1). Male individuals are then selected from the G1, and all male individuals are fertile tetraploid offspring.

[0029] In this stage, the female parent meets the conditions of double triploid silver crucian carp and the male parent meets the conditions of double diploid silver crucian carp. In the embodiment, the double triploid silver crucian carp used isobrious silver crucian carp "Zhongke No. 3", and the specific operation steps of this stage are:

[0030] S1. Trypsinization of Semen: During the breeding season, collect semen by squeezing the cloaca of male amphidiploid crucian carp. Prepare a 2.0% trypsin solution using sperm preservation medium. Mix the semen and trypsin solution at a volume ratio of 1:50 and incubate at room temperature for 15 minutes with constant agitation to dissolve the sperm plasma membrane and permeabilize the nuclear membrane.

[0031] S2. Fertilize trypsin-treated amphidiploid crucian carp sperm with silver crucian carp eggs. During the breeding season, artificially induce spawning in the albigenic silver crucian carp "Zhongke No. 3" to obtain mature eggs. The sperm treated in step S1 is quickly mixed with the eggs and poured into a water-filled porcelain dish for fertilization. A feather is used to agitate the eggs to evenly adhere to the bottom of the dish. After approximately one minute of fertilization, the dish is quickly discarded to prevent residual trypsin from damaging the membrane structure of the fertilized eggs. These fertilized eggs hatch to produce offspring (G1).

[0032] S3. Screening male individuals from the synthetic tetraploid population: During the breeding season, the male individuals are identified as tetraploid individuals by squeezing the cloaca and judging whether or not there is semen.

[0033] The second stage: creating a population of double triploid carp and crucian carp

[0034] Using a double tetraploid male fish (sexual reproduction) as the male parent and a sexually reproducing amphidiploid Yellow River carp as the female parent, a double triploid carp and crucian carp offspring G2 is obtained. In the G2, about 90% of the female individuals regain gynogenetic ability. The specific steps of this stage are:

[0035] Fertilizing sexually reproducing amphidiploid Yellow River carp eggs with sexually reproducing tetraploid crucian carp sperm: During the breeding season, female amphidiploid Yellow River carp with well-developed gonads are selected for artificial induced spawning. Mature amphidiploid Yellow River carp eggs are then dry-fertilized with sperm from male tetraploid crucian carp. Specifically, the eggs and sperm are squeezed into a water-free container, mixed evenly, and then poured into a water-filled porcelain dish for fertilization. A feather is used to agitate the eggs to evenly adhere to the bottom of the dish. The resulting fertilized eggs are then hatched to produce G2 offspring, all of which are triploid.

[0036] The third stage: creation of double triploid carp and crucian carp clone lines

[0037] The female fish of the double triploid common carp and crucian carp G2 was used as the maternal parent and the sperm of Xingguo red common carp was used to stimulate natural gynogenesis to obtain the double triploid offspring G3, which is a double triploid common carp and crucian carp clone line.

[0038] The specific steps for this stage are:

[0039] The eggs of the double triploid common carp and crucian carp were fertilized with the sperm of the Xingguo red carp: During the breeding season, female individuals with well-developed gonads were selected from the double triploid common carp and crucian carp population G2, and artificial induced spawning was performed. The eggs were then dry-fertilized with male Xingguo red carp, and the fertilized eggs were hatched. The hatched offspring G3 were all double triploid common carp and crucian carp clone lines.

[0040] Experiment 1

[0041] Crucian herpes virus infection experiment

[0042] 1. Obtaining triploid carp and crucian carp

[0043] Follow the steps in stages 1-3 above:

[0044] S1. Mature eggs of the allogeneous silver crucian carp "Zhongke No. 3" were fertilized with trypsin-treated amphidiploid crucian carp sperm, yielding approximately 500 fertilized eggs. The fry obtained from these 500 fertilized eggs were raised to sexual maturity, yielding 406 adult fish. Testing revealed that among these 406 adult fish, 24 were ditetraploid males, representing a proportion of 5.91%.

[0045] S2. A female amphidiploid Yellow River carp was selected and fertilized with a male ditetraploid carp to obtain offspring; G2; the egg fertilization rate was 94.8%, and the fertilized egg hatching rate was 92.6%.

[0046] S3. Randomly select one female individual from the G2 generation and breed it with Xingguo red common carp to obtain the offspring group G3, that is, the double triploid common carp and crucian carp clone line.

[0047] 2. Crucian carp herpes virus infection experiment

[0048] The G3 generation double triploid carp and crucian carp clone lines were artificially infected with the crucian carp herpes virus CaHV to verify their antiviral properties. The experimental process is as follows:

[0049] S1. Preparation of fish for challenge experiments: The triploid common carp and crucian carp clones, as well as the control group of heterogeneous silver crucian carp "Zhongke No. 3" and Yellow River carp fry, were raised in the same environment until they reached a size of approximately 20 g. After being brought back to the laboratory, the culture water temperature was gradually transitioned to 23°C (±1°C). The experimental fish were raised for two weeks and observed to confirm that there were no abnormalities.

[0050] S2. Intraperitoneal injection of virus: The three experimental fish were randomly divided into three parallel groups, totaling 9 groups of fish, each with about 20 to 30 fish, and injected simultaneously. The amount of CaHV filtrate injected was 0.1 ml per 10 g body weight, and the amount of virus in the filtrate was 2.9×10 8 Herpesvirus / mL, injected into the abdominal cavity from the base of the pectoral fin.

[0051] S3. Evaluation of disease resistance of each clone: ​​After virus injection, the culture water temperature was maintained at 23°C (±1°C). Water was changed daily to maintain cleanliness, and dead fish were removed promptly. The survival rate of nine fish groups injected intraperitoneally with the virus was calculated daily. The infection period was 21 days. The average survival rate of three parallel groups corresponding to each experimental group was calculated.

[0052] The results are as follows Figure 3 As shown, the average survival rate of the double triploid carp and crucian carp clones was 98.2%, significantly higher than that of the allogeneous silver crucian carp "Zhongke No. 3" (average survival rate 0%). It can be seen that the breeding route provided by this application can create a new crucian carp strain with significant resistance to crucian herpes virus (CaHV).

[0053] Experiment 2

[0054] Growth comparison experiment

[0055] The G3 generation double triploid carp and crucian carp clone line obtained and 1,000 summer flower fry of the same size of the control group albiculture silver crucian carp "Zhongke No. 3" were mixed and released into a pond of about 1 mu. After being raised for 6 months, 100 individuals were randomly selected, and the two strains were distinguished by appearance and weighed separately.

[0056] The results are as follows Figure 4 As shown, the average body weight of the double triploid carp and crucian carp clone line is 259.1g, which is significantly higher than the average body weight of the allogrous silver crucian carp "Zhongke No. 3" (150.6g). The double triploid carp and crucian carp clone line has a significant growth advantage over the allogrous silver crucian carp "Zhongke No. 3".

[0057] The above experiments prove that the breeding route provided by the present invention can create a new crucian carp strain with significant growth advantages, resistance to herpes virus, and maintained female reproductive ability, and is therefore an important way to cultivate new varieties of silver crucian carp.

[0058] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for creating a herpes virus-resistant triploid carp and crucian carp clone system with growth advantage, characterized in that: The following steps are involved: S1. Create a ditetraploid crucian carp: breed a gynogenetic ditriploid silver crucian carp mother with a sexually reproductive didiploid crucian carp father to obtain offspring G1, and select male fish from the offspring G1, which are fertile ditetraploid male fish; S2. Create a population of triploid carp and crucian carp: Use the male fish of the progeny G1 as the father and cross it with the female diploid Yellow River carp to obtain the triploid carp and crucian carp progeny G2; S3. Create a double triploid carp and crucian carp clone line: take the female fish in the offspring G2 as the mother, use the sperm of the father Xingguo red carp to stimulate the eggs of the mother, and perform natural gynogenesis to obtain the offspring G3, which is a double triploid carp and crucian carp clone line resistant to herpes virus.

2. The method for creating a herpes virus-resistant double triploid carp and crucian carp clone system with growth advantage as claimed in claim 1, characterized in that: The female parent of the gynogenetic triploid silver crucian carp in step S1 is the allogynogenetic silver crucian carp "Zhongke No. 3".

3. The method for creating a herpes virus-resistant double triploid carp and crucian carp clone system with growth advantage as claimed in claim 1, characterized in that: In step S1, the specific method of breeding is: in the crucian carp breeding season, the male semen is mixed with the trypsin solution at a volume ratio of 1:50, and the semen is treated at 25°C for 15 minutes; the female eggs are mixed with the treated semen for fertilization, and the obtained fertilized eggs continue to be hatched; the mass concentration of the trypsin solution is 2%.

4. The method for creating a method for creating a herpes virus-resistant double triploid carp and crucian carp clone system with growth advantage as claimed in claim 1, characterized in that: In the step S1, the method for selecting male fish from the offspring G1 is to squeeze the cloaca of the fish body during the breeding season. If semen flows out, it is a ditetraploid male fish.

Images