An efficient and rapid method for producing purebred oyster seeds

Abstract

The present invention discloses an efficient and rapid method for producing pure-line oyster seeds. It uses tetraploid sperm as a male source to induce gynogeny, which solves the problem that the gynogenetic line and the normal fertilized offspring are both diploid and difficult to distinguish or inaccurate to distinguish, and achieves the purpose of identifying gynogenetic offspring only by ploidy detection. Through ultra-low density culture, true gynogenetic offspring adults are obtained, solving the technical problem of the "bottleneck" of the lack of gynogenetic adults of shellfish in the world, and filling the international gap. Through two consecutive generations of gynogenetic technology, the previous status quo that gynogenetic lines could not survive was reversed, and for the first time in the world, genetically identified pure-line shellfish were obtained, prompting my country to reach the international leading level in this field.

Description

Technical Field

[0001] The invention belongs to the technical field of shellfish chromosome group engineering breeding in aquatic animal genetic improvement, and specifically relates to an efficient and rapid oyster pure line seed production method. Background Art

[0002] Pure line, also known as clone, is a general term for a system composed of genetically uniform pure combined individuals. It is generally produced by self-pollination of individuals who are homozygous for all relevant genes, or through long-term continuous inbreeding. Therefore, individuals belonging to the same pure line have the same genotype. The variation they show is the result of environmental effects and will not be inherited by offspring. Only mutations can cause new developments and changes. In fact, it is not easy to breed a pure line with strict scientific standards, so usually only focus on the purity of the relevant gene range, and believe that as long as there is no very obvious variation in other genes, it is considered a pure line.

[0003] For aquatic animals, pure line production has been successful in fish, especially by using 1-2 times of gynogenetics to obtain pure lines. However, there are no reports on pure line research in shellfish breeding. Most reports are about gynogenetic lines that are close to pure lines, but the research on gynogenetic lines of shellfish such as oysters, scallops, and abalone only stays at the embryo or larval stage, and there are no reports on gynogenetic adults. In other words, there are only embryos and sexually mature adults cannot be obtained, which means there is no breeding material, resulting in the inability to make progress in subsequent breeding work, which seriously restricts the breakthrough of oyster breeding technology and the creation of new varieties. To this end, it is necessary to construct a set of shellfish pure line preparation technology to fill the international gap, realize the "cutting" breeding model in crops, promote the zero breakthrough of my country's shellfish breeding technology in this field, and create more "new, special, and unique" new varieties. Summary of the invention

[0004] The purpose of the present invention is to create an efficient and rapid pure line seed production technology for oysters, aiming to cultivate pure line materials that can be used as parents, further carry out genetic breeding research, and provide technical guarantees for its pure line preparation and the utilization of hybrid advantages between pure lines.

[0005] The efficient and rapid pure-line oyster seed production method of the present invention is characterized by comprising the following steps:

[0006] a. Preparation of gynogenetic larvae: firstly, dissect diploid oysters, dissect and obtain eggs, obtain egg liquid, and activate the egg liquid with seawater; irradiate the sperm of tetraploid oysters with ultraviolet light, so that the sperm genetic material is inactivated, but the sperm has the conditions to stimulate fertilization, and obtain inactivated sperm; add the inactivated sperm to the activated egg liquid, and when 30-50% of the fertilized eggs release the first polar body, add cytochalasin B for treatment, and the CB concentration of the fertilized egg liquid is controlled at 0.3-0.5 mg / L, then wash the fertilized eggs, and put them into an incubation container for incubation; the fertilized eggs develop into D-shaped larvae; use flow cytometry to detect the ploidy of the larvae, and the diploids produced are all gynogenetic larvae, the triploids are all larvae with normal fertilization and the second polar body has not been suppressed, and the tetraploids are all larvae with normal fertilization and the second polar body has been suppressed;

[0007] b. Larvae ultra-low density cultivation: Select and breed gynogenetic larvae, and cultivate larvae with a diploid ratio of more than 70%. During the cultivation process, control the feeding to ensure that the larvae are full and not overfed. When the larvae develop to the eye-spot stage, the attachment base is released to collect seedlings to obtain gynogenetic spat;

[0008] c. Progeny culture and identification: After juvenile spats of the gynogenetic system are cultured, reared as juveniles and then cultured as adults, the ploidy of the progeny is identified. Among them, all diploids are gynogenetic progeny, and triploids, tetraploids and aneuploids are eliminated; the identified gynogenetic progeny are cultured;

[0009] d. Establishing a pure line through secondary gynogenesis: when the gonads of the first generation of gynogenetic offspring mature, repeat step a to produce egg fluid; use inactivated tetraploid oyster sperm to fertilize them, and when 30-50% of the fertilized eggs show the first polar body, add cytochalasin B at a concentration of 0.3-0.5 mg / L, wash the fertilized eggs, and put them into an incubation container for incubation; the fertilized eggs develop into D-shaped larvae of the second generation of gynogenetic development; use flow cytometer to detect the ploidy of the larvae, and the diploids produced are all gynogenetic larvae, the triploids are all larvae with normal fertilization and the second polar body has not been suppressed, and the tetraploids are all larvae with normal fertilization and the second polar body has been suppressed; then continue to repeat steps bc to obtain the second generation of gynogenetic offspring, and when they develop to the adult stage, use flow cytometer to screen out the diploids and eliminate individuals of other ploidy, that is, the gynogenetic line of two consecutive generations, that is, the pure line is obtained.

[0010] Preferably, the gynogenetic larvae are prepared as follows: using sperm of tetraploid oysters as a source for activating diploid eggs, first dissecting diploid oysters to screen out individuals with higher egg quality, dissecting to obtain eggs, washing the eggs 2-3 times with a 500-mesh screen silk mesh, then filtering with a 200-mesh screen silk mesh to obtain egg liquid, and activating the egg liquid with seawater for 30-60 minutes; irradiating the sperm of tetraploid oysters with ultraviolet rays to inactivate the sperm genetic material, but having the conditions for stimulating fertilization ability; adding the inactivated sperm to the activated egg liquid, and when fertilization occurs, When 30-50% of the eggs release the first polar body, the dissolved cytochalasin B is added, and the CB concentration of the fertilized egg liquid is controlled at 0.3-0.5 mg / L. After the treatment, the fertilized eggs are washed 1-2 times with a 500-mu silk screen, and placed in an incubation container for incubation; after 16-20 hours, the fertilized eggs develop into D-shaped larvae; the ploidy of the larvae is detected by flow cytometry, and the diploids produced are all gynogenetic larvae, the triploids are all larvae with normal fertilization and the second polar body has not been suppressed, and the tetraploids are all larvae with normal fertilization and the second polar body has been suppressed;

[0011] Preferably, the ultra-low density larval cultivation is as follows: gynogenetic larvae are selected with a 300-mesh silk screen, and the diploid ratio is above 70% for larval cultivation; under normal circumstances, the offspring obtained by gynogenetic development are a few thousandths to a few ten-thousandths of those obtained by normal hatching, so the amount of larvae obtained is generally small, and the cultivation density is controlled at 1 larva / 100-200ml seawater; during the cultivation process, the feeding is controlled to ensure that the larvae can eat enough and not excessive, and the other steps are the same as the conventional operation of oyster larval cultivation; after 15-25 days of cultivation, some larvae develop to the eye point stage, at which time, the attachment base is placed to collect seedlings; after about 5-7 days, the seedlings are collected to obtain gynogenetic spat;

[0012] Preferably, the offspring culture and identification are as follows: after the juvenile spats of the gynogenetic system are cultured, reared as juveniles and then cultured as adults, when the shell height of the offspring grows to 4-5 cm, the obtained offspring are sampled without damage and ploidy is identified, wherein all diploids are gynogenetic offspring, and triploids, tetraploids and aneuploids thereof are eliminated; the identified gynogenetic offspring are cultured according to conventional operations;

[0013] Preferably, the secondary gynogenesis is used to establish a pure line: when the gonads of the first generation of gynogenesis are mature, female individuals with good eggs are selected, and step a is repeated to produce egg fluid; inactivated tetraploid sperm is used to fertilize the female individuals, and when 30-50% of the fertilized eggs have the first polar body, cytochalasin B is added at a treatment concentration of 0.3-0.5 mg / L. After the treatment, the fertilized eggs are washed 1-2 times with a 500-mesh silk screen and then placed in an incubation container; after about 16-20 hours, the fertilized eggs are obtained. The second-generation gynogenetic larvae were obtained; the ploidy of the larvae was detected by flow cytometry, and the resulting diploids were all gynogenetic larvae, the triploids were all larvae with normal fertilization and the second polar body had not been suppressed, and the tetraploids were all larvae with normal fertilization and the second polar body was suppressed; then steps b to c were repeated to obtain the second-generation gynogenetic offspring, and when they developed to the adult stage, the diploids were screened out by flow cytometry, and the individuals with other ploidy were eliminated, that is, the gynogenetic line of two consecutive generations, that is, the pure line was obtained.

[0014] Preferably, the diploid oyster is a diploid Portuguese oyster, and the tetraploid oyster is a tetraploid Crassostrea gigas.

[0015] The present invention uses tetraploid sperm as a male source to induce gynogeny, solving the problem that the gynogenetic line and the normal fertilized offspring are both diploid and difficult to distinguish or inaccurate to distinguish, and achieves the purpose of identifying gynogenetic offspring only by ploidy detection. Through ultra-low density culture, true gynogenetic offspring adults are obtained, solving the "bottleneck" technical problem of the lack of gynogenetic adults in shellfish worldwide, filling the international gap. Through two consecutive generations of gynogenetic technology, the previous status quo that gynogenetic lines could not survive was reversed, and for the first time in the world, pure lines of shellfish that have been genetically identified were obtained, prompting my country to reach the international leading level in this field. As a disruptive technology, the present invention not only provides technical guarantees for pure line seed production, pure line maintenance, and hybridization between pure lines to obtain significant hybrid advantages in shellfish, but also provides valuable scientific research materials for the study of its pure line biological mechanism, and also opens up a new direction for the study of pure line biology of shellfish. DETAILED DESCRIPTION

[0016] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0017] Example 1

[0018] a. Preparation of gynogenetic larvae: In early May 2021, sperm from tetraploid long oysters was used as a source for activating diploid Portuguese oyster eggs. Fifteen diploid Portuguese oysters were dissected, and 8 individuals with higher egg quality were screened out. The eggs were dissected to obtain the eggs, which were washed twice with a 500-mesh silk screen, and then filtered with a 200-mesh silk screen to obtain egg fluid from 8 separate individuals. The egg fluid was activated with seawater for 40 minutes. The sperm of the tetraploid long oyster was exposed to ultraviolet light and the optimal conditions were found (in this embodiment, ultraviolet light was exposed for 3 minutes), that is, the sperm genetic material was inactivated, but the conditions for stimulating fertilization were met. Based on the above conditions, the inactivated sperm was added to the activated Portuguese oyster egg fluid. When the fertilized eggs had 30 -50% release of the first polar body, add dissolved cytochalasin B (CB), control the CB concentration in the fertilized egg solution at 0.5mg / L, treat for 15-20min, wash the fertilized eggs 1-2 times with a 500-mu sieve silk net, and put them into an incubation container for incubation; after about 18h, the fertilized eggs develop into D-shaped larvae; use flow cytometry to detect the ploidy of the larvae, where the diploid ratios are 100%, 100%, 100%, 96.7%, 95.4%, 90.4%, 87.5%, and 76.8%, respectively; where the diploids produced are all gynogenetic larvae, the triploids are all larvae with normal fertilization that have not yet suppressed the second polar body, and the tetraploids are all larvae with normal fertilization that have suppressed the second polar body;

[0019] b. Larvae ultra-low density cultivation: 8 gynogenetic larvae are selected with a 300-mesh silk screen, and the diploid ratio is above 70% for larval cultivation; the cultivation density is controlled at 1 larva / 100-200ml seawater; during the cultivation process, the feeding is controlled to ensure that the larvae can eat enough and not excessive, and the other steps are the same as the conventional operation of oyster larval cultivation; after about 15-25 days of cultivation, some larvae develop to the eye spot stage, at which time, the attachment base is placed for seed collection; after about 5-7 days, the seed collection is completed, and 5 gynogenetic spats are obtained, and the remaining 3 gynogenetic larvae have not yet developed eyespots and have completed metamorphosis;

[0020] c. Progeny culture and identification: Conventional techniques were used for the culture of juvenile shellfish, rearing of young shellfish and cultivation of adults of the five gynogenetic lines. When the shell height of the progeny grew to 4-5 cm, the progeny were sampled without damage and ploidy was identified. The diploid numbers of the five gynogenetic lines were 198, 207, 365, 167 and 129, respectively. During the detection process, triploids, tetraploids and aneuploids were eliminated. The identified gynogenetic progeny were cultured according to conventional operations.

[0021] d. Secondary gynogenetic development to establish pure lines: In late April 2022, when the gonads of the offspring of the five gynogenetic lines mature, three female individuals with good eggs are selected from each gynogenetic line, and step a is repeated to produce egg fluid; inactivated tetraploid long oyster sperm is used to fertilize them, and when 30-50% of the fertilized eggs have the first polar body, cytochalasin B is added at a concentration of 0.3 mg / L. After treatment for 3-5 minutes, the fertilized eggs are washed once with a 500-mesh silk screen and then placed in an incubation container; after about 16 hours, the fertilized eggs are obtained. The second-generation gynogenetic larvae were obtained; the ploidy of the larvae was detected by flow cytometry, and the diploids produced were all gynogenetic larvae, the triploids were all larvae with normal fertilization and the second polar body was not suppressed, and the tetraploids were all larvae with normal fertilization and the second polar body was suppressed; then steps bc were repeated to obtain the second-generation gynogenetic offspring; in late October 2022, the second-generation gynogenetic individuals developed to the adult stage, and the diploids were screened out by flow cytometry, and the individuals with other ploidy were eliminated, that is, the gynogenetic lines of two consecutive generations, that is, the pure lines were obtained. After this operation, a total of 3 pure lines were obtained in the first generation of 5 gynogenetic development lines, and the number of offspring of each pure line was 336, 275, and 565, respectively.

Claims

1. An efficient and rapid method for producing purebred oysters, characterized in that: The following steps are involved: a. Preparation of gynogenetic larvae: firstly, dissect diploid oysters, dissect and obtain eggs, obtain egg liquid, and activate the egg liquid with seawater; irradiate the sperm of tetraploid oysters with ultraviolet light, so that the sperm genetic material is inactivated, but the sperm has the conditions to stimulate fertilization, and obtain inactivated sperm; add the inactivated sperm to the activated egg liquid, and when 30-50% of the fertilized eggs release the first polar body, add cytochalasin B for treatment, and the CB concentration of the fertilized egg liquid is controlled at 0.3-0.5 mg / L, then wash the fertilized eggs, and put them into an incubation container for incubation; the fertilized eggs develop into D-shaped larvae; use flow cytometry to detect the ploidy of the larvae, and the diploids produced are all gynogenetic larvae, the triploids are all larvae with normal fertilization and the second polar body has not been suppressed, and the tetraploids are all larvae with normal fertilization and the second polar body has been suppressed; b. Larvae ultra-low density cultivation: Select and breed gynogenetic larvae, and cultivate larvae with a diploid ratio of more than 70%. During the cultivation process, control the feeding to ensure that the larvae are full and not overfed. When the larvae develop to the eye-spot stage, the attachment base is released to collect seedlings to obtain gynogenetic spat; c. Progeny culture and identification: After juvenile spats of the gynogenetic system are cultured, reared as juveniles and then cultured as adults, the ploidy of the progeny is identified. Among them, all diploids are gynogenetic progeny, and triploids, tetraploids and aneuploids are eliminated; the identified gynogenetic progeny are cultured; d. Establishing a pure line through secondary gynogenesis: when the gonads of the first generation of gynogenetic offspring mature, repeat step a to produce egg fluid; use inactivated tetraploid oyster sperm to fertilize them, and when 30-50% of the fertilized eggs show the first polar body, add cytochalasin B at a concentration of 0.3-0.5 mg / L, wash the fertilized eggs, and put them into an incubation container for incubation; the fertilized eggs develop into D-shaped larvae of the second generation of gynogenetic development; use flow cytometer to detect the ploidy of the larvae, and the diploids produced are all gynogenetic larvae, the triploids are all larvae with normal fertilization and the second polar body has not been suppressed, and the tetraploids are all larvae with normal fertilization and the second polar body has been suppressed; then continue to repeat steps bc to obtain the second generation of gynogenetic offspring, and when they develop to the adult stage, use flow cytometer to screen out the diploids and eliminate individuals of other ploidy, that is, the gynogenetic line of two consecutive generations, that is, the pure line is obtained.

2. The method for producing purebred oyster seeds according to claim 1, characterized in that: The gynogenetic larvae are prepared as follows: using the sperm of tetraploid oysters as a source for activating diploid eggs, first dissecting diploid oysters to screen out individuals with higher egg quality, dissecting to obtain eggs, washing the eggs 2-3 times with a 500-mesh screen silk mesh, then filtering with a 200-mesh screen silk mesh to obtain egg liquid, and activating the egg liquid with seawater for 30-60 minutes; irradiating the sperm of the tetraploid oysters with ultraviolet rays to inactivate the sperm genetic material, but having the conditions for stimulating fertilization ability; adding the inactivated sperm to the activated egg liquid, and when the fertilized eggs have When 30-50% of the first polar body is released, the dissolved cytochalasin B is added, and the CB concentration of the fertilized egg liquid is controlled at 0.3-0.5 mg / L. After treatment, the fertilized eggs are washed 1-2 times with a 500-mu sieve silk mesh and placed in an incubation container for incubation; after 16-20 hours, the fertilized eggs develop into D-shaped larvae; the ploidy of the larvae is detected by flow cytometry, and the resulting diploids are all gynogenetic larvae, the triploids are all larvae with normal fertilization and the second polar body has not been suppressed, and the tetraploids are all larvae with normal fertilization and the second polar body is suppressed.

3. The method for producing purebred oyster seeds according to claim 1, characterized in that: The ultra-low density larval cultivation is as follows: gynogenetic larvae are selected with a 300-mesh screen silk net, and the diploid ratio is above 70% for larval cultivation; the cultivation density is controlled at 1 larva / 100-200 ml of seawater; during the cultivation process, the feeding is controlled to ensure that the larvae can be full and not overfed, and after 15-25 days of cultivation, some larvae develop to the eye point stage, at which time, the attachment base is placed for seed collection; after 5-7 days, the seed collection is completed to obtain gynogenetic spat.

4. The method for producing purebred oyster seeds according to claim 1, characterized in that: The offspring culture and identification are as follows: the gynogenetic spats are cultured as juvenile spats, reared as young spats and cultured as adults. When the offspring shell height grows to 4-5 cm, the obtained offspring are sampled without damage and ploidy is identified, wherein all diploids are gynogenetic offspring, and triploids, tetraploids and aneuploids are eliminated; the identified gynogenetic offspring are cultured according to conventional operations.

5. The method for purebred oyster seed production according to claim 1, characterized in that: The secondary gynogenetic method for establishing a pure line is as follows: when the gonads of the first generation of gynogenetic offspring mature, female individuals with better eggs are selected, and step a is repeated to produce egg fluid; inactivated tetraploid sperm is used to fertilize the female individuals, and when 30-50% of the fertilized eggs have the first polar body, cytochalasin B is added at a treatment concentration of 0.3-0.5 mg / L. After the treatment, the fertilized eggs are washed 1-2 times with a 500-mesh silk screen and then placed in an incubation container; After 16-20 hours, the second generation of gynogenetic larvae were obtained; the ploidy of the larvae was detected by flow cytometry, and the resulting diploids were all gynogenetic larvae, the triploids were all larvae with normal fertilization and the second polar body had not been suppressed, and the tetraploids were all larvae with normal fertilization and the second polar body was suppressed; then steps b to c were repeated to obtain the second generation of gynogenetic offspring, and when they developed to the adult stage, the diploids were screened out by flow cytometry, and the individuals of other ploidy were eliminated, that is, the gynogenetic line of two consecutive generations, that is, the pure line was obtained.

6. The method for purebred oyster seed production according to claim 1, 2, 3, 4 or 5, characterized in that: The diploid oyster is a diploid Portuguese oyster, and the tetraploid oyster is a tetraploid long oyster.