Method for producing tetraploid oysters with different shell colors through interspecific hybridization

Abstract

The invention provides a method for producing tetraploid oysters with different shell colors through interspecific hybridization. The method comprises the following steps: firstly, selecting Fujian oysters and crassostrea gigas with common shell color, golden shell color and black shell color and excellent growth traits as parents, and respectively obtaining triploid and tetraploid individuals through a chemical induction technology; and then carrying out continuous 2-4 generations of population breeding to obtain Fujian oyster and crassostrea gigas tetraploid populations with stable ploidy and different shell color characteristics. Then, the tetraploid populations are hybridized to obtain hybrid tetraploid oysters with different shell colors, and the hybrid tetraploid oysters have three shell colors including a common shell color, a gold shell color and a black shell color. The produced tetraploid oysters have high growth speed, high ploidy stability and high survival rate, and excellent germplasm resources can be provided for the triploid oyster industry. The method not only improves the cultivation benefits of the oysters, but also has wide industrial application prospects. The method combines the technologies of selective breeding, polyploid breeding, cross breeding and the like, breaks through the limitation of a traditional breeding method, has higher breeding efficiency and application potential, is particularly suitable for oyster breeding in coastal areas of China, and promotes the oyster industry to be transformed and upgraded to a high-end consumer market.

Description

Technical Field

[0001] The invention belongs to the technical field of aquaculture breeding, and particularly relates to a method for producing tetraploid oysters with different shell colors through interspecific hybridization. Background Art

[0002] The primary aquaculture species in northern my country, Crassostrea gigas, also known as the Pacific oyster, is native to Northeast Asia and primarily distributed along the coast north of the Yangtze River. Crassostrea gigas boasts rapid growth, strong environmental adaptability, nutrient-rich nutrition, high growth and survival rates, and delicious meat. A tolerant of both salinity and temperature, it is a globally important commercial shellfish.

[0003] The main aquaculture species in the southern oyster industry is the Fujian oyster (C. angulata), which is characterized by its fast growth rate, strong heat tolerance, and year-round fertility. However, the cultivation of these two oysters presents several challenges. For example, while the long oyster grows quickly, grows large, and is resistant to transportation, it is not resistant to high temperatures and is prone to mass mortality in summer. The Fujian oyster, on the other hand, is not resistant to long-distance transportation, has a brittle shell, and is significantly smaller than the long oyster. Therefore, to address the long oyster's poor heat tolerance and the Fujian oyster's small size, hybridization between the long oyster and the Fujian oyster is being explored, resulting in a new strain with faster growth and greater heat tolerance.

[0004] Due to the release of gametes during the oyster breeding season, diploid oysters lose their soft parts and reduce their meat quality in the summer, making them difficult to market. Triploid oysters, due to their high sterility, rapid growth, and rich nutrition, have attracted much attention in the industry and have become a key component of oyster breeding. Furthermore, due to their poor fertility, triploid oysters can be marketed year-round, thus filling the summer market gap left by diploid oysters. Currently, there are two main methods for obtaining triploid oysters. One is to induce triploids from diploid oysters. However, this method suffers from high larval mortality, unstable ploidy of the triploid population, high toxicity of the inducer, and complex operation. The second method is to first induce tetraploid oysters from triploid oysters, and then produce triploids by hybridizing tetraploid oysters with diploid oysters. This method produces 100% triploid offspring and is simple to operate, making it suitable for large-scale production of triploid oysters. Furthermore, tetraploid oysters can produce fertile offspring through self-reproduction, allowing for continuous expansion and maintaining high-quality germplasm. Tetraploid oysters are crucial to the triploid oyster industry. Cultivating tetraploid strains with excellent economic traits plays a crucial role in promoting the development of the triploid oyster industry. Hybrid breeding techniques can quickly combine the superior traits of both parental lines, resulting in high-quality tetraploid germplasm resources. This provides an effective means of acquiring high-quality tetraploid germplasm resources. Therefore, cultivating hybrid tetraploid oysters with superior economic traits is imperative.

[0005] At the same time, with changing market demand for oysters and the continuous expansion of aquaculture, oyster quality is receiving increasing attention. The shell is a key factor influencing oyster quality, with its shape and color directly determining its characteristics. A regular shell shape and attractive shell color not only enhance the oyster's added value but also increase consumer purchasing intent. Oyster shell color is polymorphic, a trait determined by genetic factors and correlated with other economic traits. Therefore, in order to further enhance the genetic improvement of tetraploid oysters and increase market recognition of high-quality tetraploid oysters, it is particularly important to cultivate tetraploid oysters with different shell colors. Summary of the Invention

[0006] The object of the present invention is to provide a method for producing tetraploid oysters with different shell colors through interspecific hybridization. This method can be used to produce on a large scale hybrid tetraploid oysters with significant shell color characteristics and obvious advantages in growth rate and survival rate, thereby providing excellent tetraploid germplasm resources for the triploid oyster industry.

[0007] The present invention provides a method for producing tetraploid oysters with different shell colors through interspecific hybridization, comprising the following steps:

[0008] 1) A wild diploid population of Fujian oysters was used as a screening population. Oysters with regular shell shape, distinct left and right shell colors (gold and black), and ranking in the top 10%-15% in shell height and total weight were selected as breeding parents. This resulted in a diploid breeding population G0 of Fujian oysters with different shell colors. Fertilized eggs were treated with cytochalasin B to induce triploid Fujian oysters. The triploid Fujian oysters and the Fujian oyster breeding diploid population G1 were then used as parents. Fertilized eggs were treated with cytochalasin B to induce a basic tetraploid population G0 of Fujian oysters.

[0009] 2) Using the Fujian oyster tetraploid basic population G0 described in step 1) as a parent, oyster individuals with regular shell shape, distinct left and right shell colors (gold and black), and shell height and total weight ranking in the top 10% to 15% were selected as breeding parents. After three consecutive generations of population selection, the Fujian oyster normal shell color tetraploid population G3, Fujian oyster gold shell tetraploid G3, and Fujian oyster black shell tetraploid G3 were obtained.

[0010] 3) Using the diploid population of the new variety of long oyster "Haida No. 1" (GS-01-005-2013) as the basic population, the fertilized eggs were treated with cytochalasin B to induce the long oyster "Haida No. 1" triploid. Then, using the triploid long oyster "Haida No. 1" as the parent, the fertilized eggs were treated with cytochalasin B to induce the long oyster "Haida No. 1" tetraploid basic population G0.

[0011] 4) Using the tetraploid base population G0 of the long oyster "Haida No. 1" described in step 3) as a parent, oyster individuals ranked in the top 10%-15% in shell height and total weight were selected as breeding parents. After two consecutive generations of population selection, the long oyster "Haida No. 1" tetraploid G2 with normal shell color characteristics was obtained.

[0012] 5) Using the diploid population of the new variety of long oyster "Haida No. 2" (GS-01-007-2016) as the basic population, the fertilized eggs were treated with cytochalasin B to induce the long oyster "Haida No. 2" triploid. Then, using the triploid long oyster "Haida No. 2" as the parent, the fertilized eggs were treated with cytochalasin B to induce the long oyster "Haida No. 2" tetraploid basic population G0 with shell gold characteristics.

[0013] 6) Using the tetraploid base population G0 of the long oyster "Haida No. 2" described in step 5) as the parent, oyster individuals with golden left and right shell colors and ranking in the top 10%-15% in shell height and total weight were selected as breeding parents. After four consecutive generations of population selection, the long oyster "Haida No. 2" tetraploid G4 with black shell characteristics was obtained.

[0014] 7) Using the diploid population of the new variety of long oyster "Haida No. 3" (GS-01-007-2018) as the basic population, the fertilized eggs were treated with cytochalasin B to induce the long oyster "Haida No. 3" triploid. Then, using the triploid long oyster "Haida No. 3" as the parent, the fertilized eggs were treated with cytochalasin B to induce the long oyster "Haida No. 3" tetraploid basic population G0.

[0015] 8) Using the tetraploid base population G0 of the long oyster "Haida No. 3" described in step 7) as the parent, select oyster individuals with black left and right shell colors and shell height and total weight ranking in the 10%-15% as breeding parents. After four consecutive generations of population selection, the long oyster "Haida No. 3" tetraploid G4 is obtained.

[0016] 9) The Fujian oyster tetraploid G3 with different shell colors described in step 2) and the long oyster tetraploid with different shell colors described in steps 4), 6), and 8) are used as parents, and oyster individuals with regular shell shapes and distinct shell colors are selected as breeding parents. Long oysters and Fujian oyster tetraploids with the same shell color characteristics are paired and hybridized by incomplete diallel hybridization to cultivate tetraploid hybrid oysters with different shell colors.

[0017] 10) Optimization of related steps: The Fujian oysters and long oysters used as parents are all one-year-old oysters, the number of Fujian oysters used for screening is 2000-3000, and the number of parents used for breeding is 200-300; the number of long oysters and Fujian oysters used for induction is 200-300; the total weight of the parent individuals used for hybridization in step 9) is 30-45 g; the concentration of cytochalasin B used in the operation of inducing triploid and tetraploid Fujian oysters and long oysters is 0.5 mg / L, and treatment is started when 20%-30% of the fertilized eggs appear the first polar body under microscopic observation, and treatment is continued. The treatment time is 15-20 min; after the induction is completed, the D-shaped larvae hatched from the fertilized eggs are screened by flow cytometry to obtain triploid Fujian oysters and long oysters with a ploidy of 3N and tetraploid Fujian oysters and long oysters G0 with a ploidy of 4N; the parents used in step 9) are measured for ploidy before fertilization, and two sampling methods of hemolymph and gill filaments are adopted for each individual. Individuals with a ploidy of 4N in both measurements are selected as hybrid parents, and the number of parents used for the two hybrid combinations of each shell color is 100; after fertilization, hybrid tetraploid oysters with different shell colors are obtained by cultivation.

[0018] The present invention provides a method for producing tetraploid oysters with different shell colors based on interspecific hybridization between long oysters and Fujian oysters. The method integrates breeding technologies such as selective breeding, polyploid breeding and hybrid breeding. The shell color, shell height and total weight of Fujian oysters are used as target traits. A population breeding method is adopted to carry out the breeding of fast-growing lines of Fujian oysters with different shell colors. Then, the fast-growing lines are used to induce Fujian oyster tetraploids with different shell colors and carry out population self-propagation. Different long oyster varieties are used as base populations to induce long oyster tetraploids with different shell colors and carry out population self-propagation. Finally, the self-propagated offspring of long oysters and Fujian oyster tetraploids with different shell color combinations are hybridized, thereby producing tetraploid hybrid oysters with different shell colors on a large scale. The tetraploid oyster populations with ordinary shell color / shell gold / shell black produced by this method have the advantages of pure shell color, fast growth rate, and high survival rate. They are very suitable for aquaculture in my country's coastal areas and have broad application prospects and huge promotion potential. Moreover, as an important parent source for the triploid industry, the triploid offspring produced by hybridizing tetraploid oysters with different shell colors will have a signature shell color, faster growth rate and survival rate, thereby providing a new triploid option for the high-end oyster consumer market, increasing its visibility, thereby promoting the faster and better transformation and upgrading of my country's oyster industry to the high-end consumer market, and promoting the sustainable, healthy and green development of my country's oyster aquaculture industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 To develop a technical roadmap for producing tetraploid oysters with different shell colors based on interspecific hybridization between Crassostrea gigas and Crassostrea fujianensis;

[0020] Figure 2 The growth of hybrid tetraploid oysters with different shell colors in different sea areas;

[0021] Figure 3 Survival status of hybrid tetraploid oysters with different shell colors in sea areas;

[0022] Figure 4 Peak detection diagram for tetraploid Crassostrea gigas and Crassostrea fujianensis parents and hybrid tetraploid oysters with different shell colors; DETAILED DESCRIPTION

[0023] The present invention is further described below with reference to specific examples, but is not intended to limit the present invention.

[0024] Example 1

[0025] A method for producing hybrid tetraploid oysters with normal shell color comprises obtaining excellent tetraploid long oysters and tetraploid Fujian oysters, selecting parents, cultivating parents, artificial insemination and hatching, and larval cultivation.

[0026] 1) Obtaining tetraploid Fujian oyster (AAAA):

[0027] (a) Selection of a G0 population of standard-shelled Fujian oysters: 8,000 wild first-year-old Fujian oysters were collected, and 2,600 individuals with uniform shells were selected. The top 10% by weight of 260 individuals were selected as breeding broodstock, weighing 60-80 g and measuring 5-6 cm in shell length. D-type larvae were obtained through artificial insemination and reared in indoor cement tanks until eyed larvae, using scallop shells as a substrate. When the juvenile oysters reached 5-6 mm, they were transferred to a natural sea area for growout. Finally, a further generation of populations was selected using the same breeding objectives, seedling rearing techniques, and growout methods to obtain a G1 population of standard-shelled Fujian oysters.

[0028] (b) Obtaining the tetraploid basic population G0 of ordinary shell-colored Fujian oysters: ① Triploid induction: 100 ordinary shell-colored Fujian oysters with mature gonads were dissected to select the diploid population G1 parents, and females (135) and males (150) were separated under a microscope. Sperm and eggs were obtained from 120 pairs of males and females with good gonads. Sperm was first filtered through a 160-mesh sieve and then a 500-mesh sieve to remove impurities. Eggs were first filtered through a 120-mesh sieve and then a 300-mesh sieve. Before fertilization, the eggs were continuously rinsed with a 500-mesh sieve to remove tissue fluid, and then seawater was added for 30-60 minutes of maturation. Ten pairs of oysters were paired as a group for fertilization, and 5 to 10 sperm were controlled around each egg during fertilization; when 20% to 30% of the fertilized eggs developed the first polar body, 0.5 mg / L cytochalasin B was immediately added and the fertilized eggs were treated for 20 minutes, and then soaked in seawater (containing 1% dimethyl sulfoxide) for 30 minutes; the eggs were then collected from each group of fertilized eggs and placed in an indoor cement pool for hatching and larval cultivation. The induction rate of D-shaped larvae triploidy was detected by flow cytometry to be above 80%. The larvae were cultivated in the indoor cement pool until they became eye-spot larvae, and scallop shells were placed as attachment bases; when the oyster spat grew to 5 to 6 mm, they were moved to the natural sea area for culture. ② Tetraploid induction: One-year-old triploids of common-shell Fujian oysters were dissected, and female triploid individuals with mature gonads were selected. 190 individuals with ploidy of 3N were detected by flow cytometry. 120 individuals with well-developed eggs were selected as female parents through microscopic observation. 200 one-year-old common-shell Fujian oysters were dissected to select diploid population parents, and 120 individuals with full gonads and strong sperm motility were selected as male parents. Sperm were first filtered through a 160-mesh sieve and then a 500-mesh sieve to remove impurities. Eggs were first filtered through a 120-mesh sieve and then a 160-mesh sieve. Before fertilization, the eggs were continuously rinsed with a 500-mesh sieve to remove tissue fluid, and then seawater was added. The fertilization process lasted 60-90 minutes. Ten pairs of oysters were paired for fertilization, with 5-10 sperm surrounding each egg. When the first fertilized egg developed its first polar body, 0.5 mg / L cytochalasin B was added for 15 minutes, followed by a 30-minute soak in seawater (containing 1% dimethyl sulfoxide). The eggs were then collected from each group and placed in indoor cement tanks for hatching and larval rearing. Flow cytometry was used to determine if the tetraploid induction rate was above 50%. Larvae were reared in the cement tanks until eyed larvae were formed, and scallop shells were added as a substrate. When the juvenile oysters reached 5-6 mm, they were transferred to a natural sea area for growout. Approximately 3,000 first-year-old tetraploid individuals of the standard-shelled Fujian oyster were obtained in the sea area.

[0029] (c) Self-propagation of tetraploid population G0 of ordinary shell-colored Fujian oysters: During the oyster breeding season, the tetraploid population G0 of ordinary shell-colored Fujian oysters was induced as the basic population, and the oyster individuals ranked 10% to 15% in terms of shell height and total weight were selected as breeding parents; 100 female and 100 male individuals with ploidy 4N were screened by flow cytometry, and sperm and eggs were squeezed out respectively. The sperm was first filtered through a 160-mesh screen and then through a 500-mesh screen to remove impurities, and the eggs were first filtered through a 120-mesh screen to remove impurities. The eggs were then filtered through a 160-mesh silk screen. During fertilization, each egg was surrounded by 5-10 sperm. The fertilized eggs were then collected and placed in an indoor cement pool for incubation. When the fertilized eggs developed into D-type larvae, the larvae were collected from the entire pool and placed in a new, disinfected cement pool at a larval density of 1-2 per ml. When the larvae reached the eye point, scallop shells were added as a substrate. When the oyster spat reached 5-6 mm, they were transferred to a natural sea area for growout. The same method was used to self-propagate the tetraploid population for three consecutive generations, resulting in the production of the Fujian oyster tetraploid G3, which has a normal shell color and a stable ploidy, with growth advantages.

[0030] 2) Obtaining tetraploid long oysters (GGGG):

[0031] (a) Obtaining the tetraploid basic population G0 induced by inducing normal shell-colored long oysters: ① Triploid induction: 300 long oysters "Haida No. 1" with mature gonads were dissected and separated into females (132) and males (168) under a microscope; 120 pairs of males and females with better gonads were selected to obtain sperm and eggs, and the sperm were first filtered through a 160-mesh screen and then a 500-mesh screen to remove impurities, and the eggs were first filtered through a 120-mesh screen and then a 300-mesh screen; before fertilization, the eggs were continuously rinsed with a 500-mesh screen to remove tissue fluid, and then seawater was added for 30-60 minutes of maturation; ten pairs of oysters were paired as a group. The eggs were fertilized in groups, with 5 to 10 sperm surrounding each egg during fertilization. When 20% to 30% of the fertilized eggs developed the first polar body, 0.5 mg / L cytochalasin B was immediately added and the fertilized eggs were treated for 18 minutes, and then soaked in seawater (containing 1% dimethyl sulfoxide) for 30 minutes. The fertilized eggs of each group were then collected and placed in an indoor cement pool for hatching and larval culture. The induction rate of D-shaped larvae triploidy was detected by flow cytometry to be above 80%. The larvae were cultured in the indoor cement pool until they became eye-spot larvae, and scallop shells were placed as attachment bases. When the oyster spat grew to 5 to 6 mm, they were moved to the natural sea area for culture. ②Tetraploid induction: One-year-old triploid long oysters "Haida No. 1" were dissected, and female triploid individuals with mature gonads were selected. 150 individuals with ploidy of 3N were detected by flow cytometry. 120 individuals with well-developed eggs were selected as female parents through microscopic observation; 210 one-year-old Fujian oysters with normal shell color were dissected to select diploid group parents, and 120 individuals with full gonads and strong sperm motility were selected as male parents; sperm were first filtered through a 160-mesh screen and then a 500-mesh screen to remove impurities, and eggs were first filtered through a 120-mesh screen and then a 160-mesh screen; eggs were continuously rinsed with a 500-mesh screen to remove tissue fluid before fertilization, and then sea cucumber was added. The water was matured for 60-90 minutes. Ten pairs of oysters were paired for fertilization, with 5-10 sperm surrounding each egg. When the first fertilized egg developed its first polar body, 0.5 mg / L cytochalasin B was added for 15 minutes, followed by a 30-minute soak in seawater (containing 1% dimethyl sulfoxide). The eggs were then collected from each group and placed in indoor cement tanks for hatching and larval rearing. Flow cytometry was used to determine if the tetraploid induction rate was above 60%. Larvae were reared in the indoor cement tanks until eyed larvae were formed, and scallop shells were added as a substrate. When the juvenile oysters reached 5-6 mm, they were transferred to a natural sea area for growout. Approximately 2,000 induced tetraploid individuals of the one-year-old long oyster, "Haida No. 1," were obtained from these growouts.

[0032] (b) Self-propagation of tetraploid population G0 of ordinary shell-colored long oysters: During the oyster breeding season, the long oyster "Haida No. 1" induced tetraploid G0 was used as the basic population, and oyster individuals ranked 10% to 15% in shell height and total weight were selected as breeding parents; 100 female and male individuals with ploidy 4N were screened by flow cytometry, and sperm and eggs were squeezed out respectively. The sperm was first filtered through a 160-mesh screen and then through a 500-mesh screen to filter out impurities, and the eggs were first filtered through a 120-mesh screen. The eggs are then filtered through a 160-mesh silk screen. During fertilization, each egg is surrounded by 5-10 sperm. The fertilized eggs are then collected and placed in an indoor cement pool for incubation. When the fertilized eggs develop into D-type larvae, the larvae are collected from the entire pool and placed in a new, disinfected cement pool for incubation at a density of 1-2 larvae / ml. When the larvae develop to the eye point, scallop shells are added as a substrate. When the oyster spat grow to 5-6 mm, they are moved to a natural sea area for growout. The same method was used to self-propagate the tetraploid population for two consecutive generations, resulting in the "Haida No. 1" tetraploid G2, a long oyster with growth advantages and stable ploidy.

[0033] 3) Obtaining tetraploid hybrid oysters (GGAA, AAGG):

[0034] Obtaining normal shell color hybrid tetraploid oysters: 150 normal shell color Fujian oyster tetraploid G3 individuals and 150 long oyster "Haida No. 1" tetraploid G2 individuals with regular shell shape were selected, and 50 normal shell color Fujian oyster tetraploid G3 males and 50 normal shell color Fujian oyster tetraploid G2 females were obtained by flow cytometry detection and microscope observation for standby use; and 50 normal shell color Fujian oyster tetraploid G3 males and 50 normal shell color Fujian oyster tetraploid G2 females were obtained by the same method for standby use; by using normal shell color Fujian oyster tetraploid G3 males as males and long oyster "Haida No. 1" tetraploid G2 females as females The male individual of tetraploid G2 of long oyster "Haida No. 1" was used as the female parent and the female individual of tetraploid G3 of Fujian oyster with normal shell color was used as the female parent and the female individual of tetraploid G3 of Fujian oyster with normal shell color was used as the female parent and the female individual of tetraploid G2 of long oyster "Haida No. 1" was used as the male parent and the female individual of tetraploid G3 of Fujian oyster with normal shell color was used as the female parent and the female individual of tetraploid G2 ... 2 Hatching was carried out in cement pools. The entire artificial insemination and hatching process used filtered and sterilized seawater, maintained at a temperature of 24-25°C and a salinity of 30‰. Sufficient oxygen was maintained during the hatching process. The fertilized eggs were hatched at a density of 50 eggs / ml. After 26 hours of development, they developed into D-type larvae, which were then collected and obtained using a 300-mesh silk screen. The initial D-type larvae were cultured at a density of 10 eggs / ml, at a temperature of 24-25°C and a salinity of 30‰. Golden algae were used as the starter bait, with a daily feeding density of 5×10 3 / mL~2×10 4 The larvae were fed 4 to 6 times. As the larvae grew, the feeding amount was gradually increased. When the larvae shell length reached 150 μm to 200 μm, the larvae density was adjusted to 5 per milliliter and an appropriate amount of Chlorella vulgaris feed was added. The daily feeding density of golden algae reached 4×10 4 / mL~8×10 4 / mL, feed them 4 to 6 times, and when they develop to the eyed larvae stage, add scallop shell-attached seedlings and temporarily raise them in cement ponds for 30 to 45 days before raising them in the sea.

[0035] Example 2:

[0036] A method for producing shell-gold hybrid tetraploid oysters comprises obtaining excellent tetraploid long oysters and tetraploid Fujian oysters, selecting parents, cultivating parents, artificial insemination and hatching, and larval cultivation.

[0037] 1) Obtaining tetraploid Fujian oyster (AAAA):

[0038] (a) Selection of the Golden Fujian Oyster Population G0: 8,000 wild first-year-old Fujian oysters were collected, and 1,400 individuals with regular shell shapes and golden shells on both sides were selected. The top 15% by weight of 210 individuals were selected as breeding broodstock, weighing 60-80 g and measuring 5-6 cm in shell length. D-type larvae were obtained through artificial insemination and reared in indoor cement tanks until eyed larvae, using scallop shells as a substrate. When the juvenile oysters reached 5-6 mm, they were transferred to the natural sea for growout. Finally, a further generation of populations was selected using the same breeding objectives, seedling rearing techniques, and growout methods to obtain the Golden Fujian Oyster Diploid Population G1.

[0039] (b) Obtaining the tetraploid basic population G0 of Crassostrea gigas: ① Triploid induction: 100 Crassostrea gigas with mature gonads were dissected to select the diploid population G1 parents, and 160 females and 160 males were separated under a microscope. Sperm and eggs were obtained from 80 pairs of male and female individuals with good gonads. Sperm was first filtered through a 160-mesh sieve and then a 500-mesh sieve to remove impurities, and eggs were first filtered through a 120-mesh sieve and then a 300-mesh sieve. Before fertilization, the eggs were continuously rinsed with a 500-mesh sieve to remove tissue fluid, and then added with seawater for 30-60 minutes of maturation. Ten pairs of oysters were paired as a group for fertilization, and 5 to 10 sperm were controlled around each egg during fertilization; when 20% to 30% of the fertilized eggs developed the first polar body, 0.5 mg / L cytochalasin B was immediately added and the fertilized eggs were treated for 20 minutes, and then soaked in seawater (containing 1% dimethyl sulfoxide) for 30 minutes; the eggs were then collected from each group of fertilized eggs and placed in an indoor cement pool for hatching and larval cultivation. The induction rate of D-shaped larvae triploidy was detected by flow cytometry to be above 80%. The larvae were cultivated in the indoor cement pool until they became eye-spot larvae, and scallop shells were placed as attachment bases; when the oyster spat grew to 5 to 6 mm, they were moved to the natural sea area for culture. ②Tetraploid induction: One-year-old triploid shelled golden oysters were dissected, and female triploid individuals with mature gonads were selected. 300 individuals with ploidy of 3N were detected by flow cytometry. 150 individuals with well-developed eggs were selected as female parents through microscopic observation. 380 one-year-old golden shelled golden oysters were dissected to select diploid group parents, and 150 individuals with full gonads and strong sperm motility were selected as male parents. Sperm were first filtered through a 160-mesh screen and then a 500-mesh screen to remove impurities. Eggs were first filtered through a 120-mesh screen and then a 160-mesh screen. Before fertilization, the eggs were continuously rinsed with a 500-mesh screen to remove tissue fluid, and then seawater was added for filtration. The fertilization process lasted 60-90 minutes. Ten pairs of oysters were paired for fertilization, with 5-10 sperm surrounding each egg. When the first fertilized egg developed its first polar body, 0.5 mg / L cytochalasin B was added for 20 minutes, followed by a 30-minute soak in seawater (containing 1% dimethyl sulfoxide). The eggs were then collected from each group and placed in indoor cement tanks for hatching and larval rearing. Flow cytometry was used to determine if the tetraploid induction rate was above 50%. Larvae were reared in the cement tanks until eyed larvae were formed, and scallop shells were added as a substrate. When the juvenile oysters reached 5-6 mm, they were transferred to a natural sea area for growout. Approximately 4,500 first-year-old tetraploid individuals of Crassostrea gigas were obtained from these growouts.

[0040] (c) Self-propagation of the tetraploid population G0 of the Kejin Fujian oyster: During the oyster breeding season, the Kejin Fujian oyster tetraploid population G0 was used as the basic population, and oyster individuals ranking 10% to 15% in terms of shell height and total weight were selected as breeding parents. 150 female and 150 male individuals of 4N ploidy were screened by flow cytometry, and sperm and eggs were squeezed out respectively. The sperm were first filtered through a 160-mesh screen and then a 500-mesh screen to remove impurities, and the eggs were first filtered through a 120-mesh screen and then a 160-mesh screen to remove impurities. During the fertilization process, 5-10 sperm were controlled to surround each egg. The eggs were then collected and placed in an indoor cement pool for incubation. When the fertilized eggs developed into D-type larvae, the larvae were collected from the entire pool and placed in a new, disinfected cement pool for cultivation at a larval cultivation density of 1 to 2 per ml. When the larvae developed to the eye point, scallop shells were added as a substrate. When the oyster spat grew to 5 to 6 mm, they were moved to the natural sea area for aquaculture. The same method was used to self-propagate the tetraploid population for three consecutive generations, thus producing the tetraploid G3 of the Fujian oyster Crassostrea gigas with growth advantages and stable ploidy.

[0041] 2) Obtaining tetraploid long oysters (GGGG):

[0042] (a) Obtaining the tetraploid basic population G0 of Crassostrea gigas induced by induction: ① Triploid induction: 300 Crassostrea gigas "Haida No. 2" parents with mature gonads were dissected and separated into 125 females and 175 males under a microscope. Sperm and eggs were obtained from 100 pairs of males and females with good gonads. Sperm was first filtered through a 160-mesh sieve and then a 500-mesh sieve to remove impurities. Eggs were first filtered through a 120-mesh sieve and then a 300-mesh sieve. Before fertilization, eggs were continuously rinsed with a 500-mesh sieve to remove tissue fluid and then matured in seawater for 30-60 min. Ten pairs of oysters were paired as a group for fertilization, and 5 to 10 sperm were controlled to surround each egg during fertilization. When 20% to 30% of the fertilized eggs developed the first polar body, 0.5 mg / L cytochalasin B was immediately added and the fertilized eggs were treated for 15 minutes, and then soaked in seawater (containing 1% dimethyl sulfoxide) for 30 minutes. The eggs were then collected from each group of fertilized eggs and placed in an indoor cement pool for hatching and larval cultivation. The induction rate of D-shaped larvae triploidy was detected by flow cytometry to be above 80%. The larvae were cultivated in the indoor cement pool until they became eye-spot larvae, and scallop shells were placed as attachment bases. When the oyster spat grew to 5 to 6 mm, they were moved to the natural sea area for culture. ②Tetraploid induction: Dissect one-year-old triploid long oysters "Haida No. 2", select female triploid individuals with mature gonads, and use flow cytometry to detect 200 individuals with ploidy of 3N. Through microscopic observation, select 160 individuals with well-developed eggs as female parents; dissect 200 one-year-old golden Fujian oysters to select diploid group parents, and select 160 individuals with full gonads and strong sperm motility as male parents; sperm are first filtered through a 160-mesh sieve and then a 500-mesh sieve to remove impurities, and eggs are first filtered through a 120-mesh sieve and then a 160-mesh sieve; before fertilization, the eggs are continuously rinsed with a 500-mesh sieve to remove tissue fluid, and then seawater is added. The fertilization process lasted 60-90 minutes. Ten pairs of oysters were paired for fertilization, with 5-10 sperm surrounding each egg. When the first fertilized egg developed its first polar body, 0.5 mg / L cytochalasin B was added for 15 minutes, followed by a 30-minute soak in seawater (containing 1% dimethyl sulfoxide). The eggs were then collected from each group and placed in indoor cement tanks for hatching and larval rearing. Flow cytometry was used to determine if the tetraploid induction rate was above 60%. Larvae were reared in the cement tanks until eyed larvae were formed, and scallop shells were added as a substrate. When the juvenile oysters reached 5-6 mm, they were transferred to a natural sea area for growout. Approximately 2,500 induced tetraploid individuals of the one-year-old "Haida No. 2" oyster were obtained from this growout.

[0043] (b) Self-propagation of tetraploid population G0 of golden long oyster: During the oyster breeding season, the tetraploid G0 induced by long oyster "Haida No. 2" was used as the basic population, and oyster individuals ranked 10% to 15% in shell height and total weight were selected as breeding parents; 150 female and male individuals with ploidy of 4N were screened by flow cytometry, and sperm and eggs were squeezed out respectively. Sperm was first filtered through a 160-mesh screen and then through a 500-mesh screen to filter out impurities, and eggs were first filtered through a 120-mesh screen and then through a 120-mesh screen. The eggs were filtered through a 160-mesh silk screen. During fertilization, each egg was surrounded by 5-10 sperm. The fertilized eggs were then collected and placed in an indoor cement pool for incubation. When the fertilized eggs developed into D-type larvae, the larvae were collected from the entire pool and placed in a new, disinfected cement pool for incubation at a density of 1-2 larvae / ml. When the larvae reached the eye point, scallop shells were added as a substrate. When the oyster spat reached 5-6 mm, they were moved to a natural sea area for growout. The same method was used to self-propagate the tetraploid population for four consecutive generations, resulting in the "Haida No. 3" tetraploid G4, a long oyster with growth advantages and stable ploidy.

[0044] 3) Obtaining the Shell-Gold Hybrid Tetraploid Oyster (GGAA, AAGG):

[0045] Acquisition of Shell-Gold Hybrid Tetraploid Oysters: 100 Shell-Gold Fujian oyster tetraploid G3 individuals with regular shell shape and 100 long oyster "Haida No. 2" tetraploid G4 individuals were selected, and 50 male and female Shell-Gold Fujian oyster tetraploid G3 individuals were obtained by flow cytometry detection and microscopic observation for standby use; and 50 male and female long oyster "Haida No. 2" tetraploid G4 individuals were obtained by the same method for standby use; a hybrid combination (GGGAA) was set up with the Shell-Gold Fujian oyster tetraploid G3 male individual as the male parent and the long oyster "Haida No. 2" tetraploid G4 female individual as the female parent for hybridization, and the long oyster "Haida No. 2" was hybridized. A tetraploid G4 male ("No. 2") was used as the male parent, and a tetraploid G3 female ("Kejin Fujian oyster") was used as the female parent, creating a hybrid combination (GAAGG). Specific details included filtering sperm through a 160-mesh sieve and then a 500-mesh sieve to remove impurities, while eggs were filtered through a 120-mesh sieve and then a 300-mesh sieve. During artificial insemination, each egg was surrounded by 3-5 sperm. After fertilization, the fertilized eggs were evenly distributed into 20m2 cement pools for incubation. Filtered and sterilized seawater was used throughout the artificial insemination and incubation process, maintained at a temperature of 24-25°C and a salinity of 30‰, and sufficient oxygen was maintained during incubation. The hatching density of fertilized eggs was 50 eggs / ml. After 26 hours of development, the D-type larvae were collected and obtained using a 300-mesh silk screen. The early D-type larvae were cultured at a density of 10 eggs / ml, the culture temperature was 24-25°C, the salinity was 30‰, and golden algae were used as the starter bait. The daily feeding density was 5×10 3 / mL~2×10 4The larvae were fed 4 to 6 times. As the larvae grew, the feeding amount was gradually increased. When the larvae shell length reached 150 μm to 200 μm, the larvae density was adjusted to 5 per milliliter and an appropriate amount of Chlorella vulgaris feed was added. The daily feeding density of golden algae reached 4×10 4 / mL~8×10 4 / mL, feed them 4 to 6 times, and when they develop to the eyed larvae stage, add scallop shell-attached seedlings and temporarily raise them in cement ponds for 30 to 45 days before raising them in the sea.

[0046] Example 3

[0047] A method for producing black-shell hybrid tetraploid oysters comprises obtaining excellent tetraploid long oysters and tetraploid Fujian oysters, selecting parents, cultivating parents, artificial insemination and hatching, and larval cultivation.

[0048] 1) Obtaining tetraploid Fujian oyster (AAAA):

[0049] (a) Breeding of the diploid G0 population of black-shelled Fujian oysters: 8,000 wild first-year-old Fujian oysters were collected, and 1,800 individuals with regular shell shapes and black shells on both sides were selected. The top 15% by weight of 270 individuals were selected as breeding broodstock, weighing 60-80 g and measuring 5-6 cm in shell length. D-type larvae were obtained through artificial insemination and reared in indoor cement tanks until eyed larvae, using scallop shells as a substrate. When the juvenile oysters reached 5-6 mm, they were transferred to the natural sea for growout. Finally, a second generation of breeding was performed using the same breeding objectives, seedling rearing techniques, and growout methods to obtain the diploid G1 population of black-shelled Fujian oysters.

[0050] (b) Obtaining the tetraploid basic population G0 induced by black shelled Fujian oysters: ① Triploid induction: 300 black shelled Fujian oysters with mature gonads were dissected to select the diploid population G1 parents, and females (140) and males (160) were separated under a microscope. Sperm and eggs were obtained from 140 pairs of male and female individuals with good gonads. Sperm was first filtered through a 160-mesh sieve and then a 500-mesh sieve to remove impurities. Eggs were first filtered through a 120-mesh sieve and then a 300-mesh sieve. Before fertilization, the eggs were continuously rinsed with a 500-mesh sieve to remove tissue fluid, and then seawater was added for 30-60 minutes of maturation. Ten pairs of oysters were paired as a group for fertilization, and 5 to 10 sperm were controlled around each egg during fertilization; when 20% to 30% of the fertilized eggs developed the first polar body, 0.5 mg / L cytochalasin B was immediately added and the fertilized eggs were treated for 20 minutes, and then soaked in seawater (containing 1% dimethyl sulfoxide) for 30 minutes; the eggs were then collected from each group of fertilized eggs and placed in an indoor cement pool for hatching and larval cultivation. The induction rate of D-shaped larvae triploidy was detected by flow cytometry to be above 80%. The larvae were cultivated in the indoor cement pool until they became eye-spot larvae, and scallop shells were placed as attachment bases; when the oyster spat grew to 5 to 6 mm, they were moved to the natural sea area for culture. ② Tetraploid induction: One-year-old triploid black Fujian oysters were dissected, and female triploid individuals with mature gonads were selected. 300 individuals with ploidy of 3N were detected by flow cytometry. 150 individuals with well-developed eggs were selected as female parents through microscopic observation. 180 one-year-old black Fujian oysters were dissected to select diploid population parents, and 150 individuals with full gonads and strong sperm motility were selected as male parents. Sperm were first filtered through a 160-mesh screen and then a 500-mesh screen to remove impurities. Eggs were first filtered through a 120-mesh screen and then a 160-mesh screen. Before fertilization, the eggs were continuously rinsed with a 500-mesh screen to remove tissue fluid, and then seawater was added for filtration. The fertilization process lasted 60-90 minutes. Ten pairs of oysters were paired for fertilization, with 5-10 sperm surrounding each egg. When the first fertilized egg developed its first polar body, 0.5 mg / L cytochalasin B was added for 20 minutes, followed by a 30-minute soak in seawater (containing 1% dimethyl sulfoxide). The eggs were then collected from each group and placed in indoor cement tanks for hatching and larval rearing. Flow cytometry was used to determine if the tetraploid induction rate was above 50%. Larvae were reared in the cement tanks until eyed larvae were formed, and scallop shells were added as a substrate. When the juvenile oysters reached 5-6 mm, they were transferred to a natural sea area for growout. Approximately 3,000 first-instar black Fujian oyster tetraploid individuals were obtained from these growouts.

[0051] (c) Self-propagation of the tetraploid population G0 of black-shelled Fujian oysters: During the oyster breeding season, the tetraploid population G0 of black-shelled Fujian oysters was used as the basic population, and oyster individuals ranking 10%-15% in terms of shell height and total weight were selected as breeding parents. 100 female and male individuals with ploidy 4N were screened by flow cytometry, and sperm and eggs were squeezed out respectively. The sperm were first filtered through a 160-mesh screen and then a 500-mesh screen to remove impurities, and the eggs were first filtered through a 120-mesh screen and then a 160-mesh screen to remove impurities. During the fertilization process, 5-10 sperm were controlled to surround each egg. The eggs were then collected and placed in an indoor cement pool for incubation. When the fertilized eggs developed into D-type larvae, the larvae were collected from the entire pool and placed in a new, disinfected cement pool for cultivation at a larval cultivation density of 1-2 per ml. When the larvae developed to the eye point, scallop shells were added as a substrate. When the oyster spat grew to 5-6 mm, they were moved to a natural sea area for aquaculture. The same method was used to self-propagate the tetraploid population for three consecutive generations, thus producing the black-shelled Fujian oyster tetraploid G3 with growth advantages and stable ploidy.

[0052] 2) Obtaining tetraploid long oysters (GGGG):

[0053] (a) Obtaining the tetraploid basic population G0 induced by the black oyster: ① Triploid induction: 300 Crassostrea gigas "Haida No. 3" parents with mature gonads were dissected and separated into 120 females and 180 males under a microscope. Sperm and eggs were obtained from 100 pairs of males and females with good gonads. Sperm was first filtered through a 160-mesh sieve and then a 500-mesh sieve to remove impurities. Eggs were first filtered through a 120-mesh sieve and then a 300-mesh sieve. Before fertilization, eggs were continuously rinsed with a 500-mesh sieve to remove tissue fluid and then ripened with seawater for 30-60 min. Ten pairs of oysters were paired as a group for fertilization, and 5 to 10 sperm were controlled to surround each egg during fertilization. When 20% to 30% of the fertilized eggs developed the first polar body, 0.5 mg / L cytochalasin B was immediately added and the fertilized eggs were treated for 15 minutes, and then soaked in seawater (containing 1% dimethyl sulfoxide) for 30 minutes. The eggs were then collected from each group of fertilized eggs and placed in an indoor cement pool for hatching and larval cultivation. The induction rate of D-shaped larvae triploidy was detected by flow cytometry to be above 80%. The larvae were cultivated in the indoor cement pool until they became eye-spot larvae, and scallop shells were placed as attachment bases. When the oyster spat grew to 5 to 6 mm, they were moved to the natural sea area for culture. ②Tetraploid induction: One-year-old triploid long oysters "Haida No. 2" were dissected, and female triploid individuals with mature gonads were selected. 300 individuals with ploidy of 3N were detected by flow cytometry. 120 individuals with well-developed eggs were selected as female parents through microscopic observation; 200 one-year-old shelled golden Fujian oysters were dissected to select diploid group parents, and 120 individuals with full gonads and strong sperm motility were selected as male parents; sperm were first filtered through a 160-mesh screen and then a 500-mesh screen to remove impurities, and eggs were first filtered through a 120-mesh screen and then a 160-mesh screen; before fertilization, the eggs were continuously rinsed with a 500-mesh screen to remove tissue fluid, and then seawater was added. The fertilization process lasted 60-90 minutes. Ten pairs of oysters were paired for fertilization, with 5-10 sperm surrounding each egg. When the first fertilized egg developed its first polar body, 0.5 mg / L cytochalasin B was added for 15 minutes, followed by a 30-minute soak in seawater (containing 1% dimethyl sulfoxide). The eggs were then collected from each group and placed in indoor cement tanks for hatching and larval rearing. Flow cytometry was used to determine if the tetraploid induction rate was above 60%. Larvae were reared in the cement tanks until eyed larvae were formed, and scallop shells were added as a substrate. When the juvenile oysters reached 5-6 mm, they were transferred to a natural sea area for growout. Approximately 2,500 induced tetraploid individuals of the one-year-old "Haida No. 2" oyster were obtained from this growout.

[0054] (b) Self-propagation of tetraploid population G0 of black long oyster: During the oyster breeding season, the tetraploid G0 induced by long oyster "Haida No. 3" was used as the basic population, and oyster individuals ranked 10% to 15% in shell height and total weight were selected as breeding parents; 100 female and male individuals with ploidy of 4N were screened by flow cytometry, and sperm and eggs were squeezed out respectively. Sperm was first filtered through a 160-mesh screen and then through a 500-mesh screen to filter out impurities, and eggs were first filtered through a 120-mesh screen and then through a 100-mesh screen. The eggs were filtered through a 160-mesh silk screen. During fertilization, each egg was surrounded by 5-10 sperm. The fertilized eggs were then collected and placed in an indoor cement pool for incubation. When the fertilized eggs developed into D-type larvae, the larvae were collected from the entire pool and placed in a new, disinfected cement pool for incubation at a density of 1-2 larvae / ml. When the larvae reached the eye point, scallop shells were added as a substrate. When the oyster spat reached 5-6 mm, they were moved to a natural sea area for growout. The same method was used to self-propagate the tetraploid population for four consecutive generations, resulting in the "Haida No. 3" tetraploid G4, a long oyster with growth advantages and stable ploidy.

[0055] 3) Cultivation of black shell hybrid tetraploid oysters (GGAA, AAGG):

[0056] Cultivation of black shell hybrid tetraploid oysters: 80 black shell Fujian oyster tetraploid G3 individuals with regular shell shape and 100 long oyster "Haida No. 3" tetraploid G4 individuals were selected, and 50 male and female individuals of black shell Fujian oyster tetraploid G3 were obtained by flow cytometry detection and microscopic observation for standby use; and 50 male and female individuals of long oyster "Haida No. 3" tetraploid G4 were obtained by the same method for standby use; a hybrid combination (BGGAA) was set up with the male individual of golden shell Fujian oyster tetraploid G3 as the male parent and the female individual of long oyster "Haida No. 3" tetraploid G4 as the female parent for hybridization, and the long oyster "Haida No. 3" was hybridized. A "No. 3" tetraploid G4 male served as the male parent and a "G3" tetraploid Fujian oyster female served as the female parent, creating a hybrid combination (BAAGG). Specific details included filtering sperm through a 160-mesh sieve and then a 500-mesh sieve to remove impurities, while eggs were filtered through a 120-mesh sieve and then a 300-mesh sieve. During artificial insemination, each egg was surrounded by 3-5 sperm. After fertilization, the fertilized eggs were evenly distributed into 20m2 cement pools for incubation. Filtered and sterilized seawater was used throughout the artificial insemination and incubation process, maintained at a temperature of 24-25°C and a salinity of 30‰, and sufficient oxygen was maintained during incubation. The hatching density of fertilized eggs was 50 eggs / ml. After 26 hours of development, the D-type larvae were collected and obtained using a 300-mesh silk screen. The early D-type larvae were cultured at a density of 10 eggs / ml, the culture temperature was 24-25°C, the salinity was 30‰, and golden algae were used as the starter bait. The daily feeding density was 5×10 3 / mL~2×10 4The larvae were fed 4 to 6 times. As the larvae grew, the feeding amount was gradually increased. When the larvae shell length reached 150 μm to 200 μm, the larvae density was adjusted to 5 per milliliter and an appropriate amount of Chlorella vulgaris feed was added. The daily feeding density of golden algae reached 4×10 4 / mL~8×10 4 / mL, feed them 4 to 6 times, and when they develop to the eyed larvae stage, add scallop shell-attached seedlings and temporarily raise them in cement ponds for 30 to 45 days before raising them in the sea.

[0057] Comparative Example 1

[0058] A method for producing tetraploid Fujian oyster (CAAAA)

[0059] (a) Triploid induction: 400 wild diploid individuals of Fujian oysters with mature gonads were randomly selected as parents and separated into females (157) and males (243) using a microscope. Sperm and eggs were obtained from 140 pairs of males and females with good gonads. Sperm was filtered through a 160-mesh sieve and then a 500-mesh sieve to remove impurities, while eggs were filtered through a 120-mesh sieve and then a 300-mesh sieve. Before fertilization, eggs were continuously rinsed with a 500-mesh sieve to remove tissue fluid and then matured in seawater for 30-60 min. Ten pairs of oysters were paired as a group for fertilization, and 5 to 10 sperm were controlled to surround each egg during fertilization. When 20% to 30% of the fertilized eggs developed the first polar body, 0.5 mg / L cytochalasin B was immediately added and the fertilized eggs were treated for 15 minutes, and then soaked in seawater (containing 1% dimethyl sulfoxide) for 30 minutes. The eggs were then collected from each group of fertilized eggs and placed in an indoor cement pool for hatching and larval cultivation. The induction rate of D-shaped larvae triploidy was detected by flow cytometry to be above 60%. The larvae were cultivated in the indoor cement pool until they became eye-spot larvae, and scallop shells were placed as attachment bases. When the oyster spat grew to 5 to 6 mm, they were moved to the natural sea area for culture.

[0060] (b) Tetraploid induction: dissect the one-year-old triploid obtained in step (a), select female triploid individuals with mature gonads, and detect 100 individuals with ploidy of 3N by flow cytometry. 50 individuals with well-developed eggs are selected as female parents by microscopic observation; dissect 100 one-year-old wild diploid parents of Fujian oysters, and select 50 individuals with full gonads and strong sperm motility as male parents; sperm are first filtered through a 160-mesh sieve and then a 500-mesh sieve to remove impurities, and eggs are first filtered through a 120-mesh sieve and then a 160-mesh sieve; before fertilization, the eggs are continuously rinsed with a 500-mesh sieve to remove tissue fluid, and then seawater is added for fertilization. The fertilization time is 60-90 minutes; ten pairs of oysters are paired as a group for fertilization, and 5-10 sperm are controlled around each egg during fertilization; when the first polar body is observed to develop from the first fertilized egg, 0.5 mg / L cytochalasin B is immediately added and the fertilized egg is treated for 15 minutes, and then soaked in seawater (containing 1% dimethyl sulfoxide) for 30 minutes; the eggs are then collected from each group of fertilized eggs and placed in an indoor cement pool for hatching and larval cultivation. The tetraploid induction rate is detected by flow cytometry to be above 50%. The larvae are cultivated in the indoor cement pool until they become eye-spot larvae, and scallop shells are placed as attachment bases; when the oyster spat grow to 5-6 mm, they are moved to the natural sea area for culture. Although tetraploid Fujian oysters can be obtained by the cytochalasin B induction method, the tetraploid rate is not stable, and it drops to 40% at 120 days of age. The cost is high, the operation repeatability is poor, the process is cumbersome, and the survival rate of the induced tetraploids is low due to the toxicity of cytochalasin B during the induction process. Therefore, the number of tetraploid individuals that can be obtained is small, and the growth rate of the induced tetraploids is slow, and it is impossible to obtain enough tetraploid Fujian oysters to meet the requirements of large-scale production of triploids.

[0061] Comparative Example 2

[0062] According to the method of Comparative Example 1, tetraploid long oysters (CGGGG) were produced, and the population used was a long oyster culture population. Similar to the tetraploid Fujian oysters in Comparative Example 1, under the same culture conditions, the tetraploid rate of 120-day-old tetraploid long oysters was low, the growth rate was slow, and the survival rate was also low. The different shell color hybrid tetraploids constructed in Examples 1, 2, and 3 were 25.26% to 42.06% higher than the tetraploid oysters induced in Comparative Examples 1 and 2, and the survival rate was increased by 7.14% to 30.94%, and the tetraploid rate was always maintained at 100%. The specific statistical results are shown in Tables 1 and Figure 2 , Figure 3 .

[0063] Table 1 Characteristics of hybrid tetraploid oysters with different shell colors

[0064]

[0065] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for producing tetraploid oysters with different shell colors by interspecific hybridization, characterized in that: The following steps are included. 2.1) A wild diploid population of Fujian oysters was used as a screening population. Oyster individuals with regular shell shape, distinct left and right shell colors (gold and black), and ranking in the top 10%-15% in shell height and total weight were selected as breeding parents. After selection, Fujian oyster breeding diploid populations G0 and G1 were obtained. Fertilized eggs from the Fujian oyster breeding diploid population G0 were then treated with cytochalasin B to induce triploid Fujian oysters. Fertilized eggs from the triploid Fujian oysters and the Fujian oyster breeding diploid population G1 were then treated with cytochalasin B to induce a basic tetraploid population G0 of Fujian oysters. 3.2) The tetraploid G0 of the Fujian oyster described in step 1) was selected for three consecutive generations to obtain tetraploid G2-G3 of the Fujian oyster with different shell color characteristics, fast growth rate and stable ploidy. 4.3) Using the diploid basal population of the new Crassostrea gigas variety "Haida No. 1" (GS-01-005-2013), fertilized eggs were treated with cytochalasin B to induce triploid Crassostrea gigas "Haida No. 1." Fertilized eggs from the triploid Crassostrea gigas "Haida No. 1" were then treated with cytochalasin B to induce a tetraploid basal population, G0, of the Crassostrea gigas "Haida No. 1." 5.4) Using the tetraploid base population G0 of the long oyster "Haida No. 1" described in step 3) as a parent, two generations of population selection were performed to obtain the tetraploid G2 of the long oyster "Haida No. 1". 6.5) Using the diploid basal population of the new Crassostrea gigas variety "Haida No. 2" (GS-01-007-2016), fertilized eggs were treated with cytochalasin B to induce triploid Crassostrea gigas "Haida No. 2." Fertilized eggs from the triploid Crassostrea gigas "Haida No. 2" were then treated with cytochalasin B to induce a tetraploid basal population G0 of Crassostrea gigas with the gold shell characteristic. 7.6) Using the tetraploid base population G0 of the long oyster "Haida No. 2" described in step 5) as the parent, four generations of population selection were performed to obtain the long oyster "Haida No. 2" tetraploid G4 with the shell gold characteristic. 8.7) Using the diploid basal population of the new Crassostrea gigas variety "Haida No. 3" (GS-01-007-2018), fertilized eggs were treated with cytochalasin B to induce triploid Crassostrea gigas "Haida No. 3". Fertilized eggs from the triploid Crassostrea gigas "Haida No. 3" were then treated with cytochalasin B to induce a tetraploid basal population G0 of Crassostrea gigas "Haida No. 3". 9.8) Using the tetraploid base population G0 of the long oyster "Haida No. 3" described in step 7) as the parent, four generations of population selection were performed to obtain the tetraploid long oyster "Haida No. 3" G4. 10.9) The Fujian oyster tetraploid G3 of different shell colors described in step 2) and the long oyster tetraploid of different shell colors described in steps 4), 6), and 8) are used as parents. Oyster individuals with regular shell shapes and distinct shell color characteristics are selected as breeding parents. Long oysters and Fujian oyster tetraploids with the same shell color characteristics are paired and hybridized using an incomplete diallel hybridization method to produce tetraploid hybrid oysters with different shell colors.

11. The method for producing tetraploid oysters with different shell colors by interspecific hybridization according to claim 1, characterized in that: Step 9) The shell colors include ordinary shell colors, gold and black.

12. The method for producing tetraploid oysters with different shell colors by interspecific hybridization according to claim 1, characterized in that: The total weight of the parent individuals used for hybridization in step 9) is 30-45 g.

13. The method for producing tetraploid oysters with different shell colors by interspecific hybridization according to claim 1, characterized in that: The concentration of cytochalasin B in the operation of inducing triploid and tetraploid Fujian oysters and Crassostrea gigas was 0.5 mg / L.

14. The method for producing tetraploid oysters with different shell colors by interspecific hybridization according to claim 1, characterized in that: In the process of inducing triploid and tetraploid Fujian oysters and Crassostrea gigas, the treatment time of cytochalasin B is 15-20 minutes.

15. The method for producing tetraploid oysters with different shell colors by interspecific hybridization according to claim 1, characterized in that: After the induction of triploid and tetraploid Fujian oysters and long oysters is completed, the induced D-shaped larvae are screened by flow cytometry to obtain triploid Fujian oysters and long oysters with a ploidy of 3N and tetraploid Fujian oysters and long oysters G0 with a ploidy of 4N.

16. The method for producing tetraploid oysters with different shell colors by interspecific hybridization according to claim 1, characterized in that: The parents in step 9) were subjected to ploidy determination before fertilization. For each individual, two sampling methods, hemolymph and gill filament, were adopted. Individuals with a ploidy of 4N in both determinations were selected as hybrid parents.

17. The method for producing tetraploid oysters with different shell colors by interspecific hybridization according to claim 7, characterized in that: The number of parents used in the two hybrid combinations of each shell color is 100.

18. The method for producing tetraploid oysters with different shell colors by interspecific hybridization according to claim 1, characterized in that: The Fujian oysters and long oysters used as parents are both one-year-old oysters. The number of Fujian oyster groups used for screening includes 2,000 to 3,000, and the number of parents used for breeding is 200 to 300.

19. The method for producing tetraploid oysters with different shell colors by interspecific hybridization according to claim 1, characterized in that: The number of parents used for induction of Crassostrea gigas and Crassostrea fujianis ranges from 200 to 300.

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