Method for producing tetraploid of hongkong oyster and kumamoto oyster and obtaining triploid by hybridization

Abstract

This invention relates to a method for producing tetraploid Hong Kong oysters and Kumamoto oysters and for obtaining triploids through hybridization, belonging to the field of aquatic seedling breeding. This invention provides a method for cultivating tetraploid Hong Kong oysters and tetraploid Kumamoto oysters, and also provides a method for hybridizing female Kumamoto oyster diploids and male Kumamoto oyster tetraploids to obtain Kumamoto-Fraxin hybrid triploids, and a method for hybridizing female Hong Kong oyster diploids and male Kumamoto oyster tetraploids to obtain Kumamoto-Fraxin hybrid triploids. The methods of this invention can obtain a large number of purebred tetraploid oysters, which can be normally cultured to adulthood. Triploids are obtained by hybridizing tetraploid oysters with diploids. The method of this invention for preparing tetraploid oysters has higher rates of fertilized egg hatching, D-type tetraploid ploidization rate, eye-spot stage survival, 1-year survival, and 1-year tetraploid ploidization rate than existing technologies. Simultaneously, Kumamoto-Fraxin hybrid triploid oysters and Kumamoto-Fraxin hybrid triploid oysters were obtained and cultured to adulthood.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic breeding of aquaculture species, and particularly relates to a method for producing tetraploid Crassostrea hongkongensis and Crassostrea sikamea and obtaining triploid through hybridization. BACKGROUND

[0002] Both Crassostrea hongkongensis and Crassostrea sikamea belong to Mollusca, Bivalvia, Pterimorphia, Ostreoida, Ostreidae and Crassostrea, and are both of a semi-saltwater type.

[0003] Crassostrea hongkongensis is naturally distributed in the southern sea areas of China, is suitable for high-temperature and low-salt environments, and is an important economic species for aquaculture in Fujian, Guangdong and Guangxi regions, has a large body size and grows rapidly. However, many problems have occurred in the long-term artificial breeding process of Crassostrea hongkongensis, mainly including inbred degeneration, scale death for unknown reasons and slow growth.

[0004] Crassostrea sikamea is rich in natural resources in China, is widely distributed in the gulf and estuary areas south of Nantong, Jiangsu, is suitable for high-temperature and medium-high-salt sea areas, and is mainly distributed in Zhejiang province for aquaculture. Compared with Crassostrea hongkongensis, Crassostrea sikamea has a smaller body size, but has delicate and sweet meat, is deeply loved by local consumers, and also has high economic value.

[0005] Triploid oysters have three sets of chromosomes, exhibit biological characteristics of sterility or high sterility, have advantages of fast growth, high yield and stable annual glycogen content, and also solve the problem that ordinary diploid oysters cannot be marketed due to weight loss and transparency after the breeding period in summer. In addition, triploid oysters are obtained through hybridization of tetraploid and diploid oysters, and thus the problem of inbred degeneration is avoided. After years of promotion and market selection, the aquaculture scale of triploid Crassostrea angulata (Qianjianshi No. 1) has accounted for more than 70% of the aquaculture scale of northern oysters, and triploid Fujian oysters and triploid hybrid oysters (Crassostrea angulata X Fujian oyster) have also been promoted in China, with the aquaculture scale expanding year by year and being deeply recognized by the majority of aquaculture farmers. Crassostrea hongkongensis and Crassostrea sikamea are also important economic shellfishes for aquaculture in China, but there is no large-scale aquaculture of triploid Crassostrea hongkongensis and triploid Crassostrea sikamea on the market, because there is a lack of tetraploid Crassostrea hongkongensis and tetraploid Crassostrea sikamea required for large-scale production of triploids, and there is no report on the obtaining of tetraploid Crassostrea hongkongensis and tetraploid Crassostrea sikamea.

[0006] Different scholars have carried out preliminary studies on interspecific hybridization of diploid Kumamoto oysters and diploid Hong Kong oysters, and there is no report on the production of triploid oysters by hybridization of Kumamoto oysters and Hong Kong oysters in China. Stable acquisition of Hong Kong oyster tetraploid and Kumamoto oyster tetraploid is the prerequisite for the production of triploid, and tetraploid oysters can be obtained by inhibiting polar body release with normally fertilized diploid oocytes, or by inhibiting polar body release with triploid oyster oocytes and normal diploid sperm (CN1144454A, CN109730008A), and the preparation method of Hong Kong oyster tetraploid has also been reported in 2017 (CN107494358A). However, the current production methods of Hong Kong oyster tetraploid and Kumamoto oyster tetraploid have limitations such as instability, extremely low survival rate, low polyploidization rate and unstable ploidy, so there is no report on the acquisition of tetraploid Hong Kong oyster and tetraploid Kumamoto oyster. SUMMARY

[0007] The technical problem solved by the present application is to provide a method for producing Hong Kong oyster tetraploid and Kumamoto oyster tetraploid and obtaining triploid by hybridization, and to provide excellent Hong Kong oyster tetraploid, Kumamoto oyster tetraploid, Kumamoto oyster triploid and Hong Kong oyster triploid for oyster culture.

[0008] The present application is realized by the following technical solutions:

[0009] A method for producing Hong Kong oyster tetraploid, the method being that first stimulating triploid Hong Kong oysters with high temperature of 30 DEG C and change of salinity for 7 days, the initial salinity being 8 ‰, increasing 2 ‰ per day to salinity of 16 ‰, then culturing the triploid Hong Kong oysters to sexual maturity at water temperature of 25 DEG C, arranging the diameters of the oocytes of the triploid female oysters from large to small, fertilizing the oocytes ranked in the front 1 / 3 with sperm of diploid Hong Kong oysters, and treating the fertilized oocytes with 0.45-0.75 mg / L cytochalasin B (CB) or 35-75 mg / L 6-dimethylaminopurine (6DMAP) for 16 minutes from 5 minutes after fertilization to inhibit the release of the first polar body; culturing the larvae at room temperature of 25 DEG C and salinity of 15 ‰, and obtaining Hong Kong oyster tetraploid after the larvae attach and metamorphose and are cultured on the sea.

[0010] Further, after the triploid Hong Kong oysters are stimulated with high temperature of 30 DEG C and change of salinity for 7 days, the temperature is directly reduced to 25 DEG C and the salinity remains unchanged at 16 ‰.

[0011] The present application also provides a method for obtaining triploid by hybridization of Kumamoto oyster diploid and Hong Kong oyster tetraploid (Kumamoto 2N ♀ X Hong Kong 4N ♂), the method being parent culture, artificial insemination and hatching, and larval culture.

[0012] The parent culture: in order to synchronize the gonad development of the Kumamoto oysters and the Hong Kong oysters, the Kumamoto oysters are made to mature 35-45 days earlier than the Hong Kong oysters when the parent gonad maturation is carried out, the gonad maturation work of the Hong Kong oysters is started according to the gonad development of the Kumamoto oysters, the triploid Hong Kong oysters are stimulated by high temperature of 30 DEG C and salinity change for 7 days, the initial salinity is 10 ‰, and the salinity is increased by 2 ‰ every day to 18 ‰, then the water temperature is decreased by 1 DEG C every day until 25 DEG C, and the triploid Hong Kong oysters are cultured for 30-40 days until the gonad matures; the initial temperature of the diploid Kumamoto oysters is 16 DEG C, and then the temperature is gradually increased to 27 DEG C, the initial salinity is 24-26 ‰, the salinity is adjusted to normal seawater salinity of 16-18 ‰ after 15 days, and the diploid Kumamoto oysters are cultured until the gonad matures;

[0013] The artificial insemination and hatching: the female Kumamoto oysters ranked in the top 10% are selected in descending order, and the ova are collected, the collected ova are soaked in seawater for 40-60 minutes for maturation; the male Hong Kong oysters ranked in the top 10% are selected in descending order, and the sperm are obtained; the insemination is carried out at the proportion of 200-300 active sperm per ovum, the hatching is carried out, the temperature is 25 DEG C, the salinity is 18 ‰, and the hatching density of the fertilized eggs is 50 eggs per milliliter;

[0014] The larva culture: the early larva culture density is 8-12 per milliliter, the temperature is 25 DEG C, the salinity is 18 ‰, and the chrysis is used as the opening bait; with the growth of the larvae, the larva culture density is adjusted, and the bait feeding amount should be gradually increased; when the shell length of the larvae is more than 150 microns, the algae including ulothrix, small balls and diatoms are added; before the larvae are hatched, the temperature and the salinity are respectively decreased by 0.5 DEG C and 0.5 ‰ every day, and are adjusted to the temperature and the salinity which are different from those of the culture sea area by ≤3.

[0015] Further, in the parent culture, when the gonad tissue fluid of the Kumamoto oysters becomes less and the ovum cells are clear, the gonad maturation work of the Hong Kong oysters is started.

[0016] The application also provides a method for producing the triploid Kumamoto oysters, the method is that the triploid Kumamoto oysters are stimulated by high temperature of 30 DEG C and salinity change for 7 days, the initial salinity is 30 ‰, and the salinity is decreased by 2 ‰ every day until 20 ‰, then the triploid Kumamoto oysters are cultured in water with the temperature of 25 DEG C until the gonad matures, the ova of the triploid female oysters are arranged in descending order, and the top 1 / 3 of the ova and the sperm of the diploid Kumamoto oysters are fertilized; the fertilized eggs are treated by 0.40-0.60 mg / L cytochalasin B (CB) or 30-60 mg / L 6-dimethylaminopurine (6DMAP) for 15 minutes from 5 minutes after the fertilization, and the first polar body emission is inhibited; the indoor culture temperature is 25 DEG C, the salinity is 20, and the triploid Kumamoto oysters are obtained by culturing the larvae after the metamorphosis to the adult oysters on the sea.

[0017] Further, after the 30℃ high temperature and salinity change stimulation of the C. rubescens tetraploid for 7 days, the temperature is directly reduced to 25℃, and the salinity remains unchanged at 20‰.

[0018] The application also provides a method for obtaining triploid by hybridization of C. hongkongensis diploid and C. rubescens tetraploid (C. hongkongensis 2N ♀ X C. rubescens 4N ♂ hybrid combination), which comprises parent cultivation, artificial insemination and hatching, and larva cultivation.

[0019] The parent cultivation comprises the following steps: first, stimulating the C. rubescens tetraploid with 30℃ high temperature and salinity change for 7 days, with an initial salinity of 28‰, and then reducing the salinity to 18‰ by 2‰ per day, and reducing the water temperature by 1℃ per day until the temperature is reduced to 25℃, and then cultivating the C. rubescens tetraploid until the gonad is mature; and second, cultivating the C. hongkongensis diploid parent at an initial temperature of 16℃, and then gradually increasing the temperature to 25℃, and cultivating the C. hongkongensis diploid parent until the gonad is mature, with an initial salinity of 12-14‰, and then adjusting the salinity to normal seawater salinity of 16-18‰ after 15 days.

[0020] The artificial insemination and hatching comprises the following steps: first, selecting the female oysters of C. hongkongensis in the top 10% according to size from large to small, collecting the ova, and then soaking the ova in seawater for 40-60 minutes for maturation; second, selecting the male oysters of C. rubescens tetraploid in the top 10% according to size from large to small, and then obtaining the sperm; and third, performing insemination and hatching at a ratio of 200-300 active sperm per ovum, with a temperature of 25℃, a salinity of 18, and a density of 50 ova per milliliter.

[0021] The larva cultivation comprises the following steps: first, cultivating the early-stage larvae at a density of about 10 per milliliter, with a temperature of 25℃ and a salinity of 18‰, and using chrysophyceae as the opening feed; second, adjusting the cultivation density of the larvae and gradually increasing the amount of feed as the larvae grow; and third, adding algae including ulothrix, coccolithophore and diatom when the shell length of the larvae is greater than 150μm; and fourth, adjusting the temperature and salinity to be within a difference of ≤3 from the temperature and salinity of the breeding sea area by reducing the temperature and salinity by 0.5℃ and 0.5‰ per day, respectively, before the larvae hatch.

[0022] Further, in the parent cultivation, the gonad maturation of the C. hongkongensis is started when the gonad tissue fluid of the C. rubescens tetraploid is observed to be less and the sperm activity is high.

[0023] Compared with the prior art, the application has the following beneficial effects: (1) the application adopts a unique method for promoting the maturation of the triploid C. hongkongensis and the triploid C. rubescens, which is stimulated by 30℃ seawater and salinity change, so that the gonad development is faster, and more and better ova are obtained; and (2) the prior art method adopts naturally matured triploid C. hongkongensis and C. rubescens, which cannot obtain a large number of high-quality ova.

[0024] (2) The selection of large egg of the first 1 / 3 triploid Hong Kong oysters and triploid Kumamoto oysters is beneficial to the survival of tetraploid oysters because the large egg has high maturity degree. The prior art does not screen the size of the egg. The key point of using the triploid Hong Kong oysters and triploid Kumamoto oysters to induce tetraploid oysters is that a large number of eggs cannot be obtained. The two innovations enable the present application to obtain and utilize a large number of high-quality triploid Hong Kong oyster eggs and triploid Kumamoto oyster eggs, obtain a large number of purebred tetraploid oysters, and normally breed the tetraploid oysters to adults, thereby providing a seed source for large-scale production of triploid Hong Kong oysters and triploid Kumamoto oysters, and laying a foundation for hybridization of Hong Kong oysters and Kumamoto oysters.

[0025] (3) The triploid Hong Kong oysters and triploid Kumamoto oysters can be obtained by inhibiting the second polar body discharge after fertilization of the diploid sperm and diploid egg, but the triploidization rate of the artificially induced hybrid triploid is unstable, the hatching rate of the fertilized egg is low, and the survival rate of the larvae is low, which cannot be applied to large-scale production. The present application uses the tetraploid Hong Kong (Kumamoto) oysters and the diploid Kumamoto (Hong Kong) oysters to obtain the hybrid triploid, uses the high-concentration sperm of the tetraploid Hong Kong (Kumamoto) oysters to improve the fertilization rate of the Kumamoto (Hong Kong) oyster eggs, and finds a most suitable hybridization fertilization salinity. The present application provides a new method for producing excellent Hong Kong oyster hybrid triploids.

[0026] (4) The large-scale production of triploid Hong Kong oysters can provide a large number of high-quality seedlings for Hong Kong oyster culture in Fujian, Guangdong, Guangxi and other regions of China, improve the yield of Hong Kong oysters, and is expected to solve the problems of seedling degradation and large-scale death in high-temperature periods in recent years. The triploidization of Kumamoto oysters can also provide a new strain with fast growth speed, high yield and sweet taste for the industry, and promote the development of Kumamoto oyster breeding industry. The Hong Kong oyster (large oyster) and the Kumamoto oyster can be hybridized to obtain a hybrid triploid, which can maintain the sweet taste of the Kumamoto oyster and utilize the advantages of the Hong Kong oyster (large oyster) and interspecific hybridization to obtain a new hybrid with fast growth speed. In addition, due to the sterility of the triploid, the meat quality of the triploid oyster during the spawning season is still good.

[0027] (5) Compared with the prior art, the present application significantly improves the hatching of the fertilized egg, the D-type tetraploidization rate and the survival rate at the eye spot stage, and obtains 1-year-old tetraploid oysters after sea area cultivation. At the same time, the Hong Kong oyster interspecific hybrid triploid and the Kumamoto oyster interspecific hybrid triploid are obtained, and the yield at the harvest period is significantly improved compared with the ordinary Kumamoto oyster.

[0028] Specific implementation cases

[0029] The following implementation cases are illustrative and demonstrative of the present application, but not limiting.

[0030] Example 1: Obtaining of tetraploid Hong Kong oysters

[0031] In January 2021, 120 identified 2-year-old artificially induced triploid Hong Kong oysters and 100 2-year-old diploid Hong Kong oysters were taken. The diploid oysters were placed in the initial temperature 16℃ and salinity 16 of the parent oyster culture pool for routine cultivation. The triploid oysters were placed in the initial temperature 16℃ and initial salinity 8 of the culture pool. The triploid Hong Kong oysters were cultivated in the following way: first, static for one day, then increase 7℃ every day from the second day, reach 30℃ on the third day, and then increase salinity by 2‰ every day to salinity 16‰ from the third day, keep at 30℃ for 7 days, then reduce the temperature to 25℃ from the eighth day, feed 8 times a day, and change 100% of the water. The triploid Hong Kong oysters and the diploid Hong Kong oysters reached sexual maturity on the 35th day.

[0032] Open 120 triploid Hong Kong oysters, get 72 female oysters, and take the eggs after dissection and washing, then put them into 72 beakers respectively. Measure the diameter of 10 oocytes from each beaker and take the average value. Mix the oocytes in the first 24 beakers with the largest oocyte diameter, get a total of 660 million oocytes, and dilute to 10,000 oocytes per milliliter. Open 89 diploid Hong Kong oysters, get 36 male oysters, and take the sperm from the largest 12 diploid Hong Kong oysters after dissection, then filter and mix equally. From 5 minutes after fertilization, treat the fertilized eggs with CB (0.48 mg / L) to inhibit the first polar body, then rinse after 16 minutes, and then put them into 1 m 3 of water to hatch. After hatching, put them into D-type cloth pools at 10 per milliliter, and cultivate them according to the normal Hong Kong oyster diploid larva cultivation method. All the treatment operations are carried out in seawater with a salinity of 16‰ at 25℃, filtered with sand and disinfected with ultraviolet light.

[0033] Develop to the creeping larva stage, put into oyster shell strings to attach to the seedlings, and adjust the temperature and salinity to differ by ≤3 from the temperature and salinity of the cultivation sea area before hatching. Follow the existing Hong Kong oyster cultivation method to cultivate. The cultivation results are as follows.

[0034] Table 1 Comparison of the cultivation results of tetraploid Hong Kong oysters between the present example and the prior art

[0035]

[0036] Note: The tetraploid Hong Kong oyster adult is obtained for the first time through the technology of the present case. The values of the prior art are the optimal values obtained by experiments, and no juvenile or adult is obtained.

[0037] Example 2: Obtaining tetraploid Kumamoto oysters

[0038] In January 2021, 250 2-year-old artificially induced triploid Kumamoto oysters and 100 2-year-old diploid Kumamoto oysters were identified. The diploid Kumamoto oysters were placed in a parent oyster cultivation tank with an initial temperature of 16°C and a salinity of 20, and were cultivated according to the conventional method. The triploid Kumamoto oysters were placed in a cultivation tank with an initial temperature of 16°C and an initial salinity of 30. The cultivation method for triploid Hong Kong oysters was as follows: first, static for one day, then increase the temperature by 7°C every day from the second day, reach 30°C on the third day, and then decrease the salinity by 2‰ every day from the third day until the salinity reaches 20. Maintain at 30°C for 7 days, then decrease the temperature to 25°C, and maintain every day. Feed 8 times a day, and change the water by 100%. The triploid Kumamoto oysters and the diploid Kumamoto oysters reached sexual maturity on the 50th day.

[0039] Open 240 triploid Kumamoto oysters, obtain 150 female oysters, and disarticulate to obtain eggs. After washing, place the eggs in 150 beakers, measure the diameter of 10 eggs from each beaker, and take the average value. Mix the eggs in the top 50 beakers with the largest egg diameter, and obtain a total of 8.25 million eggs, diluted to 10,000 eggs / mL. Open 90 diploid Kumamoto oysters, obtain 40 male oysters, and disarticulate the largest 12 diploid Kumamoto oysters to obtain sperm. After filtration, mix an equal amount. From 5 minutes after fertilization, treat the fertilized eggs with CB (0.55 mg / L) to inhibit the first polar body, then rinse after 15 minutes, and then place in 1 m 3 of water to hatch. After hatching, place 10 D-type larvae per mL in a beaker, and cultivate according to the normal Hong Kong oyster diploid larva cultivation method. All treatment operations are performed in seawater filtered with sand and disinfected with ultraviolet light at 25°C and a salinity of 20.

[0040] Develop to the creeping larva stage, and place in oyster shell strings to attach to seedlings. Before hatching, decrease the temperature and salinity by 0.5°C and 0.5, respectively, every day, and adjust to a temperature and salinity that differs by ≤3 from the temperature and salinity of the cultivation sea area. Cultivate according to the existing Hong Kong oyster cultivation method. The cultivation results are as shown in Table 2.

[0041] Table 2 Comparison of the cultivation results of tetraploid Kumamoto oysters between the present example and the prior art

[0042]

[0043] Note: The present example is the first time to obtain tetraploid Kumamoto oyster adult oysters. The values of the prior art are the optimal values obtained by experiments, and no juvenile oysters or adult oysters were obtained.

[0044] Example 3 Obtaining of hybrid triploid oysters (Kumamoto 2N♀ X Hong Kong 4N♂):

[0045] In April 2022, 1000 four-ploid Hong Kong oysters of 15 months old were identified by flow cytometry, and the largest 100 (10%) were selected as seed oysters. At the same time, 1000 wild diploid Kumamoto oysters were selected, and the largest 100 (10%) were selected as seed oysters. In order to synchronize the gonadal development of four-ploid Hong Kong oysters and diploid Kumamoto oysters, the Kumamoto oysters were warmed up to promote maturation in advance. The Kumamoto oysters were placed in water at 16°C and a salinity of 20 for 2 days, then the temperature was increased by 1°C per day, and the temperature was maintained at 27°C. After 15 days of cultivation, the salinity was adjusted to 18, and the gonads were matured for spawning. When the gonadal tissue fluid of Kumamoto oysters was observed to be less and the egg cells were clear, the gonadal maturation of Hong Kong oysters was started. The four-ploid Hong Kong oysters were placed in water at 16°C and a salinity of 10, and the gonadal maturation of Hong Kong oysters was started when the gonadal tissue fluid of Kumamoto oysters was observed to be less and the egg cells were clear or the sperm activity was high. The maturation method was as follows: the temperature was increased to 24°C on the first day, to 30°C on the second day, and the salinity was increased by 2‰ per day to a salinity of 18, which was maintained at 30°C for 7 days. On the 8th day, the temperature was reduced to 25°C, and the water temperature was reduced by 1°C per day to 25°C. The Kumamoto oysters and four-ploid Hong Kong oysters were managed in the same way every day, with 8 times of feeding and 100% of water replacement.

[0046] After the Kumamoto oysters and four-ploid Hong Kong oysters were synchronized to sexual maturity, 91 Kumamoto oysters were opened, and 56 female individuals were identified under a microscope, obtaining 400 million eggs. After the eggs were collected, they needed to be soaked in seawater for 40-60 minutes for maturation. 85 four-ploid Hong Kong oysters were opened, and 33 male individuals were identified under a microscope, obtaining sperm, which was filtered with a 300 mesh net. High-concentration sperm, i.e., 300 active sperm per egg, was used for insemination. The entire insemination and hatching process used filtered and ultraviolet disinfected seawater at a temperature of 25°C and a salinity of 18, with sufficient oxygen during hatching. The inseminated eggs were hatched at a density of 50 eggs per milliliter, and developed to D type after 24 hours. The early D type larvae were cultured at a density of 10 per milliliter at a temperature of 25°C and a salinity of 18, with golden algae as the opening feed, and the daily feeding density was 5x10 4 4 ​4-6 times, and the amount of feed should be gradually increased as the larvae grow; when the shell length of the larvae is more than 150 μm, adjust the density of the larvae to 5 per milliliter, and add an appropriate amount of Isochrysis and Chlorella, and feed them 6-8 times, until the larvae reach the creeping stage, and then put them into the shell strings of scallops, adjust the temperature and salinity of the culture pond after attachment, and reduce the temperature by 0.5°C per day until it reaches 22°C, and then put them into the sea for cultivation after 10 days. The statistics of the cultivation results of the hybrid triploid oysters of Xiongxiang are as follows: the hatching rate of fertilized eggs is 18%, the triplication rate of D-type larvae is 100%, the survival rate during the incubation stage in the workshop is 55%, and the survival rate of one-year-old marine cultivation is 67%. The hybrid triploid oysters of Xiongxiang maintain the delicate and delicious taste of Xiongben oysters, and under the same cultivation conditions, compared with ordinary Xiongben oysters, the shell height of the hybrid triploid oysters of Xiongxiang is increased by 27-33%, the fresh weight is increased by 56-67%, and the survival rate is increased by 11-16%. At the same time, due to the highly sterile characteristics of triploids, the hybrid triploid oysters of Xiongxiang can be marketed all year round.

[0047] Example 4: Obtaining of the hybrid triploid oysters of Xiang Xiong (Xiang Xiong hybrid combination (Hong Kong 2N ♀ X Xiongben 4N ♂)):

[0048] In April 2022, 1000 four-ploid Xiongben oysters of 15 months old were identified by flow cytometry, and the largest 100 (10%) were selected as seed oysters. At the same time, 700 diploid Hong Kong oysters were selected, and the largest 70 (10%) were selected as seed oysters. In order to synchronize the gonadal development of the four-ploid Xiongben oysters and the diploid Hong Kong oysters, the four-ploid Xiongben oysters were promoted to maturity in advance, with water temperature of 16°C and salinity of 28. The promotion method was as follows: the first day, the temperature was raised to 24°C, the second day, the temperature was raised to 30°C, the third day, the salinity was reduced by 2‰ to 18, and the temperature was kept at 30°C for 7 days, and then the temperature was reduced to 25°C, and the water temperature was reduced by 1°C per day. The culture was carried out until the gonadal maturity. The Hong Kong oysters were kept at water temperature of 16°C and salinity of 14. When the gonadal tissue of the Xiongben oysters was observed under a microscope, the gonadal tissue was observed to be less and the sperm activity was high, the Hong Kong oysters were promoted to maturity, the temperature was raised by 1°C per day, the temperature was raised to 25°C, and the culture was carried out for 15 days, and then the salinity was adjusted to 18, and the gonadal maturity was obtained. The daily management methods of the Xiongben oysters and the four-ploid Hong Kong oysters were the same, and the feed was fed 8 times a day, and the water was changed by 100%.

[0049] After the tetraploid O. formosanus and the diploid O. hongkongensis were developed to sexual maturity, 58 O. hongkongensis were opened, and 30 male individuals were identified under a microscope, and 600 million eggs were obtained. The eggs need to be soaked in seawater for 40-60 minutes to mature. 86 O. formosanus were opened, and 39 male individuals were identified under a microscope, and sperm were obtained. The impurities were filtered with a 300 mesh net. High concentration of sperm, i.e. 300 active sperm per egg, was used for insemination. Filtered and ultraviolet disinfected seawater was used for the whole insemination and hatching process, with a temperature of 25℃ and a salinity of 18. Oxygen was kept sufficient during hatching. The hatching density of fertilized eggs was 50 eggs per milliliter, and the development to D type was achieved after 24 hours. The cultivation method was the same as that of the O. formosanus and O. hongkongensis hybrid combination. The results of the O. formosanus and O. hongkongensis hybrid triploid oyster cultivation are as follows: the fertilized egg hatching rate was 16%, the D type larva triploidization rate was 100%, the survival rate during the workshop cultivation stage was 49%, and the survival rate during the one-year sea area cultivation was 63%. The O. formosanus and O. hongkongensis hybrid triploid oyster maintained the delicate taste of O. formosanus, and under the same cultivation conditions, there was no significant difference in growth and survival between the O. formosanus and O. hongkongensis hybrid combination and the O. formosanus and O. hongkongensis hybrid combination.

Claims

1. A method for producing tetraploid Crassostrea hongkongensis, characterized in that, The method is to stimulate triploid Hong Kong oysters with 30 DEG C high temperature and salinity change for 7 days, initial salinity 8 ‰, increasing 2 ‰ per day to salinity 16 ‰, then culturing the triploid Hong Kong oysters to sexual maturity at water temperature 25 DEG C, arranging the oocytes of triploid female oysters from large to small, selecting the top 1 / 3 oocytes and the sperm of diploid Hong Kong oysters for fertilization, inhibiting the first polar body discharge by treating the fertilized eggs with 0.45-0.75 mg / L cytochalasin B or 35-75 mg / L 6-dimethylaminopurine for 16 minutes from 5 minutes after fertilization, culturing the larvae in the room at temperature 25 DEG C and salinity 15 ‰, and obtaining tetraploid Hong Kong oysters by culturing the metamorphosed larvae to adult oysters in the sea.

2. The method for producing tetraploid of Crassostrea hongkongensis according to claim 1, characterized in that, After the triploid Hong Kong oysters are stimulated with 30 DEG C high temperature and salinity change for 7 days, the temperature is directly reduced to 25 DEG C and the salinity is kept unchanged at 16 ‰.

3. A method for obtaining triploid by crossing male individuals in female Kumamoto oysters diploid and Hong Kong oysters tetraploid cultured by the method of claim 1, the method comprising parent culture, artificial insemination and hatching and larva culture; characterized in that, In the parent culture, when the gonadal tissue fluid of the Kumamoto oysters becomes less and the oocyte is clear, the gonadal maturation of the Hong Kong oysters is started. In the artificial insemination and hatching, the top 10% of the Kumamoto oyster females are selected from large to small, the oocytes are collected, and the collected oocytes are matured by soaking in seawater for 40-60 min; the top 10% of the Hong Kong oyster tetraploid males are selected from large to small, and the sperm is obtained; the insemination is performed at a ratio of 200-300 active sperm per oocyte, the hatching is performed at temperature 25 DEG C and salinity 18 ‰, and the hatching density of the fertilized eggs is 50 eggs per milliliter. In the parent culture, when the gonadal tissue fluid of the Kumamoto oysters becomes less and the oocyte is clear, the gonadal maturation of the Hong Kong oysters is started.

4. The method of claim 3, wherein the step of obtaining the triploid is performed by the method of claim 1 or 2. In the parent culture, when the gonadal tissue fluid of the Kumamoto oysters becomes less and the oocyte is clear, the gonadal maturation of the Hong Kong oysters is started.

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