A method for breeding a new hybrid triploid oyster line with high growth rate and high resistance

Abstract

The application discloses a breeding method of a hybrid triploid oyster new strain which is fast-growing and high-resistant. The hybrid triploid oyster new strain is obtained through the following technical links: breeding of a high-resistant and fast-growing diploid strain, establishment of a high-resistant and fast-growing tetraploid induction group strain, breeding of a high-resistant and fast-growing tetraploid new strain, breeding of a high-resistant and fast-growing hybrid diploid new strain, batch production of a high-resistant and fast-growing hybrid triploid new strain, and the like, combined use of population selection breeding, family selection breeding, stress breeding, hybrid breeding and polyploid breeding technologies. The hybrid triploid oyster new strain is fast-growing, high-resistant, adaptive to the habitat of North China sea area, poor in fertility, good in quality and high in fullness degree throughout the year, and can effectively solve the problems of high mortality, slow growth, high breeding risk and low benefit of Pacific oysters in North China sea area, and has remarkable popularization significance and value.

Description

Technical Field

[0001] This invention belongs to the field of shellfish genetic breeding technology in marine agriculture, specifically involving a method for cultivating a new hybrid triploid oyster strain that is fast-growing and highly resistant to adverse conditions. Background Technology

[0002] Pacific oysters, also known as long oysters, are an economically important shellfish cultured in the high-salinity waters of the Pacific Ocean. In China, they are mainly cultivated in the northern waters of Liaoning, Shandong, Hebei, and Jiangsu provinces, with the southernmost distribution in the waters off Ningde, Fujian. Mortality rates are high in the South China Sea. As a low-temperature, high-salinity species, they play a pivotal role in aquaculture in the North China Sea and have significant economic value. Geographical brands of Pacific oysters include Rushan oysters, Rongcheng oysters, Dalian oysters, Rizhao oysters, and Lushun oysters, renowned for their delicious meat and once widely loved by farmers and consumers. However, traditional farming of diploid Pacific oysters resulted in problems such as poor quality during the breeding season, increased water content after the breeding season, poor taste, and high mortality. The industrial promotion of triploid Pacific oysters solved these problems. The period from 2018 to 2022 was the peak season for triploid Pacific oyster farming, with a continuous expansion of the farming area, bringing significant economic benefits to farmers.

[0003] However, since 2022, due to factors such as rising sea temperatures, global warming, changes in water quality, and increased farming density, the mortality rate of Pacific oysters (including triploids) has continued to rise. In August and October of 2023 and 2024, large-scale mortality occurred in most areas of the North China Sea, affecting both diploid and triploid Pacific oysters, with mortality rates reaching over 90%. This severely dampened farmers' enthusiasm for oyster farming and hindered industry development. The industry urgently needs to cultivate new triploid oyster strains with strong resilience suitable for northern waters. On the other hand, compared to Portuguese oysters (Fujian oysters), Pacific oysters grow more slowly, requiring more than two years to reach marketable size. Farmers also hope to cultivate new, faster-growing triploid oyster strains suitable for northern waters. Therefore, it is necessary to cultivate a new, fast-growing, and highly resilient hybrid triploid oyster strain that is adapted to the northern environment and comparable in quality to Pacific oysters.

[0004] New diploid Pacific oyster and Portuguese oyster strains with high stress resistance and rapid growth were developed through population selection and family selection techniques. Then, polyploid breeding techniques were used to cultivate tetraploid Pacific oysters and Portuguese oysters with high stress resistance and rapid growth, respectively. Hybrid breeding techniques were then used to cross these tetraploid Pacific oysters and Portuguese oysters, and fast-growing tetraploids were selected from the offspring. Simultaneously, diploid male Pacific oysters and female Portuguese oysters were crossed to obtain hybrid diploid offspring. Finally, the fast-growing tetraploid males and hybrid diploid females were used to produce a new hybrid triploid oyster strain with strong stress resistance and rapid growth on a large scale. By combining selective breeding, hybrid breeding, and polyploid breeding techniques, a new triploid oyster strain with rapid growth, strong stress resistance, high survival rate, and good plumpness was developed, effectively addressing the problems faced by the Pacific oyster industry. In addition, the poor physical condition of three-fold seedlings can effectively protect seed rights and avoid pollution of marine biological gene pools. Controllable seed rights can, on the one hand, motivate germplasm researchers, R&D companies, and promotion companies, and on the other hand, regulate the amount of seedlings produced to increase the amount of aquaculture, thereby improving the plumpness of the oysters and promoting the quality improvement, efficiency enhancement, and sustainable development of the oyster industry. Summary of the Invention

[0005] The purpose of this invention is to provide an excellent breeding method for cultivating a new hybrid triploid oyster strain that is highly resistant to adverse conditions, grows rapidly, has a high survival rate, poor fertility, good quality, stable traits, and can be marketed year-round.

[0006] The method for cultivating a new fast-growing and highly resistant hybrid triploid oyster strain of the present invention includes the following steps:

[0007] Cultivate highly resistant and fast-growing diploid strains of oysters A and B, then further establish new tetraploid strains of oysters A and B that are highly resistant and fast-growing. Next, screen and establish new tetraploid strains of oysters A and B that are highly resistant and fast-growing. Cross the highly resistant and fast-growing diploid strains of oysters A and B to obtain new hybrid diploid strains that are highly resistant and fast-growing. Cross the new tetraploid strains of oysters with the new hybrid diploid strains to obtain new hybrid triploid oyster strains that are fast-growing and highly resistant.

[0008] Preferably, oyster A is Pacific oyster and oyster B is Portuguese oyster.

[0009] Preferably, the specific steps are as follows:

[0010] a. Cultivation of highly resilient and rapidly growing diploid strains: Multiple geographical populations of diploid Pacific oysters and Portuguese oysters were collected. Multiple individuals from each population were temporarily reared in the same seawater environment to induce gonadal maturation. At gonadal maturity, shell height was measured in each individual within each population and arranged by size. Gonads were dissected and sexes identified. Males and females with higher shell heights were selected and artificially mixed for fertilization to obtain F1 juveniles from each population. F1 juveniles were then placed in the main rearing area with higher mortality rates, and their shell height and survival rate were tracked. Those exhibiting good growth and survival performance were selected. The optimal geographical population is the group of individuals from the best geographical population. Diploid individuals from the optimal geographical population are brought to maturity in the same environment. Shell height is measured at gonadal maturity and the individuals are arranged from highest to lowest. They are then dissected and sexed. Male and female individuals with the highest shell height are selected to establish several families. Juvenile oysters from these families are simultaneously placed in the same sea area. Their shell height and survival rate are tracked and measured. The families with the best and most balanced growth and survival performance are selected. These are the highly resilient and fast-growing diploid strains, which are the Pacific oyster strains and the Portuguese oyster strains.

[0011] b. Establishment of a highly resilient and rapidly growing tetraploid induction population: In the optimal seawater environment, male and female individuals of a highly resilient and rapidly growing diploid strain of Pacific oysters were mixed-fertilized. After fertilization, an inducing agent was used to induce incubation, and the fertilized eggs were placed in seawater for hatching to obtain highly resilient and rapidly growing triploids. Highly resilient and rapidly growing triploid female individuals with better shell shape and larger shell height were selected and arranged from largest to smallest shell height. The female individuals with better gonadal development were selected first. At the same time, male individuals of a highly resilient and rapidly growing diploid strain with larger shell height and higher sperm activity were selected. After mixed-fertilization in the optimal seawater environment, an inducing agent was used to induce incubation, and the fertilized eggs were placed in seawater for hatching. This is the highly resilient and rapidly growing tetraploid induction population. Highly resilient and rapidly growing tetraploid induction populations of Pacific oysters and Portuguese oysters were obtained respectively.

[0012] c. Cultivation of a new tetraploid strain with high stress resistance and rapid growth: The tetraploid induced population with high stress resistance and rapid growth from b is cultured in a sea area with high mortality. Its ploidy changes, shell height and survival rate are tracked. Tetraploids are selected at the gonadal maturity stage, and shell height is arranged from high to low. Sex is identified, and male and female individuals with high shell height are selected separately. They are mixed and fertilized to establish F1 tetraploids. F1 tetraploids are cultured in a sea area with high mortality. At the gonadal maturity stage, Pacific oyster tetraploids and Portuguese oyster tetraploids are identified separately. They are dissected and arranged according to shell height from large to small. Female Pacific oyster tetraploids and male Portuguese oyster tetraploids with high shell height are selected. They are artificially mixed and fertilized to obtain a new tetraploid strain with high stress resistance and rapid growth.

[0013] d. Cultivation of new hybrid diploid strains with high stress resistance and rapid growth: From strain a, which has high stress resistance and rapid growth, Pacific oyster diploids and Portuguese oyster diploids with full gonads and good shell shape are selected. The sexes are identified by artificial dissection. Male Pacific oyster diploids and female Portuguese oyster diploids are selected separately. They are then arranged from highest to lowest shell height. Male Pacific oyster diploids and female Portuguese oyster diploids with higher shell heights are selected. They are then artificially mixed and fertilized to obtain hybrid F1 diploids. The hybrid F1 diploids are cultured in sea areas with high mortality rates. When the gonads reach maturity, individuals are randomly selected and arranged from highest to lowest shell height. Male and female individuals with higher shell heights are then artificially mixed and fertilized to obtain larvae. This is the new hybrid diploid strain with high stress resistance and rapid growth.

[0014] e. Mass production of new hybrid triploid lines with high stress resistance and rapid growth: The new tetraploid line with high stress resistance and rapid growth from c and the new hybrid diploid line with high stress resistance and rapid growth from d are placed in seawater with a salinity of 25-28‰ and a gradually increasing temperature to promote maturation; during the gonadal maturity period, the tetraploid males and diploid females are artificially dissected and identified, and mixed for fertilization to obtain new hybrid triploid lines with high stress resistance and rapid growth.

[0015] Preferably, the mixed fertilization described in e involves artificially mixing and incubating the cells in seawater with a salinity of 28‰ and a temperature of 24~26℃. This results in a new hybrid triploid strain that is highly resistant to adverse conditions and grows rapidly.

[0016] Further optimization includes the following steps:

[0017] a. Cultivation of highly resilient and rapidly growing diploid strains: At least five different geographical populations of diploid Pacific oysters and Portuguese oysters were collected, with at least 500 individuals obtained from each population. These individuals were temporarily housed in the same marine environment to induce gonadal maturation. At gonadal maturity, the shell height of each individual in each population was measured and arranged by size. The gonads were dissected and sexes identified. The top 10% of male and female individuals in terms of shell height were selected, and then artificially mixed and fertilized to obtain F1 juveniles from each population. The F1 juveniles were then placed in the main culture area with a high mortality rate, and their shell height and survival rate were tracked. Geographical populations with good growth and survival performance were selected. The optimal geographical population was selected. 1000 diploid individuals from the optimal geographical population were brought to maturity in the same environment. Shell height was measured at gonadal maturity and arranged from highest to lowest. The individuals were dissected and sexed. The top 30 male and female individuals in terms of shell height were selected. Following the principle of 1-to-1 fertilization, 30 families were established. Juvenile oysters from the 30 families were simultaneously placed in the same sea area. Their shell height and survival rate were tracked and measured. The families with the best and most balanced growth and survival performance were selected. These are the highly resilient and fast-growing diploid strains, which are the Pacific oyster strains and the Portuguese oyster strains.

[0018] b. Establishment of a highly resilient and rapidly growing tetraploid induction population: In the optimal seawater environment, one male and 20 female individuals from the highly resilient and rapidly growing diploid strain described in a above-mentioned section were mixed-fertilized. 13-16 minutes after fertilization, a prepared induction agent was added and treated for 18-22 minutes. The induction agent was then washed off, and the fertilized eggs were placed in seawater for incubation to obtain highly resilient and rapidly growing triploids. 500 highly resilient and rapidly growing triploid female individuals with good shell shape and large shell height were selected and arranged according to shell height from largest to smallest. The top 15 female individuals with good gonadal development were selected, and three male individuals with larger shell height and higher sperm motility were selected as high stress-resistant and fast-growing diploid strains. After mixed fertilization in the optimal seawater environment, the fertilized eggs were treated with an inducer for 23-30 minutes from 16 to 20 minutes after fertilization. Then the inducer was washed off and the fertilized eggs were placed in seawater for incubation. This is the high stress-resistant and fast-growing tetraploid inducer population. High stress-resistant and fast-growing tetraploid inducer populations of Pacific oyster and Portuguese oyster were obtained respectively.

[0019] c. Cultivation of a new high-resistance and fast-growing hybrid tetraploid strain: The high-resistance and fast-growing tetraploid induced population from b was cultured in a sea area with a high mortality rate. Its ploidy changes, shell height, and survival rate were tracked. At the gonadal maturity stage, 200 tetraploids were selected and arranged from high to low shell height. Sex was identified, and the top 10 male and female individuals with the highest shell height were selected. They were then mixed and fertilized to establish the F1 tetraploid strain. The F1 tetraploid strain was cultured in a sea area with a high mortality rate. At the gonadal maturity stage, 300 Pacific oyster tetraploids and 300 Portuguese oyster tetraploids were identified. They were dissected and arranged from high to low shell height. The top 30 female Pacific oyster tetraploids and the top 30 male Portuguese oyster tetraploids with the highest shell height were selected. They were then artificially mixed and fertilized to obtain a hybrid tetraploid strain, which is the new high-resistance and fast-growing tetraploid strain.

[0020] d. Cultivation of new hybrid diploid strains with high stress resistance and rapid growth: From strain a, which has high stress resistance and rapid growth, 500 Pacific oyster diploids and 500 Portuguese oyster diploids with full gonads and good shell shape were selected. They were dissected and identified as male and female. Male Pacific oyster diploids and female Portuguese oyster diploids were selected separately. They were then arranged from highest to lowest shell height. The top 20 Pacific oyster diploid males and Portuguese oyster diploid females with the highest shell height were selected and artificially mixed and fertilized to obtain hybrid F1 diploids. The hybrid F1 diploids were cultured in sea areas with high mortality rates. When the gonads matured, 1000 individuals were randomly selected and arranged from highest to lowest shell height. The top 50 males and females with the highest shell height were then artificially mixed and fertilized to obtain larvae. This is the new hybrid diploid strain with high stress resistance and rapid growth.

[0021] e. Mass production of highly stress-resistant and rapidly growing hybrid triploid strains: The highly stress-resistant and rapidly growing tetraploid strain from c and the highly stress-resistant and rapidly growing hybrid diploid strain from d are placed together in seawater with a salinity of 25-28‰ and a gradually increasing temperature to promote maturation; during the gonadal maturity period, tetraploid males and diploid females are artificially dissected and identified, artificially mixed and fertilized, and then incubated in seawater with a salinity of 28‰ and a temperature of 24-26℃. This is the highly stress-resistant and rapidly growing hybrid triploid strain.

[0022] Preferably, the collection of diploid oysters in step a requires at least 5 different geographical populations. The geographical populations of Pacific oysters and Portuguese oysters collected must be those that have undergone molecular identification to confirm that their genetic relationships are significantly different. At the same time, the selected diploid geographical populations of Pacific oysters must be from the main aquaculture areas in North China, and the selected diploid geographical populations of Portuguese oysters must be from the main aquaculture areas in South China.

[0023] Preferably, the process of temporarily raising and maturing gonads in the same seawater environment and maturing in the same environment, as described in step a, specifically refers to the following: for Pacific oysters, this environment has a salinity of 24-28‰ and a temperature of 14-26°C, with the temperature gradually increasing from 14°C to 26°C and maintained at 26°C; for Portuguese oysters, this environment has a salinity of 26-30‰ and a temperature of 22-28°C, with the temperature gradually increasing from 22°C to 28°C and maintained at 28°C.

[0024] Preferably, the geographical populations with good growth and survival performance mentioned in step a refer to the geographical populations that are ranked in the top 3 in terms of both shell height and survival rate, based on the average shell height and average survival rate from high to low.

[0025] Preferably, the establishment of several families in step a is based on the principle of 1-to-1 fertilization, selecting the top 30 male and female individuals with larger shell heights, and strictly comparing them in the order from front to back, with one male artificially fertilizing one female to form one family.

[0026] Preferably, step a, which involves selecting the family with the best and most balanced growth and survival performance, means arranging the shell height and cumulative survival rate of each family from high to low, first selecting the family with the highest survival rate, and then evaluating whether its shell height is in the top 5. If it is, it is the family with the best and most balanced growth and survival performance; if not, it is replaced in order.

[0027] Preferably, the main aquaculture areas, sea areas, and sea areas with high mortality rates mentioned in steps a, c, and d, as well as the same sea area in step a, refer to sea areas in the North China Sea where the mortality rate of Pacific oysters exceeds 50%, and sea areas in the South China Sea where the mortality rate of Portuguese oysters exceeds 30%.

[0028] Preferably, the inducing agent in step b is cytochalasin B or N6,N6-dimethylaminopurine.

[0029] Preferably, the incubation in seawater and the optimal seawater environment mentioned in step b specifically refer to incubating Pacific oyster larvae in filtered seawater with a salinity of 26-28‰ and a temperature of 24-26℃, and incubating Portuguese oyster larvae in filtered seawater with a salinity of 27-29‰ and a temperature of 26-28℃.

[0030] Preferably, the triploid female with better gonads mentioned in step b refers to one with full gonads, more than 500,000 eggs, more than 60% of the eggs being unblemished and pear-shaped or oval, and the eggs being rich in nutrients and relatively dark.

[0031] Preferably, when screening male and female individuals as described in steps a to d, careful observation under a microscope is necessary to strictly screen out hermaphroditic individuals and prevent accidental fertilization.

[0032] Preferably, during the mixed fertilization process described in steps a to e, strict procedures must be followed. Before extracting eggs from female individuals, they must be soaked in fresh water for 1 minute. The instruments used during fertilization must be rinsed with fresh water. Before fertilization, the system must be checked under a microscope to check for any accidental fertilization. Accidental fertilization must be strictly avoided.

[0033] Preferably, the seawater temperature gradually increased in step e specifically refers to gradually increasing the temperature from 14°C to 26°C, maintaining the water temperature at 14°C for the first 7 days, increasing the temperature by 1°C per day from day 8 to 16, increasing the temperature by 0.5°C per day from day 17 to 25, and maintaining the temperature at 26°C in the later stage.

[0034] Preferably, in step e, the number of breeding shells to be used can be selected based on the number of seedlings to be cultivated. This refers to using tetraploid males and diploid females according to the number of seedlings to be produced. Specifically, the average number of eggs per female individual needs to be determined quantitatively, and the number of male individuals to be used is determined according to the number of sperm around each egg.

[0035] This invention combines population selection breeding, family selection breeding, stress selection breeding, hybridization breeding, and polyploid breeding techniques. By collecting different geographical populations, it cultivates new diploid Pacific oysters and Portuguese oysters with high stress resistance and rapid growth through population selection and family selection techniques. Then, it applies polyploid breeding techniques to cultivate tetraploid Pacific oysters with high stress resistance and rapid growth, and tetraploid Portuguese oysters with high stress resistance and rapid growth, respectively. Furthermore, it uses hybridization breeding techniques to cross the tetraploid Pacific oysters and tetraploid Portuguese oysters, selecting fast-growing tetraploids from the offspring. Simultaneously, it conducts diploid breeding of Pacific oysters... Hybrid diploid offspring were obtained by crossing male oysters with female Portuguese oysters. Finally, by using fast-growing tetraploid males and hybrid diploid females, a new hybrid triploid oyster strain with strong stress resistance and fast growth was produced on a large scale. This successfully cultivated a new hybrid triploid oyster strain with fast growth, strong stress resistance, high survival rate, stable traits, and poor fertility. It effectively solved the industrial problems of high mortality rate, poor stress resistance, and slow growth of Pacific oysters in the North China Sea, and helped to improve the quality and efficiency of the oyster farming industry in the North China Sea and promote its sustainable and healthy development.

[0036] This invention is primarily based on the industry problems of Pacific oyster diploids and triploids in recent years, such as a sharp decline in survival rate, poor stress resistance, slow growth, high farming risk, and low economic benefits. At the same time, interspecific triploid hybrids, which have low innovation (can be easily replicated), also have problems such as easy replication, difficulty in protecting species rights, low uniqueness, and unstable traits. Therefore, by scientifically and effectively combining population selection, family selection, stress selection, hybridization breeding, and polyploid breeding techniques, a new fast-growing and stress-resistant hybrid triploid oyster strain is cultivated to solve the above problems. The new hybrid triploid oyster strain cultivated by this invention combines the characteristics of Pacific oysters, such as adaptability to the North China Sea, good plumpness, and hard shell, with those of Portuguese oysters, such as rapid growth, strong resistance, and high survival rate. It exhibits the superposition of breeding advantages, hybrid vigor, and triploid advantages, and has the advantages of rapid growth, strong resistance, adaptation to the North China Sea habitat, high survival rate, poor fertility, good quality, and high plumpness throughout the year. It has important industrial promotion and application value and potential. The new hybrid triploid oyster strain obtained by this invention has a 391.14%~465.30% higher survival rate, a 57.24%~59.64% higher shell height, and a 126.77%~158.01% higher total weight than diploid Pacific oysters; and a 141.62%~204.38% higher survival rate, a 29.03%~33.87% higher shell height, and a 51.34%~64.43% higher total weight than triploid Pacific oysters.

[0037] The new hybrid triploid oyster strain cultivated in this invention not only possesses advantages such as rapid growth, strong stress resistance, adaptation to the North China Sea habitat, high survival rate, low fertility, good quality, and good plumpness, but also utilizes parent lines that have undergone specific modification (stress selection or hybridization), resulting in high recognizability, strong innovation (simple replication is difficult), good seed rights protection, and stable traits. Seedling companies are more willing to commercialize and promote this technology. Therefore, this invention has advantages such as great potential for industrial application, high innovation, good seed rights protection, and strong operability. Detailed Implementation

[0038] The following examples illustrate in detail the cultivation method of a new hybrid triploid oyster strain that is fast-growing and highly resistant to adverse conditions provided by the present invention, but are not intended to limit the scope of the invention.

[0039] Example 1

[0040] a. Cultivation of highly resilient and rapidly growing diploid strains: In April 2017, six different geographical populations of Pacific oysters were collected from sea areas including Lushun, Guanglu Island, and Zhuanghe in Dalian, Liaoning Province; Rizhao, Yantai, and Rongcheng in Shandong Province; and Weihai, Shandong Province. For each population, 673, 724, 617, 733, 782, and 689 individuals were collected, respectively. Gonadal maturation was induced in seawater with a salinity of 26-28‰ and a temperature of 14-26℃ (the temperature was gradually increased from 14℃ to 26℃ and maintained at 26℃). Dead individuals were selected at designated times and locations, and the oysters were fed sufficient Chaetoceros algae and microalgae. The maturation cycle of *Chlorella vulgaris* is 55 days. After dissection and microscopic observation of gonadal development, and finding that the gonads of each population were full and mature, 500 individuals were randomly selected from each population. Their shell height was measured with calipers, and they were arranged from highest to lowest shell height. Dissection and microscopic examination were then used to identify males and females, accurately selecting each individual and discarding hermaphroditic individuals. Then, the top 10% of male and female individuals by shell height were selected. The female individuals were immersed in fresh water for 1 minute, strictly avoiding sperm contamination. The eggs from the selected female individuals were then collected, mixed, and soaked until over 80% of the eggs were round or oval. Microscopic examination revealed no accidental contamination. Sperm from each male individual was collected, soaked in seawater to activate them, and mixed thoroughly when sperm motility was good. Artificial fertilization was then performed with 2-3 sperm around each egg; these were the F1 generation for each geographic population. After attachment, the F1 generation from six geographic populations was simultaneously deployed in a high-mortality sea area in Laizhou, Yantai, and their growth and survival rates were tracked and compared. At day 360, the top three geographic populations in terms of shell height were Rizhao (Shandong), Guanglu Island (Liaoning), and Zhuanghe (Liaoning), while the top three populations in terms of survival rate were Zhuanghe (Liaoning), Laizhou (Shandong), and Weihai (Shandong). The best geographical population was the Zhuanghe population in Liaoning Province, which showed the best and most balanced growth and survival. From this population, 1000 individuals with regular shell shapes and larger size were randomly selected and matured using the gonadal maturation method described above. During gonadal maturity, the top 30 male and female individuals by shell height were selected and fertilized one-to-one according to shell height from front to back, resulting in 30 families. After attachment, the juvenile oysters from these 30 families were simultaneously released into a high-mortality area in Laizhou, Shandong Province. One family with good and balanced growth and survival was selected, which became the highly resilient and fast-growing diploid strain of Pacific oyster.

[0041] Following the steps and methods described above, geographical populations of Portuguese oysters from five different sea areas—Taishan in Guangdong, Beihai in Guangxi, Zhangpu, Zhao'an in Fujian, and Lianjiang in Fujian—were collected. For each population, 936, 946, 878, 952, and 1043 individuals were collected, respectively. Gonadal maturation was induced in an environment with a salinity of 27–28‰ and a temperature of 22–28℃ (the temperature was gradually increased from 22℃ to 28℃ and maintained at 28℃). Dead individuals were selected at specific times and locations. Sufficient amounts of Chaetoceros and Microchlorophyllaria were provided, and the maturation period was 46 days. Following the same breeding steps as the Pacific oysters described above, growth and survival rates were obtained. The best-performing and most balanced geographical population was the Beihai, Guangxi population, which was considered the optimal geographical population. From this optimal geographical population, 1000 individuals with regular shell shapes and larger sizes were randomly selected and matured using the aforementioned gonadal maturation method. During gonadal maturity, the top 30 male and female individuals by shell height were selected, and fertilization was performed one-to-one according to shell height from front to back, resulting in 30 families. After attachment, the juvenile oysters from these 30 families were simultaneously placed in the high-mortality area of ​​the Beihai Zhulin Salt Field in Guangxi. One family with good and balanced growth and survival was selected; this is the highly resilient and rapidly growing diploid strain of the Portuguese oyster.

[0042] b. Establishment of a highly resilient and rapidly growing tetraploid induced population: In June 2020, in the optimal seawater environment of 27.3‰ salinity and 25.6℃, one male and 20 female individuals from the highly resilient and rapidly growing diploid Pacific oyster strain mentioned in a above were mixed-fertilized. Sixteen minutes after fertilization, a prepared 0.45 mg / mL cytochalasin B inducer was added and treated for 22 minutes. The inducer, some bad eggs, and impurities were then washed away using a 500-mesh silk screen. The fertilized eggs were then incubated in seawater with a salinity of 26.8‰ and a temperature of 25.8℃ to obtain highly resilient and rapidly growing triploid Pacific oysters. Pluripotency was detected by flow cytometry on the second day of D-type larvae, with a triploid rate of 90.47%. Triploid larvae were meticulously cultured, and when 45% of the larvae showed eyespots, they were placed on a scallop shell attachment substrate. Juvenile oysters were cultured in Laizhou, Shandong Province. On day 340, 500 highly resilient and rapidly growing triploid females with good shell shape and large shell height were selected. They were then arranged from largest to smallest shell height, and the top 15 females with the best gonadal development were selected. At the same time, 3 males of a highly resilient and rapidly growing diploid strain with large shell height and high sperm activity were also selected. After fertilization in the optimal seawater environment of 26.6‰ salinity and 25.5℃, the fertilized eggs were treated with a prepared 0.45 mg / mL cytochalasin B inducer for 30 minutes 20 minutes after fertilization. Then, the inducer, some bad eggs, impurities, etc. were washed away through a 500-mesh silk screen, and the fertilized eggs were put into seawater for incubation. This is the highly resilient and rapidly growing tetraploid induced population of Pacific oysters.

[0043] Meanwhile, in the optimal seawater environment of 28.2‰ salinity and 26.7℃, one male and 20 female individuals of the highly resilient and rapidly growing diploid strain of Portuguese oyster (a) were taken and mixed-fertilized. Thirteen minutes after fertilization, a prepared 0.55 mg / mL cytochalasin B inducer was added and treated for 18 minutes. The inducer, some bad eggs, and impurities were then washed away using a 500-mesh silk screen. The fertilized eggs were then incubated in seawater with a salinity of 28.5‰ and a temperature of 27.7℃ to obtain highly resilient and rapidly growing triploid Portuguese oysters. Pluripotency was detected by flow cytometry on the second day of D-type larvae, with a triploidity rate of 94.33%. Triploid larvae were then carefully cultured, and oyster shell attachment substrates were introduced when 45% of the larvae showed eye spots. In Beihai, Guangxi, oysters were cultured. On day 280, 500 highly resilient and rapidly growing triploid females with good shell shape and large shell height were selected. These females were then arranged from largest to smallest shell height, and the top 15 females with the best gonadal development were selected. At the same time, 3 males from a highly resilient and rapidly growing diploid strain with large shell height and high sperm activity were also selected. After fertilization in the optimal seawater environment of 27.9‰ salinity and 27.7℃, the fertilized eggs were treated with a prepared 0.55 mg / mL cytochalasin B inducer for 24 minutes, 16 minutes after fertilization. The inducer, some bad eggs, and impurities were then washed away through a 500-mesh silk screen, and the fertilized eggs were placed in seawater for incubation. This is the highly resilient and rapidly growing tetraploid induced population of Portuguese oysters.

[0044] c. Cultivation of new tetraploid hybrid strains with high stress resistance and rapid growth: Highly stress-resistant and rapidly growing tetraploid induced populations of Pacific oysters (b) were cultured in the sea area of ​​Laizhou, Shandong Province, while highly stress-resistant and rapidly growing tetraploid induced populations of Portuguese oysters were cultured in Beihai, Guangxi Province. Their ploidy changes, shell height, and survival rate were tracked. During the gonadal maturity period in June, ploidy was sequentially identified using flow cytometry, and 200 tetraploids were selected and arranged according to shell height from highest to lowest. Sex was determined using a microscope, and the top 10 male and female individuals by shell height were selected for mixed fertilization. Pacific oyster F1 tetraploids and Portuguese oyster F1 tetraploids were established separately. The Pacific oyster F1 tetraploids were cultured in Laizhou, Shandong, and the Portuguese oyster F1 tetraploids were cultured in Beihai, Guangxi. Their mortality rate was tracked. At the gonadal maturity stage, individual individuals were examined by flow cytometry, and 300 Pacific oyster tetraploids and 300 Portuguese oyster tetraploids were identified. They were dissected and arranged according to shell height from largest to smallest. The sex was determined by microscopy, and the top 30 female Pacific oyster tetraploids and the top 30 male Portuguese oyster tetraploids by shell height were selected. Artificial mixing and fertilization were carried out in seawater with a salinity of 27.2‰ and a temperature of 25.8℃ to obtain hybrid tetraploids, which are new hybrid tetraploid strains with high stress resistance and rapid growth.

[0045] d. Cultivation of a new hybrid diploid strain with high stress resistance and rapid growth: In June 2021, 500 Pacific oyster diploids and 500 Portuguese oyster diploids with full gonads and good shell shape were selected from strain a, which has high stress resistance and rapid growth. The sexes were identified by artificial dissection. Male Pacific oyster diploids and female Portuguese oyster diploids were selected separately. They were then arranged from highest to lowest shell height. The top 20 Pacific oyster diploid males and Portuguese oyster diploid females by shell height were selected and artificially mixed for fertilization to obtain hybrid F1 diploids. The hybrid F1 diploids were cultured in Beihai, Guangxi. 1000 individuals were randomly selected at gonadal maturity and arranged from largest to smallest shell height. The top 50 males and females by shell height were then artificially mixed for fertilization to obtain larvae. This is the new hybrid diploid strain with high stress resistance and rapid growth.

[0046] e. Mass production of highly stress-resistant and rapidly growing hybrid triploid strains: In January 2023, 400 individuals from the highly stress-resistant and rapidly growing hybrid tetraploid strain c and 1500 individuals from the highly stress-resistant and rapidly growing hybrid diploid strain d were randomly selected. These strains were uniformly placed in a salinity environment of 27.3‰, with the temperature gradually increased (from 14℃ to 26℃, maintaining a constant temperature of 14℃ for the first 7 days, increasing by 1℃ daily from days 8 to 16, and increasing by 0.5℃ daily from days 17 to 25). The gonads were matured in seawater at a temperature of 26°C for 48 days. Microscopic observation revealed that the gonads were full, the sperm were active, and the proportion of round or oval eggs was relatively high. At this point, the gonads were mature. The tetraploid males and diploid females were identified by artificial dissection and flow cytometry and microscopy. Twelve tetraploid males and 520 diploid females were artificially mixed and fertilized, and then incubated in seawater with a salinity of 28‰ and a temperature of 25.6°C. This is the new hybrid triploid strain that is highly resistant to adverse conditions and grows rapidly.

[0047] Simultaneously, control groups of diploid and triploid Pacific oysters were established. Three groups—a new hybrid triploid strain with high stress resistance and rapid growth, diploid Pacific oysters, and triploid Pacific oysters—were simultaneously cultured in two sea areas: Laizhou, Shandong, and Jinshitan, Dalian, Liaoning (due to the inability of Portuguese oysters to withstand low temperatures and survive in northern seas, control groups of diploid and triploid Portuguese oysters were not included). Shell height, total weight, and survival rate were tracked. The results showed that at day 360, in the Laizhou, Shandong sea area, compared to diploid Pacific oysters, the new hybrid triploid strain with high stress resistance and rapid growth exhibited a 57.24% increase in shell height, a 158.01% increase in total weight, and a 391.14% increase in survival rate; compared to triploid Pacific oysters, the new hybrid triploid strain with high stress resistance and rapid growth exhibited a 33.87% increase in shell height, a 64.43% increase in total weight, and a 204.38% increase in survival rate. In the Jinshitan sea area of ​​Liaoning, compared with diploid Pacific oysters, the new hybrid triploid strain with high stress resistance and rapid growth showed a 59.64% increase in shell height, a 126.77% increase in total weight, and a 465.30% increase in survival rate; compared with triploid Pacific oysters, the new hybrid triploid strain with high stress resistance and rapid growth showed a 29.03% increase in shell height, a 51.34% increase in total weight, and a 141.62% increase in survival rate.

[0048] The main aquaculture areas, sea areas, and sea regions with high mortality rates mentioned in steps a, c, and d, as well as the same sea area mentioned in step a, refer to the sea areas in North China where the mortality rate of Pacific oysters exceeds 50%, and the sea areas in South China where the mortality rate of Portuguese oysters exceeds 30%.

[0049] The triploid female with good gonads mentioned in step b refers to one with full gonads, more than 500,000 eggs, more than 60% of the eggs being unblemished and pear-shaped or oval, and the eggs being rich in nutrients and relatively dark.

[0050] When screening male and female individuals as described in steps a to d, careful observation under a microscope is necessary to strictly screen out hermaphroditic individuals and prevent accidental fertilization.

[0051] During the mixed fertilization process described in steps a to e, strict procedures must be followed. Before extracting eggs from female individuals, they must be soaked in fresh water for 1 minute. The instruments used during fertilization must be rinsed with fresh water. Before fertilization, the individuals must be examined under a microscope to check for any accidental fertilization. Accidental fertilization must be strictly avoided.

Claims

1. A method for cultivating a new hybrid triploid oyster strain that is fast-growing and highly resistant, characterized in that, Includes the following steps: Cultivate highly resistant and fast-growing diploid strains of oysters A and B, then further establish new tetraploid strains of oysters A and B that are highly resistant and fast-growing. Next, screen and establish new tetraploid strains of oysters A and B that are highly resistant and fast-growing. Cross the highly resistant and fast-growing diploid strains of oysters A and B to obtain new hybrid diploid strains that are highly resistant and fast-growing. Cross the new tetraploid strains of oysters with the new hybrid diploid strains to obtain new hybrid triploid oyster strains that are fast-growing and highly resistant.

2. The method according to claim 1, characterized in that, The oyster A mentioned is the Pacific oyster, and the oyster B mentioned is the Portuguese oyster.

3. The method according to claim 2, characterized in that, The specific steps are as follows: a. Cultivation of highly resilient and rapidly growing diploid strains: Multiple geographical populations of diploid Pacific oysters and Portuguese oysters were collected. Multiple individuals from each population were temporarily reared in the same seawater environment to promote gonad maturation. At gonadal maturity, the shell height of each individual in each population was measured and arranged by size. The gonads were dissected and the sex was identified. Male and female individuals with higher shell heights were selected and then artificially mixed and fertilized to obtain F1 juveniles from each population. The F1 juveniles were placed in the main rearing area with a high mortality rate. Their shell height and survival rate were tracked, and the geographical population with better growth and survival performance was selected as the optimal geographical population. Diploid individuals from the optimal geographical population were brought to maturity in the same environment. Shell height was measured at gonadal maturity and arranged from highest to lowest. The individuals were dissected and sexed, and male and female individuals with higher shell heights were selected to establish several families. Juvenile oysters from different families were simultaneously placed in the same sea area, and their shell height and survival rate were tracked and measured. The families with the best and most balanced growth and survival performance were selected, which are the highly resilient and fast-growing diploid strains. This yielded highly resilient and fast-growing diploid strains of Pacific oysters and Portuguese oysters. b. Establishment of a highly resilient and rapidly growing tetraploid induction population: In the optimal seawater environment, male and female individuals of a highly resilient and rapidly growing diploid strain of Pacific oysters were mixed-fertilized. After fertilization, an inducing agent was used to induce incubation, and the fertilized eggs were placed in seawater for hatching to obtain highly resilient and rapidly growing triploids. Highly resilient and rapidly growing triploid female individuals with better shell shape and larger shell height were selected and arranged from largest to smallest shell height. The female individuals with better gonadal development were selected first. At the same time, male individuals of a highly resilient and rapidly growing diploid strain with larger shell height and higher sperm activity were selected. After mixed-fertilization in the optimal seawater environment, an inducing agent was used to induce incubation, and the fertilized eggs were placed in seawater for hatching. This is the highly resilient and rapidly growing tetraploid induction population. Highly resilient and rapidly growing tetraploid induction populations of Pacific oysters and Portuguese oysters were obtained respectively. c. Cultivation of a new tetraploid strain with high stress resistance and rapid growth: The tetraploid induced population with high stress resistance and rapid growth from b is cultured in a sea area with high mortality. Its ploidy changes, shell height and survival rate are tracked. Tetraploids are selected at the gonadal maturity stage, and shell height is arranged from high to low. Sex is identified, and male and female individuals with high shell height are selected separately. They are mixed and fertilized to establish F1 tetraploids. F1 tetraploids are cultured in a sea area with high mortality. At the gonadal maturity stage, Pacific oyster tetraploids and Portuguese oyster tetraploids are identified separately. They are dissected and arranged according to shell height from large to small. Female Pacific oyster tetraploids and male Portuguese oyster tetraploids with high shell height are selected. They are artificially mixed and fertilized to obtain a new tetraploid strain with high stress resistance and rapid growth. d. Cultivation of new hybrid diploid strains with high stress resistance and rapid growth: From strain a, which has high stress resistance and rapid growth, Pacific oyster diploids and Portuguese oyster diploids with full gonads and good shell shape are selected. The sexes are identified by artificial dissection. Male Pacific oyster diploids and female Portuguese oyster diploids are selected separately. They are then arranged from highest to lowest shell height. Male Pacific oyster diploids and female Portuguese oyster diploids with higher shell heights are selected. They are then artificially mixed and fertilized to obtain hybrid F1 diploids. The hybrid F1 diploids are cultured in sea areas with high mortality rates. When the gonads reach maturity, individuals are randomly selected and arranged from highest to lowest shell height. Male and female individuals with higher shell heights are then artificially mixed and fertilized to obtain larvae. This is the new hybrid diploid strain with high stress resistance and rapid growth. e. Mass production of new hybrid triploid lines with high stress resistance and rapid growth: The new tetraploid line with high stress resistance and rapid growth from c and the new hybrid diploid line with high stress resistance and rapid growth from d are placed in seawater with a salinity of 25-28‰ and a gradually increasing temperature to promote maturation; during the gonadal maturity period, the tetraploid males and diploid females are artificially dissected and identified, and mixed for fertilization to obtain new hybrid triploid lines with high stress resistance and rapid growth.

4. The method according to claim 3, characterized in that, The mixed fertilization mentioned in step e involves artificially mixing and incubating the cells in seawater with a salinity of 28‰ and a temperature of 24~26℃. This results in a new hybrid triploid strain that is highly resistant to adverse conditions and grows rapidly.

5. The method according to claim 3, characterized in that, Includes the following steps: a. Cultivation of highly resilient and rapidly growing diploid strains: Collect at least 5 different geographical populations of diploid Pacific oysters and Portuguese oysters, obtaining at least 500 individuals from each population. Temporarily raise them in the same seawater environment and promote gonad maturation. At gonadal maturity, measure the shell height of each individual in each population and arrange them by size. Dissect the gonads and identify males and females. Select the top 10% of male and female individuals by shell height, and then artificially mix and fertilize them to obtain F1 juveniles from each population. Place the F1 juveniles in the main culture area with high mortality rate, and track their shell height and survival rate. Select the geographical population with better growth and survival performance, which is the optimal geographical population. One thousand diploid individuals from the optimal geographical population were brought to maturity in the same environment. Shell height was measured at gonadal maturity and arranged from tallest to shortest. The individuals were dissected and sexed, and the top 30 male and female individuals in terms of shell height were selected. Fertilization was carried out on a one-to-one basis to establish 30 families. Juvenile oysters from these 30 families were simultaneously placed in the same sea area, and their shell height and survival rate were tracked and measured. The families with the best and most balanced growth and survival performance were selected. These are the highly resilient and fast-growing diploid strains, which are the Pacific oyster strains and the Portuguese oyster strains. b. Establishment of a highly resilient and rapidly growing tetraploid induction population: In the optimal seawater environment, one male and 20 female individuals from the highly resilient and rapidly growing diploid strain described in a above-mentioned section were mixed-fertilized. 13-16 minutes after fertilization, a prepared induction agent was added and treated for 18-22 minutes. The induction agent was then washed off, and the fertilized eggs were placed in seawater for incubation to obtain highly resilient and rapidly growing triploids. 500 highly resilient and rapidly growing triploid female individuals with good shell shape and large shell height were selected and arranged according to shell height from largest to smallest. The top 15 female individuals with good gonadal development were selected, and three male individuals with larger shell height and higher sperm motility were selected as high stress-resistant and fast-growing diploid strains. After mixed fertilization in the optimal seawater environment, the fertilized eggs were treated with an inducer for 23-30 minutes from 16 to 20 minutes after fertilization. Then the inducer was washed off and the fertilized eggs were placed in seawater for incubation. This is the high stress-resistant and fast-growing tetraploid inducer population. High stress-resistant and fast-growing tetraploid inducer populations of Pacific oyster and Portuguese oyster were obtained respectively. c. Cultivation of a new high-resistance and fast-growing hybrid tetraploid strain: The high-resistance and fast-growing tetraploid induced population from b was cultured in a sea area with a high mortality rate. Its ploidy changes, shell height, and survival rate were tracked. At the gonadal maturity stage, 200 tetraploids were selected and arranged from high to low shell height. Sex was identified, and the top 10 male and female individuals with the highest shell height were selected. They were then mixed and fertilized to establish the F1 tetraploid strain. The F1 tetraploid strain was cultured in a sea area with a high mortality rate. At the gonadal maturity stage, 300 Pacific oyster tetraploids and 300 Portuguese oyster tetraploids were identified. They were dissected and arranged from high to low shell height. The top 30 female Pacific oyster tetraploids and the top 30 male Portuguese oyster tetraploids with the highest shell height were selected. They were then artificially mixed and fertilized to obtain a hybrid tetraploid strain, which is the new high-resistance and fast-growing tetraploid strain. d. Cultivation of new hybrid diploid strains with high stress resistance and rapid growth: From strain a, which has high stress resistance and rapid growth, 500 Pacific oyster diploids and 500 Portuguese oyster diploids with full gonads and good shell shape were selected. They were dissected and identified as male and female. Male Pacific oyster diploids and female Portuguese oyster diploids were selected separately. They were then arranged from highest to lowest shell height. The top 20 Pacific oyster diploid males and Portuguese oyster diploid females with the highest shell height were selected and artificially mixed and fertilized to obtain hybrid F1 diploids. The hybrid F1 diploids were cultured in sea areas with high mortality rates. When the gonads matured, 1000 individuals were randomly selected and arranged from highest to lowest shell height. The top 50 males and females with the highest shell height were then artificially mixed and fertilized to obtain larvae. This is the new hybrid diploid strain with high stress resistance and rapid growth. e. Mass production of highly stress-resistant and rapidly growing hybrid triploid strains: The highly stress-resistant and rapidly growing tetraploid strain from c and the highly stress-resistant and rapidly growing hybrid diploid strain from d are placed together in seawater with a salinity of 25-28‰ and a gradually increasing temperature to promote maturation; during the gonadal maturity period, tetraploid males and diploid females are artificially dissected and identified, artificially mixed and fertilized, and then incubated in seawater with a salinity of 28‰ and a temperature of 24-26℃. This is the highly stress-resistant and rapidly growing hybrid triploid strain.

6. The method according to claim 3 or 5, characterized in that, The collection of diploid oysters as described in step a requires at least 5 different geographical populations. The geographical populations of Pacific oysters and Portuguese oysters collected must be those that have undergone molecular identification to confirm that their genetic relationships are significantly different. At the same time, the selected diploid geographical populations of Pacific oysters must be from the main aquaculture areas in North China, and the selected diploid geographical populations of Portuguese oysters must be from the main aquaculture areas in South China.

7. The method according to claim 3 or 5, characterized in that, The temporary rearing and gonad maturation in the same seawater environment described in step a, and the maturation in the same environment, specifically, for Pacific oysters, this environment is a salinity of 24~28‰ and a temperature of 14~26℃, with the temperature gradually increased from 14℃ to 26℃ and maintained at 26℃; for Portuguese oysters, this environment is a salinity of 26~30‰ and a temperature of 22~28℃, with the temperature gradually increased from 22℃ to 28℃ and maintained at 28℃.

8. The method according to claim 3 or 5, characterized in that, The selection of geographical populations with good growth and survival performance in step a refers to arranging them from highest to lowest based on shell height and survival rate, with the top 3 geographical populations having both shell height and survival rate. Preferably, the establishment of several families in step a involves selecting the top 30 male and female individuals with the largest shell heights according to the principle of 1-to-1 fertilization, and performing artificial fertilization on a single male and a single female in a strict order from front to back, thus forming one family. Preferably, the selection of the family with the best and most balanced growth and survival performance in step a involves arranging the shell height and cumulative survival rate of each family from highest to lowest, first selecting the family with the highest survival rate, and then assessing whether its shell height is in the top 5. If it is, it is the family with the best and most balanced growth and survival performance; if not, it is replaced in order.

9. The method according to claim 3 or 5, characterized in that, The main breeding areas, sea areas, and sea areas with high mortality rates mentioned in steps a, c, and d, as well as the same sea area mentioned in step a, refer to sea areas in the North China Sea where the mortality rate of Pacific oysters exceeds 50%, and sea areas in the South China Sea where the mortality rate of Portuguese oysters exceeds 30%. Preferably, the inducing agent mentioned in step b is cytochalasin B or N6,N6-dimethylaminopurine. Preferably, the incubation in seawater and the optimal seawater environment mentioned in step b specifically refer to incubating Pacific oyster larvae in filtered seawater with a salinity of 26-28‰ and a temperature of 24-26℃, and incubating Portuguese oyster larvae in filtered seawater with a salinity of 27-29‰ and a temperature of 26-28℃.

10. The method according to claim 3 or 5, characterized in that, The seawater temperature gradually increased in step e specifically refers to gradually increasing the temperature from 14℃ to 26℃, maintaining a constant water temperature of 14℃ for the first 7 days, increasing the temperature by 1℃ per day from day 8 to 16, increasing the temperature by 0.5℃ per day from day 17 to 25, and maintaining a temperature of 26℃ in the later stage.