A method for breeding a new line of fast-growing, low-salt and high-temperature-adaptable three-way hybrid oysters

Abstract

The application constructs a hybrid basic population GA F0 by group hybridization of long oyster as female parent and Fujian oyster as male parent; the GA F0 is used as a basis, shell height selection is combined with high-temperature heat stress screening, continuous multi-generation population breeding is carried out, and the GA breeding population GA F6 with rapid growth and high-temperature tolerance is obtained; the RGA F0 is constructed by hybridization of near Jiang oyster as female parent and GA F6 as male parent; the RGA F0 is used as a basic population, shell height selection is combined with low-salt and high-temperature combined stress screening, continuous multi-generation population breeding is carried out, and the fast-growing, low-salt and high-temperature-adaptable oyster three-way hybrid new strain is obtained. The application establishes an oyster three-way hybrid breeding procedure, so that the breeding population has the characteristics of rapid growth, low-salt tolerance and high-temperature tolerance, and has a good industrial application prospect.

Description

Technical Field

[0001] This invention belongs to the field of shellfish breeding technology, specifically relating to a method for cultivating a new three-way hybrid oyster strain that is fast-growing, low-salt, and highly adaptable to high temperatures. Background Technology

[0002] Oysters are important marine aquaculture species, holding a significant position in my country's marine aquaculture industry. In recent years, the scale of oyster farming in my country has continued to expand, and the level of industrialization has been continuously improving. In 2024, oyster production reached 7.2525 million tons (China Fisheries Statistical Yearbook, 2025). The oysters farmed along my country's coast are mainly of the genus *Crassostrea*, including the giant oyster (*Crassostrea gigas*), the Fujian oyster (*Crassostrea angulata*), and the ark oyster (*Crassostrea ariakensis*). Giant oysters are mainly distributed along the coast north of the Yangtze River in my country, possessing advantages such as rapid growth, large size, strong environmental adaptability, and delicious meat. The Fujian oyster, mainly produced in Fujian and other southern coastal areas, is characterized by its small size, rapid growth, and strong heat resistance. The ark oyster is mainly distributed in estuaries and nearshore brackish waters, exhibiting strong tolerance to salinity fluctuations and maintaining good feeding and growth performance even under low to medium salinity conditions.

[0003] To meet the industry's demand for high-yield, stable-yield, and high-quality seedlings, hybridization breeding and selective breeding technologies have been integrated and developed in recent years, becoming an important direction for shellfish genetic improvement. Selective breeding can obtain stable and superior lines through continuous generations of targeted screening. Hybridization breeding can integrate the superior traits of the parents and utilize hybrid vigor to improve overall production performance.

[0004] During periods of concentrated summer rainfall or increased runoff, salinity in aquaculture areas often drops sharply, frequently compounded by high summer temperatures, creating a complex stress environment. This often disrupts oyster osmotic pressure regulation and metabolic balance, leading to limited feeding and growth, decreased resilience, and in severe cases, mass mortality during the summer, posing a significant risk to aquaculture production. Currently, purebred or hybrid populations primarily cultivated using Pacific oysters and Fujian oysters, while possessing certain growth and survival advantages, still face challenges under the combined stress of high summer temperatures and sharp salinity drops. These limitations hinder the full realization of these growth advantages and result in decreased survival rates, failing to meet the oyster industry's demand for seedlings with "rapid growth and high survival rates."

[0005] Therefore, there is an urgent need to establish a breeding method that can be implemented on a large scale, combining the three-way hybridization of Pacific oyster, Fujian oyster and oyster with targeted population selection, so that the selected population can still maintain rapid growth and significantly improve the summer survival rate in the combined stress environment of low salinity and high temperature, thereby creating a new three-way hybrid oyster strain that is more suitable for promotion. Summary of the Invention

[0006] To overcome the defects and shortcomings of the prior art, this invention provides a method for cultivating a new three-way hybrid oyster strain that is fast-growing, low-salt, and highly adaptable to high temperatures.

[0007] A method for cultivating a new three-way hybrid oyster strain that is fast-growing, low-salt, and highly adaptable to high temperatures, characterized by comprising the following steps:

[0008] (1) Construction of the basic population of GA: The new variety of Pacific oyster “Haida No. 1” (GS-01-005-2013) was used as the female parent and Fujian oyster was used as the male parent to carry out population hybridization and obtain the basic population of Pacific oyster × Fujian oyster GA F0;

[0009] (2) GA population selection: Based on the GA F0 population, individuals with shell height in the top 20% are selected as primary parents; before the gonads develop, the primary parents are subjected to high temperature stress to select for high temperature tolerance, and the surviving individuals are retained as preferred parents; the preferred parents are artificially inseminated, and after hatching and cultivation, GA F1 is obtained; the population is continuously selected for 6 generations to obtain the GA F6 population.

[0010] (3) Construction of the three-way hybrid basic population: The three-way hybrid basic population RGA F0 was obtained by hybridization with Crassostrea lizard as the female parent and GA F6 as the male parent;

[0011] (4) RGA population selection: Based on RGA F0, individuals with shell height in the top 20% are selected as primary parents; before the gonads develop, the primary parents are subjected to low salt and high temperature synergistic stress, and low salt and high temperature tolerance selection is carried out, and the surviving individuals are retained as preferred parents; the preferred parents are artificially inseminated, and RGA F1 is obtained after hatching and cultivation; after four generations of continuous selection, a new three-way hybrid line RGA F4 is obtained.

[0012] Furthermore, the Fujian oysters mentioned in step (1) above refer to commercial populations farmed in Zhangzhou, Fujian; dissecting the long oysters, selecting female individuals with well-developed gonads, uniformly developed eggs, and dark-colored yolks as the maternal parent; dissecting the Fujian oysters, selecting male individuals with strong sperm motility, using molecular markers for species identification, and retaining purebred Fujian oysters as the paternal parent; the number of each parent parent is no less than 50.

[0013] Furthermore, the high-temperature resistance selection in step (2) above is carried out before the gonads develop, in order to reduce the damage of heat shock to gametes and improve the fertilization success rate; the high-temperature resistance selection is divided into 3 steps: artificially heat shocking the shell-high initial selected parents at 34°C for 96 hours to achieve an initial mortality rate of 25–30%; transferring the surviving individuals to an outdoor pond (25–30°C) for temporary rearing for 7–15 days, using natural heat shock to subject the surviving individuals to high-temperature stress, and controlling the cumulative heat shock mortality rate to about 50%; transferring the surviving individuals to an indoor cement pond (21–23°C) for temporary rearing to restore vitality.

[0014] Furthermore, the oysters mentioned in step (3) above are commercially farmed in Qingdao, Shandong; female individuals with well-developed gonads, uniform egg development, and dark yolk color are selected by dissecting the oysters, and species identification is performed using molecular markers, and purebred oysters are retained as the maternal parent; GA F6 is dissected, and male individuals with strong sperm motility are selected as the paternal parent; the number of each parent is not less than 50.

[0015] In step (4) above, the selection for low salt tolerance and high temperature tolerance is carried out before the gonads develop, in order to reduce the damage of stress to gametes and improve the fertilization success rate. The selection for low salt tolerance and high temperature tolerance is divided into three steps: applying artificial stress of 15‰ salinity and 34℃ water temperature to the initial selected population with high shells for 96 hours to achieve an initial mortality rate of 25-30%; transferring the surviving individuals to an outdoor pond (salinity 15-20‰, temperature 25-30℃) for temporary rearing for 7-15 days, and continuing to subject the surviving individuals to low salt and high temperature stress, with the cumulative mortality rate controlled at around 50%; and transferring the surviving individuals to an indoor cement pond for temporary rearing to restore vitality.

[0016] The beneficial effects of the present invention are as follows: (1) The present invention utilizes the fast growth genetic basis of the Pacific oyster “Haida No. 1” and the high temperature adaptation genetic basis of the Fujian oyster. By constructing GA hybrids and conducting continuous multi-generation high-intensity population selection, the paternal system can achieve directional improvement in growth and high temperature resistance traits and has high stability; (2) The present invention uses the Pacific oyster as the maternal parent and further introduces the low salt adaptability genetic background of the Pacific oyster to construct the RGA ternary hybrid population. Combined with low salt and high temperature stress screening, it can significantly improve the growth rate and summer survival rate of the offspring. It is suitable for promotion and application in aquaculture areas such as Shandong and Jiangsu and has good industrial application prospects. Attached Figure Description

[0017] Figure 1 This is a technical roadmap for the cultivation of a new three-way hybrid line that is fast-growing, low-salt, and highly adaptable to high temperatures, according to the present invention. Detailed Implementation

[0018] The following embodiments further illustrate the cultivation method of a new three-way hybrid oyster strain that is fast-growing, low-salt, and highly adaptable to high temperatures, provided by the present invention, but are not intended to limit the invention.

[0019] Example:

[0020] (1) Construction of the GA basic population (GA F0): 500 12th generation Pacific Ocean oysters "Haida No. 1" were collected from Rongcheng City, Shandong Province, and 500 cultured Fujian oysters were collected from Zhangzhou City, Fujian Province. Shell height was selected to obtain 120 Pacific Ocean oysters and 120 Fujian oysters as candidate parents. The two types of parents were cultured separately in ponds for one month to promote maturity. During the maturation period, a mixture of diatoms and flat algae was fed three times a day, the water was changed once a day, and the temperature was controlled at 18℃ to achieve synchronous gonadal development and maturation. After the gonads matured, they were dissected and examined. Those with well-developed gonads and eggs were selected. Female *Crassostrea gigas* with well-developed eggs and dark yolk color were used as the maternal parent; male *Crassostrea kwangsiensis* with vigorous sperm motility were selected, and species identification was performed using COI primers. Purebred *Crassostrea kwangsiensis* were retained as the paternal parent; each parent had no fewer than 50 individuals. Artificial insemination was carried out according to standard procedures. After hatching and selection of the best fertilized eggs, larvae were raised using standard methods. When the larvae reached the eyespot larval stage, they were placed on an attachment substrate. Once the juveniles reached a certain size, they were transferred to the sea area of ​​Rongcheng City, Shandong Province, and raised using the hanging rope method to obtain a basic population of female *Crassostrea gigas* × male *Crassostrea kwangsiensis* (GA F0).

[0021] (2) Selection and breeding of GA population: 3,000 GA F0 adult scallops were collected from Rongcheng City, Shandong Province and transferred to a seedling farm for temporary rearing; the top 20% of individuals by shell height were selected to obtain 600 GA F0 individuals; heat resistance selection was carried out, and the GA F0 individuals with the initial shell height were placed in seawater at 34℃ for heat shock for 96 hours, with a mortality rate of about 25-30%; the surviving individuals were transferred to an outdoor pond (25-30℃) for outdoor high temperature stress, and the surviving individuals were used as the preferred GA F0 broodstock (the cumulative mortality rate was controlled at about 50%); the surviving individuals were transferred to an indoor cement pond (21℃) for temporary rearing for 15 days, and when no more individuals died, the broodstock were prepared for maturation. The preferred GA F0 broodstock were placed in a pond for maturation culture for 1 month. During the maturation period, they were fed a sufficient amount of diatom and flat algae mixture every day, the water was changed once a day, and the temperature was 18℃. After gonadal maturity, oval-shaped, dense female individuals and male individuals with active sperm were selected through dissection and microscopy, with no fewer than 50 individuals of each sex. Artificial insemination was performed using conventional methods, and the larvae were raised using standard methods. The juveniles were then transferred to Rongcheng City, Shandong Province, for rearing using the hanging rope method, resulting in the selected breeding population GA F1. The above steps were repeated for six consecutive generations of population selection, focusing on shell height and heat resistance, to obtain the stable, fast-growing, heat-resistant hybrid population GA F6.

[0022] (3) Construction of the three-way hybrid basic population (RGA F0): 500 cultured adult oysters of Crassula ovata were collected from Qingdao City, Shandong Province, and 500 adult GA F6 oysters were collected from Rongcheng City, Shandong Province and transferred to the seedling farm for temporary rearing; shell height was selected to obtain 120 Crassula ovata and 120 GAF6 oysters as candidate parents, and the species of Crassula ovata were identified by COI. Two candidate parent oysters were cultured separately in ponds for one month to promote maturation. During this period, they were fed a mixture of diatoms and flat algae three times a day, with water changes once a day and the temperature controlled at 18℃ to achieve synchronous gonadal development and maturation. After gonadal maturation, female Crassula ovata 'Oriole' with well-developed gonads, uniform egg development, and dark yolk color were selected for species identification using COI primers. Purebred Crassula ovata 'Oriole' was retained as the maternal parent. Male individuals with active sperm and high sperm motility, GAF6, were selected as the paternal parent. Each parent had no fewer than 50 individuals. Artificial insemination was performed according to standard procedures. After hatching and selection of the best fertilized eggs, larvae were raised using standard methods. When the larvae reached the eyespot larval stage, they were placed on an attachment substrate. Once the juveniles reached a certain size, they were transferred to the sea area of ​​Rongcheng City, Shandong Province, for trellising using the rope method to obtain a three-way hybrid population of Crassula ovata 'Oriole' ♀ × GAF6 ♂ (RGAF0).

[0023] (4) RGA population selection (obtaining new RGA strains): 3,000 adult RGA F0 scallops were collected from the sea area of ​​Rongcheng City, Shandong Province and transferred to a seedling farm for temporary rearing. The top 20% of individuals by shell height were selected to obtain 600 RGA F0 individuals with initial shell height selection. Screening for tolerance to low salinity and high temperature was carried out. The individuals with initial shell height selection were placed under artificial stress conditions of 15‰ salinity and 34℃ for 96 consecutive hours, with a mortality rate of about 25-30%. After the stress ended, the surviving individuals were transferred to an outdoor pond (salinity 15-20‰, temperature 25-30℃) for temporary rearing for 15 days for further screening under the continued fluctuation of low salinity and high temperature environment. The surviving individuals were used as the preferred parents of RGA F0 (the cumulative mortality rate was controlled at about 50%). The surviving individuals were collected and transferred to an indoor cement pond (21℃) for temporary rearing for 15 days to restore vitality. The selected RGAF0 parent oysters were cultured separately in a pond for one month to promote maturation. During this period, they were fed a sufficient amount of a mixture of diatoms and flat algae daily, with water changes once a day at a temperature of 18℃. After gonadal maturity, oval-shaped, dense female individuals and male individuals with active sperm were selected by dissection and microscopy, yielding 50 RGA F0 females and 50 RGA F0 males. Artificial fertilization was performed using conventional methods. After hatching and selection, the fertilized eggs were raised as larvae using conventional methods. The juveniles were transferred to the sea area of ​​Rongcheng City, Shandong Province, and raised using the rope-hanging method to obtain the selected breeding population RGA F1. The above steps were repeated to select four generations of populations for shell height, low salinity tolerance, and high temperature tolerance, resulting in a stable, fast-growing, low-salinity, and high-temperature-adaptable oyster three-way hybrid strain, RGAF4.

[0024] (5) Comparative test

[0025] The aforementioned three-way hybrid line RGA F4 was set as the experimental group, and a control group of Pacific oysters with the same number of parents was constructed. Both groups were bred using conventional culture methods. The experimental and control groups were transferred to three culture sites in Weihai City, Shandong Province; Qingdao City, Shandong Province; and Lianyungang City, Jiangsu Province. The hanging rope method was used for culture, and the oyster productivity data are recorded in Table 1.

[0026] As shown in Table 1, the new three-way hybrid oyster strain RGA F4 bred in this invention exhibited superior growth and summer survival rates under aquaculture conditions in Weihai, Qingdao, and Lianyungang. The shell height of RGA F4 was 54.20–58.40 mm, which was 12.09–20.98% higher than the control group (44.80–52.10 mm); the wet weight of RGA F4 was 14.05–15.05 g, which was 12.31–21.12% higher than the control group (11.60–13.40 g); and the summer survival rate of RGA F4 was 80.83–85.00%, which was 21.43–44.78% higher than the control group (55.83–70.00%). In the low-salinity sea area of ​​Lianyungang, the growth and survival rate of the control group decreased more significantly, while RGA F4 maintained a higher wet weight and summer survival rate, indicating that this new strain has stronger adaptability and stability to the combined stress of low salinity and high summer temperatures. In summary, the new RGA F4 strain bred in this invention exhibits rapid growth and a significantly improved summer survival rate, demonstrating good production application value and promising prospects for promotion.

[0027] Table 1. Comparison of production performance between the rapid-growing, low-salt, and high-temperature-adaptable oyster three-way hybrid strain and the control group. Weihai City RGA F4 58.40 37.10 18.20 15.05 85.00 control group 52.10 33.10 16.30 13.40 70.00 Qingdao RGA F4 56.30 35.90 17.70 14.60 82.50 control group 49.00 31.30 15.40 12.55 66.67 Lianyungang City RGA F4 54.20 34.60 17.10 14.05 80.83 control group 44.80 28.90 14.20 11.60 55.83

[0028] The above description is only used to illustrate the technical solutions and features of the present invention, and is not intended to limit the scope of protection of the present invention. Any simple modifications or equivalent substitutions made by those skilled in the art to the technical solutions of the present invention should fall within the scope of protection of the technical solutions of the present invention.

Claims

1. A method for cultivating a new three-way hybrid oyster strain that is fast-growing, low-salt, and highly adaptable to high temperatures, characterized in that, The steps include the following: (1) Construction of the basic population of GA: The new variety of Pacific oyster "Haida No. 1" (GS-01-005-2013) was used as the female parent and Pacific oyster was used as the male parent to carry out population hybridization and obtain the basic population of Pacific oyster × Pacific oyster GA F0; (2) GA population selection: Based on the GA F0 population, individuals with shell height in the top 20% are selected as primary parents; before the gonads develop, the primary parents are subjected to high temperature stress to select for high temperature tolerance, and the surviving individuals are retained as preferred parents; the preferred parents are artificially inseminated, and after hatching and cultivation, GA F1 is obtained; the population is continuously selected for 6 generations to obtain the GA selection population GAF6; (3) Construction of the three-way hybrid basic population: The three-way hybrid basic population RGA F0 was obtained by hybridization with Crassula lizarda as the female parent and GA F6 as the male parent; (4) RGA population selection: Based on RGA F0, individuals with shell height in the top 20% are selected as primary parents; before the gonads develop, the primary parents are subjected to low salt and high temperature synergistic stress, and low salt and high temperature tolerance selection is carried out, and the surviving individuals are retained as preferred parents; the preferred parents are artificially inseminated, and RGA F1 is obtained after hatching and cultivation; after four generations of continuous selection, a new three-way hybrid line RGA F4 is obtained.

2. The method according to claim 1, characterized in that, The Fujian oysters mentioned in step (1) are commercially farmed in Zhangzhou, Fujian. Dissect the long oysters and select female individuals with well-developed gonads, uniform egg development, and dark yolk color as the maternal parent. Dissect the Fujian oysters and select male individuals with strong sperm motility. Use molecular markers for species identification and retain purebred Fujian oysters as the paternal parent. The number of each parent parent shall not be less than 50.

3. The method according to claim 1, characterized in that, In step (2), the high-temperature resistance selection is carried out before the gonads develop in order to reduce the damage of heat shock to gametes and improve the fertilization success rate. The high-temperature resistance selection is divided into three steps: the initial selected parents with high shells are subjected to artificial heat shock at 34°C for 96 hours to achieve an initial mortality rate of 25-30%; the surviving individuals are transferred to an outdoor pond (25-30°C) for temporary rearing for 7-15 days, and natural heat shock is used to subject the surviving individuals to high-temperature stress, with the cumulative heat shock mortality rate controlled at around 50%; the surviving individuals are transferred to an indoor cement pond (21-23°C) for temporary rearing to restore vitality.

4. The method according to claim 1, characterized in that, The oysters mentioned in step (3) are commercially farmed in Qingdao, Shandong. Female individuals with well-developed gonads, uniform egg development, and dark yolk color are selected by dissecting the oysters. Molecular markers are used for species identification, and purebred oysters are retained as the maternal parent. GA F6 is dissected, and male individuals with strong sperm motility are selected as the paternal parent. The number of each parent is no less than 50.

5. The method according to claim 1, characterized in that, In step (4), the low-salt and high-temperature tolerance selection is carried out before the gonads develop, in order to reduce the damage of stress to gametes and improve the fertilization success rate. The low-salt and high-temperature tolerance selection is divided into three steps: applying artificial stress of 15‰ salinity and 34℃ water temperature to the shell-high initial selection population for 96 hours to achieve an initial mortality rate of 25-30%; transferring the surviving individuals to an outdoor pond (salinity 15-20‰, temperature 25-30℃) for temporary rearing for 7-15 days, and continuing to subject the surviving individuals to low-salt and high-temperature stress, with the cumulative mortality rate controlled at around 50%; and transferring the surviving individuals to an indoor cement pond for temporary rearing to restore vitality.

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