Artificial cultivation method of low-temperature-resistant Yunjin hybrid spots

Abstract

The invention relates to an artificial cultivation method of low-temperature-resistant Yunjin hybrid spots, and the method belongs to the technical field of aquatic product distant hybridization breeding, and the method comprises the following steps: (1) low-temperature-resistant grouper group breeding and grouper blue-body group breeding; (2) collecting and storing sperms of epinephelus coioides; (3) epinephelus moara reproduction regulation and ovum acquisition; (4) artificial preparation and incubation of hybrid fertilized eggs; compared with the prior art, the fertilization rate of the bred fries is increased by 2.91%, the hatching rate is increased by 9.58%, and the malformation rate is reduced by 11.33%. The survival rate of hybrid seeds is increased by 69.56% compared with that of purebred epinephelus moara, the low-temperature semi-lethal tolerance temperature of new germplasm seeds is 5.8 DEG C, meanwhile, 'Yunjin hybrid spots' have better hybrid growth advantages, and the weight of 7-month-old seeds is increased by 79.72% compared with that of female parent epinephelus moara; compared with that before breeding, the weight gain rate of the hybrid at the same time is increased by 23.20% compared with that of the female parent epinephelus moara.

Description

Technical Field

[0001] This invention relates to a method for the artificial cultivation of low-temperature resistant cuminite hybrids, which belongs to the field of aquatic distant hybridization breeding technology. Background Technology

[0002] With the continuous expansion of the grouper aquaculture industry, the shortage of fast-growing and stress-resistant seedlings has become particularly prominent. Distant hybridization breeding technology can integrate the genomes of distantly related species to obtain hybrid offspring with large-scale genotypic and phenotypic variation, resulting in significant hybrid vigor in growth, survival rate, and stress resistance. Currently, all approved new grouper varieties are hybrids, accounting for over 70% of the market. However, grouper are tropical and subtropical fish, and broodstock breeding, fertilized egg production, and commercial fish production are mostly concentrated in aquaculture companies along the southern coast of my country. While northern aquaculture areas in my country possess complete industrialized and deep-sea aquaculture facilities, the low temperatures and overwintering costs have limited the large-scale farming and industrialization of this valuable grouper variety. Therefore, combining population selection, reproductive regulation, and distant hybridization breeding technologies to develop new fast-growing and cold-resistant grouper germplasm can further revitalize the grouper breeding and industrial development, providing high-quality, suitable varieties for industrialized and deep-sea aquaculture in my country.

[0003] Blue-bodied large-spotted grouper ( Epinephelus tukula Belonging to the genus *Gastrodon*, commonly known as the golden grouper, it is mainly distributed in the Indian Ocean to the Pacific Ocean, growing up to 2 meters in length and reaching a maximum weight of 100 kg. The natural and farmed populations of the blue-bodied large-spotted grouper are very small, and breeding broodstock under aquaculture conditions is difficult, exhibiting bottlenecks such as long maturation time for broodstock, low sperm count in males, low maturity rate in females, and difficulty in the survival of artificially bred fry. These problems severely restrict the large-scale farming of large groupers and the development and utilization of their fast-growing traits. The cloud-patterned grouper (…) E. moara This species belongs to the small to medium-sized grouper family and is tolerant of low temperatures, with an optimal temperature range of 8–35 °C. However, it still faces bottlenecks limiting the industry's development, such as low survival rate of purebred fry and slow growth rate. Both of these grouper species exhibit spatial, temporal, and interspecies reproductive isolation, and no natural hybridization has been observed.

[0004] The inventors' team first broke through the hybridization of Epinephelus merra female and Epinephelus lanceolatus male to obtain hybrid offspring in 2019 (Tang et al., Development and growth of hybrid offspring of Epinephelus merra (♀) and Epinephelus lanceolatus (♂), Journal of Guangdong Ocean University, 2019, 39: 27-34), but the above hybrid parents have not been strictly group selected, resulting in a high rate of deformity (24.55%) of the hybrid offspring, and the temperature tolerance advantage is not obvious compared with the parents (half lethal temperature 7℃), the survival rate of the breeding seedlings is low, and large-scale popularization and cultivation has not been carried out. Therefore, on the basis of the previous research, the present application further integrates group selection, genome matching, reproductive regulation and distant hybrid breeding technology, uses large-sized Epinephelus lanceolatus as the male parent and hybridizes with low-temperature selected Epinephelus merra female, in order to break through and cultivate high-quality hybrid offspring with fast growth, low temperature tolerance and high survival rate, and further improve the coverage rate of grouper good strains.

[0005] Both of the above two kinds of grouper are protogynous hermaphrodites, and the female matures first. Through temperature regulation and sperm freezing preservation technology research and development, the sperm of Epinephelus lanceolatus can be used for artificial seedling production all year round, but the large-scale egg production technology of its female has not been completely broken through, and there is a problem of low survival rate of pure seedlings. At present, there is a lack of cultivable seedlings in the market. The artificial propagation technology of Epinephelus merra is relatively mature, and batch egg production can be realized in the north and south of China. Therefore, by using the fast-growing effect of Epinephelus lanceolatus through distant hybrid breeding technology, the growth rate and survival rate of Epinephelus merra can be further improved, the breeding cycle and overwintering cost can be reduced, the breeding income can be effectively improved, and the sustainable development of grouper seed industry and industry can be promoted. SUMMARY

[0006] The technical problem to be solved by the present application is to provide a breeding method for artificially hybridizing Epinephelus merra female and large-sized Epinephelus lanceolatus male to cultivate a new strain of Epinephelus merra and Epinephelus lanceolatus hybrid, aiming to solve the problems of slow growth of Epinephelus merra, low survival rate of seedlings, narrow temperature range and lack of breakthrough new strains.

[0007] In order to solve the above technical problems, the present application is realized by the following technical scheme: A method for artificially breeding low-temperature-resistant Epinephelus merra and Epinephelus lanceolatus hybrid, the method comprising (1) low-temperature-resistant group selection of Epinephelus merra and group selection of Epinephelus lanceolatus; (2) sperm collection and preservation of Epinephelus lanceolatus; (3) reproductive regulation of Epinephelus merra and obtaining of eggs; (4) artificial preparation and hatching of hybrid fertilized eggs; The low-temperature-resistant group selection of Epinephelus merra in (1) comprises: a. Group selection: selecting Epinephelus merra with an age of 5 years or more, a body weight of 7 kg or more and a total length of 50 cm or more from different geographical groups as a backup breeding group, and the selection intensity is 5%; b. Low temperature selection: The low temperature resistant selection was carried out on the reserve breeding population for more than 2 generations, and the core breeding population with fast growth and low temperature resistance was screened, and the selection intensity was 10%.

[0008] Further, the low temperature resistant group of Epinephelus merra in (1) is selected and bred. The breeding fry of the basic population (G0) is raised to 12 months and 24 months, and the low temperature resistant (normal swimming) individuals are screened by the method of gradually reducing the temperature (reducing 1℃ per day, and the minimum is 10℃) to construct the G1 breeding population, and the selection intensity is 4.5% and 5.5% respectively. The breeding fry of the G1 population is raised to 24 months, and the low temperature resistant selection is carried out again, and the selection intensity is 10%, to construct the G2 breeding population. The female fish of the low temperature resistant G2 population of Epinephelus merra is cultured to promote maturity.

[0009] The population selection of Epinephelus maculatus in (1) is as follows: collecting blue body large spotted grouper fish of different genetic backgrounds for breeding, and selecting fast-growing, non-deformed, and well-proportioned blue body large spotted grouper fish as parents after two generations of selection.

[0010] Further, different genetic backgrounds of blue body large spotted grouper fish are collected, and the coefficient of variation of phenotypic traits is controlled to be less than or equal to 12.47% to construct a basic breeding population (G0). The fry bred by the basic population (G0) is grown to 24 months, and fast-growing, non-deformed, and well-proportioned fish are selected to construct a G1 breeding population, and the selection intensity is 10%. The G1 breeding population is artificially bred to grow into adult fish, and the coefficient of variation of phenotypic traits is controlled to be less than or equal to 11.96%. The F2 generation fry is bred by the G1 population, and is raised to 24 months, and fast-growing, non-deformed, and well-proportioned G2 breeding population is selected and constructed, and the selection intensity is 10%. The G2 population is raised to adult fish, and the coefficient of variation of phenotypic traits is controlled to be less than or equal to 9.87%. Fast-growing, non-deformed blue body large spotted grouper fish are selected as parents.

[0011] The sperm collection and preservation of blue body large spotted grouper fish in (2) is as follows: a. Cultivation environment: indoor factory workshop cultivation, water temperature is 22~28℃, dissolved oxygen is more than 6 mg / L, salinity is 28~31‰, water flow exchange is 8m 3 / h; b. Reproduction regulation and sperm collection: in March of the next year, the water temperature of the male fish is strictly controlled to be 23~26℃, the water flow exchange is increased to 10m 3 / h, and the ice fresh fish mixed with nutritional enhancer (vitamin C and vitamin E mixed at 1:1, content is 5g / kg) is fed to promote the development of testis; 48h before sperm collection, stop feeding, and after anesthesia, the parent fish, the fresh sperm is collected by artificial method, and the sperm is preserved in a low temperature environment at 4℃ in the dark.

[0012] Said (3) cloud stone fish reproductive regulation and egg acquisition: in late October every year, the selected cloud stone fish is transferred to the offshore net cage for intensive cultivation, and in early March, the well-developed parent fish is transferred to the factory workshop for intensive cultivation, the female fish with the gonad development to the IV stage is selected, the chorionic gonadotropin (HCG, 350 IU / kg fish weight) is injected on the back, and at the same time, the water flow exchange amount is increased to 10 m 3 / h to stimulate the maturation of female gonad; Said (4) artificial preparation and hatching of hybrid fertilized eggs: a. Hybrid insemination: the mature cloud stone fish eggs are artificially taken, and at the same time, the blue body large spot grouper sperm (the mirror examination activity is greater than 90%) is artificially inseminated according to the sperm egg volume ratio of 1:800, 2-3 times the volume of 22-24 DEG C seawater is added, and after standing and filtering, the fertilized eggs are transferred to the incubator for hatching; b. Hatching and cultivation: when hatching, the dissolved oxygen is 6-7 mg / L, the water temperature for cultivation is 23-25 DEG C, and the salinity is 28-30 ‰; in order to reduce the risk of nervous necrosis virus and improve the survival rate of cloud gold hybrid grouper fry, the water is not changed during the early cultivation of the fry, the ozone and ultraviolet rays are used to strictly disinfect and kill the fresh seawater to carry out SPF (specific pathogen free) environment cultivation before spawning, and external pathogen infection is avoided during the period; the hatching is transferred to the rearing pond for cultivation at the tail bud stage, the water temperature for cultivation is 22-26 DEG C, the salinity is 28-31 ‰, the dissolved oxygen is greater than 6 mg / L, and the water flow exchange speed is 4-6 m 3 / h after 28 days; during the period, the fish meal is used for fry intensive cultivation.

[0013] The technical principle of the application is: by using the growth and temperature tolerance traits of the strictly selected male blue body large spot grouper and the female cloud stone fish, the hybrid new germplasm "cloud gold hybrid grouper" with fast growth, low temperature tolerance, low deformity rate and high survival rate is bred through reproductive regulation, distant hybridization and SPF environment fry cultivation.

[0014] The beneficial effects of the application compared with the prior art are as follows: 1. The application uses the growth and phenotype traits of the hybrid parents selected through two generations of strict selection to create the new germplasm "cloud gold hybrid grouper", and carries out SPF environment cultivation in the early stage of fry cultivation. Compared with the prior art (Tang Jiang et al., 2019), the fertilization rate of the fry bred by the application is increased by 2.91%, the hatching rate is increased by 9.58%, and the deformity rate is reduced by 11.33%. The survival rate of the hybrid fry is increased by 69.56% compared with the purebred cloud stone fish. At present, 1 million fry have been bred and popularized, which breaks through the large-scale propagation of the new germplasm "cloud gold hybrid grouper" and improves the coverage rate of grouper good seeds.

[0015] 2、 The low-temperature selected Epinephelus merra was used to develop the new germplasm of low-temperature tolerant "Yunjin hybrid grouper". The low-temperature semi-lethal tolerance temperature of the new germplasm fry was 5.8℃, which was 1.7℃ lower than that of the parent Epinephelus fuscoguttatus and 1.2℃ lower than that before selection (Tang et al., 2019), showing better low-temperature tolerance performance and expanding the culture space of warm-water grouper. At the same time, "Yunjin hybrid grouper" has better hybrid growth advantage, and the body weight of 7-month-old is increased by 79.72% compared with that of the parent Epinephelus merra; compared with before selection (Tang et al., 2019), the weight gain rate of the hybrid at the same period is increased by 23.20% compared with that of the parent Epinephelus merra, and the low-oxygen lethal point of Yunjin hybrid grouper is 0.47 mg / L, which is significantly lower than that of Epinephelus merra (0.53 mg / L); it provides a fast-growing, low-temperature tolerant and suitable breed for China's land-based factory and deep-sea aquaculture. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 The 6-month-old grouper hybrid new germplasm Yunjin hybrid grouper was cultured. DETAILED DESCRIPTION

[0017] The technical solutions of the present application will be described clearly and completely below in combination with specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0018] Example 1: Parent fish screening and intensive breeding (1) Parent fish selection a. Low-temperature tolerant female Epinephelus merra selection: 1500 wild and cultured basic breeding populations from different regions were collected, with an average weight of 5.96 kg. The parent individuals with large body size, no deformity and strong body were selected to construct the basic breeding population (G0) of Epinephelus merra, and the artificial breeding fry was cultured on a large scale. The low-temperature tolerance traits were selected at 12-month-old and 24-month-old fry, respectively, and the fast-growing and low-temperature tolerant individuals were selected to construct the G1 breeding population of 800 Epinephelus merra, with selection intensities of 4.5% and 5.5%, respectively. The G1 population was intensively bred for 5 years to reach sexual maturity, with an average weight of 6.74 kg. The F2 generation fry (24-month-old) bred was selected for low-temperature tolerance again, with a selection intensity of 10%, to construct the G2 breeding population with low-temperature tolerance. The female fish reached sexual maturity after 5 years of artificial breeding, with an average weight of 7.16 kg, and the artificial spawning was used for the large-scale production of Yunjin hybrid grouper new germplasm.

[0019] b. Fast-growing male Epinephelus fuscoguttatus selection: 1100 wild and cultured Epinephelus fuscoguttatus from different regions were collected, with an average weight of 20.16 kg and a phenotypic trait variation coefficient of 12.47%, to construct the basic breeding population (G0).

[0020] The artificial breeding of the foundation population (G0) seedlings is grown to 24 months of age, and the fast-growing, non-deformed, and well-proportioned fish are selected to construct the G1 breeding population with a selection intensity of 10%. The G1 breeding population is artificially bred for 6 years to reach 800 individuals, with an average weight of 21.26 kg and a phenotypic trait variation coefficient of 11.96%.

[0021] The F2 generation seedlings are bred using the G1 population, and the fast-growing, non-deformed, and well-proportioned G2 breeding population is constructed by selecting the fish at 24 months of age with a selection intensity of 10%. The G2 population is artificially bred for 6 years to reach 600 parent individuals, with an average weight of 22.37 kg and a phenotypic trait variation coefficient of 9.87%. Fast-growing, non-deformed male blue body large spotted grouper fish are selected for the production of new hybrid grouper species.

[0022] (2) Intensified cultivation a. Cultivation environment: Blue body large spotted grouper fish are cultivated in indoor factory workshops with a water depth of 1.5-2 m, a water temperature of 22-28℃, a dissolved oxygen of 6 mg / L or more, a salinity of 28-31‰, and a water flow exchange of 8 m 3 / h. The Epinephelus merra is intensively cultivated in offshore cages combined with indoor factory conditions, with a water depth of 2-3 m and an indoor factory workshop water temperature of 23-26℃.

[0023] b. Male blue body large spotted grouper fish cultivation: In March of the following year, the water temperature for male fish cultivation is strictly controlled at 23-26℃, the water flow exchange is increased to 10 m 3 / h, and the fish are fed with vitamin mixture injected ice-fresh fish for nutritional enhancement, with a feeding amount of 2%-3% of the fish body weight. Forty-eight hours before sperm collection, the fish are stopped from feeding, and the parent fish are anesthetized before mature sperm is obtained by pressing the abdominal cavity. The sperm with a microscope viability of more than 90% is stored at 4℃ in a low-temperature, light-free environment and used for hybrid new species production on the same day.

[0024] c. Female Epinephelus merra cultivation: In late October of each year, the selected Epinephelus merra are transferred to offshore cages for intensive cultivation. Through tidal force, photoperiod, water temperature, and water flow stimulation, the development of the ovary is promoted. During this period, the fish are fed with small and mackerel fish for nutritional enhancement, with a feeding amount of 3%-4% of the fish body weight. In early March, the well-developed parent fish are transferred to the factory workshop for intensive cultivation. The female fish with developed gonads to stage IV are selected and injected with human chorionic gonadotropin (HCG, 350 IU / kg fish body weight) on the back. At the same time, the water flow exchange is increased to 10 m 3 / h to stimulate the maturation of female gonads. After 48 hours of hormone effect, mature eggs are collected by pressing the abdomen for the large-scale production of hybrid new species.

[0025] Example 2: Hybridization and fertilization of Epinephelus merra female and Epinephelus fuscoguttatus male a. Artificial insemination: Select well-developed female Epinephelus merra for hybrid and purebred grouper seed cultivation, divide the collected eggs into two equal parts, and simultaneously inseminate Epinephelus fuscoguttatus sperm and Epinephelus merra sperm with a sperm-egg volume ratio of 1:800, which has a mirror examination of more than 90% viability. After stirring evenly, 2-3 times the volume of 22-24℃ seawater is added, and after standing for 8 minutes, the floating eggs are transferred to the incubator for incubation. The fertilization rate, hatching rate and deformity rate of the hybrid have no significant difference with those of the purebred Epinephelus merra (Table 1). Compared with the previous research results (Tang et al., 2019), the fertilization rate of the seed cultivated in the present application is increased by 2.91%, the hatching rate is increased by 9.58%, and the deformity rate is decreased by 11.33%, achieving large-scale production of new germplasm seed.

[0026] Table 1 Comparison of hatching of hybrid new germplasm Epinephelus merra (♀) x Epinephelus fuscoguttatus (♂) ; b. Incubation and cultivation: The incubation water temperature is 23-25℃, the dissolved oxygen is 6-7 mg / L, and the salinity is 28-30‰; the incubation is transferred to the rearing pond for cultivation at the tail bud stage, and the egg density is 3.8-5.2 g / m 3 . To prevent viral diseases during rearing, fresh seawater filtered by ozone and ultraviolet disinfection is added to the rearing pond, and the seawater is not changed during early cultivation. The hatching rate of the hybrid is 85.26±4.37%, which has no significant difference with that of the purebred Epinephelus merra (89.74±3.58%) (Table 1).

[0027] c. Mitochondrial genome analysis: The mitochondrial genome size of the hybrid Epinephelus merra is 16405 bp, containing 13 mRNAs, and the GC content is 45.31%. The base composition is: A: 26.21%, T: 28.49%, C: 16.16%, G: 29.15%, and the mitochondrial composition follows the maternal inheritance rule.

[0028] Example 3: Comparison of survival rate of hybrid new germplasm In this example, a total of 3.6 kg of hybrid and purebred fertilized eggs were obtained, and the fertilization rate and hatching rate of the hybrid Epinephelus merra had no significant difference with those of the purebred Epinephelus merra. The feeding activity of the hybrid Epinephelus merra was significantly better than that of the purebred Epinephelus merra seed, and by the age of 6 months, the survival quantity of the hybrid seed was 78,000, and that of the purebred Epinephelus merra was 46,000, and the survival rate of the hybrid seed was increased by 69.56% compared with that of the purebred Epinephelus merra.

[0029] The results showed that the hybrid offspring of Epinephelus merra (♀) x Epinephelus lanceolatus (♂) had high fertilization rate and hatching rate, and the metamorphosis of the larvae and juveniles developed normally. The survival rate of the hybrid offspring was significantly higher than that of the purebred Epinephelus merra.

[0030] Example 4: Test of growth traits of hybrid new germplasm Through family construction, the hybrid line of Epinephelus merra (♀) x Epinephelus lanceolatus (♂) and the pure line of Epinephelus merra were obtained. Comparative breeding experiments of growth traits were carried out in the same environment, and the growth traits were compared and analyzed.

[0031] After 7 months of comparative breeding, the total length of the hybrid group and the control group reached 21.98±0.40 cm and 17.87±0.31 cm respectively, and the total length of the hybrid group was 1.23 times that of the purebred.

[0032] The body weight of the 7-month-old hybrid group and the control group was measured, and the body weight of the hybrid group was 145.30±5.83 g, which was significantly higher than that of the purebred Epinephelus merra (80.85±4.50 g), and the body weight of the hybrid group was increased by 79.72% compared with the purebred Epinephelus merra. Compared with the mother Epinephelus merra before selection (Tang et al., 2019), the body weight of the hybrid group was increased by 23.20% at the same time, showing a significant hybrid growth advantage and a better morphological appearance (Tang et al., 2019). Figure 1 .

[0033] Example 5: Test of low oxygen tolerance traits of hybrid new germplasm Oxygen consumption rate determination: Select an experimental tank with a volume of 5 L as a respiratory chamber, and measure the oxygen consumption rate of 6-month-old Epinephelus merra and hybrid Epinephelus merra at a water temperature of 26.0±0.5 ℃. After the experimental fish is in good condition for 1 h, slowly fill the respiratory chamber with water and seal it with plastic wrap. The experiment lasts for 2 h. Sample and quickly fix the dissolved oxygen concentration before and after the experiment. The oxygen consumption rate of the hybrid Epinephelus merra is 0.24 mg / L, which is significantly higher than that of the Epinephelus merra (0.17 mg / L).

[0034] Asphyxia point determination: At a water temperature of 26.0±0.5 ℃, place 6-month-old Epinephelus merra and hybrid Epinephelus merra in a 5 L respiratory chamber. After the experimental fish is in good condition for 1 h, start the experiment. The sealing operation is the same as above. Sample and quickly fix the dissolved oxygen concentration before the experiment starts. The dissolved oxygen concentration at 50% fish death is taken as the asphyxia point of the fish. The lethal point of low oxygen for the hybrid Epinephelus merra is 0.47 mg / L, which is significantly lower than that of the Epinephelus merra (0.53 mg / L), showing better low oxygen tolerance traits.

[0035] Example 6: Test of low temperature tolerance of hybrid new germplasm (1) Acute cooling experiment: A cylindrical water tank with a diameter of 2 m and a height of 50 cm was selected for the test of low temperature tolerance of 6-month-old blue body large grouper and Yunjin hybrid grouper new germplasm. The experimental fish were temporarily raised in the water tank for 7 days, during which they were normally fed, with water temperature of 26.0±0.5 ℃, salinity of 28-29‰, pH of 7-8, and dissolved oxygen of >6 mg / L. After the temporary raising, ice sea water was slowly added to the water tank system, and the temperature was lowered at a rate of 1 ℃ / h. The behavior of the two kinds of grouper was observed, and the temperature at which 50% of the experimental fish turned over (lost balance) was taken as the half lethal temperature of the experimental fish. It was determined that the half lethal temperature of Yunjin hybrid grouper was 5.8 ℃, which was 1.7 ℃ lower than that of blue body large grouper (7.5 ℃) and 1.2 ℃ lower than that before selection, showing better low temperature tolerance.

[0036] (2) Chronic cooling experiment: Using the above experimental conditions, the slow cooling method of 1 ℃ / day was used to carry out the test of stop-feeding temperature and low temperature tolerance of blue body large grouper and Yunjin hybrid grouper new germplasm. It was determined that the stop-feeding temperature of Yunjin hybrid grouper was 14 ℃, which was 2 ℃ lower than that of blue body large grouper; the half lethal temperature was 6.5 ℃, which was 3 ℃ lower than that of blue body large grouper (9.5 ℃), showing more significant low temperature tolerance. Compared with the gold tiger hybrid grouper new variety widely promoted in China (Aquaculture, 2023, 562: 738751), the stop-feeding temperature of Yunjin hybrid grouper new germplasm was 2 ℃ lower than that of the new variety of grouper, and the half lethal temperature was 2.5 ℃ lower. In addition, compared with the hybrid varieties (Yunlong grouper, pearl gentian and green dragon grouper) bred with saddle grouper as the hybrid father (Table 2), Yunjin hybrid grouper was still significantly lower than the similar breeding varieties, which was attributed to the selection of Yunlong grouper and blue body large grouper two low temperature tolerant varieties, combined with population selection, environmental regulation, distant hybridization and other breeding techniques, to break through and breed Yunjin hybrid grouper new germplasm with fast growth and low temperature tolerance. The breakthrough breeding of this new germplasm will further expand the scale of grouper culture and reduce the cost of overwintering, providing suitable breeding varieties for China's land-based factory and deep sea aquaculture.

[0037] Table 2 Comparison of low temperature half lethal temperature of common breeding species of grouper (Fisheries Science Progress, 2017, 38: 70-76) ; The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Those skilled in the art can make several improvements without departing from the principles of the present application, and these improvements should be considered within the scope of protection of the present application.

Claims

1. A method for artificially cultivating low-temperature resistant cumulonimbus hybrid spots, characterized in that, The methods include (1) low-temperature tolerant group selection of cloud-patterned grouper and group selection of blue-bodied large-spotted grouper; (2) sperm collection and preservation of blue-bodied large-spotted grouper; (3) reproductive regulation and egg acquisition of cloud-patterned grouper; and (4) artificial preparation and hatching of hybrid fertilized eggs. The low-temperature tolerant grouping of the cloud-patterned grouper mentioned in (1) is as follows: a. Population selection: Select grouper that are 5 years or older, weigh over 7 kg, and have a total length of over 50 cm from different geographical populations as a reserve breeding population, with a selection intensity of 5%; b. Low-temperature selection: Conduct low-temperature tolerance selection for more than two generations of the reserve breeding population, and select core breeding populations that combine fast growth and low-temperature tolerance, with a selection intensity of 10%; The cold-resistant breeding of the cloud-patterned grouper was carried out as follows: When the basic population G0 breeding fry reached 12 and 24 months of age, cold-resistant individuals were selected to construct the G1 breeding population by lowering the temperature by 1°C daily, down to a minimum of 10°C, with selection intensities of 4.5% and 5.5%, respectively. When the breeding fry of the G1 population reached 24 months of age, cold-resistant selection was carried out again with a selection intensity of 10%, constructing the G2 breeding population. The female cloud-patterned grouper from the cold-resistant G2 population underwent accelerated maturation cultivation. In the above (1), the blue-bodied large-spotted grouper population selection and breeding: adult blue-bodied large-spotted grouper with different genetic backgrounds were collected for breeding, and male blue-bodied large-spotted grouper with fast growth, no deformities and symmetrical body shape were selected as parents after two generations of selection and breeding. The aforementioned breeding of blue-bodied large-spotted grouper involves: collecting blue-bodied large-spotted grouper from different genetic backgrounds, controlling the coefficient of variation of phenotypic traits to below 12.47%, and constructing a basic breeding population G0. Fry bred from the basic population G0 are selected for adulthood at 24 months of age, characterized by rapid growth, no deformities, and symmetrical body shape, to construct a G1 breeding population with a selection intensity of 10%. The G1 breeding population is then artificially raised to adulthood, with the coefficient of variation of phenotypic traits controlled to below 11.96%. F2 generation fry are bred from the G1 population and raised to 24 months of age, again selected to construct a G2 breeding population characterized by rapid growth, no deformities, and symmetrical body shape, with a selection intensity of 10%. The G2 population is then cultured to adulthood, with the coefficient of variation of phenotypic traits controlled to below 9.87%. From this population, male blue-bodied large-spotted grouper that grow rapidly and are free of deformities are selected as parent fish.

2. The method for artificially cultivating low-temperature resistant cumin hybrid spots according to claim 1, characterized in that, (2) Collection and preservation of sperm from blue-bodied large-spotted grouper: a. Cultivation Environment: Indoor factory-style cultivation, with a cultivation water temperature of 22~28℃, dissolved oxygen above 6 mg / L, salinity of 28~31‰, and water exchange rate of 8m³ / h. 3 / h; b. Reproductive regulation and sperm collection: In March of the following year, the water temperature for male fish rearing was strictly controlled at 23-26℃, and the water exchange rate was increased to 10m. 3 / h, and feed fresh fish mixed with nutritional fortifiers to promote testicular development; 48 hours before semen collection, stop feeding, anesthetize the parent fish, collect fresh semen artificially, and preserve the sperm in a low temperature environment of 4℃ in the dark.

3. The method for artificially cultivating low-temperature resistant cumin hybrid spots according to claim 1, characterized in that, The reproductive regulation and egg acquisition of the spotted grouper mentioned in (3) are as follows: In late October each year, the selected spotted grouper are transferred to offshore net cages for intensive cultivation. In early March, well-developed parent fish are transferred to a factory workshop for intensive cultivation. Female fish with gonads developed to stage IV are selected and injected with human chorionic gonadotropin (hCG) on their backs. At the same time, the water exchange rate is increased to 10 m. 3 / h stimulates the maturation of the female fish's gonads.

4. The method for artificially cultivating low-temperature resistant cumin hybrid spots according to claim 1, characterized in that, The aforementioned (4) artificial preparation and hatching of hybrid fertilized eggs: a. Hybridization and insemination: Mature grouper eggs are artificially collected, and blue-bodied large-spotted grouper sperm is used for artificial insemination at a sperm-to-egg volume ratio of 1:

800. 2-3 times the volume of seawater at 22-24℃ is added, and after static filtration, the fertilized eggs are transferred to an incubator for incubation. b. Hatching and rearing: During hatching, dissolved oxygen should be 6-7 mg / L, rearing water temperature 23-25℃, and salinity 28-30‰. No water changes are needed during the early rearing period of the fry. Before releasing the eggs, the fresh seawater should be strictly disinfected using ozone and ultraviolet light. From hatching to the tail bud stage, the fry are transferred to a rearing pond for rearing. The rearing water temperature should be 22-26℃, salinity 28-31‰, and dissolved oxygen greater than 6 mg / L. After 28 days of age, the water exchange rate should be 4-6 m / s. 3 / h; during this period, fish fry are intensified and cultivated using fish paste.

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