A method for preparing high temperature resistant oyster germplasm

Abstract

The present invention belongs to the technical field of shellfish genetic breeding, and specifically, relates to a method for preparing high-temperature-resistant oyster germplasm. Parent shellfish that have been temporarily cultured until the gonads begin to develop are used as samples, and are subjected to short-term acute stress in high-temperature seawater. The short-term acute stress is then repeated during the development of the parent shellfish's gonads. After repeated stimulation, a group germplasm of high-temperature-resistant germplasm is constructed by group mating using artificial dissection and insemination, thereby obtaining oyster germplasm with improved high-temperature resistance. The method of the present invention was used to evaluate high-temperature resistance through summer sea area and indoor high-temperature stress experiments, and the survival rate of the high-temperature-resistant germplasm was increased by 33.2%-36.9%, thereby obtaining oyster germplasm with improved high-temperature resistance. Compared with traditional breeding methods, this method has the advantages of strong practicality, significant effect, and easy promotion. The present invention can quickly and efficiently obtain oyster germplasm with significantly enhanced high-temperature resistance, significantly reducing the risk of large-scale death in the oyster industry during the high temperature period in summer.

Description

Technical Field

[0001] The invention belongs to the technical field of shellfish genetic breeding, and in particular relates to a method for preparing high-temperature-resistant oyster germplasm. Background Art

[0002] Oysters are the pillar industry of shellfish in my country and the largest farmed shellfish in the world. Oysters are usually farmed in open seas. Against the backdrop of global warming, extreme climate events such as abnormally high sea temperatures are frequent, and the challenges posed to the oyster industry are becoming increasingly prominent. In recent years, large-scale deaths of oysters in the summer have occurred frequently, posing a major threat to the oyster industry. The causes of large-scale deaths in the summer are relatively complex, and high temperatures are the trigger for this problem. Therefore, improving the high temperature resistance of oysters is a direct way to solve this problem. Classical genetic improvement can improve the high temperature resistance of oysters to a certain extent, but there are problems such as long breeding cycles and high costs. Therefore, the oyster industry urgently needs a method for preparing germplasm that can quickly and scalably improve the high temperature resistance of oysters. Summary of the Invention

[0003] In order to cope with the challenges brought to the oyster industry by the frequent occurrence of extreme climate events such as abnormally high sea temperatures, solve the current dilemma of the industry's lack of rapid and large-scale cultivation of high-temperature resistant oyster germplasm, and reduce the risk of large-scale death of oysters in summer, a method for preparing high-temperature resistant oyster germplasm is provided.

[0004] To achieve the above purpose, the present invention adopts the following technical solutions:

[0005] A method for preparing high-temperature-resistant oyster germplasm comprises taking broodstock temporarily cultured until gonad development begins as samples, subjecting them to short-term acute stress in high-temperature seawater, and then repeating the short-term acute stress during gonad development of the broodstock. After repeated stimulation, group germplasm of high-temperature-resistant germplasm is constructed by group mating with artificial dissection and insemination, thereby obtaining oyster germplasm with improved high-temperature resistance.

[0006] The method of the present invention is used to evaluate the high temperature resistance through summering experiments in sea areas and indoor high temperature stress experiments, and the survival rate of high temperature resistant germplasm is increased by 33.2%-36.9%.

[0007] Specifically:

[0008] (1) Collection of broodstock: Select diploid oysters with intact shells, appropriate size, and undeveloped gonads as candidate broodstock;

[0009] (2) Temporary screening: After the oysters are grouped and placed in the broodstock culture pond for temporary culture for 5-7 days, the oysters in the culture pond with a mortality rate of less than 10% during the temporary culture period are selected as oyster germplasm samples for improving high temperature resistance;

[0010] (3) Ripening stress: The water temperature of the broodstock culture tank used as the sample is raised by 0.5-1.5°C every day from the natural water temperature until the broodstock gonads begin to develop. The oysters whose gonads begin to develop are then placed in high-temperature seawater for the first short-term acute stress. After the high-temperature stress, the oysters are taken out and returned to room temperature, and then the samples are placed in the culture tank to continue the gonad ripening.

[0011] (4) Germplasm enhancement: the above samples were subjected to gonadal maturation and gonadal development, and the short-term acute stress of step (3) was repeated 1-4 times;

[0012] (5) Germplasm preparation: The samples with good gonad development after the above germplasm enhancement are used to construct a group of high-temperature resistant germplasm by artificial dissection and insemination in a large group mating method, that is, to obtain oyster germplasm with improved high-temperature resistance.

[0013] The oysters selected in step (1) are oysters collected from a sea area at a natural seawater temperature of 1-8°C, with an individual weight greater than 50g, a soft body weight ratio of greater than 15% of the individual weight, and undeveloped gonads.

[0014] The environmental conditions of the broodstock culture pond during the temporary rearing period in step (2) are as follows: the seawater temperature is 8-10°C, the temporary rearing density is 10-15 kg / m 3 .

[0015] In step (3), the water temperature of the broodstock culture pond used as the sample is controlled at 12-13°C (natural water temperature), and then increased by 0.5-1.5°C every day until the broodstock gonads begin to develop.

[0016] After the gonads of the parent shellfish begin to develop, they are placed in a seawater stress tank with a water temperature of 37-39°C and a salinity of 30 for 1-3 hours. The density of the sample during the stress period does not exceed 100 kg / m 3 , maintain sufficient inflation and perform a short-term acute stress treatment.

[0017] The oysters after the ripening stress in step (4) are placed in an oyster cultivation pond with a water temperature range of 15-22° C., and then the short-term acute stress in step (3) is repeated 1-4 times; the time interval between the two stresses is greater than 72 hours.

[0018] The short-term acute stress in step (4) is to place the oysters in a seawater stress tank with a water temperature of 37-39°C and a salinity of 30 for 1-3 hours, and the density of the sample during the stress period does not exceed 100 kg / m 3 , keep it adequately inflated as a short-term acute stress treatment.

[0019] Beneficial effects of the present invention

[0020] The present invention is based on the plasticity mechanism of oysters' resistance to short-term high-temperature stress environments during gonadal development, and utilizes the transgenerational memory of oysters to environmentally induced epigenetic-related resistance enhancement, and adopts multiple short-term stresses to significantly improve the oysters' high-temperature resistance level. Compared with traditional breeding methods, this method significantly improves the utilization efficiency of parent shellfish, shortens the cycle of germplasm preparation, and significantly improves the high-temperature resistance of oysters. This method has the advantages of strong practicality, significant effects, and easy promotion. Through the present invention, oyster germplasm with significantly enhanced high-temperature resistance can be obtained quickly and with high throughput, significantly reducing the risk of large-scale mortality in the oyster industry during the high temperature period in summer. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a photo of a tissue section of the gonad of a long oyster provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0022] The present invention will be further described below in conjunction with specific examples. It should be understood that the following examples are provided to further illustrate the present invention, rather than to limit the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalents fall within the scope limited by the appended claims of the application.

[0023] Example 1:

[0024] Broodstock collection: In January 2022, the seawater temperature was monitored at 5°C in the Huangdao Sea Area of Qingdao City, Shandong Province. Diploid long oysters with intact shells, individual weight greater than 50g, and soft body weight accounting for 15%-25% of individual body weight were selected from the Guzhenkou Bay oyster breeding area as broodstock. The gonadal tissues of 10 individuals were randomly selected for tissue sections. The diploid oysters with undeveloped gonadal development stages were selected as candidate broodstock ( Figure 1 ).

[0025] Temporary screening: 4,000 oysters were collected, cleaned of surface attachments, and then divided into 5 groups and placed in 1,000L broodstock culture ponds for temporary culture for 7 days. Feed was given regularly every day. The seawater temperature was 8-10℃ and the temporary culture density was 15kg / m 3 ; Oysters in the cultivation ponds with a mortality rate of less than 10% during temporary rearing were selected as oyster germplasm samples for improving high temperature resistance.

[0026] Ripening stress: The water temperature in the broodstock rearing pond was raised by 1°C per day from the natural temperature of 8°C. When the water temperature reached 12°C (i.e., until the gonads of the broodstock began to develop), 500 broodstock were randomly selected and bred using conventional gonad ripening methods as the control group.

[0027] The remaining individuals were subjected to the first short-term acute stress as the experimental group of high-temperature resistant germplasm. The stress method was to place the oysters of the experimental group of high-temperature resistant germplasm in a seawater stress tank with a volume of 700 liters, a water temperature of 38-39℃, and a salinity of 30 for 1 hour. The density of the samples was maintained at about 50 kg / m during the stress period. 3 After high-temperature stress, remove the oysters from the stress tank and place them in a dry place for 2 hours until they return to room temperature (room temperature is 10-20°C). Then, place the samples back into the broodstock culture tank to continue gonadal maturation.

[0028] Germplasm reinforcement: After the stress treatment in the above steps, the samples are placed back into the parent shellfish culture tank and subjected to short-term acute stress twice again when the water temperature is 15℃-19℃. Each short-term acute stress period is 38-39℃ and the salinity is 30. The samples are treated in the seawater stress tank for 1 hour. The density of the samples is maintained at about 50kg / m 3 , maintain sufficient inflation, and the time interval between each stress and the previous stress is greater than 72 hours.

[0029] Germplasm Preparation: After germplasm enrichment, oysters were placed back into the parent maturation pond. When the water temperature reached 24°C, 60-100 broodstock with well-developed gonads were selected as breeding groups. The sex of each individual was determined, and sperm or eggs from each individual were collected in 500ml beakers. The eggs were matured in seawater for 1 hour, and sperm-contaminated individuals were removed. Sperm from 30 individuals and eggs from 30 individuals were collected. The eggs were mixed in equal amounts and then divided into 30 equal portions. Each of the 30 eggs was fertilized with sperm from 30 individuals to ensure an equal number of offspring from each individual. After cleavage in each group, the 30 fertilized beakers were mixed and incubated. Both the experimental and control groups of heat-resistant germplasm were established through group mating. Seedlings were cultured using conventional methods, with the density of oyster spat controlled at 15-20 per substrate.

[0030] The above-mentioned seedlings with high temperature resistance were placed in the Yantai sea area for summer survival test. Specifically, the oyster juveniles of the experimental group and the control group were fixed on 4m long culture ropes, with the spacing of the attachment bases being 15-20cm. Conventional culture was carried out using the rope clamping method, with the spacing of the culture ropes being 30-50cm. In September and October in autumn, 3 culture ropes were randomly selected, and the number of surviving and dead oysters was counted, and then the summer survival rate of each group was calculated.

[0031] The heat-resistant germplasm prepared using this method had a 75.10% survival rate during the summer heat period, compared to 54.84% for the control group that did not use this method. The heat-resistant germplasm's survival rate in the summer heat period at sea was increased by 36.9%. In an indoor test of heat tolerance using an acute heat stress test at 42°C for one hour, the heat-resistant germplasm prepared using this method had a 39.59% survival rate, compared to 29.73% for the control group that did not use this method. The heat-resistant germplasm's survival rate during the indoor acute heat stress test was increased by 33.2%.

[0032] Comparative Example:

[0033] Collection of broodstock: In January 2021, the seawater temperature was monitored at 5.2℃ in the Huangdao sea area of Qingdao City, Shandong Province. Diploid long oysters with intact shells, individual weight greater than 50g, and soft body weight accounting for 15%-25% of individual body weight were selected from the Guzhenkouwan oyster farming area as broodstock. The gonad tissues of 10 individuals were randomly taken for tissue sections, and the diploid oysters whose gonad development stages were all in the undeveloped stage were selected as alternative broodstock.

[0034] Temporary screening: 1800 oysters were collected, and after removing the surface attachments and washing them clean, they were divided into 3 groups and placed in 500L broodstock culture ponds for temporary culture for 6 days. Feeding was done regularly every day. The seawater temperature was 8-10℃ and the temporary culture density was 10kg / m 3 ; Oysters from the cultivation ponds where no individuals died during the temporary rearing period were selected as oyster germplasm samples for improving high temperature resistance.

[0035] Ripening stress: The water temperature of the broodstock rearing pond was raised by 0.5°C per day from the natural water temperature of 8°C. When the water temperature reached 12°C (i.e. until the broodstock gonads began to develop), 100 broodstock were randomly selected and bred using conventional gonad ripening methods as the control group.

[0036] The remaining individuals were subjected to the first short-term acute stress as the experimental group of high-temperature resistant germplasm. The stress method was to place the oysters of the experimental group of high-temperature resistant germplasm in a 700-liter seawater stress tank with a water temperature of 38-39°C and a salinity of 30 for 1.5 hours when the water temperature rose to 19°C. The density of the samples was maintained at about 50 kg / m during the stress period. 3 After high-temperature stress, remove the oysters from the stress tank and place them in a dry place for 2 hours until they return to room temperature. Then, place the samples back into the broodstock culture tank to continue gonadal maturation.

[0037] Germplasm preparation: After stress treatment, the samples were returned to the broodstock culture tank. When the water temperature reached 24°C, 60-100 broodstock with well-developed gonads were selected as breeding groups. The sex of each individual was determined, and sperm or eggs from each individual were collected in 500ml beakers. The eggs were matured in seawater for 1 hour, and sperm-contaminated individuals were removed. Sperm from 30 individuals and eggs from 30 individuals were collected. The eggs were mixed in equal amounts and then divided into 30 equal portions. The sperm from 30 individuals was then fertilized with each of the 30 eggs, ensuring that each individual produced an equal number of offspring. After cleavage in each group, the 30 fertilized beakers were mixed and incubated. Both the experimental and control groups of heat-resistant germplasm were established through group mating. Seedlings were cultured using conventional methods, with the density of oyster spat controlled at 15-20 per substrate.

[0038] The seedlings of the experimental group and the control group were placed in the Jiaonan and Rongcheng sea areas for summer testing. The survival rates of the experimental group during the summer high temperature period in the Jiaonan and Rongcheng sea areas were 22.7% and 62.7% respectively, and the survival rates of the control group during the summer high temperature period were 21.2% and 59.6% respectively. There was no significant difference in the survival rates of the two groups during the summer high temperature period in the two sea areas.

[0039] It can be seen from the above embodiments that the survival rate of the high temperature resistant germplasm in Example 1, which strictly adopts the method of this patent, during the summer high temperature period and the survival rate of indoor high temperature stress are significantly higher than those of the control group, but there is no significant difference in the survival rate of the experimental group and the control group in the comparative example during the summer high temperature period in the two sea areas. The main technical difference between Examples 1 and 2 is that the comparative example lacks the germplasm enhancement step, which shows that germplasm enhancement can improve the high temperature resistance of oysters; therefore, only by completing all the steps under the specific conditions specified in this patent can oyster germplasm with improved high temperature resistance be obtained.

[0040] The above are only specific embodiments of the invention, but the design concept of the present invention is not limited thereto. Any non-substantial changes to the present invention using the concept shall be deemed as an infringement of the protection scope of the present invention.

Claims

1. A method for preparing high temperature resistant oyster germplasm, characterized by: Using broodstock that have been temporarily reared until gonadal development begins as samples, they are subjected to short-term acute stress in high-temperature seawater. This short-term acute stress is then repeated during gonadal development. Following repeated stimulation, group mating with artificial dissection and insemination is used to construct a high-temperature-resistant germplasm population, thereby obtaining oyster germplasm with improved high-temperature resistance. Specifically: (1) Collection of broodstock: Select diploid oysters with intact shells, appropriate size, and undeveloped gonads as candidate broodstock; (2) Temporary screening: After the oysters are grouped and placed in the broodstock culture pond for temporary culture for 5-7 days, the oysters in the culture pond with a mortality rate of less than 10% during the temporary culture period are selected as oyster germplasm samples for improving high temperature resistance; (3) Ripening stress: The water temperature of the broodstock culture tank used as the sample is raised by 0.5-1.5°C every day from the natural water temperature until the broodstock gonads begin to develop. The oysters whose gonads begin to develop are then placed in high-temperature seawater for the first short-term acute stress. After the high-temperature stress, the oysters are taken out and returned to room temperature, and then the samples are placed in the culture tank to continue the gonad ripening. (4) Germplasm enhancement: the above samples were subjected to gonadal maturation and gonadal development, and the short-term acute stress of step (3) was repeated 1-4 times; (5) Germplasm preparation: The samples with good gonad development after the above germplasm enhancement are used to construct a group of high-temperature resistant germplasm by artificial dissection and insemination in a large group mating method, that is, to obtain oyster germplasm with improved high-temperature resistance; After the gonads of the parent shellfish begin to develop, they are placed in a seawater stress tank with a water temperature of 37-39°C and a salinity of 30 for 1-3 hours. The density of the sample during the stress period does not exceed 100 kg / m 3 , maintain sufficient inflation and perform a short-term acute stress treatment.

2. The method for preparing high temperature resistant oyster germplasm according to claim 1, characterized in that: The oysters selected in step (1) are oysters collected from a sea area at a natural seawater temperature of 1-8°C, with an individual weight greater than 50g, a soft body weight ratio of greater than 15% of the individual weight, and undeveloped gonads.

3. The method for preparing high temperature resistant oyster germplasm according to claim 1, characterized in that: The environmental conditions of the broodstock culture pond during the temporary rearing period in step (2) are as follows: the seawater temperature is 8-10°C, the temporary rearing density is 10-15 kg / m 3 .

4. The method for preparing high temperature resistant oyster germplasm according to claim 1, characterized in that: In step (3), the water temperature of the broodstock culture pond used as the sample is controlled at 12-13°C (natural water temperature), and then increased by 0.5-1.5°C every day until the broodstock gonads begin to develop.

5. The method for preparing a high temperature resistant oyster germplasm according to claim 3, characterized in that: The oysters after the ripening stress in step (4) are placed in an oyster cultivation pond with a water temperature range of 15-22° C., and then the short-term acute stress in step (3) is repeated 1-4 times; the time interval between the two stresses is greater than 72 hours.

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