An identification method for the phenotype of turbot selected for low-salt tolerance

Abstract

The present invention discloses a method for identifying the phenotype of turbot with low-salt tolerance, belonging to the field of selective breeding of economic traits of aquatic animals. The method is as follows: Prepare a culture pond, inject fresh water, and adjust the water temperature, dissolved oxygen to be consistent with the environment where the test population is located. After the test turbot population has fasted for 72 hours, place it in the fresh water pond for cultivation, and record the initial time. Check the color change of the pigmented side of the fish body every hour. After black stripes appear, record the appearance time of the black stripes. Immediately return the experimental fish to the normal salinity environment after the recording is completed, and the body color of the fish will recover. The determination of the phenotype of turbot with high-efficiency low-salt tolerance is realized according to the formula. The individual low-salt tolerance breeding phenotype values are arranged from large to small, and the individuals with the top rankings are selected. The phenotype identification method provided by the present invention can achieve more efficient and accurate results, and cause no damage to the tested individuals, laying a foundation for subsequent genetic evaluation and selective breeding of this trait.

Description

Technical Field

[0001] The present invention belongs to the field of breeding of economic traits of aquatic animals, and particularly relates to a method for identifying the phenotype of low-salt tolerance breeding of turbot. Background Art

[0002] In the breeding of aquatic animals, the breeding of stress resistance traits is an important aspect of the breeding of environmental adaptability and a practical manifestation of environmental adaptability breeding. It can not only improve the survival rate of aquatic animals, ensure the sustainable development of the aquatic industry, but also guarantee the quality and safety of aquatic products and enrich the resources of aquatic animals. At present, a small number of fine fish varieties with stress resistance traits have been bred in China. However, it should also be noted that the progress of stress resistance breeding is slow and difficult to meet the development needs. One of the main reasons is the lack of high-efficiency breeding phenotypes. The phenotypes currently used in stress resistance breeding basically focus on the survival status and survival time in adversity. On the one hand, it is time-consuming and laborious to obtain phenotypes, and the individuals after obtaining phenotypes cannot be used as broodstock for cultivation. On the other hand, these two phenotypes are difficult to become continuous quantitative traits that conform to the normal distribution, seriously affecting the breeding progress. Therefore, screening suitable breeding phenotypes is the top priority of stress resistance breeding. Turbot is an important marine cultured fish in China. In recent years, due to market demand and the guidance of environmental protection policies, the aquaculture area has expanded to the tidal flats and inland salt lake areas, putting forward higher requirements for its low-salt tolerance traits. Cultivating fine turbot varieties adapted to low-salt environments can better meet the development needs of the turbot breeding industry. Therefore, it is urgent to identify efficient breeding phenotypes of turbot with low-salt tolerance. Summary of the Invention

[0003] In view of the above technical problems, the present invention provides a method for identifying the phenotype of low-salt tolerance breeding of turbot, which is simple and easy to operate and will not cause harm to experimental individuals.

[0004] The present invention is realized through the following technical solutions:

[0005] A method for identifying the phenotype of low-salt tolerance breeding of turbot, the specific steps of the method are as follows:

[0006] First step, prepare a culture pond, inject fresh water, and adjust the water temperature, dissolved oxygen to be the same as the environment where the test group is located; the fresh water pond is the same as the culture pond where the test group is located, which can ensure that other environments are the same except for salinity to the greatest extent;

[0007] Second step, after the test turbot group has fasted for 72-96 hours, place it in the fresh water pond for cultivation, and record the initial time;

[0008] Third step, check the color change of the pigmented side of the fish body every hour. After black stripes appear (as shown in the appendix Figure 1) Record the appearance time of the black stripes. Immediately after the recording is completed, return the experimental fish to the normal salinity environment, and the body color of the fish will recover;

[0009] Step 4: Measure the phenotype of efficient low-salinity tolerance breeding of turbot according to the formula. The formula is: individual low-salinity tolerance breeding phenotype = the time when black stripes appear on each fish - the initial time. Arrange the individual low-salinity tolerance breeding phenotype values from largest to smallest, and select the individuals with the top rankings.

[0010] Further, in the first step, the slightly flowing water in the fresh water pool is the same as that in the breeding pool where the population to be tested is located.

[0011] Further, in the second step, the weight of the turbot individual is above 20 g.

[0012] Further, in the third step, after the recording of the time when black stripes appear is completed, classify the population to be tested according to the length of time, and retain the group with a longer time as the candidate parent for continued cultivation.

[0013] Further, in the third step, if the weight of the turbot individual is above 150 g, after the recording of the time when black stripes appear is completed, the turbot individual can be marked, which can better correspond to the phenotype after cultivating the broodstock.

[0014] Further, in the fourth step, the recording of the individual low-salinity tolerance breeding phenotype is in hours as the unit.

[0015] Advantages of the present invention compared with the prior art: The present invention innovatively proposes to use body color change as the stress resistance breeding phenotype, provides an efficient method for identifying the low-salinity tolerance breeding phenotype of turbot, overcomes the problems that the survival state and survival time are generally used as phenotypes in stress resistance traits, resulting in fish death or fish body damage, and also solves the problem that the fish cannot develop into broodstock for offspring cultivation due to damaged phenotype measurement. The method is simple to operate, and the appearance of black stripes is visible to the naked eye. Compared with the prior art for checking the survival state, using the phenotype identification method provided by the present invention can achieve more efficient and accurate results, and there is no damage to the tested individuals, laying a solid foundation for subsequent genetic evaluation and selective breeding of this trait. Brief Description of the Drawings

[0016] Figure 1 Turbot individuals after the appearance of black stripes;

[0017] Figure 2 Normal distribution graph of the time trait when black stripes appear. Detailed Embodiments

[0018] The technical solutions of the present invention will be further explained below through embodiments in combination with the drawings, but the protection scope of the present invention is not limited by any form of the embodiments.

[0019] Example 1 A method for identifying the low-salt tolerance breeding phenotype of turbot, the specific steps of the method are as follows: Step 1, preparation of turbot test population: The turbot test population was from Shandong Kehe Ocean High-Tech Co., Ltd., 300 strong, undamaged, and vigorous young fish were selected, with a body length of 11.9 cm ± 0.7 cm and a body weight of 27.5 ± 4.6 g, and were cultured in an indoor breeding pond (12 m 2 ) were temporarily maintained for 2 weeks, with water temperature of 17℃, salinity of 30ppt, and dissolved oxygen>6mg / L.

[0020] Step 2: Freshwater pool preparation: Select a breeding pond on the side of the seawater pool where the population to be tested is located, and inject an equal amount of fresh water with a water temperature of 17°C, a salinity of 0ppt, and dissolved oxygen>6mg / L.

[0021] Step 3: Low-salt tolerance test: Take out all the test groups, put them in the fish frame, and then pour them into the freshwater pool at the same time. The experiment starts and the start time is recorded. Check the fish every hour to see if there is any change in the color of the eye side. When the turbot individual has black stripes (such as the attached Figure 1 ), record the time. After recording, cut a small piece of tail fin for further analysis, such as GWAS analysis. After sampling, the turbot individuals were immediately returned to the 30ppt culture pond, and the body color of the turbot individuals gradually returned to normal. No feeding operation was performed during the experiment until all individuals had black stripes, and the experiment ended.

[0022] The fourth step is to determine the low-salt tolerance and efficient breeding phenotype of turbot according to the formula (individual low-salt tolerance breeding phenotype = time for each fish to have black stripes - initial time).

[0023] Step 5: After the experiment, the low-salt tolerance breeding phenotypes of all individuals were statistically analyzed. After KS testing, it was found that the trait basically conformed to the normal distribution (P>0.05 was significant). Figure 2 As shown, on the one hand, this phenotype can indirectly reflect that the low-salt tolerance trait is a quantitative trait, which conforms to the characteristics of the normal distribution of quantitative traits. On the other hand, the use of this phenotype can distinguish individuals well, laying the foundation for further breeding work.

[0024] Example 2: A method for identifying the phenotype of turbot with low-salt tolerance. The specific steps of the method are as follows: The turbot individuals transferred to the 30 ppt aquaculture pond in the low-salt tolerance experiment in Example 1 were continuously reared for 60 days at a water temperature of 17 °C, a salinity of 30 ppt, and a dissolved oxygen > 6 mg / L. During the experiment, they were fed twice a day until satiation, and dead fish were removed in a timely manner. After the experiment, the survival rate was counted and the weight data were measured. The experimental results showed that the survival rate was 90%, and the weight was 39.66 ± 7.2 g, which was similar to the aquaculture results under normal conditions. Therefore, using the trait measurement method provided by the present invention, the turbot individuals after obtaining the traits can return to normal and can continue to be cultivated into broodstock, solving the problem that the individuals after obtaining the phenotype by the previous traditional methods cannot continue to be cultivated as reserve broodstock.

[0025] Example 3: A method for identifying the phenotype of turbot with low-salt tolerance. The specific steps of the method are as follows: First step: Construction of turbot family groups: The turbot broodstock were from Tianyuan Aquatic Products Co., Ltd. During the breeding period, 15 female fish and 15 male fish were selected from 100 reserve broodstock for one-to-one pairing, and 15 full-sib families were successfully established. After 5 months of cultivation, the juvenile fish of the families grew to 20 - 40 g. 100 healthy, uninjured, and highly active juvenile fish were randomly selected from each family and separately reared in a 1 m 3 aquaculture bucket at a water temperature of 17 °C, a salinity of 30 ppt, a dissolved oxygen > 6 mg / L, and flowing water culture for a culture period of 2 weeks to enable them to fully adapt to the environment.

[0026] Second step: Measurement of body color phenotypic traits in the fresh water stress experiment: After the temporary rearing ended, the seawater in the bucket was drained, and fresh water was introduced as the water source. The flow rate of each aquaculture bucket was controlled to be the same using a flow meter. The water temperature was 17 °C, the salinity was 0 ppt, the dissolved oxygen > 6 mg / L, and flowing water culture was carried out. The body color change of the eyed side of the fish was checked every hour. When black stripes appeared on the turbot individuals (as shown in the appendix Figure 1 ), the time was recorded. After the recording was completed, the turbot individuals were immediately returned to the 30 ppt aquaculture pond, and the body color of the turbot individuals gradually returned to normal. No feeding operation was carried out during the experiment until black stripes appeared on all individuals, and the experiment ended. According to the formula (individual low-salt tolerance breeding phenotype = time when black stripes appeared on each fish - initial time), the body color phenotypic traits of all families were statistically analyzed to achieve the determination of the high-efficiency breeding phenotype of turbot with low-salt tolerance. The families were ranked according to the results.

[0027] Step 3. Low-salt aquaculture experiment: Continue to use the above-mentioned aquaculture barrels. Adjust the flow rates of fresh water and seawater in advance through a flowmeter, control the salinity at 10 ppt. Select 3 families with extreme traits from 15 families according to the body color phenotype data, randomly select 100 tails per family. After measuring the body weight data, transfer them into the aquaculture barrels for feeding experiments. During the experiment, the water temperature is 17 °C, the dissolved oxygen > 6 mg / L, and it is a flowing water aquaculture. The experimental period is 60 days. Feed twice a day during the experiment, feed to satiety, and promptly remove dead fish. After the experiment, count the survival rate and measure the body weight data.

[0028] Step 4. Comparative analysis of family body color phenotype data with low-salt tolerance growth and survival rate phenotypes: As shown in Table 1, for the 2 families (3, 7, 9) with good body color phenotype performance, their survival rate, body length, and body weight are significantly better than those of the 2 families (4, 11, 14) with poor body color phenotype performance. This further indicates that the phenotypes and measurement methods provided by the present invention can achieve efficient and accurate effects.

[0029] Table 1 Comparison of family body color phenotype data with low-salt tolerance growth and survival rate phenotypes

[0030]

Claims

1. A method for identifying the phenotype of turbot selected for low-salt tolerance, characterized in that, The specific steps of the method are as follows: First step: Prepare a breeding pond, inject fresh water, and adjust the water temperature, dissolved oxygen to be consistent with the environment where the test population is located; Second step: After the test turbot population has fasted for 72 - 96 hours, place it in the fresh water pond for breeding, and record the initial time; Third step: Check the color change of the pigmented side of the fish body every hour. After black stripes appear, record the appearance time of the black stripes. Immediately return the experimental fish to the normal salinity environment after the recording is completed; Fourth step: Determine the phenotype of high - efficiency low - salinity tolerance breeding of turbot according to the formula. The formula is: individual low - salinity tolerance breeding phenotype = time when black stripes appear on each fish - initial time. Arrange the individual low - salinity tolerance breeding phenotype values from large to small, and select the individuals with the top rankings.

2. The identification method of the low-salt tolerance breeding phenotype of turbot according to claim 1, characterized in that, In the first step, there is gentle flowing water in the breeding pond.

3. The identification method of the low-salt tolerance breeding phenotype of turbot according to claim 1, characterized in that, In the second step, the weight of each turbot individual is above 20 g.

4. The identification method of the low-salt tolerance breeding phenotype of turbot according to claim 1, characterized in that, After the recording of the appearance time of the black stripes is completed, classify the test population according to the length of time, and retain the group with a longer time as the candidate parent for continued cultivation.

5. The identification method of the low-salt tolerant breeding phenotype of turbot according to claim 1, characterized in that, If the weight of the turbot individual is above 150 g, after the recording of the appearance time of the black stripes is completed, mark the turbot individual.

6. The identification method of turbot phenotype resistant to low salinity according to claim 1, characterized in that, In the fourth step, the recording of the individual low - salinity tolerance breeding phenotype is in hours as the unit.

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