Preparation method of novel high-temperature-resistant and fast-growing paralichthys olivaceus triploid strain

Abstract

The invention belongs to the technical field of genetic breeding, and particularly relates to a preparation method of a novel high-temperature-resistant and fast-growing paralichthys olivaceus triploid strain. According to the method, high-temperature resistance and body weight are used as screening indexes, a high-temperature-resistant commercial line and a fast-growing commercial line of paralichthys olivaceus are constructed by using the technologies of population breeding, gynogenesis and the like, then the commercial lines are used as male and female parents, mature sperms and eggs are collected for insemination, triploid induction is performed, and high-temperature resistance and growth traits are measured, so that the high-temperature-resistant commercial line of paralichthys olivaceus is obtained. And obtaining a triploid new strain with two characters of high temperature resistance and fast growth at the same time. According to the method, the triploid paralichthys olivaceus fries with high-temperature-resistant and fast-growing characters can be efficiently induced and generated in batches, and high practicability is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic breeding, and in particular relates to a method for preparing a new triploid strain of flounder that is resistant to high temperatures and grows fast. Background Art

[0002] Triploid fish, due to their sterility or low fertility, may offer growth advantages and effectively prevent precocious puberty, overcoming growth stagnation and decreased meat quality during gonadal development. This not only facilitates the production of large-sized seedlings and improves aquaculture economics, but also can be used in reproductive operations and for the safety control of introduced species. However, due to the poor fertility of triploids, it is difficult to obtain fertile tetraploid adults, making direct breeding through subculture difficult. Therefore, aside from potential growth advantages at different developmental stages, selection for other economic traits, such as stress resistance, is not possible. Complementary line breeding involves first obtaining specialized lines with specific economic traits, then hybridizing them to fully exploit hybrid vigor. This can produce offspring that inherit the high-quality traits of both parents. It also avoids inbreeding depression that can occur after high-level selection, maintains genetic diversity in the population, and improves the production traits and adaptability of future generations.

[0003] Currently, breeding of new, heat-resistant varieties of a few fish species is underway to meet the needs of aquaculture production. These new diploid varieties not only possess heat tolerance but also rapid growth, effectively improving aquaculture production efficiency and reducing costs. As mentioned earlier, triploid fish, due to their sterile or low-fertility gonads, are unable to reach sexual maturity and reproduce. This can reduce growth stagnation during the reproductive period and high mortality rates during farming and transportation, and also facilitate the production of large commercial fish. Triploid fish obtained through hybridization of complementary lines may possess both heterosis and triploid vigor, resulting in superior economic traits such as growth rate and stress resistance, meeting the needs of intensive aquaculture and deep-sea cage culture. Therefore, the development of fast-growing, heat-resistant triploid fish will promote the diversification of aquaculture, expand the aquaculture range, address global warming trends, enhance the industry's competitiveness, and contribute to the ecological safety of aquaculture waters. Similarly, no new fish species have been reported to date.

[0004] flounder ( Paralichthysolivaceus ), also known as brown flounder, belongs to the order Pleuronectiformes, family Paralichthyidae, genus Paralichthyidae ( Paralichthys ), a commercial marine species in my country and an important aquaculture and stocking fish. Triploidy has been successfully induced in Japanese flounder both domestically and internationally, with attention focused on growth and fertility traits. However, in addition to growth, stress resistance traits such as high-temperature tolerance are also urgently needed in aquaculture. Summary of the Invention

[0005] The purpose of the present invention is to comprehensively utilize the advantages of the two methods of triploid and matching line hybridization, provide an effective method for inducing the preparation of a new triploid strain of flounder that is heat-resistant and grows fast by matching line, and cultivate a triploid fish with hybrid advantage and sterility.

[0006] In order to achieve the above-mentioned object, the present invention adopts the following specific technical scheme: a method for preparing a new triploid strain of flounder that is resistant to high temperatures and grows fast, wherein high temperature resistance and body weight are used as screening indicators respectively, and a high temperature-resistant complementary strain and a fast-growing complementary strain are constructed by utilizing group breeding and gynogenetic techniques. Then, using the complementary strains as parents, mature sperm and eggs are collected and fertilized, and triploid induction is performed. The high temperature resistance and growth trait are measured to obtain a new triploid strain that has both high temperature resistance and fast growth.

[0007] Preferably, the number of generations of continuous gynogenetic selection is more than two when constructing the high-temperature resistant complementary lines and the fast-growing complementary lines.

[0008] Preferably, the triploid new strain is obtained by using a high-temperature-resistant complementary strain as the male parent and a fast-growing complementary strain as the female parent, and fertilizing mature sperm and eggs and then inducing cold shock.

[0009] Preferably, the screening and determination of high temperature resistance traits are carried out more than twice during the breeding cycle, and families with a high average upper limit of high temperature resistance and a high survival rate under constant high temperature are considered to have high temperature resistance traits; families with high temperature resistance characteristics in each measurement are identified as high temperature resistant strains.

[0010] Preferably, two high-temperature acute experiments are used to determine the high-temperature resistance trait. Method 1: continuously increase the temperature, observe and record the lethal temperature of each fish, and calculate the average upper limit of high-temperature resistance of each family / group; Method 2: determine a constant high temperature based on the half-lethal temperature of the unselected control group, and calculate the survival rate of each family after a period of time at the constant high temperature.

[0011] Preferably, the cold shock induction condition is to place the fertilized eggs in 0-1°C seawater for cold shock treatment 5 minutes after fertilization to inhibit the release of the second polar body. The treatment time is 45 minutes. After the treatment is completed, the fertilized eggs are moved into constant temperature seawater for continued cultivation; the fertilization water temperature and the incubation water temperature are both 15°C.

[0012] The advantages and positive effects of the method of the present invention are as follows: A method for efficiently producing a new triploid strain of flounder that is resistant to high temperatures and grows fast is provided. For the first time, a triploid with complex traits is prepared by combining triploid induction technology and matching line hybridization technology. Due to the poor fertility of triploids, it is difficult to directly select and breed for traits other than growth, and it is difficult to aggregate excellent traits. The traditional triploid breeding method is to first induce tetraploids, and then hybridize the mature tetraploids with diploids to obtain triploids. However, tetraploid adults have not yet been obtained in most marine fish, and triploid breeding has not yet been achieved. The method of the present invention has opened up a new technical approach for the breeding of triploids with complex traits in marine fish. By selecting and breeding the parents, matching lines with high temperature resistance and rapid growth traits are obtained respectively, and then the hybrid vigor is utilized to carry out large-scale production and cultivation of triploids of flounder that are resistant to high temperatures and grow fast.

[0013] A method for evaluating the heat resistance of Japanese flounder is provided. Currently, there is no unified standard for evaluating heat resistance in fish. Based on the actual situation of Japanese flounder, this method combines two acute high-temperature experiments to screen for heat resistance. Furthermore, studies have shown that fish size has a certain impact on heat resistance. Therefore, to accurately assess heat resistance, we conducted two heat resistance screenings on Japanese flounder seedlings and commercial fish before reaching market size to evaluate the stability of heat resistance during the cultivation period.

[0014] The established method for inducing high-temperature-resistant and fast-growing triploids allows for efficient production of triploid flounder fry in large quantities, significantly improving their heat tolerance and growth performance, making them highly practical. Furthermore, triploids are characterized by low or infertility, which allows them to devote more energy to growth, resulting in larger adult fish, making them suitable for intensive aquaculture and deep-sea cage culture. It also helps protect the rights of new strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a ploidy detection diagram of the flow cytometer provided in Example 2 of the present invention; wherein (a) is a common diploid group; (b) is a compound triploid group; Figure 2 The growth traits of the compound triploid flounder at one year old provided in Example 2 of the present invention; Figure 3 These are the test results of the upper limit of high temperature resistance of the composite triploid flounder at 2 months and 1 year old provided in Example 2 of the present invention; wherein, (a) 2 months old; (b) 1 year old. DETAILED DESCRIPTION

[0016] The present invention discloses a method for cultivating high-temperature-resistant and fast-growing triploid flounder. Those skilled in the art can refer to the present disclosure and appropriately modify the process parameters to achieve the desired effect. It should be noted that all similar substitutions and modifications obvious to those skilled in the art are considered encompassed by the present invention. The following examples further illustrate specific embodiments of the present invention. It should be noted that the specific embodiments described herein are intended only to illustrate and explain the present invention and are not intended to limit the present invention.

[0017] Example 1: Construction of high temperature resistant and fast growing system Establishment of a breeding base population: Wild flounder are collected from the Weihai waters of Shandong Province for domestication and cultivation. Individuals with standard body shape, normal coloration, and well-developed gonads are selected from these individuals to establish a breeding base population.

[0018] F0 generation population construction: Individuals in the breeding stock were electronically tagged with PITs (passive integrated transponders) and cultured at a high temperature of 27.0 ± 0.5°C for two months. Individuals with positive weight gain and in the top 50% of their specific growth rate (SGR) were selected as candidate broodstock for gonadal maturation. In-stock breeding was performed using mixed mating. 40,000 fry were selected for breeding. Two in-stock selections were conducted at two and six months of age, selecting individuals with normal coloration, no deformities, and large size for further breeding.

[0019] Individuals were screened for heat resistance at 6 and 14 months of age, with a total selection rate of approximately 10% for these two heat resistance screenings. A third growth selection was conducted at 18 months of age. The total selection rate after these three heat resistance screenings and three selections was approximately 1%, establishing the F0 generation population.

[0020] Construction of high temperature resistant matching lines: Using the F0 generation as parents, the sexually mature female parents were induced to undergo meiotic gynogenetic development. 60 gynogenetic families were initially established, and 20 families were screened for further cultivation.

[0021] About 10,000 fry were selected from each family, and three family selections were conducted when they were raised to 2, 6, and 18 months of age.

[0022] At 6 and 14 months of age, gynogenetic families were screened for heat tolerance through acute high-temperature experiments. First, the water temperature was gradually increased at a rate of 1°C / h to 31.0°C, then at a rate of 0.2°C / h. The lethal temperature of each fish was recorded, and the average upper temperature limit of heat tolerance for each family was calculated. Second, the water temperature was maintained at a high temperature (31.5 ± 0.2°C), and the survival rate after 24 hours was calculated. Finally, the water temperature was gradually increased at a rate of 1°C / h to 31.0°C, then at a rate of 0.2°C / h to 31.5°C. The water temperature was then maintained constant for 24 hours, and the survival rate of each group was calculated. Two heat-tolerant F1 families were identified.

[0023] Using the two selected heat-tolerant F1 families as parents, gynogenesis was induced again to obtain second-generation gynogenetic families. At 1-3 months of age, the chicks were cultured at 20.0 ± 0.5°C and 28.0 ± 0.5°C, respectively, to induce male and female phenotypes. Within-family selection was repeated three times at 2, 6, and 18 months of age.

[0024] At 6 and 14 months of age, two high temperature acute experiments were conducted to screen high temperature resistant families, and one high temperature resistant family with stable traits was obtained to form the F2 generation high temperature resistant matching system.

[0025] Construction of fast-growing complementary lines: Growth trait testing was performed on the 20 gynogenetic families obtained in the previous screening. Three family selections were conducted at 2, 6, and 18 months of age.

[0026] At 10 months of age, 300 fish from each family were fluorescently labeled and then mixed-cultured. At 18 months of age, the weight of the mixed-cultured individuals was measured, and their growth was tracked. Five fast-growing F1 families were screened.

[0027] The five fast-growing families of the selected F1 generation were used as parents, and gynogenetic induction was performed again to obtain the second-generation gynogenetic families. The male and female phenotypes were induced using temperature control, and three family selections were performed at 2, 6, and 18 months of age.

[0028] According to the evaluation method of F1 generation growth traits, their body weights were measured at 18 months of age, and three families with significant growth advantages were screened to form the F2 generation fast-growing matching system.

[0029] Example 2: Construction of a new triploid strain of flounder that is heat-resistant and fast-growing The high-temperature-resistant complementary line and the fast-growing complementary line of flounder were used as parent stocks, and the pseudo-male and female fish of the complementary lines were bred and matured at a ratio of 2:1.

[0030] Select broodstock with well-developed glands, artificially collect mature fast-growing female eggs and high-temperature resistant pseudo-male sperm, and select eggs with uniform size, transparency, smooth surface, and a single oil ball and semen with good vitality for artificial insemination.

[0031] Sperm and eggs were mixed in a volume ratio of 1:100, and activated by adding 15°C seawater to complete fertilization.

[0032] The cold shock method was used to induce triploidy in the complementary hybridization: the fertilized eggs were incubated in 15°C seawater for 5 minutes, then placed in 0-1°C seawater for 45 minutes, and then taken out and continued to be incubated in 15°C seawater for the conventional post-incubation period.

[0033] During the hatching and fry stage, the ploidy of the triploid induced group was detected by flow cytometry. The DNA content was measured by flow cytometry (Partec, Germany). Diploids were used as controls. The results showed that the DNA content of the induced flounder was 1.5 times that of the diploid, and the triploid rate was 100%. Figure 1 shown.

[0034] At one year old, 15 individuals were randomly selected from the triploid population and the unselected diploid control population. After fin ploidy was determined, their weight, total length, body length and other body size traits were measured. The results showed that triploids had a significant growth advantage, with weight and total length traits significantly higher than those of the unselected diploid control group (P<0.0001). Figure 2 shown.

[0035] At the same time, their heat tolerance was assessed using acute high-temperature experiments. At two months and one year of age, individuals were randomly selected from the triploid-induced group and the unselected diploid control group. Fins were removed to identify ploidy, and then fluorescently labeled. The water temperature was gradually increased using a heating device. Two acute high-temperature experiments were conducted: the lethal temperature of each fish was recorded, and the average upper temperature of the heat tolerance of the triploid-induced group and the diploid control group was calculated as the upper temperature of each group. The second experiment maintained the fish at a high temperature (31.5 ± 0.2°C), and the survival rate of each group was calculated after 24 hours.

[0036] like Figure 3 The results showed that the upper temperature tolerance limit of the induced triploid group at 2 months and 1 year old was 2.8℃ and 1.6℃ higher than that of the unselected diploid control group, respectively (P<0.0001), and the survival rate after 24 hours at high temperature (31.5 ± 0.2℃) was increased by more than 40%.

Claims

1. A method for preparing a new triploid strain of flounder that is resistant to high temperatures and grows fast, characterized in that: Using high temperature resistance and body weight as screening indicators respectively, high temperature resistant complementary lines and fast-growing complementary lines were constructed. Then, using the complementary lines as parents, mature sperm and eggs were collected and fertilized, and triploid induction was performed. Through high temperature resistance and growth trait tests, a new triploid variety with both high temperature resistance and fast growth traits was obtained.

2. The preparation method according to claim 1, characterized in that The parental matching lines used for the preparation of new triploid strains are constructed through two or more consecutive generations of gynogenetic induction, purification, and breeding.

3. The preparation method according to claim 1, characterized in that The new triploid strain is obtained by using a high-temperature-resistant complementary strain as the male parent and a fast-growing complementary strain as the female parent, and is fertilized with mature sperm and eggs and then induced by cold shock.

4. The preparation method according to claim 3, characterized in that The cold shock induction condition is to place the fertilized eggs in 0-1℃ seawater for cold shock treatment 5 minutes after fertilization to inhibit the release of the second polar body. The treatment time is 45 minutes. After the treatment is completed, the fertilized eggs are moved into constant temperature seawater for continued cultivation; the fertilization water temperature and the incubation water temperature are both 15℃.

5. The preparation method according to claim 1, characterized in that The screening and determination of high temperature resistance traits are carried out more than twice during the breeding cycle. Families with a high average upper limit of high temperature resistance and a high survival rate under constant high temperature are considered to have high temperature resistance traits; families that have high temperature resistance traits in each test are identified as high temperature resistant strains.

6. The preparation method according to claim 5, characterized in that Two acute high-temperature experiments were used to determine the heat resistance trait. Method 1: continuously increase the temperature, observe and record the lethal temperature of each fish, and calculate the average upper limit of heat resistance for each family / group; Method 2: determine a constant high temperature based on the half-lethal temperature of the unselected control group, and calculate the survival rate of each family after a period of constant high temperature.

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