Method for breeding lateolabrax japonicus by utilizing ARTP mutagenesis technology

Abstract

The invention discloses a method for breeding lateolabrax japonicus by utilizing an ARTP mutagenesis technology, which comprises the following steps: taking fertilized eggs of lateolabrax japonicus, carrying out ARTP mutagenesis treatment on the fertilized eggs, then hatching to obtain fish fries, and carrying out fry culture to obtain the lateolabrax japonicus. The ARTP mutagenesis treatment conditions are as follows: the radio frequency power is 120 to 220 W; the starting time is 30 to 90 minutes; the treatment time is 5 to 15 minutes. A lateolabrax japonicus fertilized egg is mutated by utilizing an ARTP mutagenesis technology, the mutated lateolabrax japonicus is screened, and the lateolabrax japonicus mutant with excellent characters is screened out.

Description

Technical Field

[0001] The present invention relates to the technical field of aquaculture, in particular to a method for selecting and breeding Japanese seabass using the atmospheric room temperature plasma (ARTP) mutagenesis technology. Background Art

[0002] Japanese seabass (Lateolabrax maculatus) is the third largest mariculture variety in China. Japanese seabass has become an important pillar of the aquaculture industry due to its delicious meat and strong market demand. However, with the rapid development of the aquaculture industry, the problem of lack of improved varieties has become increasingly prominent, which has become the main bottleneck restricting the healthy development of the Japanese seabass aquaculture industry. Germplasm resources are the basis of breeding work, which is directly related to the potential and effect of variety improvement. At present, the genetic diversity of Japanese seabass germplasm resources in China is relatively low, and the genetic differences are small, resulting in a shortage of germplasm resources, which undoubtedly increases the difficulty of genetic breeding of Japanese seabass. Therefore, innovating germplasm resources and improving genetic diversity have become the urgent needs of Japanese seabass genetic breeding work.

[0003] The atmospheric room temperature plasma (ARTP) mutagenesis breeding technology has emerged as the times require. With its advantages such as high mutagenesis mutation rate and rich phenotypic diversity, it has become an important means for innovating biological germplasm resources. The ARTP technology can generate plasma under atmospheric pressure and room temperature conditions, directly act on the DNA of organisms, and induce genetic variation, providing the possibility for quickly and effectively improving the Japanese seabass variety. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for selecting and breeding Japanese seabass using the ARTP mutagenesis technology, which uses the ARTP mutagenesis technology to mutagenize Japanese seabass fertilized eggs, and screens the mutagenized Japanese seabass to screen out Japanese seabass mutants with excellent traits.

[0005] Specifically, the method for selecting and breeding Japanese seabass using the ARTP mutagenesis technology is as follows: take Japanese seabass fertilized eggs, perform ARTP mutagenesis treatment on them, then incubate to obtain fry, and perform seedling cultivation, thus obtaining the desired result.

[0006] The present invention selects induction conditions according to the evaluation of fertilization rate, hatching rate, survival rate and mutation rate, and comprehensively considers factors such as hatching rate and mutation rate. The ARTP mutagenesis treatment conditions are as follows: the radio frequency power is 120 - 220 W; the starting time is 30 - 90 min; the treatment time is 5 - 15 min.

[0007] Furthermore, the optimal induction conditions are: the radio frequency power is 120 W; the starting time is 30 min; the treatment time is 10 min.

[0008] Furthermore, in the ARTP mutagenesis treatment, the distance between the plasma source and the sample stage is fixed at 3 mm; the purity of helium gas is ≥99.999%.

[0009] During the seedling cultivation process of the present invention, by screening the growth rate and other aspects of the mutated Japanese seabass after mutagenesis, mutated individuals of Japanese seabass with excellent traits are selected. Preferably, mutated individuals of Japanese seabass with a weight growth rate more than 1.316 times that of wild-type individuals are selected.

[0010] Furthermore, the present invention continues to cultivate the selected mutated Japanese seabass individuals until sexual maturity to obtain mature males and females of the F0 generation, constructs a full-sib family of the F1 generation, and selects the F1 generation individuals with excellent growth traits and the largest breeding value as parents for intensive cultivation.

[0011] Take the selected F1 generation individuals with excellent growth traits and the largest breeding value, expand the reproduction under the condition of avoiding inbreeding, and conduct four consecutive generations of selection. At the F4 generation, individuals with relatively distant genetic relationships and relatively independent are divided into two large groups, namely group A and group B. The offspring group obtained by the orthogonal pairing of group A and group B is the new strain of Japanese seabass.

[0012] In the present invention, during the selection of the F1-F4 generations, from the cultivation of fry to commercial fish, when the total length of the fry reaches 10 cm, according to the growth and survival rate of each family, individuals with excellent growth rates are selected and continue to be cultured for 6 months. Then, according to the growth and survival rate of each family, the breeding value of the individuals is evaluated, and individuals with excellent growth traits and the largest breeding value are selected.

[0013] Cultivation conditions of the present invention: indoor cement pond, cultivation density 500 tails / m 3 , cultivation water temperature 20°C - 22°C, pH 8.0 - 8.2, light intensity 500 lx - 800 lx, dissolved oxygen greater than 5 mg / L.

[0014] Advantages of the present invention:

[0015] The present invention uses the ARTP mutagenesis technology to study the mutagenesis effects of different mutagenesis parameters on Japanese seabass fertilized eggs, screens and optimizes the mutagenesis conditions, establishes a set of efficient ARTP mutagenesis technology systems for Japanese seabass, and obtains a high mutation efficiency of Japanese seabass.

[0016] The mutated Japanese seabass screened by the present invention has a fast growth rate; through multiple generations of breeding, individuals with heritable traits can be screened and further used for genetic breeding.

[0017] The present invention screens out individuals with excellent traits from the mutant population for the cultivation of reserve broodstock, breeds offspring, and further applies the obtained offspring population by family or population selection. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It shows the embryonic morphological changes of Japanese seabass fertilized eggs after ARTP irradiation.

[0019] Figure 2 GO analysis of genes corresponding to specific SNPs of Lateolabrax maculatus mutants.

[0020] Figure 3 GO analysis of genes corresponding to specific InDels of Lateolabrax maculatus mutants.

[0021] Figure 4 Sampling analysis of each growth index of the selected breeding group and the control group of Lateolabrax maculatus;

[0022] Note: A. PCA analysis of each growth data of the selected breeding group and the control group; B - D. Comparison of body weight, body length and body height between the selected breeding group and the control group.

[0023] Figure 5 Analysis of body weight, body length and body height of the selected breeding group and the control group of Lateolabrax maculatus at different times. Specific implementation manners

[0024] To make the objectives, technical solutions and advantages of the embodiments of this application clearer, the following will, with reference to the accompanying drawings in the embodiments of this application, describe the technical solutions in the embodiments of this application completely to fully understand the objectives, effects and application prospects of the present invention. The following embodiments are only used to clarify the present invention and are not used to limit the scope of application of the present invention. However, without departing from the spirit and essence of the present invention, any modification or replacement made to the methods, steps or culture conditions of the present invention belongs to the scope of the present invention.

[0025] 1. ARTP mutagenesis technology for Lateolabrax maculatus fertilized eggs

[0026] (1) Acquisition and quality control of fertilized eggs: Select healthy Lateolabrax maculatus parents with mature gonads (female fish body weight ≥ 3 kg, male fish ≥ 1.5 kg). Gently squeeze the abdomens of female and male Lateolabrax maculatus, and squeeze the mature oocytes and sperm into a clean beaker respectively for artificial fertilization. After artificial fertilization, place the fertilized eggs in an incubation barrel for incubation. Water quality conditions are: water temperature 22 ± 0.5 °C, salinity 30‰, pH 8.2, nitrite 0, ammonia nitrogen 0, DO ≥ 6 mg / L. Lighting conditions are: photoperiod 12 h light / 12 h dark, light intensity 600 - 800 lx.

[0027] (2) ARTP treatment:

[0028] 1) Equipment debugging: Use the ARTP mutagenesis system (ARTP - B, Wuxi Yuanqing Tianmu Biotechnology Co., Ltd.) to carry out ATRP mutagenesis on Lateolabrax maculatus fertilized eggs. The ARTP mutagenesis system is debugged as follows: the distance between the plasma source and the sample stage is fixed at 3 mm; the purity of helium gas ≥ 99.999%.

[0029] 2) ARTP mutagenesis treatment: Set different mutagenesis parameters (radio frequency power, starting time, treatment time) to treat fertilized eggs. Radio frequency power: 120W, 180W, and 220W; starting time: 30 min, 60 min, and 90 min after fertilization; treatment time: ARTP irradiation time 5 min, 10 min, and 30 min. This experiment was a three-factor and three-level experiment. The L9(33) orthogonal array was selected and 9 experiments were carried out ( Figure 1 ). Use a 200-mesh sieve to collect high-quality fertilized egg samples suspended in the upper and middle layers of the water body with intact egg membranes and uniform egg diameters (1.1 - 1.3 mm). Disperse 400 - 600 fertilized eggs evenly in the sample slot for mutagenesis treatment. During the treatment, maintain the temperature of the treatment area at 22 °C through a circulating water cooling system.

[0030] 3) Incubation after mutagenesis: Transfer to an incubation barrel, slightly aerate (dissolved oxygen ≥ 6 mg / L), water temperature 22 ± 1.0 °C, salinity 30‰. Incubate according to the conventional incubation method of Japanese seabass, and evaluate the hatching rate, mutation rate, and survival rate 3 days after hatching (Table 1). Considering the hatching rate, mutation rate, and survival rate comprehensively, the best induction conditions were selected: S1Q1C2, that is, radio frequency power 120W; starting time 30 min; treatment time 10 min.

[0031] Table 1 Experimental indexes obtained from each experiment and the best levels selected for each factor

[0032]

[0033] 2. Construction of Japanese seabass mutant population

[0034] Using 30 min as the starting time, 120W as the radio frequency power, and 10 min as the treatment time as the mutagenesis conditions, continuously treat multiple batches of Japanese seabass fertilized eggs, and then incubate them (the basic incubation conditions are the same as those after mutagenesis incubation above). Conduct multi-level screening on the mutants, and eliminate individuals with abnormal hatching times (compared with the control group, the hatching time exceeds 6 hours); 3 days after hatching, eliminate deformed individuals with bent body shapes. Then use the conventional seedling raising method to cultivate the fry of the control group and the treatment group. Cultivation conditions: indoor cement pond, cultivation density 500 tails / m 3For cultivation, the water temperature is 20°C - 22°C, pH is 8.0 - 8.2, light intensity is 500 lx - 800 lx, and dissolved oxygen is greater than 5 mg / L. Seedling feeding: After the yolk sac disappears, rotifers are fed for 10 consecutive days; from the 11th day, nauplii of Artemia are added; from the 28th day, small copepods are added; after 40 days, larger copepods are fed; from the 50th day, compound feed is fed. After 60 days of cultivation, the total length of the fry in the experimental group and the control group is about 3.0 cm, and the weight is about 0.25 - 0.35 g. Through cultivation, a total of 3128 mutants with a total length of about 3.0 cm are obtained. Corresponding to the number of mutants, the control group retains the same number (randomly retains 3128) of Japanese sea bass fry for the next-stage comparative test.

[0035] Transfer the mutants and the Japanese sea bass fry in the control group to the net cages for cultivation. Hang 4 net cages (length × width × height = 2 m × 2 m × 1.2 m) in the same pond. The control group and the experimental group each put 2 net cages, with the same density, and 1564 fry are placed in each net cage. On the day after stocking, commercial compound feed is fed, and the feeding weight of each net cage is the same. On the 60th, 120th, 180th, and 240th days after hatching, the weights and body lengths of the Japanese sea bass in the control group and the experimental group are measured respectively. After 240 days of comparative test, a total of 1766 mutants survive, and it is screened that the weights of 752 mutants are more than 31.6% faster than the average value of the control group, and among them, the weights of 165 mutants are significantly higher than the largest individual in the control group.

[0036] Table 3 Comparison of the weights of mutants and the control group 240 days after hatching

[0037]

[0038] Using the method of whole-genome resequencing, nucleotide variation analysis is carried out on 9 control group samples and 13 mutant samples. Compared with the control group, the average number of specific SNP sites in mutants is 490881, and the average SNP mutation rate is 0.1438%. The average number of specific InDel sites is 213925, and the average InDel mutation rate is 0.2362%.

[0039] Table 4 Detection of SNPs and InDels in mutants

[0040]

[0041] 3. Cultivation of mutant reserve parents and selection of offspring

[0042] Transfer the 165 mutants to the offshore net cages and continue to cultivate until sexual maturity. After cultivation, a total of 90 mature males and 18 mature females of the F0 generation are obtained. Select 18 individuals with excellent growth traits from the 90 males and construct 18 full-sib families of the F1 generation through artificial insemination with the 18 females.

[0043] From fry cultivation to commercial fish harvest, two rounds of selection and elimination were carried out: The first round was when the fry were cultivated until they reached a total length of 10 cm. The growth and survival rates of each family line were compared, and 10 family lines with excellent comprehensive traits, totaling more than 2,000 individuals, were selected. After being marked with electronic chips, they were cultured in the same pond. The second round was after continuing to culture for 6 months. 300 tails were randomly sampled for measurement, the trait data of all individuals were recorded, the growth and survival rates of each family line were compared, the breeding values of individuals were evaluated, and 500 individuals with excellent comprehensive traits and the largest breeding values were selected as the parents for the next year for intensive cultivation.

[0044] For the 500 individuals selected and reserved in the previous year, their pedigrees were identified by microsatellite paternity testing technology (Patent ZL202111229080.4). Under the condition of avoiding inbreeding, 100 F2 generation family lines were paired and constructed. The same method was used for continuous selection for 4 generations. At the F4 generation, individuals with relatively distant and independent genetic relationships were divided into two major groups, Group A and Group B. The filial generation group was obtained by orthogonal pairing of Group A and Group B, which was the new strain of Japanese seabass.

[0045] After 8 months of pond culture, the results of PCA analysis of growth data showed that the new strain of Japanese seabass in the selected breeding group was significantly separated from the control group of Japanese seabass, indicating a significant growth difference between the two. The average weight of the selected breeding group of Japanese seabass was 434.0 g, and the average body length was 29.2 cm; the average weight of the non-selected control group was 367.1 g, and the body length was 26.5 cm. Compared with the non-selected group, the weight, body length, and body height of the selected breeding group of Japanese seabass increased by 18.2%, 10.4%, and 10.6% respectively.

Claims

1. A method for breeding Lateolabrax japonicus using ARTP mutagenesis technology, characterized in that: The fertilized eggs of sea bass are taken, subjected to ARTP mutagenesis treatment, and then hatched to obtain fry, and the fry are cultivated to obtain the obtained fish; the ARTP mutagenesis treatment conditions are: radio frequency power of 120-220W; start time of 30-90min; treatment time of 5-15min.

2. The method for breeding Lateolabrax using ARTP mutagenesis technology according to claim 1, wherein: The ARTP mutagenesis treatment conditions are: radio frequency power 120W; start time 30min; treatment time 10min.

3. The method for breeding Lateolabrax using ARTP mutagenesis technology according to claim 1 or 2, characterized in that: In the ARTP mutagenesis treatment, the distance between the plasma source and the sample stage is fixed at 3 mm; the purity of the helium gas is ≥ 99.999%.

4. The method for breeding Lateolabrax using ARTP mutagenesis technology according to claim 1, wherein: During the seedling cultivation process, mutant individuals of Lateolabrax japonicus whose weight growth rate was 1.316 times that of wild-type individuals were selected.

5. The method for breeding Lateolabrax using ARTP mutagenesis technology according to claim 1 or 4, characterized in that: The selected mutant individuals of striped seabass are cultivated until they reach sexual maturity to obtain mature males and females of the F0 generation, to construct F1 generation full-sib families, and to select F1 generation individuals with excellent comprehensive traits and the greatest breeding value as parents for intensive cultivation.

6. The method for breeding Lateolabrax using ARTP mutagenesis technology according to claim 5, characterized in that: Take the selected F1 generation individuals with excellent comprehensive traits and the largest breeding value, expand the reproduction under the condition of avoiding inbreeding, and carry out breeding for four consecutive generations. In the F4 generation, the individuals with distant genetic relationship and relatively independent are divided into two major groups, namely group A and group B. The offspring group obtained by orthogonal pairing of group A and group B is the new strain of striped seabass.

7. The method for breeding Lateolabrax using ARTP mutagenesis technology according to claim 6, wherein: In the breeding of F1-F4 generations, from the process of fry cultivation to commercial fish, when the fry are cultivated to a total length of 10 cm, individuals with excellent comprehensive traits are selected based on the growth and survival rate of each family. After continuing to cultivate for 6 months, the breeding value of the individuals is evaluated based on the growth and survival rate of each family, and individuals with excellent comprehensive traits and the largest breeding value are selected.

8. The method for breeding Lateolabrax japonicus utilizing ARTP mutagenesis technology according to claim 1, 6 or 7, characterized in that, Conditions: Indoor cement pool, breeding density 500 fish / m 3 The cultivation water temperature is 20℃~22℃, pH 8.0~8.2, light intensity is 500lx~800lx, and dissolved oxygen is greater than 5mg / L.

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