Method for selecting grass carp parents with moderate immune level

Abstract

The invention relates to the technical field of grass carp resistance breeding, and particularly discloses a selection method of grass carp parents with moderate immune level, which is technically characterized by comprising the following steps: S1, building a parent breeding group, and detecting the expression level of parent background immune protein; s2, dividing the parents into three groups with certain selection intensity according to the difference of the background immune protein expression levels of the parents; s3, breeding in the three parent groups to generate filial generations, and detecting the resistance of each filial generation to GCRV infection; and S4, determining a parent population capable of obtaining high-resistance offspring. In the Yangtze River water system grass carp stock seed parents, based on background immune protein expression level difference grouping, the resistance of each group of offspring to GCRV infection is detected, and a parent population capable of obtaining high-resistance offspring is determined, so that a reliable parent population library is established for grass carp disease-resistant stress-resistant breeding; the method is a key technical basis for subsequent breeding of new varieties of high-resistance grass carp.

Description

Technical Field

[0001] The invention relates to the technical field of grass carp resistance breeding, and in particular to a method for selecting grass carp parents with a moderate immunity level. Background Art

[0002] Grass carp is a major freshwater fish species farmed in my country, with a total of 5.9 million tons of farmed grass carp in 2022. As the aquaculture model changes from extensive to intensive, the density of aquatic animal farming has increased significantly compared with the past, and the deterioration of living environment conditions and the mixing of populations have caused fish disease outbreaks. When water quality deteriorates, water temperature changes greatly, and fish resistance is low, viral grass carp hemorrhagic disease and bacterial enteritis, red skin disease, gill rot and other infectious diseases are prone to outbreaks. Among them, grass carp hemorrhagic disease caused by infection with grass carp reovirus (GCRV) is the most serious. The disease is prevalent in a wide area, has a long epidemic season, and has high morbidity and mortality rates, causing an economic loss of 15 billion yuan to my country's grass carp industry each year.

[0003] In recent years, there have been new research progress in this field. Studies have found that there are obvious differences in antibody titers among rainbow trout individuals, which are subject to complex genetic control and can be used as a standard for individual differentiation and breeding; additive genetic variance analysis showed that it is feasible to breed rainbow trout for resistance to viral hemorrhagic septicemia; with the help of ELISA and monoclonal antibody technology to screen fish growth hormone levels, transgenic carp with rapid growth can be screened out, greatly shortening the breeding cycle; rainbow trout with different levels of serum lysozyme activity were selected, and infection experiments with Aeromonas Salmonella and Vibrio anguillarum were carried out before and after vaccination. There were significant differences in the mortality rates of the two strains in the two experiments, indicating that serum lysozyme activity may have the potential to be used as a selection standard for fish disease resistance.

[0004] Disease resistance and stress resistance are micro-effect polygenic quantitative traits. It is difficult to obtain significant target trait phenotypes through traditional hybridization. Molecular marker-assisted breeding is difficult to quickly obtain target trait populations due to the large number of trait-determining genes. The difficulty of genomic selection lies in the lack of reference populations. Therefore, the present invention aims to provide a method for selecting grass carp parents with moderate immunity levels to solve the above problems. Summary of the invention

[0005] The purpose of the present invention is to solve the problem of how to select and cultivate excellent disease-resistant grass carp new varieties based on individual differences in the background immunity level of grass carp populations, and provide a method for selecting grass carp parents with moderate immunity levels. The present invention divides the original grass carp parents in the Yangtze River system into groups based on the differences in the expression levels of background immune proteins, detects the resistance of each group of offspring to GCRV infection, and determines the parent group from which highly resistant offspring can be obtained, thereby laying a reliable parent group library for the disease-resistant and stress-resistant breeding of grass carp, and is the key technical foundation for the subsequent breeding of new highly resistant grass carp varieties.

[0006] The above-mentioned purpose of the present invention is achieved like this:

[0007] The scheme of the present invention provides a method for selecting grass carp parents with moderate immunity level, comprising the following steps:

[0008] S1. Build a parent breeding population and detect the expression level of parent background immune proteins;

[0009] S2, according to the differences in the expression levels of the background immune proteins of the parents, the parents were divided into three groups with a certain selection intensity;

[0010] S3, breeding offspring in the three parent groups respectively, and testing the resistance of each group of offspring to GCRV infection;

[0011] S4. Determine the parent population that can produce highly resistant progeny.

[0012] Furthermore, the specific steps of constructing a parent breeding population in step S1 and detecting the expression level of the parent background immune protein are as follows:

[0013] (1) Selecting healthy and sexually mature grass carp of 4-5 years old as parents;

[0014] (2) Collect serum from the parents and detect the background expression level of the parents.

[0015] Furthermore, in step S2, the specific method of dividing the parents into three groups with a certain selection intensity according to the difference in the expression level of the background immune protein of the parents is:

[0016] According to the comprehensive expression level of the background immune protein of the parents, the parents in the same batch were divided into three subgroups: high, medium and low according to the selection intensity of the top 20%, middle 60% and bottom 20% of the background expression level of the parents;

[0017] Furthermore, the specific steps of breeding and producing offspring in the three parent groups in step S3, and detecting the resistance of each group of offspring to GCRV infection are:

[0018] (1) After intensive cultivation of the high, medium and low parent groups, artificial reproduction is performed within each group to produce offspring;

[0019] (2) The offspring of each group were cultured under the same conditions, and the autumn leaf individuals of each subgroup were artificially infected with GCRV;

[0020] (3) Select offspring with similar physiques from each group and place them in a recirculating aquaculture system for 7 days to relieve the stress of the fry. Then gradually increase the temperature until the water temperature stabilizes at 30±1℃ and the experimental grass carp eats normally before starting the GCRV artificial infection experiment.

[0021] (4) Set up 4 replicates for each group of offspring, dilute the venom 1000 times, soak for 15 min, and then add reserve water at a temperature of 30±1℃ to maintain the normal circulation water level of the system. The water temperature is stable at 30±1℃ during the entire attack period;

[0022] (5) Observe the feeding and health status of the offspring in each group, and record the mortality and symptoms;

[0023] (6) The challenge experiment was terminated when no dead fish appeared in each group of offspring for 3 days, and the survival rate of the offspring in each group was calculated.

[0024] Furthermore, the specific method for determining the parent population from which highly resistant progeny can be obtained in step S4 is:

[0025] The differences in survival rates of the progeny in each group in the GCRV artificial infection experiment were combined with the background expression levels of the parents in each group to evaluate the resistance levels of the parents.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1. The grass carp parent population selected by the method of the present invention can be used as an excellent germplasm resource for breeding highly resistant grass carp, and the method of the present invention is expected to quickly screen highly resistant grass carp parents, which can greatly improve the accuracy and efficiency of grass carp disease-resistant breeding;

[0028] 2. The method of the invention can obtain a highly resistant grass carp parent group, and the offspring produced by the highly resistant grass carp parents bred by the method have excellent disease resistance and survival ability, which is convenient for effectively promoting the development of grass carp farming. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is a scatter plot of the comprehensive activity level of multi-factor proteins in the serum of parents from the Yangtze River and Xiangjiang River basins in the embodiment of the present invention;

[0030] Figure 2 is the survival rate of the two-phase challenge experiment in the embodiment of the present invention;

[0031] Figure 3 is the relationship between the parental background expression level and the survival rate of the first-stage challenge progeny in the embodiment of the present invention;

[0032] Figure 4 It is the relationship between the parental background expression level and the survival rate of the second-stage challenge offspring in the embodiment of the present invention. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0034] The implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0035] As shown in the drawings, a preferred embodiment of the present invention is provided.

[0036] Embodiment: The embodiment of the present invention provides a method for selecting grass carp parents with moderate immunity level, which method specifically comprises the following steps:

[0037] 1) Establish a parent breeding population and detect the expression level of parent background immune proteins, which specifically includes the following implementation steps:

[0038] ① In March 2021, we went to the original breeding farm of the four major carps in the Yangtze River in Laohe, Shishou, Hubei, and the original breeding farm of aquatic products in Hunan Province to collect breeding materials, and collected serum and fin samples from parents;

[0039] ② In March 2021, 173 grass carp parents from the Yangtze River Basin and 92 grass carp parents from the Xiangjiang River Basin were collected;

[0040] ③ The parent population of the original Yangtze River grass carp used was raised in Tianjiahu Fishery in Huarong County, Yueyang City, Hunan Province, and the parent population of the original Xiangjiang River grass carp used was raised in the Water Science Institute of Xiangyin City, Hunan Province and the Aquatic Seed Farm of Zixing City;

[0041] ④ In 2022, the protein activity levels of the four immune factors were detected by ELISA method. It was found that the scatter plots of C3, Bf, LZM, MBL and the comprehensive expression levels of the four factors in 173 Yangtze River grass carp parents and 92 Xiangjiang River grass carp parents were roughly the same, all showing a distribution with a wide middle and pointed ends, which conforms to the normal distribution characteristics, indicating that there are subgroups with high or low expression levels of the four-factor molecules in the population. Figure 1 As shown, it is a scatter plot of the comprehensive activity levels of multi-factor proteins in the serum of parents in the Yangtze River and Xiangjiang River basins.

[0042] 2) According to the differences in the expression levels of the background immune proteins of the parents, the parents were divided into three groups with a certain selection intensity. The specific method is as follows:

[0043] According to the comprehensive expression levels of four immune molecules in the serum of 173 Yangtze grass carp parents in 2022, the 173 Yangtze River parents were divided into Yangtze River Group A (37 fish), Yangtze River Group B (99 fish), and Yangtze River Group C (37 fish) according to high level 20%, medium level 60%, and low level 20%.

[0044] 3) Producing offspring in the three parent groups respectively, and testing the resistance of each group of offspring to GCRV infection; the specific implementation steps are:

[0045] ① Carry out inter-subgroup combination for the parents of each Yangtze River A, B, and C subgroups, and artificially breed and produce offspring groups AA, BB, and CC

[0046] ② Based on the survival and sexual maturity of each group of parents, there will be 60,000 fry in each of the three subpopulations of the Yangtze River A, B and C in 2022.

[0047] 4) Determine the parent population that can produce highly resistant progeny. The specific implementation steps are:

[0048] ① After the offspring of each subgroup were raised to autumn, two GCRV challenge experiments were carried out and the mortality rate was counted.

[0049] ②In the first phase of the attack, three experimental groups (Yangtze River A, B, and C groups) were set up, with four replicates in each group. Forty experimental grass carps were placed in each aquaculture system and temporarily kept in the recirculating water aquaculture system for 7 days to relieve the stress of the fry. The temperature was gradually increased until the water temperature stabilized at 30±1℃ and the experimental grass carps fed normally. The experiment was started. At the beginning of the experiment, the water level was lowered to 5cm, 18mL of venom was added to each system (the venom was diluted 1000 times according to the system volume), and the system was soaked for 15min. Then, reserve water with a water temperature of 30±1℃ was added to maintain the normal circulating water level of the system. The feeding and health status of the experimental fish were observed, the death and symptoms were recorded, and the dying experimental fish were sampled.

[0050] ③ In the second phase of the challenge, three replicates were set up for each group. 40 experimental grass carps were placed in each culture system. The venom was diluted 500 times and soaked for 10 minutes. Other operations were the same as the first phase of the challenge.

[0051] ④ The mortality of the three groups A, B, and C of the Yangtze River in the two phases of infection were similar, and the survival rate of group B was significantly higher than that of the other two groups; in the first phase of GCRV artificial infection experiment, the survival rate of group B was 55.6%, the survival rate of group A was 35%, and the survival rate of group C was 45%; in the second phase of GCRV artificial infection experiment, the survival rate of group B of the Yangtze River was 51.7%, the survival rate of group A was 27.5%, and the survival rate of group C was 26.7%. Overall, the survival rate of the offspring of group B of the Yangtze River was significantly higher than that of the offspring of groups A and C of the Yangtze River, indicating that group B of the Yangtze River has higher resistance to GCRV.

[0052] like Figure 2 As shown, it is the survival rate of the two-phase challenge experiment. (Note: a. The survival rate results of the first phase of the offspring challenge experiment in 2022. b. The survival rate results of the second phase of the challenge experiment in 2022)

[0053] ⑤ From the comprehensive expression levels of the four factors in the serum of the three groups of Yangtze River A, B, and C combined with the survival rate of the offspring in the two phases of virus attack, it is shown that the higher or lower the background expression level of the parent protein, the higher the mortality rate of the offspring. When the background expression level of the grass carp parent protein is moderate, the higher the survival rate of the offspring (such as Figure 3 As shown, it is the relationship between the parental background expression level and the survival rate of the first-stage challenge progeny; Figure 4 As shown, it is the relationship between the parental background expression level and the survival rate of the second-stage challenged offspring).

[0054] In summary, it is difficult to obtain significant target trait phenotypes through hybridization in traditional technologies, and it is difficult to quickly obtain target trait populations through molecular marker-assisted breeding due to the large number of trait-determining genes. The difficulty of genome selection lies in the lack of reference populations. The method of the present invention overcomes the above-mentioned prior art problems, and by grouping the original parents of grass carp in the Yangtze River system based on differences in background immune protein expression levels, and detecting the resistance of each group of offspring to GCRV infection, the parent population from which highly resistant offspring can be obtained is determined, which lays a reliable parent population library for disease-resistant and stress-resistant breeding of grass carp, and provides a key technical foundation for the subsequent selection and breeding of new highly resistant grass carp varieties.

[0055] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for selecting grass carp parents with moderate immunity level, It is characterized in that The following steps are involved: S1. Build a parent breeding population and detect the expression level of parent background immune proteins; S2, according to the differences in the expression levels of the background immune proteins of the parents, the parents were divided into three groups with a certain selection intensity; S3, breeding offspring in the three parent groups respectively, and testing the resistance of each group of offspring to GCRV infection; S4. Determine the parent population that can produce highly resistant progeny.

2. The method for selecting grass carp parents with moderate immunity level according to claim 1, It is characterized in that The specific steps of constructing a parent breeding population in step S1 and detecting the expression level of the parent background immune protein are as follows: (1) Select healthy and sexually mature grass carp of 4-5 years old as parents; (2) Collect serum from the parents and detect the background expression level of the parents.

3. The method for selecting grass carp parents with moderate immunity level according to claim 1, It is characterized in that In step S2, the specific method of dividing the parents into three groups with a certain selection intensity according to the differences in the expression levels of the background immune proteins of the parents is: According to the comprehensive expression level of the parental background immune protein, the parents in the same batch were divided into three subgroups: high, medium, and low according to the selection intensity of the top 20%, middle 60%, and bottom 20% of the parental background expression level; 4. The method for selecting grass carp parents with moderate immunity level according to claim 1, It is characterized in that The specific steps of breeding and producing offspring in the three parent groups in step S3, and detecting the resistance of each group of offspring to GCRV infection are as follows: (1) After intensive cultivation of the three groups of high, medium and low parents, artificial reproduction is carried out within each group to produce offspring; (2) The offspring of each group were cultured under the same conditions, and the autumn leaflets of each subgroup were artificially infected with GCRV; (3) Select offspring with similar physiques from each group and place them in a recirculating aquaculture system for 7 days to relieve the stress of the fry. Then gradually increase the temperature until the water temperature stabilizes at 30±1℃ and the experimental grass carp eat normally before starting the GCRV artificial infection experiment. (4) Set up 4 replicates for each group of offspring, dilute the venom 1000 times, soak for 15 minutes, and then add reserve water at a temperature of 30±1℃ to maintain the normal circulation water level of the system. The water temperature is stable at 30±1℃ during the entire attack period; (5) Observe the feeding and health status of the offspring in each group, and record the mortality and symptoms; (6) The challenge experiment was terminated when no dead fish appeared in the offspring of each group for 3 days, and the survival rate of the offspring of each group was calculated.

5. The method for selecting grass carp parents with moderate immunity level according to claim 1, It is characterized in that The specific method for determining the parent population from which highly resistant progeny can be obtained in step S4 is: The differences in survival rates of the progeny in each group in the GCRV artificial infection experiment were combined with the background expression levels of the parents in each group to evaluate the resistance levels of the parents.

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