Breeding method of excellent urechis unicinctus strain with ammonia nitrogen tolerance

Abstract

The invention discloses a breeding method of an excellent urechis unicinctus strain with ammoniacal nitrogen tolerance. The breeding method comprises the following steps: (1) establishing and breeding a family; (2) calculating a semi-lethal concentration value by ammonia nitrogen concentration gradient stress; (3) constructing a semi-lethal concentration stress environment to count survival time of individuals of different families; (4) establishing a genetic evaluation model to estimate ammonia nitrogen tolerance and a body weight breeding value; (5) evaluating a comprehensive breeding value and screening an F1-generation excellent family as a parent for reserving seeds; and (6) F2 generation family construction and subculture selection. According to the method, a wild group of the urechis unicinctus is used as a basic group, and an ammoniacal nitrogen stress environment is constructed for successive generation breeding, so that the bred new strain has the characteristics of high ammoniacal nitrogen stress resistance, high growth speed and high survival rate in industrial aquaculture. The method is easy to operate and high in practicability, and the improved urechis unicinctus variety with excellent characters can be bred through multi-generation breeding.

Description

Technical Field

[0001] The invention belongs to the field of aquatic genetic breeding, and in particular relates to a method for breeding a new strain of Echinops monocyclicus that is tolerant to ammonia nitrogen. Background Art

[0002] Urechis unicinctus, commonly known as "sea intestines", is a common benthic organism in the intertidal and subtidal zones along the Yellow Sea and Bohai Sea in China. It has delicious meat, rich in protein, amino acids and trace elements, and has high nutritional and health value. In addition, various bioactive substances such as peptides and fatty acids extracted from the body wall and internal organs of Urechis unicinctus have the effects of enhancing the body's immunity, anti-tumor and thrombus dissolution, and can be used as raw materials for extracting marine medicinal active substances and manufacturing health products. Urechis unicinctus is a filter-feeding benthic animal that feeds on suspended particulate organic matter in the water layer. On the other hand, the current breeding model of Urechis unicinctus is mainly based on intensive breeding, which is very likely to cause excess and enrichment of nitrogen-containing organic matter in the breeding environment, thereby increasing the concentration of ammonia nitrogen, exceeding the tolerance limit of Urechis unicinctus and ultimately leading to a decrease in its production and economic losses.

[0003] In aquaculture, ammonia nitrogen refers to the ammonia nitrogen in inorganic nitrogen, which is one of the toxic factors in aquaculture water. Ammonia nitrogen consists of ionized ammonia (NH 4 + ) and non-ionic ammonia (NH 3 ) are composed of two substances. Under certain conditions, the two substances can be transformed into each other in water: The water temperature, dissolved oxygen, pH and salinity in the aquaculture environment will affect the concentration of ionized ammonia and non-ionized ammonia. Among them, non-ionized ammonia is very toxic. When the concentration of non-ionized ammonia in the water exceeds a certain range, it will cause aquatic animals to be unable to excrete ammonia normally, causing metabolic disorders and blood ammonia poisoning. In addition, non-ionized ammonia is uncharged and has an extremely small radius. It can fuse with the phospholipid bilayer on the cell membrane and diffuse into the body, affecting or even destroying the hydrolysis reaction of the aquatic enzyme system and the functional stability of the membrane organs. While ionized ammonia is very low in toxicity to aquatic organisms, it can cause changes in the aquaculture environment to indirectly affect the survival of aquatic organisms, such as causing eutrophication of water bodies to produce algal blooms, consuming a large amount of dissolved oxygen and causing water hypoxia. During the breeding process, ammonia nitrogen mainly comes from bait residues and metabolic waste accumulation. With the increase of breeding density and feeding rate, a large amount of residual bait, feces and dead and decayed aquatic plants and animals are deposited at the bottom of the breeding pond. These nitrogen-containing organic matter can be decomposed by heterotrophic bacteria to produce a large amount of ammonia nitrogen, causing hypoxia, poisoning and death of aquatic organisms.

[0004] The present invention aims at the intensive breeding mode commonly existing in the monocyclic insect industry and the current lack of germplasm resources with tolerance to ammonia nitrogen. The present invention uses quantitative genetics and family breeding methods to screen and cultivate new excellent varieties of monocyclic insects with fast growth rate, high meat quality and strong tolerance to ammonia nitrogen. It has very important scientific significance and industrial value in terms of efficient utilization of breeding environment resources and improvement of breeding survival rate. Summary of the invention

[0005] The purpose of the present invention is to provide a method for selecting and breeding ammonia nitrogen-tolerant strains of Spinella monocyclica and producing seeds in view of the current situation that there are no excellent strains tolerant to ammonia nitrogen stress factors in the industrial breeding of Spinella monocyclica, so as to cultivate a new variety of Spinella monocyclica with excellent growth traits and strong ammonia nitrogen tolerance.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A method for breeding ammonia nitrogen tolerant strain of Echinops monocyclicus, characterized in that the method steps are:

[0008] 1) Family establishment and cultivation: wild Echinops spp. with mature gonads in natural waters were collected as breeding populations, and robust Echinops spp. individuals with a body weight 15-20% greater than the average body weight and full nephridial tubes were selected as breeding parents. The F1 generation full-sib family of Echinops spp. was established using the Nest mating design method according to the principle of one male mating with three females. After hatching in the incubator, the F1 generation full-sib family was subsequently cultivated in a specific area according to the factory seedling cultivation model;

[0009] 2) Calculation of half-lethal concentration value under ammonia nitrogen concentration gradient stress: An acute attack experiment was used to explore the 72h half-lethal concentration value of Monochamus monocygnus under ammonia nitrogen stress conditions. After the F1 generation seedlings were cultivated to 200 days of age, the ammonia nitrogen concentration gradient experimental groups were set up with 2g / L, 1g / L, 0.8g / L, 0.6g / L, 0.4g / L and a normal filtered seawater control group (0g / L). Each group was set up with three parallel groups and the attack lasted for 120h, during which the death of the insects was recorded every 8h. The linear regression method was used to calculate the half-lethal ammonia nitrogen concentration value of Monochamus monocygnus through the survival rate at 72h under each ammonia nitrogen concentration gradient stress condition;

[0010] 3) Construct a semi-lethal concentration stress environment to count the survival time of individuals from different families: Use the 72h semi-lethal ammonia nitrogen concentration value obtained from the investigation to prepare seawater of corresponding concentration for aquaculture stress. 60 insects were randomly selected from each family, weighed and placed in an independent incubator for stress culture. At the same time, 60 monocyclic spiny worms were randomly selected from the experimental system, weighed and placed in an incubator of the same specifications for culture using filtered seawater as a control group. The interior of the incubator was isolated with micro-isolation plates to ensure that each insect was in a separate area, and all incubators were placed in a culture pool for aquaculture stress. For the experimental group, 72h semi-lethal ammonia nitrogen concentration seawater was used for unified culture, and the stress continued until all individuals died, and the survival time of each individual was counted. For the control group, filtered seawater was used for unified culture, and observation was stopped after the experimental group completed the stress statistics;

[0011] 4) Establish a genetic evaluation model to estimate ammonia nitrogen tolerance and body weight breeding values: The heritability of ammonia nitrogen tolerance trait was determined by the survival time of the individuals of the monocypris punctatus under the stress condition of 72h semi-lethal ammonia nitrogen concentration, and the breeding value of individual ammonia nitrogen tolerance and the average breeding value of family ammonia nitrogen tolerance were estimated based on the heritability of the ammonia nitrogen tolerance trait. The heritability of the body weight trait was determined by the body weight of the individuals of the monocypris punctatus under the stress condition, and the breeding value of the individual body weight trait and the average breeding value of family weight were estimated based on the heritability of the body weight trait. The best linear unbiased estimation method (BLUP method) of animal models was used to estimate genetic parameters and breeding values.

[0012] Establishment of genetic parameter estimation model for ammonia nitrogen stress tolerance: i =μ+α i +е i

[0013] where y i is the ammonia nitrogen tolerance survival time of the i-th test individual, μ is the mean ammonia nitrogen tolerance survival time of all test individuals, α i is the additive genetic effect of ammonia nitrogen tolerance survival time of the i-th test individual, е i is the random residual of the ammonia nitrogen tolerance survival time of the i-th test individual.

[0014] The heritability calculation formula for the above animal model is as follows:

[0015]

[0016] in, is the additive genetic variance of ammonia nitrogen tolerance survival time, is the residual variance of ammonia nitrogen tolerance survival time. The calculation method of body weight heritability is the same as that of

[0017] At the same time, the genetic and phenotypic correlations among the traits (rx，y ) is calculated as follows:

[0018]

[0019] When calculating genetic correlation, σ xy is the additive genetic covariance between ammonia nitrogen tolerance and body weight traits, and is the additive genetic variance of the two traits. When calculating phenotypic correlation, σ xy is the phenotypic covariance between the ammonia nitrogen tolerance trait and the body weight trait, and is the phenotypic variance of the two traits;

[0020] 5) Evaluate the comprehensive breeding value and select the F1 generation excellent families as parents for seed retention: weight the average breeding value of ammonia nitrogen tolerance and the average breeding value of body weight traits of different families at a ratio of 1:1 to obtain the comprehensive breeding value of each family under ammonia nitrogen stress conditions. Use the comprehensive breeding value to sort and retain the top 5 families as candidate families for ammonia nitrogen tolerance breeding. The selected candidate families will be retained at a seed retention rate of 3-5% and continue to be raised to adulthood;

[0021] 6) Construction of F2 generation families and selection of successive generations: The candidate families for breeding with ammonia nitrogen tolerance of the F1 generation are cultured to the stage of gonadal maturity, and the strong and vigorous individuals of the monocyprids with weight in the top 15-20% are selected for mating and breeding with the same family source and different family sources to construct the F2 generation families, and the inbreeding coefficient is controlled within 0.1. After that, the F2 generation families are continuously selected for 4-5 generations with strong ammonia nitrogen tolerance and excellent weight traits as breeding indicators, and the healthy male and female monocyprids with distant kinship, large size and strong vitality are selected from the families finally obtained as excellent varieties for industrial breeding applications.

[0022] Preferably, the wild population of Echinops monocyclica in natural sea areas in step 1) is a wild population in Qinhuangdao sea area, which is artificially caught and transported to a breeding base at low temperature for selection. The process of establishing a family is to use in vitro fertilization technology to pair male and female individual parents, dissect the male and female parents and take out the kidney ducts in a dry and clean dissection tray, and after all parents are dissected, the kidney ducts are cut into pieces and diluted with seawater, and then sprinkled into an incubator for fertilization and hatching. During the family seedling cultivation process, the fertilized eggs in the incubator are first hatched and cultivated until they are in the early stage of metamorphosis benthic life, that is, cultivated to about 25 days old (water temperature range: 17-19℃); then, the embryos of the single-ringed worms in the development period before metamorphosis benthic life are removed from the incubator and transferred to a specific area of ​​the indoor breeding pond for cultivation until they reach the seedling size stage in industrial breeding, about 100 days old (water temperature range: 17-19℃); finally, the single-ringed worms of each family are filtered out from the indoor breeding pond and transferred to the outdoor pond for further cultivation until they are 200 days old (water temperature range: 18-22℃). In order to avoid mixing between families, independent boxes are used for breeding during the incubator incubation process, 100-mesh silk screens are used to isolate the breeding areas of each family during the indoor breeding pond breeding process, and 20-mesh silk screens are used to isolate the breeding areas of each family during the outdoor pond breeding process. In addition, each time the individual families of the monocyclic worms are transferred between different breeding environments, the breeding density of all families is controlled at the same level. The basic method of feeding twice a day, changing water once, and removing dirt from the bottom every three days is maintained throughout the cultivation process, and appropriate adjustments are made according to the specific situation during the process. The conditions for breeding and filtering seawater are: the dissolved oxygen content is greater than 5mg / L, and the salinity is 31-32ppt. The feeding conditions are: feeding a mixture of spirulina and marine red yeast (100 million / ml) for the first 100 days of age, and then feeding a spirulina powder solution filtered through a 300-mesh silk sieve, and the standard is that the culture water becomes clear 3-4 hours after feeding.

[0023] Preferably, the individuals of the monocyclic thorn worms in the screening process of the half-lethal concentration of ammonia nitrogen stress for 72 hours in step 2) are randomly selected in the entire cultivation system. The ammonia nitrogen stress experimental seawater is obtained by dissolving ammonia chloride in aquaculture filtered seawater after calculating and weighing according to a certain experimental concentration. Each ammonia nitrogen concentration stress experimental group and control group are set up with three parallels, and at least 10 monocyclic thorn worms participate in the stress process in each parallel. The ammonia nitrogen concentration gradient of the experimental group can be set accordingly according to the tolerance of different aquatic economic species.

[0024] Preferably, in step 3), the 72h semi-lethal ammonia nitrogen stress environment of the whole family is constructed, and the ammonia nitrogen stress experimental group and the control group are placed in a separate culture pool for treatment to ensure the consistency of experimental conditions such as temperature. During the period, the survival of the monocyclic thorns is observed every 4 hours and the dead individuals are fished out in time. A small amount of feeding and a water change operation are performed once a day. When feeding, specific adjustments are made according to the number of surviving insects and the vitality state. When changing water, the experimental group uses 72h ammonia nitrogen semi-lethal concentration seawater for replacement, and the control group chooses natural filtered seawater for replacement. The amount of water change is half of the volume of seawater in the culture box.

[0025] Preferably, in step 4), the genetic parameter estimation of the ammonia nitrogen stress tolerance trait and the body weight trait is achieved by using ASReml-R 4.0 software.

[0026] Preferably, in step 5), the F1 generation parents are retained for seed by weighting the average breeding value of the ammonia nitrogen tolerance trait of each family calculated under ammonia nitrogen stress conditions and the average breeding value of the family for the body weight trait in a ratio of 1:1 to obtain a comprehensive breeding value and using it for sorting, retaining the top 5 families as candidate families for ammonia nitrogen tolerance breeding, and retaining the selected families at a seed retention rate of 3-5%.

[0027] Preferably, the family construction and breeding methods in the F2 generation family and the successive selection in step 6) are consistent with the operation methods in the F1 generation family construction and subsequent breeding. The parents in the F2 generation family construction are F1 generation seed individuals, which are used for pairing and breeding of the same family source and different family sources, and the inbreeding coefficient is controlled within 0.1. In the successive selection, the continuous selection of the successive families is carried out with strong ammonia nitrogen tolerance and excellent body weight traits as breeding indicators.

[0028] In summary, the present invention selects a wild population of Echinops monocyclica as a basic breeding population, and uses an ammonia nitrogen stress environment to stress the population, thereby screening out families with large genetic improvement space and breeding potential. The screened families are kept and propagated, and after multiple generations of breeding, new strains of Echinops monocyclica with excellent growth traits and strong ammonia nitrogen tolerance can be cultivated. The breeding method is simple and practical, highly operable, and has high industrial application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Survival curves of Echinops monocylindrica under different ammonia nitrogen concentration gradient stress conditions

[0030] Figure 2 Survival curve of Echinops monocylindrica at 72 h under different ammonia nitrogen concentration gradient stress conditions

[0031] Figure 3Scatter plot of weight-survival time distribution of different individuals of Echinops monocylindrica under 72h semi-lethal ammonia nitrogen concentration stress Figure 4 Body weight and survival time of half-sib families of Echinops monocytogenes from different paternal lines under 72h semi-lethal ammonia nitrogen concentration stress Figure 5 Body weight and survival time of full-sib families of different maternal lines of Echinops monocytogenes under 72h semi-lethal ammonia nitrogen concentration stress DETAILED DESCRIPTION

[0032] In order to explain the purpose and technical solution details of the present invention more clearly, specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. The following embodiments are used to further illustrate the present invention, but should not be construed as fixing or limiting the present invention. The operating methods and technical means recorded in the embodiments can be modified or replaced in other species without departing from the spirit or scope of the present invention to achieve the breeding goal.

[0033] 1. Family establishment and cultivation

[0034] Wild mature parents of Echinops monocyclica in the Qinhuangdao sea area were collected, and individuals with good physiological conditions and abundant nephridia were selected and transported at low temperature to Shandong Dayi Luyu Marine Technology Co., Ltd. as the basic breeding population. Individuals with a body weight greater than 15-20% of the average body weight (the selected individuals in this embodiment weighed more than 120 g) were selected from the basic breeding population as parents for in vitro fertilization and passage, and 24 full-sib families of Echinops monocyclica (8 heads ♂×24 heads ♀) were established using the Nest mating design. The preliminary preparation stage is as follows: 24 incubators (length × width × height: 60cm × 50cm × 35cm) are cleaned by physical means, and then disinfected with a certain concentration of potassium permanganate solution. After cleaning and disinfection, fresh filtered seawater is injected for standby use; the in vitro fertilization process is as follows: first, the parents are classified according to gender and cultured in a 12-hour aquaculture filtered seawater environment. After the vitality of the parents is restored, they are dissected and the renal tubes are neatly placed in a dry and clean dissection tray. After all the parents are dissected, the renal tubes are transferred to 24 different 50ml centrifuge tubes according to different parent individuals. The renal tubes are cut into pieces and diluted with seawater and then sprinkled into the incubator for fertilization and hatching. During fertilization, the density of the egg cells in the incubator is ensured to be 8-10 / ml, and the number of sperms around each egg cell is about 5-8; the family cultivation process is as follows: the fertilized eggs in the incubator are hatched and cultivated until they are in the early stage of metamorphosis benthic life, and in this example, they are cultivated to 25 days of age (the water temperature during this cultivation period is between 17-18°C); the embryos of the single-ringed worms in the development period before metamorphosis benthic life are removed from the incubator and transferred to a specific area of ​​the indoor breeding pond for cultivation until they reach the seedling size stage in industrial breeding, and in this example, they are cultivated to 100 days of age (the water temperature during this cultivation period is between 17-19°C); after reaching the seedling size, the single-ringed worms of each family are filtered out from the indoor breeding pond and transferred to an outdoor pond for continued cultivation until they are 200 days of age (the water temperature during this cultivation period is between 18-22°C). In order to avoid the mixing of families, the incubator incubation process is an independent box culture, the indoor culture pond culture process is to use 100 mesh silk screen to isolate the culture area of ​​each family, and the outdoor pond culture process is to use 20 mesh silk screen to isolate the culture area of ​​each family. In addition, each time the individual of the single-ringed spiny worm family is transferred between different culture environments, the culture density of all families is controlled at the same level. The basic method of feeding twice a day, changing water once, and sucking and removing dirt once every three days is maintained throughout the culture process. Appropriate adjustments are made according to specific circumstances during the process. The conditions for aquaculture filtration seawater are: dissolved oxygen content greater than 5mg / L, and salinity is 31-32ppt. The feeding conditions are: feeding a mixture of spirulina and marine red yeast (100 million / ml) for the first 100 days of age, and then feeding a spirulina powder solution filtered by a 300-mesh silk screen, and the standard is that the culture water becomes clear 3-4h after feeding. Due to problems such as family loss and seed retention during the breeding process, a total of 20 families were used to participate in subsequent ammonia nitrogen stress breeding.

[0035] 2. Calculation of the 72h half-lethal concentration of Echinops monocyrthospermum under different ammonia nitrogen concentration gradient stress

[0036] After the F1 generation seedlings were cultivated to 200 days of age in the outdoor pond, an acute challenge experiment was used to explore the 72-hour semi-lethal ammonia nitrogen concentration value of the monocypris punctatus under ammonia nitrogen stress conditions. Five ammonia nitrogen concentration gradient experimental groups (2g / L, 1g / L, 0.8g / L, 0.6g / L, 0.4g / L, the experimental seawater was obtained by dissolving ammonium chloride in aquaculture filtered seawater in a certain proportion) and a normal filtered seawater control group (0g / L) were set up. Three parallels were set up for each group, and 10 monocypris punctatus individuals were randomly selected from each parallel to participate in the stress. The 72-hour semi-lethal ammonia nitrogen concentration exploration experiment was carried out in the breeding pond of the breeding workshop. The challenge was continued for 120 hours. During this period, the death of the insect body was recorded every 8 hours, and the survival rate of the monocypris punctatus at each ammonia nitrogen concentration gradient at different time points was counted ( Figure 1 ), the water temperature of the experimental system in the breeding pond was between 17.5-19℃ during the exploration. The linear regression method was used to calculate the survival curve equation ( Figure 2 ), and the 72h semi-lethal ammonia nitrogen concentration of the monocyclic spiny worm population under ammonia nitrogen stress conditions was calculated using the survival curve equation to be 1.390 g / L.

[0037] 3. Construct a 72-hour semi-lethal ammonia nitrogen concentration stress environment to calculate the survival time of individuals from different families

[0038] A sufficient number of stress incubators (length × width × height: 28cm × 19.5cm × 16.5cm) were prepared and cleaned and disinfected. Each incubator was isolated into 30 independent areas with micro-isolation boards. 60 individuals of the monocyclic thorn were randomly selected from each family, weighed and numbered, and placed in an independent space in the incubator according to the individual number for ammonia nitrogen stress. The control group consisted of 60 monocyclic thorns randomly selected from the experimental system containing 20 full-sib families, and the same treatment was taken and cultured with filtered seawater. Each family and the control group used two incubators to construct a stress environment, and all 42 incubators were placed in a culture pool for culture, and the water temperature of the culture environment was between 17.5-19℃. The experimental group used the 72h semi-lethal ammonia nitrogen concentration seawater obtained in the study for stress culture, and continued the stress until all individuals died. During the period, they were observed every 4h and the survival time of each individual was counted. The control group used filtered seawater for unified culture. During the entire stress process, a small amount of feeding was carried out once a day (feeding was fine-tuned according to the number of surviving insects and their vitality to avoid polluting the stress environment with bait residues) and a water change operation was performed (the amount of water changed was half of the volume of the water in the culture tank, the ammonia nitrogen stress experimental group was replaced with filtered seawater with an ammonium chloride concentration of 1.390 g / L, and the control group was replaced with normal filtered seawater). The phenotypic parameters of the entire experimental system and each family are shown in Tables 1 and 2. It can be seen that the survival time of the single-ringed thorn insect body in the stress system is quite different: the difference is significant at the individual level, with the minimum survival time being 48h and the maximum survival time being 232h. The average survival time of each single-ringed thorn insect individual is 118.37±1.33h (Table 1); the survival time of the single-ringed thorn insect body is also quite different at the family level, with the minimum survival time being 87.77±3.35h and the maximum survival time being 169.53±5.56h (Table 2), indicating that the tolerance of single-ringed thorn insects to ammonia nitrogen under different genetic backgrounds is significantly different. In addition, in terms of weight traits, it can also be seen from Tables 1 and 2 that there are certain differences between different individuals and families. Afterwards, the weight and survival time data of the single-ringed worms in the stress system were used to make a scatter plot for analysis. It can be seen that under stress conditions, the survival time of the single-ringed worms has a tendency to increase with the increase of weight ( Figure 3 ), indicating that there is a certain correlation between the two. At the same time, it can be clearly found that the phenotypes of the survival time of ammonia chloride tolerance of different individuals in the 0-1g weight range are somewhat different, indicating that genetic factors have a certain influence on the adversity tolerance of the monocyclic spiny worm. Using the survival time data and weight data of different paternal half-sib families and maternal full-sib families for graphical analysis, it can be seen that in terms of phenotypic data analysis, among the paternal half-sib families: the ammonia nitrogen tolerance of the No. 1, No. 4 and No. 8 half-sib families is stronger ( Figure 4); among maternal full-sib families: 1-2, 1-3, 4-1, 4-2 and 8-2 full-sib families have stronger tolerance to ammonia nitrogen ( Figure 5 ), that is, from the analysis of the phenotypic parameters of the tolerance of the monocyclic family, the above families have better tolerance to ammonia nitrogen, and the above families are preliminarily marked.

[0039] Table 1 Phenotypic data of survival time and body weight of individuals of Echinococcus monocygnus under 72h semi-lethal ammonia nitrogen concentration stress

[0040] Characteristics Mean Standard error Minimum Maximum Coefficient of variation Weight / g 0.89 0.01 0.25 4.25 55.77％ Survival time / h 118.37 1.33 48 232 38.87％

[0041] Table 2 Phenotypic data of the survival time and body weight of each full-sib family of Echinops monocytogenes under 72h semi-lethal ammonia nitrogen concentration stress (mean ± standard error)

[0042]

[0043]

[0044] 4. Establish a genetic evaluation model to estimate genetic parameters and breeding values ​​for ammonia nitrogen tolerance and body weight

[0045] The survival time and body weight of the individuals of the monocyclic worms collected during the above-mentioned ammonia nitrogen stress process were used to analyze the genetic parameters. i =μ+α i +е i

[0046] where y i is the ammonia nitrogen tolerance survival time of the i-th test individual, μ is the mean ammonia nitrogen tolerance survival time of all test individuals, α i is the additive genetic effect of ammonia nitrogen tolerance survival time of the i-th test individual, е i is the random residual of the ammonia nitrogen tolerance survival time of the i-th test individual.

[0047] The heritability calculation formula of this animal model is as follows:

[0048]

[0049] in, is the additive genetic variance of ammonia nitrogen tolerance survival time, is the residual variance of ammonia nitrogen tolerance survival time. The calculation method of body weight heritability is the same as that of

[0050] At the same time, the genetic and phenotypic correlations between ammonia nitrogen tolerance and body weight traits (r x，y ) is calculated as follows:

[0051]

[0052] When calculating genetic correlation, σ xy is the additive genetic covariance between ammonia nitrogen tolerance and body weight traits, and is the additive genetic variance of the two traits. When calculating phenotypic correlation, σ xy is the phenotypic covariance between the ammonia nitrogen tolerance trait and the body weight trait, and is the phenotypic variance of the two traits.

[0053] In terms of genetic parameter calculation and analysis: the estimated heritability of the ammonia nitrogen tolerance trait of the monocyclic aphid was 0.2127±0.0776, indicating that it has a certain breeding potential. The estimated heritability of the weight trait of the monocyclic aphid was 0.7244±0.1559, which belongs to a high heritability level, indicating that the weight trait also has the potential for further breeding (Table 3). In addition, the genetic and phenotypic correlations between the two were 0.7986±0.0809 and 0.4856±0.0414 (Table 3), both of which belong to a high correlation level. This result shows that the weight trait of the monocyclic aphid can be used to indirectly select its ammonia nitrogen tolerance trait. This result also provides theoretical guidance for the comprehensive breeding of ammonia nitrogen tolerance and growth traits of the monocyclic aphid.

[0054] Table 3 Genetic parameters of the survival time and body weight of Echinops monocylindrica under 72h semi-lethal ammonia nitrogen concentration stress (the bold diagonal line is heritability, the line above the diagonal line is genetic correlation, and the line below the diagonal line is phenotypic correlation)

[0055]

[0056]

[0057] 5. F1 generation parent seed preservation

[0058] The BLUP method (best linear unbiased estimation method) was used to estimate the individual breeding values ​​of E. monocylindrica using the heritability of ammonia nitrogen tolerance and body weight traits, and then the average breeding value of the family was determined based on the individual breeding values. The average breeding values ​​of ammonia nitrogen tolerance and body weight traits of all families were weighted at a ratio of 1:1 to obtain the comprehensive breeding values ​​of all families under ammonia nitrogen stress conditions (Table 4). The top five full-sib families were sorted using the comprehensive breeding values, and they were: 1-3, 4-2, 8-2, 7-3, and 1-2 full-sib families (arranged in order of ranking). This result was basically consistent with the results of the previous analysis of the stress tolerance phenotypic data, with only the 4-1 full-sib family being different. The reason for this analysis was that the ammonia nitrogen tolerance of E. monocylindrica individuals in this family was quite different, that is, the genetic advantage of this family was low. Comprehensive analysis determined that the full-sib families No. 1-2, No. 1-3, No. 4-2, No. 7-3, and No. 8-2 would be candidate families for ammonia nitrogen tolerance breeding, and they would continue to be bred to adulthood at a seed retention rate of 3-5%.

[0059] Table 4 Breeding values ​​of ammonia nitrogen tolerance, body weight and weighted comprehensive breeding values ​​of each full-sib family of Echinops monocyrthosiphon under 72h semi-lethal ammonia nitrogen concentration stress

[0060]

[0061] 6. F2 generation family construction and succession selection

[0062] The seed individuals in the five ammonia nitrogen tolerance breeding candidate families selected in the F1 generation were cultured to the stage of gonadal maturity, and the strong male and female individuals of the monocyprid with large size and strong vitality were selected for pairing breeding between the same family source and different family sources to construct the F2 generation family, and the inbreeding coefficient was controlled within 0.1. In the subsequent breeding process, the F2 generation family was continuously selected for 4-5 generations with strong ammonia nitrogen tolerance and excellent weight traits as breeding indicators. In the final family, strong male and female individuals of the monocyprid with distant kinship and excellent weight traits were selected as excellent varieties for industrial breeding applications.

[0063] The above embodiments are only used to illustrate the technical solutions in the present invention and are not used to limit the present invention. For professionals in the field, the methods and technical means in the above embodiments can be modified or replaced to achieve the breeding goals. However, equivalent changes made according to the claims of the present invention still fall within the scope of protection required by the present invention.

Claims

1. A method for breeding ammonia nitrogen tolerant strain of Echinops monocyclica, characterized in that Quantitative genetics and family breeding methods are used together to screen and cultivate new industrial breeding varieties with excellent growth traits and strong tolerance to ammonia nitrogen. The breeding method comprises the following steps: 1) Family establishment and cultivation: wild Echinops spp. with mature gonads in natural waters were collected as breeding populations, and robust Echinops spp. individuals with a body weight 15-20% greater than the average body weight and full nephridial tubes were selected as breeding parents. The F1 generation full-sib family of Echinops spp. was established using the Nest mating design method according to the principle of one male mating with three females. After hatching in the incubator, the F1 generation full-sib family was subsequently cultivated in a specific area according to the factory seedling cultivation model; 2) Calculation of half-lethal concentration value under ammonia nitrogen concentration gradient stress: An acute attack experiment was used to explore the 72h half-lethal concentration value of Monochamus monocygnus under ammonia nitrogen stress conditions. After the F1 generation seedlings were cultivated to 200 days of age, the ammonia nitrogen concentration gradient experimental groups were set up with 2g / L, 1g / L, 0.8g / L, 0.6g / L, 0.4g / L and a normal filtered seawater control group (0g / L). Each group was set up with three parallel groups and the attack lasted for 120h, during which the death of the insects was recorded every 8h. The linear regression method was used to calculate the half-lethal ammonia nitrogen concentration value of Monochamus monocygnus through the survival rate at 72h under each ammonia nitrogen concentration gradient stress condition; 3) Construct a semi-lethal concentration stress environment to count the survival time of individuals from different families: Use the 72h semi-lethal ammonia nitrogen concentration value obtained from the investigation to prepare seawater of corresponding concentration for aquaculture stress. 60 insects were randomly selected from each family, weighed and placed in an independent incubator for stress culture. At the same time, 60 monocyclic spiny worms were randomly selected from the experimental system, weighed and placed in an incubator of the same specifications for culture using filtered seawater as a control group. The interior of the incubator was isolated with micro-isolation plates to ensure that each insect was in a separate area, and all incubators were placed in a culture pool for aquaculture stress. For the experimental group, 72h semi-lethal ammonia nitrogen concentration seawater was used for unified culture, and the stress continued until all individuals died, and the survival time of each individual was counted. For the control group, filtered seawater was used for unified culture, and observation was stopped after the experimental group completed the stress statistics; 4) Establish a genetic evaluation model to estimate ammonia nitrogen tolerance and body weight breeding values: The heritability of ammonia nitrogen tolerance trait was determined by the survival time of the individuals of the monocypris punctatus under the stress condition of 72h semi-lethal ammonia nitrogen concentration, and the breeding value of individual ammonia nitrogen tolerance and the average breeding value of family ammonia nitrogen tolerance were estimated based on the heritability of the ammonia nitrogen tolerance trait. The heritability of the body weight trait was determined by the body weight of the individuals of the monocypris punctatus under the stress condition, and the breeding value of the individual body weight trait and the average breeding value of family weight were estimated based on the heritability of the body weight trait. The best linear unbiased estimation method (BLUP method) of animal models was used to estimate genetic parameters and breeding values. Establishment of genetic parameter estimation model for ammonia nitrogen stress tolerance: i =μ+α i +е i where y i is the ammonia nitrogen tolerance survival time of the i-th test individual, μ is the mean ammonia nitrogen tolerance survival time of all test individuals, α i is the additive genetic effect of ammonia nitrogen tolerance survival time of the i-th test individual, е i is the random residual of the ammonia nitrogen tolerance survival time of the i-th test individual. The heritability calculation formula for the above animal model is as follows: in, is the additive genetic variance of ammonia nitrogen tolerance survival time, is the residual variance of ammonia nitrogen tolerance survival time. The calculation method of body weight heritability is the same as that of At the same time, the genetic and phenotypic correlations among the traits (r x，y ) is calculated as follows: When calculating genetic correlation, σ xy is the additive genetic covariance between ammonia nitrogen tolerance and body weight traits, and is the additive genetic variance of the two traits. When calculating phenotypic correlation, σ xy is the phenotypic covariance between the ammonia nitrogen tolerance trait and the body weight trait, and is the phenotypic variance of the two traits; 5) Evaluate the comprehensive breeding value and select the F1 generation excellent families as parents for seed retention: weight the average breeding value of ammonia nitrogen tolerance and the average breeding value of body weight traits of different families at a ratio of 1:1 to obtain the comprehensive breeding value of each family under ammonia nitrogen stress conditions. Use the comprehensive breeding value to sort and retain the top 5 families as candidate families for ammonia nitrogen tolerance breeding. The selected candidate families will be retained at a seed retention rate of 3-5% and continue to be raised to adulthood; 6) Construction of F2 generation families and selection of successive generations: The candidate families for breeding with ammonia nitrogen tolerance of the F1 generation are cultured to the stage of gonadal maturity, and the strong and vigorous individuals of the monocyprids with weight in the top 15-20% are selected for mating and breeding with the same family source and different family sources to construct the F2 generation families, and the inbreeding coefficient is controlled within 0.

1. After that, the F2 generation families are continuously selected for 4-5 generations with strong ammonia nitrogen tolerance and excellent weight traits as breeding indicators, and the healthy male and female monocyprids with distant kinship, large size and strong vitality are selected from the families finally obtained as excellent varieties for industrial breeding applications.

2. The method according to claim 1, characterized in that In step 1), the family is established by using in vitro fertilization technology to pair male and female individual parents. The male and female parents are dissected and the kidney tubes are taken out in a dry and clean dissection tray. After all the parents are dissected, the kidney tubes are cut into pieces and diluted with seawater, and then splashed into the incubator for fertilization and hatching. In the process of family seedling cultivation, the fertilized eggs in the incubator are first hatched and cultivated to the early stage of metamorphosis benthic life, that is, cultivated to about 25 days old (water temperature range: 17-19°C); then, before the embryos of the single-ringed spiny worms are about to develop into metamorphosis benthic life, they are moved out of the incubator and cultured in a specific area of ​​the indoor culture pond to 100 days old, that is, the seedling size stage in industrial cultivation (water temperature range: 17-19°C); finally, the larvae of each family are filtered out from the indoor culture pond and transferred to the outdoor pond for continued cultivation to 200 days old (water temperature range: 18-22°C). In order to avoid mixing between families, the incubator incubation process is an independent box culture, the indoor culture pond culture process is to use 100 mesh silk screen to isolate the culture area of ​​each family, and the outdoor pond culture process is to use 20 mesh silk screen to isolate the culture area of ​​each family. In addition, each time the monocyclic thorn family is transferred to a different culture environment, the culture density of all monocyclic thorn families is controlled at the same level. The basic method of feeding twice a day, changing water once, and sucking and removing dirt once every three days is maintained throughout the culture process, and appropriate adjustments are made according to the specific situation during the process. The conditions for aquaculture filtration seawater are: dissolved oxygen content greater than 5mg / L, and salinity is 31-32ppt. The feeding conditions are: feeding a mixture of spirulina and marine red yeast (100 million / ml) for the first 100 days of age, and then feeding a spirulina powder solution filtered by a 300 mesh silk screen, and the standard is that the culture water becomes clear 3-4h after feeding.

3. The method according to claim 1, characterized in that In step 2), the individuals of the monocyclic stingray in the screening process of the half-lethal concentration of ammonia nitrogen stress for 72 hours are randomly selected in the entire cultivation system. The ammonia nitrogen stress experimental seawater is obtained by dissolving ammonia chloride in aquaculture filtered seawater after calculating and weighing according to a certain experimental concentration. Each ammonia nitrogen concentration stress experimental group and control group are set up with three parallels, and at least 10 monocyclic stingrays participate in the stress process in each parallel. The ammonia nitrogen concentration gradient of the experimental group can be set accordingly according to the tolerance of different aquatic economic species.

4. The method according to claim 1, characterized in that In step 3), a 72-hour semi-lethal ammonia nitrogen stress environment is constructed for the entire family. The ammonia nitrogen stress experimental group and the control group are placed in a separate culture pool for treatment to ensure the consistency of experimental conditions such as temperature. During the period, the survival of the monocyclic thorns is observed every 4 hours and the dead individuals are promptly fished out. A small amount of feeding and a water change operation are performed once a day. When feeding, specific adjustments are made according to the number of surviving insects and the vitality state. When changing water, the experimental group uses 72-hour ammonia nitrogen semi-lethal concentration seawater for replacement, and the control group chooses natural filtered seawater for replacement. The amount of water change is half of the volume of seawater in the culture box.

5. The method according to claim 1, characterized in that: The genetic parameter estimation of the ammonia nitrogen stress tolerance trait and the body weight trait in step 4) was achieved by using ASReml-R 4.0 software.

6. The method according to claim 1, characterized in that The methods for family construction and seedling cultivation in steps 5) and 6) are the same as those in step 1).

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