CN117837531A - Breeding method of new urechis unicinctus strain resistant to low salt - Google Patents
Breeding method of new urechis unicinctus strain resistant to low salt Download PDFInfo
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Abstract
The invention discloses a breeding method of a new urechis unicinctus strain tolerant to low salt, which comprises the following steps: (1) family establishment and cultivation; (2) calculation of low salt stress semi-lethal conditions; (3) Low salinity stress statistics of different family individual phenotype data; (4) Establishing a genetic evaluation model to estimate salinity tolerance and weight breeding values; (5) Evaluating the comprehensive breeding value and screening out excellent F1 generation families as parents for seed reservation; (6) F2 generation family construction and subculture selection. By the method, a new urechis unicinctus strain with strong low salt tolerance and excellent growth characteristics can be bred. The new line screened by the method is suitable for being popularized in low-salinity water area cultivation, and solves the problems that the existing urechis unicinctus is low in survival rate, weak in low-salinity resistance and the like in low-salinity sea area cultivation and restricts industrial development.
Description
Technical Field
The invention belongs to the field of aquatic product genetic breeding, and particularly relates to a breeding method of a new urechis unicinctus strain tolerant to low salt.
Background
Urechis unicinctus (Urechis unicinctus) is a link animal living in a U-shaped cave in the intertidal zone and is mainly distributed on the coasts of yellow and Bohai sea in Russia, korean, japan and China. Urechis unicinctus contains rich essential amino acids, glycosaminoglycans and unsaturated fatty acids of human bodies, and has high nutritive value. In addition, the composition also has active substances such as polypeptide, plasmin, macromolecular protein and the like with antioxidant, thrombolytic and in-vitro anti-tumor effects, and has potential medicinal value. Urechis unicinctus is evaluated by agricultural rural authorities as a good and heterogeneous resource of ten aquatic products in 2022 years, and has wide industrial development prospect. In recent years, due to the excessive fishing of wild resources of urechis unicinctus, the yield of the urechis unicinctus is drastically reduced, the market price is greatly improved and can reach 200 yuan/kg at maximum, and the contradiction between supply and demand is increasingly prominent. On the other hand, the current artificial breeding and cultivation of urechis unicinctus are in a rapid development stage, and the main breeding mode is to collect wild populations as parents for breeding, and genetic improvement is not carried out yet. The fine variety breeding is one of important keys for determining the continuous and rapid development of the urechis unicinctus breeding industry, and the excellent variety with strong stress resistance can be cultivated through artificial directional breeding, so that the environmental stress resistance of the urechis unicinctus can be enhanced, and the breeding survival rate can be improved.
Salinity is one of important environmental factors in the living condition of marine organisms, influences various biological processes such as growth and development, energy metabolism, osmotic adjustment, immune defense and the like of the marine organisms, and even researches show that the salinity can influence the flavor and the meat quality of the marine organisms. Unlike the deep open sea with relatively stable salinity, the environments of the intertidal zone, estuary and land-based seawater ponds and the like can severely increase the frequency of salinity fluctuation under the influence of the factors such as tides, rainfall, high-temperature drought or human activities and the like, so that the salinity of the seawater is changed to cause different degrees of damage to marine organisms. On the other hand, it is counted that there is 3067 hectare of inland low-salt water in our country, some of which have been used for aquaculture, but most of which are in a barren state for a long time. At present, the low-salinity water-area culture species in China mainly comprise Litopenaeus vannamei, and the problems of single cultivar, low water area utilization rate and the like exist. The development of novel high-value culture varieties has important significance for utilizing wide low-salt water resources in China. The urechis unicinctus has high economic value and wide salinity tolerance range, and is an aquatic economic species with extremely low-salinity water-domain cultivation development prospect. The novel excellent urechis unicinctus strain with strong low-salt tolerance and high growth speed is cultivated by a quantitative genetics means and a family breeding method, and the method has important significance for improving the artificial breeding yield of urechis unicinctus, expanding the breeding space of urechis unicinctus, efficiently utilizing low-salt water resources and finally assisting the development of the artificial breeding industry of urechis unicinctus.
Disclosure of Invention
The invention aims to provide a breeding method of a new variety of urechis unicinctus with low salt tolerance, aiming at the current situation that the existing urechis unicinctus breeding industry does not have the new variety of low salt tolerance. The novel urechis unicinctus strain with excellent growth characteristics and strong low-salt resistance can be cultivated by the method, and a solid foundation is laid for the rapid, stable and sustainable development of the urechis unicinctus industry.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a breeding method of a new variety of urechis unicinctus resistant to low salt is characterized by comprising the following steps:
1) Family establishment and cultivation: collecting a wild urechis unicinctus population with mature gonad development in a natural sea area as a breeding parent, selecting urechis unicinctus adults with vigorous vitality and full kidney pipes with weight between 120g and 150g (individuals with weight larger than 15-20% of average weight), and establishing an F1 generation isotactic cell family of urechis unicinctus according to a principle of male and female mating by adopting a nest mating design method. Carrying out subsequent culture on F1 generation isotactic cell families according to an industrial seed culture mode;
2) Low salt stress semi-lethal condition calculation: after F1 generation seedlings are cultivated to 200 days old, setting low salinity gradient experimental groups to 7.5 permillage, 10 permillage, 12.5 permillage and 15 permillage, and exploring a 72-hour semi-lethal salinity value of urechis unicinctus under a low salinity stress condition by adopting an acute toxicity attack experiment. The semi-lethal salinity stress exploration experiment is provided with four low salinity gradient experiment groups and a normal seawater control group (32%o), and each group is provided with three parallel groups. Continuously attacking the insects until all individuals die, recording the death condition of the insects every 8 hours, and calculating the semi-lethal salinity by using a linear regression method according to the survival rate of 72 hours under each salinity gradient;
3) Low salinity stress: and (5) culturing and stressing by using the explored 72h semi-lethal low-salinity seawater. 60 insects are randomly selected in each family, the insects are placed in an independent incubator for salinity stress after weighing, and the inside of the incubator is isolated by a miniature isolation plate to ensure that each insect is in an area. Placing all the incubators in a culture pond, uniformly culturing by using semi-lethal low-salinity seawater, continuously stressing until all individuals die, and counting the survival time of each individual;
4) Salinity tolerance and body weight breeding value determination: determining the salt tolerance genetic force by using the survival time of the individual under the condition of low salt stress, and estimating the individual low salinity tolerance breeding value and the family low salinity tolerance average breeding value according to the salt tolerance genetic force. And determining the body weight character genetic force by using the body weight values of the individuals participating in the stress condition, and estimating the body weight breeding value and the average family body weight breeding value according to the body weight character genetic force. The breeding value was estimated by using the BLUP method (optimal linear unbiased estimation method). Genetic and phenotypic correlations between traits are estimated based on low salt stress survival times and body weight parameters.
Establishing a low-salt stress tolerance genetic parameter estimation model: y is i ＝μ+α i +е i
Wherein y is i For the low salt tolerance survival time of the ith test individual, μ is the mean, α, of the low salt tolerance survival times of all test individuals i Low salt tolerance survival time additive genetic effects, e.g., for the ith test individual i Low salt tolerance survival time random residuals for the ith test individual.
The genetic force calculation formula of the animal model is as follows:
wherein,additive genetic variance for low salt tolerance survival time, < >>Tolerance time to live residual variance for low salt. The weight genetic force calculation method is the same as that described above.
At the same time, genetic and phenotypic relativity (r x，y ) The calculation is as follows:
in calculating genetic correlations, σ xy For additive genetic covariance between low salt tolerance traits and body weight traits,andis the additive genetic variance of the two traits. In calculating phenotype correlations, σ xy Is a phenotypic covariance between low salt tolerance trait and body weight trait, ++>And->Is the phenotypic variance;
5) And F1, parent seed reserving: and weighting the low salinity tolerance average breeding value and the weight average breeding value of each family according to the proportion of 1:1 to obtain the comprehensive breeding value of all families under the condition of low salinity stress. And sequencing by utilizing the comprehensive breeding values, and reserving families ranked in the top 5 as low-salt breeding candidate families. Continuously breeding the selected families to adults according to the seed retention rate of 3-5%;
6) F2 generation family construction and subculture selection: and (3) breeding the F1 generation reserved seed low-salt breeding candidate family to the gonad maturation period, screening out robust urechis unicinctus individuals with weight of 15-20% and vigorous vigor, carrying out pairing breeding of the same family source and different family sources to construct an F2 generation family, and controlling the inbred coefficient within 0.1. And then, continuously selecting F2 generation families by using strong low-salt tolerance and excellent weight character as breeding indexes, and selecting healthy urechis unicinctus male and female individuals with far genetic relationship, large individuals and pink body color from the finally obtained families as excellent strains to industrially popularize and apply.
Preferably, the wild urechis unicinctus population in the natural sea area in the step 1) is a wild urechis unicinctus population in the Qin dynasty island sea area, and the urechis unicinctus population is transported to a breeding base for selection at low temperature after being manually caught. The family offspring seed cultivating process is to pair by using in vitro insemination technology, dissect the parent and get the kidney tube in the dry clean dissecting tray, after all parents dissect, cut the kidney tube and dilute with sea water, spill into the incubator to fertilize and hatch. The family offspring seed is cultivated by firstly hatching fertilized eggs in an incubator until the fertilized eggs are converted into the prophase of the metamorphosis benthic life, and the age is about 25 days (18-19.5 ℃). Then, the strain is transferred into a specific area of a culture pond to be cultured until the seedling grows to the size of about 100 days old (18-20 ℃), just before the strain grows to the life of the metamorphosis benthic life. Finally, the larvae of each family are filtered and removed and transferred to an outdoor pond for further culture until the age of 200 days (19-23 ℃). In order to avoid hybridization between families, the hatching process of the incubator is independent box body cultivation, the cultivation process of the cultivation pond is to isolate each family area by using 100-mesh bolting silk, and the outdoor pond cultivation process is to isolate each family area by using 20-mesh bolting silk. In addition, the culture density of all families is controlled to be the same level every time family individuals in different culture environments are transferred. The whole culture process adopts the basic method of twice feeding/day, once water changing/day and once bottom sucking/3 days for culture, and the process is properly adjusted according to specific conditions.
Preferably, the low salt stress semi-lethal condition screening process in step 2) performs a gradient stress experiment for randomly selecting individuals in all families. The experimental seawater is obtained by mixing fresh water into the culture seawater, three parallels are arranged on each salinity gradient, at least 10 insects participate in the stress process under each parallels, and the dead individuals are observed every 8 hours in the exploration process and fished out in time. The 72h semi-lethal low salinity obtained by exploration is 10 per mill.
Preferably, in the step 3), for the low salt stress condition, the low salt stress experimental group and the control group are placed in a single culture pond for treatment, so that consistency of experimental conditions such as temperature and the like is ensured. During the period, the survival condition is observed every 4 hours, dead individuals are fished out in time, and a small amount of feeding and water changing operation are carried out every day. When the feeding is performed, fine adjustment is performed according to the survival number and the vitality state of the insects, 10 permillage of salinity of seawater is selected for replacement when water is changed (32 permillage of salinity of natural seawater is selected for a control group), and the water changing amount is half of the volume of the culture box.
Preferably, the genetic parameter estimation of the low salt tolerance trait and the body weight trait in step 4) is both achieved by ASReml software.
Preferably, in the step 5), F1 generation parent reservation is to weight the average salt resistance and the breeding value of the body weight character of each family calculated under the condition of low salt stress according to the proportion of 1:1 to obtain a comprehensive breeding value, order the comprehensive breeding value, and reserve the families ranked in the top 5 as low salt breeding candidate families. And then the selected families are reserved according to the seed reserving rate of 3-5%.
Preferably, the family breeding method in the F2 generation family and the secondary selection in the step 6) is consistent with the F1 generation construction operation method. In the family construction, the parent is F1 generation reserved individuals to carry out paired breeding of the same family source and different family sources, and the inbred coefficient is controlled within 0.1. In the secondary selection, the continuous selection is carried out on the passage families by taking the low-salt tolerance capability as a breeding index and the excellent weight property.
In conclusion, the invention selects the wild urechis unicinctus population as the basic population, and screens the families with larger genetic improvement space and breeding potential through low-salt tolerance breeding. And (3) carrying out seed reserving and propagation on the family with excellent performance to obtain a new strain with excellent growth character and strong salinity tolerance. The breeding method is simple and practical, has strong operability, and can cultivate fine urechis unicinctus seeds with excellent low-salt tolerance property through multi-generation breeding.
Drawings
FIG. 1 is a diagram showing the survival curves of urechis unicinctus under different low salinity stress conditions
FIG. 2 is a graph showing the survival of urechis unicinctus at 72h under different low salinity stress conditions
FIG. 3 is a scatter plot of survival time distribution of individuals with different weights of urechis unicinctus under 72h semi-lethal low salinity stress condition
FIG. 4 is the weight and survival time of urechis unicinctus male parent half-sibling families under 72h of semi-lethal low salinity stress conditions
FIG. 5 is the weight and survival time of the urechis unicinctus maternal isotactic cell line under 72h semi-lethal low salinity stress conditions
Detailed Description
For a clearer explanation of the objects and technical scheme details of the present invention, the technical scheme of the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments:
1. family establishment and cultivation
The mature parents of wild urechis unicinctus in Qinghai island are collected, and individuals with good physiological conditions and rich kidney pipes are selected and transported to urechis unicinctus biological technology limited company in Qinghai island at low temperature to serve as basic breeding groups. In the basic breeding population, individuals with the weight of 120g-150g, namely, the weight of more than 15-20% of the average weight are selected for in vitro insemination and passage, and 24 isocytoblast families (8 female) of urechis unicinctus are established by adopting the nest mating design. The in vitro insemination process is as follows: firstly, performing restorative culture on parents in a seawater culture environment for 12 hours according to male-female classification, dissecting all the parents after the vitality of the parents is restored, cutting kidney pipes obtained by dissection, placing the kidney pipes into a 50ml centrifuge tube, diluting with seawater, and then sprinkling the kidney pipes into an incubator (length, width, height: 60cm, 50cm, 35 cm) for fertilization and hatching. The density of the egg cells in the incubator is ensured to be 8-10 per ml during insemination, and the number of sperms around each egg cell is about 5-8. The family cultivation process is as follows: fertilized eggs in the incubator are hatched and cultivated to a period of about 25 days old (18-19.5 ℃) before transformation into an allergic benthic life. Transferring the strain to a specific area of a culture pond for cultivation until the strain emerges at about 100 days old (18-20 ℃) just before the strain develops to the life of the metamorphosis benthonic species. After reaching the size of emergence, the larvae of each family are filtered and taken out and transferred to an outdoor pond to be continuously cultured until the age of 200 days (19-23 ℃). The hatching process of the incubator is independent box body culture, the culture pond culture process is to isolate each family area by using 100-mesh bolting silk, and the outdoor pond culture process is to isolate each family area by using 20-mesh bolting silk. In addition, the culture density of all families is controlled to be the same level every time family individuals in different culture environments are transferred. The whole culture process adopts the basic method of twice feeding/day, once water changing/day and once bottom sucking/3 days for culture, and the process is properly adjusted according to specific conditions. Due to the problems of environmental mutation, manual operation and the like in the cultivation process, 3 isotactic families are lost, and 20 families participate in subsequent salinity stress.
2. Calculation of low salt stress semi-lethal conditions
After F1 offspring seeds are cultivated to 200 days old in an outdoor pond, an acute low-salt stress experiment is adopted to explore the semi-lethal salinity value of urechis unicinctus for 72 hours under the condition of low-salt stress. Four low salinity gradient experiment groups (7.5%o, 10%o, 12.5%o and 15%o) and a normal sea water control group (32%o) are arranged, three parallel groups are arranged, 10 insects are randomly selected for each parallel group to participate in stress, and a semi-lethal salinity stress exploration experiment is carried out in a culture pond of a culture workshop (17-19 ℃). The challenge was continued for 120h, and the death of the insects was recorded every 8h during which time the survival rate of the insects at different time nodes at each salinity gradient was counted (fig. 1). And calculating a semi-lethal salinity survival curve equation (figure 2) by using a linear regression method through the survival rate of 72h under each salinity gradient, and calculating through a trend line equation to obtain the semi-lethal salinity value of 10 per mill of 72h under the condition of low salinity stress of urechis unicinctus.
3. Low salinity stress
60 insects in each family are randomly selected, weighed and placed in an independent incubator (length x width x height: 60cm x 50cm x 35 cm) for salinity stress exploration, and the inside of the incubator is isolated by a miniature isolation plate, so that each insect is ensured to be in one area. And (3) using the explored 72h semi-lethal low-salinity seawater to stress, placing all 20 incubators in a culture pond to culture (17-19 ℃), continuously stress until all individuals die, observing every 4h during the period, and counting the survival time of each individual. In the whole stress process, a small amount of feeding is carried out once daily (fine adjustment is carried out according to the survival quantity and the activity state of the insects during feeding) and one water changing operation is carried out (the water changing quantity is half of the volume of the culture box, the low-salt experimental group is used for changing 10 permillage of salinity seawater, and the control group is used for changing 32 permillage of salinity normal seawater). The phenotype parameters of individuals and families in the whole experimental system are shown in tables 1 and 2, and the survival time of urechis unicinctus bodies in the stress system can be seen to have larger difference: the difference on individual level is remarkable, the minimum survival time is 40h, the maximum survival time is 220h, and the average survival time of each urechis unicinctus individual is 116.7408 +/-34.9905 h (table 1); the survival time of urechis unicinctus bodies also has a large difference on the family level, the minimum survival time is 97.4000 +/-4.3234 hours, and the maximum survival time is 156.6000 +/-4.7925 hours (table 2). In addition, it can be seen in tables 1 and 2 that there is also a significant difference in weight parameters between different families. By performing a scatter plot analysis of the survival time data of urechis unicinctus individuals of different weights throughout the stress system, it can be seen that the individual survival time has a tendency to increase with weight gain (fig. 3). By using the survival time data and the weight data of different father half-sibling families and mother isotactic siblings for mapping analysis, the father half-sibling families can be seen: the salt tolerance of the half-sibling No. 1, the half-sibling No. 4, the half-sibling No. 7 and the half-sibling No. 8 families is stronger (figure 4); between maternal isotactic families: the salt tolerance of the whole siblings 1-2, 1-3, 4-1, 4-2, 4-3, 7-3 and 8-3 (figure 5) shows that the above families are better in low salinity tolerance and are initially marked.
TABLE 1 tolerance survival time and weight trait phenotype data for urechis unicinctus individuals under 72h semi-lethal low salinity stress conditions
| Traits (3) | Mean value of | Standard error of | Minimum value | Maximum value | Coefficient of variation/% |
| Weight/g | 0.8099 | 0.0114 | 0.2716 | 3.1788 | 48.83％ |
| Survival time/h | 116.7408 | 1.0101 | 40 | 220 | 29.97％ |
TABLE 2 tolerance survival time and weight trait phenotype data (mean.+ -. Standard error) for urechis unicinctus each isotactic cell line under semi-lethal low salinity stress conditions 72h
4. Salinity tolerance and weight genetic parameter determination
And (3) carrying out genetic parameter analysis by using the survival time and the weight value of the urechis unicinctus individuals counted in the low-salt stress process. Establishing a low-salt tolerance genetic parameter estimation model: y is i ＝μ+α i +е i
Wherein y is i For the low salt tolerance survival time of the ith test individual, μ is the mean, α, of the low salt tolerance survival times of all test individuals i Low salt tolerance survival time additive genetic effects, e.g., for the ith test individual i Low salt tolerance survival time random residuals for the ith test individual.
The genetic force calculation formula of the animal model is as follows:
wherein,additive genetic variance for low salt tolerance survival time, < >>Tolerance time to live residual variance for low salt. The weight genetic force calculation method is the same as that described above.
At the same time, genetic and phenotypic correlation between low salt tolerance and body weight traits (r x，y ) The calculation formula is as follows:
sigma when calculating genetic correlations xy For additive genetic covariance between low salt tolerance traits and body weight traits,and->Is the additive genetic variance of the two traits. In calculating phenotype correlations, σ xy Is a phenotypic covariance between low salt tolerance trait and body weight trait, ++>And->Is a phenotype squareAnd (3) difference.
The method is characterized by comprising the following steps of: the genetic power estimated value of the low-salt stress survival character of urechis unicinctus is 0.1602 +/-0.0639, which shows that the urechis unicinctus has certain breeding potential. The estimated value of the genetic force of the weight character of urechis unicinctus is 0.7244 +/-0.1559, which belongs to the higher genetic force level and shows that the selection breeding of the weight character also has certain advantages (table 3). Meanwhile, the survival time and the variation coefficient of the body weight are 29.97% and 48.83% (table 1) respectively, which further shows that the breeding basis of the urechis unicinctus low-salt tolerance and the body weight property is good. In addition, the genetic and phenotypic correlation coefficients between low salt tolerance and body weight traits were 0.8877.+ -. 0.0530 and 0.4879.+ -. 0.0401 (Table 3), respectively, and the high correlation between the two suggests that indirect selection between the two traits is possible.
TABLE 3 tolerance survival time and weight trait genetic parameters of urechis unicinctus under 72h semi-lethal low salinity stress conditions (genetic correlation on diagonal, phenotypic correlation under diagonal)
| Traits (3) | Low salt | Weight of body |
| Low salt | 0.1602±0.0639 | 0.8877±0.0530 |
| Weight of body | 0.4879±0.0401 | 0.7244±0.1559 |
Seed reserving of F1 generation parent
According to the low-salt tolerance character and the genetic force of the weight character, the BLUP method (optimal linear unbiased estimation method) is utilized to estimate the individual breeding value of urechis unicinctus, and then the average breeding value of the family is determined according to the individual breeding value. The average breeding values of the salt resistance and the body weight characteristics of each family were weighted according to a ratio of 1:1 to obtain comprehensive breeding values of all families under the condition of low salt stress (Table 4). Sequencing by using comprehensive breeding values, and screening to obtain families ranked in the top 5 families as follows: 4-1, 4-2, 7-3, 1-3, 4-3 (ordered by ranking) have correspondence to a family that is initially determined based on the phenotypic data. And (5) taking the selected five families as low-salt breeding candidate families, and continuing to cultivate the plants to adults according to the seed reserving rate of 3-5%.
Table 4 72h Low salt tolerance trait, weight trait and weighted comprehensive Breeding value of urechis unicinctus of each isotactic cell line under semi-lethal low salinity stress condition
F2 generation family construction and subculture selection
And breeding reserved individuals in the five low-salt breeding candidate families screened by the F1 generation until the gonad maturation period, selecting robust urechis unicinctus male and female individuals with large individuals and strong vitality, and carrying out pairing breeding between the same family sources and different family sources to construct an F2 generation family, wherein the inbreeding coefficient is controlled within 0.1. In the subsequent breeding process, the F2 generation family is continuously selected for 4-5 generations by taking the low-salt tolerance capability as a breeding index, and healthy male and female urechis unicinctus individuals with far relativity, good weight property and pink body color are selected from the finally obtained family to be industrially popularized and applied as good strains.
The above embodiments are only for illustrating the technical solution of the present invention. The methods and techniques of operation described herein may be implemented in other embodiments without departing from the spirit or scope of the invention, however, all such modifications and substitutions are intended to be within the scope of the invention.
Claims (6)
1. A breeding method of a new urechis unicinctus line with low salt tolerance is characterized in that a quantitative genetics means and family breeding combined action is adopted to screen and cultivate a new urechis unicinctus line with excellent growth characters and strong low salt tolerance;
the breeding method comprises the following steps:
1) Family establishment and cultivation: collecting a wild urechis unicinctus population with mature gonad development in a natural sea area as a breeding parent, selecting urechis unicinctus adults with vigorous vitality and full kidney pipes with weight between 120g and 150g (individuals with weight larger than 15-20% of average weight), and establishing an F1 generation isotactic cell family of urechis unicinctus according to a principle of male mating and female mating by adopting a nest mating design method; carrying out subsequent culture on F1 generation isotactic cell families according to an industrial seed culture mode;
2) Low salt stress semi-lethal condition calculation: after F1 generation seedlings are cultivated to 200 days old, setting a low salinity gradient experimental group to be 7.5 permillage, 10 permillage, 12.5 permillage and 15 permillage, and exploring a 72-hour semi-lethal salinity value of urechis unicinctus under a low salinity stress condition by adopting an acute toxicity attack experiment; the semi-lethal salinity stress exploration experiment is provided with four low salinity gradient experiment groups and a normal seawater control group (32%o), and each group is provided with three parallel groups; continuously attacking the insects until all individuals die, recording the death condition of the insects every 8 hours, and calculating the semi-lethal salinity by using a linear regression method according to the death rate of 72 hours under each salinity gradient;
3) Low salinity stress: performing cultivation stress by using the explored 72h semi-lethal low-salinity seawater; 60 insects are randomly selected in each family, the insects are placed in an independent incubator for salinity stress after weighing, and the inside of the incubator is isolated by a miniature isolation plate to ensure that each insect is in one area; placing all the incubators in a culture pond, uniformly culturing by using semi-lethal low-salinity seawater, continuously stressing until all individuals die, and counting the survival time of each individual;
4) Salinity tolerance and body weight breeding value determination: determining salt tolerance genetic force by using survival time of individuals under the condition of low salt stress, and estimating individual low salinity tolerance breeding values and family low salinity tolerance average breeding values according to the salt tolerance genetic force; determining the genetic transmission of the body weight character by using the weight values of the individuals participating in the stress condition, and estimating the weight breeding value of the individuals and the average breeding value of the family body weight according to the genetic transmission of the body weight character; the breeding value is estimated by using BLUP method (optimal linear unbiased estimation method); estimating genetic and phenotypic correlations between traits based on low salt stress survival times and weight parameters;
establishing a low-salt stress tolerance genetic parameter estimation model: y is i ＝μ+α i +е i ；
Wherein y is i For the low salt tolerance survival time of the ith test individual, μ is the mean, α, of the low salt tolerance survival times of all test individuals i Low salt tolerance survival time additive genetic effects, e.g., for the ith test individual i Low salt tolerance survival time random residuals for the ith test individual;
the genetic force calculation formula of the animal model is as follows:
wherein,additive genetic variance for low salt tolerance survival time, < >>Tolerance time to live residual variance for low salt; weight genetic force calculation method is the same as that above;
at the same time, genetic and phenotypic relativity (r x，y ) The calculation is as follows:
in calculating genetic correlations, σ xy For additive genetic covariance between low salt tolerance traits and body weight traits,and->Additive genetic variance of two traits; in calculating phenotype correlations, σ xy Is a phenotypic covariance between low salt tolerance trait and body weight trait, ++>And->Is the phenotypic variance;
5) And F1, parent seed reserving: weighting the low salinity tolerance average breeding value and the weight average breeding value of each family according to the proportion of 1:1 to obtain comprehensive breeding values of all families under the condition of low salinity stress; sequencing by utilizing the comprehensive breeding values, and reserving families ranked in the top 5 as low-salt breeding candidate families; continuously breeding the selected families to adults according to the seed retention rate of 3-5%;
6) F2 generation family construction and subculture selection: breeding F1 generation reserved seed low-salt breeding candidate families to the gonad maturation period, screening robust urechis unicinctus individuals with weight of 15-20% and vigorous vigor from the candidate families, carrying out pairing breeding of the same family sources and different family sources to construct F2 generation families, and controlling the inbred coefficient within 0.1; and then, continuously selecting F2 generation families by using strong low-salt tolerance and excellent weight character as breeding indexes, and selecting healthy urechis unicinctus male and female individuals with far genetic relationship, large individuals and pink body color from the finally obtained families as excellent strains to industrially popularize and apply.
2. The method according to claim 1, wherein in step 1), the family is established to pair male and female parents by using an in vitro insemination technology, the parents are dissected and kidney pipes are taken in a dry and clean dissecting disc, after all the parents are dissected, the kidney pipes are sheared and diluted by seawater, and the kidney pipes are splashed into an incubator for fertilization and hatching; the family offspring seed is cultivated by firstly hatching fertilized eggs in an incubator until the fertilized eggs are converted into the prophase of the allergic benthonic life, and the age is about 25 days (18-19.5 ℃); then transferring the strain to a specific area of a culture pond to culture until the strain emerges to the size of about 100 days old (18-20 ℃) immediately before the strain develops to the life of the allergic benthonic species; finally, filtering and taking out the larvae of each family, transferring the larvae to an outdoor pond, and continuously culturing the larvae until the larvae of each family are 200 days old (19-23 ℃); in order to avoid hybridization between families, the hatching process of the incubator is independent box body cultivation, the cultivation process of the cultivation pond is to isolate each family area by using 100-mesh bolting silk, and the outdoor pond cultivation process is to isolate each family area by using 20-mesh bolting silk; in addition, each time family individuals in different culture environments are transferred, the culture densities of all families are controlled to be the same level; the whole culture process adopts the basic method of twice feeding/day, once water changing/day and once bottom sucking/3 days for culture, and the process is properly adjusted according to specific conditions.
3. The method of claim 1, wherein the low salt stress semi-lethal condition screening process of step 2) performs a gradient stress experiment for randomly selecting individuals throughout the system; the low salinity stress experimental seawater is obtained by doping fresh water into the culture seawater, three parallels are arranged in each salinity condition, and at least 10 insects participate in the stress process under each parallels; the 72h semi-lethal low salinity obtained by exploration is 10 per mill.
4. The method according to claim 1, wherein in step 3), for low salt stress conditions, the low salt stress experimental group and the control group are placed in a single culture pond for treatment, so as to ensure consistency of experimental conditions such as temperature; observing survival condition every 4 hours, fishing out dead individuals in time, and carrying out small-amount feeding and water changing operation every day; when the feeding is performed, fine adjustment is performed according to the survival number and the vitality state of the insects, 10 permillage of salinity of seawater is selected for replacement when water is changed (32 permillage of salinity of natural seawater is selected for a control group), and the water changing amount is half of the volume of the culture box.
5. The method according to claim 1, wherein the genetic parameter estimation of the low salt tolerance trait and the body weight trait in step 4) is achieved by ASReml software.
6. The method according to claim 1, wherein the method of step 6) for family construction and larval rearing is identical to the method of step 1).
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