CN117461583A - Breeding method of high-salt-tolerant new urechis unicinctus line - Google Patents
Breeding method of high-salt-tolerant new urechis unicinctus line Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/40—Culture of aquatic animals of annelids, e.g. lugworms or Eunice
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Abstract
The invention discloses a breeding method of a new urechis unicinctus strain with high salt tolerance, which comprises the following steps: (1) basic breeding population introduction; (2) F1 generation family construction and seed cultivation; (3) calculation of high salt stress semi-lethal conditions; (4) Stress by using high salinity semi-lethal conditions and counting survival time data; (5) Establishing a genetic evaluation model to estimate high salinity tolerance characters and weight character breeding values; (6) Determining comprehensive breeding values according to the amphoteric breeding values, and screening out excellent F1 generation families for seed reservation; (7) And constructing an F2 generation family and performing subculture selection by utilizing the seed reserving high-salt breeding candidate family. The method can be used for breeding new urechis unicinctus lines with strong high salt tolerance and growth character advantages, and solves the problems of low survival rate, poor economic index and the like of urechis unicinctus cultivation in a high-salt water area and serious restrictions on industrial development in the prior art.
Description
Technical Field
The invention belongs to the field of aquatic product genetic breeding, and particularly relates to a breeding method of a novel urechis unicinctus strain capable of tolerating high 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. The urechis unicinctus has delicious taste, is evaluated 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 an important environmental factor for growth, metabolism and reproduction of aquatic organisms, and can affect their enzymatic activity, energy distribution, metabolic rate and secretion and distribution of related hormones. Thus, aquatic organisms require higher physiological costs to accommodate extensive salinity. As important physicochemical factors in the urechis unicinctus culture water body, high-temperature drought weather, seasonal rainfall, tidal activity and the like in the natural world can cause the seawater salinity of the culture area to fluctuate, the salinity change can influence the physiological and biochemical reaction of the urechis unicinctus, for example, the external low osmotic pressure environment easily causes the water absorption swelling of cells, and the high osmotic pressure environment can influence the activities of substances such as osmotic adjusting enzymes, immune adjusting enzymes and the like. Studies have shown that the adoption of proper breeding and screening methods and salinity domestication modes can greatly improve the survival rate of aquatic organisms in a high-salt environment. On the other hand, the coastal high-salinity water area of the yellow Bohai sea in China is rich in resources, and the area of the high-salinity water area only reaches 1300km in the coastal of the peninsula in Liaodong and the peninsula in Shandong 2 The distribution area of the high-salinity water area is wide and most of the high-salinity water area is not industrially applied. The urechis unicinctus has high economic value and wide salinity tolerance range, and has great prospect of high-salinity water-domain cultivation and developmentIs an aquatic economic species. The method has the advantages that the novel excellent urechis unicinctus variety with strong high salt tolerance and high growth speed is screened and cultivated through the quantitative genetics means, and the method has important significance for widening the development space of artificial urechis unicinctus cultivation, promoting the high-value utilization of coastal high salt water resources of the Bohai sea in China and promoting the development scale of the artificial urechis unicinctus cultivation industry.
Disclosure of Invention
The invention aims to provide a breeding method of a new variety of urechis unicinctus with high salt tolerance, aiming at the current situation that the existing urechis unicinctus breeding industry does not have the new variety of high salt tolerance. The novel urechis unicinctus strain with excellent growth characteristics and strong high-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 urechis unicinctus strain tolerant to high salt is characterized by comprising the following steps:
1) Basic breeding population introduction: taking multiple tails of wild urechis unicinctus groups from natural sea areas, and selecting urechis unicinctus individuals with excellent phenotypic characters and rich renal tubes as basic breeding groups;
2) F1 generation family construction and offspring cultivation: selecting a urechis unicinctus adult with mature gonad development and full kidney tube, and establishing an F1-generation male parent half-sibling family of urechis unicinctus and a female isotactic-sibling family by adopting a nest mating design method (one male mating and three female). Culturing factory seedlings after hatching the larvae of each family of the F1 generation;
3) Calculation of high salt stress semi-lethal conditions: after F1 generation seedlings are cultivated to 200 days old, an acute toxicity attack experiment is adopted to explore the salinity value of the semi-lethal for 72 hours under the condition of high salt stress of urechis unicinctus. The high salinity gradient experimental groups are 47.5 permillage, 50 permillage, 52.5 permillage and 55 permillage, and the experimental seawater is obtained by dissolving sea salt in the culture seawater. A high salinity gradient experimental group and a normal seawater control group (32%o) are arranged together, and three groups are arranged in parallel. Continuously attacking for 120 hours, recording death conditions of the insects once every 8 hours during the period, and calculating semi-lethal salinity by using a linear regression method according to the survival rate of 72 hours under each salinity gradient;
4) High salinity stress: and (5) stress is carried out by utilizing the explored seawater with the semi-lethal high salinity value for 72 hours. In the high salinity stress process, 60 insects in each family are randomly selected, the weight of the insects is weighed, the insects are placed in independent incubators (the incubators are isolated by miniature isolating plates to ensure that each insect is in one area) for stress, all the incubators are placed in one culture pond and are uniformly cultured by using semi-lethal high salinity seawater, and the survival time of each individual is counted;
5) Salinity tolerance and body weight genetic parameter determination: and carrying out genetic parameter analysis by using the survival time and the weight value of the urechis unicinctus individuals counted in the high salinity stress process. Determining the salinity tolerance genetic force according to the survival time of the individuals under the salinity stress, and estimating individual salinity tolerance breeding values and family salinity tolerance average breeding values according to the salinity tolerance genetic force. And determining the genetic transmission of the body weight character according to the body weight of the test individual, and estimating the body weight breeding value of the individual and the average breeding value of the family body weight according to the genetic transmission of the body weight character. The estimation of the breeding value was performed by using the BLUP method (optimal linear unbiased estimation method). Genetic and phenotypic correlations between traits are estimated based on salinity stress survival time and body weight trait parameters.
For the high-salinity tolerance character, establishing a high-salinity tolerance genetic parameter estimation model: y is i ＝μ+α i +е i
Wherein y is i For the high salt tolerance survival time of the ith test individual, μ is the mean, α, of the high salt tolerance survival times of all test individuals i For the i-th test individual to have a high salt-tolerant survival time additive genetic effect, e.g. i High salt resistant survival time random residuals for the ith test individual.
The genetic force calculation formula of the animal model is as follows:
wherein,for high salt-tolerant survival time additive genetic variance, < >>Is the variance of the high salt-tolerant time-to-live residual. The weight genetic force calculation method is the same as that described above.
At the same time, genetic and phenotypic correlation between high salt tolerance and body weight traits (r x，y ) The calculation is as follows:
in calculating genetic correlations, σ xy For additive genetic covariance between high salt tolerance and body weight traits,andis the additive genetic variance of the two traits. In calculating phenotype correlations, σ xy Is the phenotypic covariance between the high salt tolerance trait and the body weight trait, +.>And->Is the phenotypic variance;
6) And F1, parent seed reserving: and weighting the breeding values of the average salt resistance and the weight property of each family according to the ratio of 1:1 to obtain the comprehensive breeding value of all families under the high-salt condition. Sequencing by using comprehensive breeding values, reserving families ranked in the top 5 as high-salt breeding candidate families, and continuously breeding the selected families to adults according to a seed reserving rate of 3-5%;
7) F2 generation family construction and subculture selection: and for the high-salt tolerance character, breeding the high-salt breeding candidate family of the F1 generation reserved seeds to the gonad maturation period, screening out robust urechis unicinctus individuals with large individuals and strong vitality, and carrying out paired breeding of the same family source and different family sources to construct an F2 generation family, wherein the inbreeding coefficient is controlled within 0.1. And then, continuously selecting F2 generation families by using high 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.
Preferably, in the step 2), the family offspring seed cultivating process is to pair parents by utilizing an in-vitro insemination technology, dissect and take kidney tubes from the parents in a dry and clean dissecting tray, cut the kidney tubes after all the parents are dissected, dilute the kidney tubes with seawater, spill the kidney tubes into a cultivating box for fertilization and hatching, and transfer the kidney tubes into a specific area of a cultivating pool for cultivation before the cultivating is converted into an abnormal benthic life (25 days, 18-19.5 ℃). After the larvae are cultivated to the size of emergence (100 days, 18-20 ℃), the larvae of each family are filtered and taken out and transferred to an outdoor pond for cultivation (200 days, 19-23 ℃) and are isolated by bolting silk in all cultivation processes, so that the families are prevented from being mixed. The culture process adopts basic method culture of twice feeding/day (feeding spirulina liquid during indoor culture pond culture period and feeding filtered spirulina powder during outdoor pond culture period), once water changing/day (water changing volume is half of culture pond or pond volume), and once bottom sucking/3 days, and the process is properly adjusted according to specific conditions.
Preferably, the high salt stress semi-lethal condition screening process in step 3) performs a gradient stress experiment for randomly selecting individuals in all families. Every salinity gradient all sets up three parallels, and every parallel down has 10 at least worm bodies to participate in the stress process, observes once every 8 hours in the exploration process, in time drags out dead individual. The 72h semi-lethal high salinity obtained by exploration is 53 per mill.
Preferably, the high salinity stress exploration system in the step 4) is carried out in a single culture pond, and the seawater in the culture environment is 53 per mill. During the period, the survival condition is observed every 4 hours, dead individuals are fished out in time, a small amount of feeding is carried out every day (fine adjustment is carried out according to the survival quantity and the activity state of the insects during feeding), and water changing operation is carried out once (the water changing quantity is half of the volume of the culture box, 53 permillage of salinity seawater is replaced by a high-salt experimental group, and 32 permillage of salinity normal seawater is replaced by a control group).
Preferably, the genetic parameter determinations of the high salt tolerance trait and the body weight trait in step 5) are both processed using ASReml software.
Preferably, in the step 6), F1 generation parent seed reserving is to reserve families with comprehensive breeding values ranked in the top 5 as high-salt breeding candidate families, and the selected families are continuously cultivated to adults according to the seed reserving rate of 3-5%, wherein the cultivation process is outdoor pond cultivation.
Preferably, the family breeding method in the F2 generation family and the secondary selection in the step 7) is consistent with the in-vitro insemination operation of the F1 generation. F2 generation family is constructed as the paired breeding of the same family source and different family sources, and the inbreeding coefficient is controlled within 0.1. In the secondary selection, continuous breeding is carried out by taking high salt tolerance and excellent weight property as breeding indexes.
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 high-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 high salinity tolerance. The breeding method is simple and practical, has strong operability, and can cultivate fine urechis unicinctus seeds with excellent high-salt tolerance property through multi-generation breeding.
Drawings
FIG. 1 is a diagram showing the survival curves of urechis unicinctus under different conditions of high salinity stress
FIG. 2 is a graph showing the survival of urechis unicinctus at 72h under different high salinity stress conditions
FIG. 3 is the weight and survival time of urechis unicinctus male parent half-sibling families under 72h of semi-lethal high salinity stress conditions
FIG. 4 is the weight and survival time of the urechis unicinctus maternal isotactic cell line under 72h semi-lethal high 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. basic breeding population introduction
The mature parent of wild urechis unicinctus is caught in the Qinghai island sea area, and an individual which has good physiological condition, rich kidney tubes, pink body wall, no damage and quick response to external stimulus is transported to the urechis unicinctus biological technology limited company in Qingdao island sea at low temperature to serve as a basic breeding group for carrying out passage operation.
Construction of F1 generation family and cultivation of seedlings
In the basic breeding population, the urechis unicinctus parents with body weight 15-20% greater than average weight and good gonad development and strong vitality are selected for in-vitro insemination and passage, and 24 isocytoblast families (8 female x 24 female) of urechis unicinctus are established by adopting nest mating design. The parents are cultivated in a recovery way under a seawater environment for 12 hours according to the classification of male and female, all the parents are dissected after the vitality of the parents is recovered, the dissected kidney tubes are cut and placed in a 50ml centrifuge tube, diluted by seawater and sprayed into a cultivation box (length, width, height: 60cm, 50cm, 35 cm) for fertilization and hatching, the cultivation is continued for 25 days (18-19.5 ℃) before the cultivation is converted into an abnormal benthonic living, the abnormal benthonic living is transferred into a specific area of a cultivation pond for cultivation until the abnormal benthonic living is 100 days old (18-20 ℃), and after the emergence size is reached, the larvae of each family are filtered, taken out and transferred into an outdoor pond (19-23 ℃) for subsequent cultivation. In order to avoid sample mixing among families in the cultivation process, the hatching process of the incubator is independent box 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. The culture process adopts basic method culture of twice feeding/day (feeding spirulina liquid during indoor culture pond culture period and feeding filtered spirulina powder during outdoor pond culture period), once water changing/day (water changing volume is half of culture pond or pond volume), and once bottom sucking/3 days, 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.
3. Calculation of high salt stress semi-lethal conditions
After F1 offspring seeds are cultivated to 200 days old in an outdoor pond, an acute high-salt stress experiment is adopted to explore the semi-lethal salinity value of urechis unicinctus for 72 hours under the high-salt stress condition. The high salinity gradient experimental groups are 47.5 permillage, 50 permillage, 52.5 permillage and 55 permillage, and the experimental seawater is obtained by dissolving sea salt in the culture seawater. Four high salinity gradient experimental groups and a normal seawater control group (32%o) were set, each group was set with three parallels, each parallel had 10 worms involved in stress (19-19 ℃ C.). Continuously attacking the insects for 120 hours, recording death conditions of the insects every 8 hours, and counting survival rates of the insects at each time node under different high salinity gradient conditions (figure 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 53 per mill of 72h under the condition of high salinity stress of urechis unicinctus.
4. High salinity stress
60 insects in each family are randomly selected, weighed and placed in an independent incubator for subsequent stress exploration. And 20 incubators are used for culturing, and the interior of each incubator is isolated by a miniature isolating plate, so that each insect body is ensured to be in one area. Stress is carried out by utilizing the explored seawater with the semi-lethal salinity of 72 hours, all incubators are placed in a culture pond for culture (19-20 ℃), stress is continued until all individuals die, and the survival time of each individual is counted by observing every 4 hours. 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 high-salt experimental group is used for changing 53 permillage of salinity seawater, and the control group is used for changing 32 permillage of salinity normal seawater). The phenotypic parameters of the individuals and the families in the whole experimental system are shown in tables 1 and 2. Can be seen that the survival time of urechis unicinctus bodies in the stress system has larger difference: the difference on individual level is remarkable, the minimum survival time is 36h, the maximum survival time is 440h, and the average survival time of each urechis unicinctus individual is 236.5933 +/-3.0796 h (table 1); the survival time of urechis unicinctus bodies also has a large difference on the family level, the minimum survival time is 112.5333 +/-8.2374 hours, and the maximum survival time is 381.2667 +/-6.0763 hours (table 2). Likewise, for the body weight parameters, a significant difference between the different families can be seen in tables 1 and 2. By using the survival time data and the weight data of different father-unit cell families and mother-unit isotactic cell families of urechis unicinctus, the diagram analysis can be seen between the father-unit half-unit cell families: the salt tolerance of the families of the No. 4 half sibling, the No. 7 half sibling and the No. 8 half sibling is stronger (figure 3); between maternal isotactic families: the salt tolerance of the families of the No. 4-1 whole siblings, the No. 7-3 whole siblings and the No. 8-3 isotactic siblings is stronger (figure 4), which shows that the families are better in high salinity tolerance, so the families are primarily marked.
TABLE 1 tolerance survival time and weight trait phenotype data for urechis unicinctus individuals under 72h semi-lethal high salinity stress conditions
| Traits (3) | Mean value of | Standard error of | Minimum value | Maximum value | Coefficient of variation/% |
| Weight/g | 0.8368 | 0.0143 | 0.2334 | 3.7334 | 59.02％ |
| Survival time/h | 236.5933 | 3.0796 | 36 | 440 | 45.09％ |
TABLE 2 tolerance survival time and weight trait phenotype data (mean.+ -. Standard error) for urechis unicinctus each isotactic cell line under semi-lethal high salinity stress conditions 72h
5. Salinity tolerance and weight genetic parameter determination
And carrying out genetic parameter analysis by using the survival time and the weight value of the urechis unicinctus individuals counted in the high salinity stress process. Establishing a high salinity tolerance genetic parameter estimation model: y is i ＝μ+α i +е i
Wherein y is i For the high salt tolerance survival time of the ith test individual, μ is the mean, α, of the high salt tolerance survival times of all test individuals i For the i-th test individual to have a high salt-tolerant survival time additive genetic effect, e.g. i High salt resistant survival time random residuals for the ith test individual.
The genetic force calculation formula of the animal model is as follows:
wherein,for high salt-tolerant survival time additive genetic variance, < >>Is the variance of the high salt-tolerant time-to-live residual. The weight genetic force calculation method is the same as that described above.
At the same time, genetic and phenotypic correlation between high 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 high salt tolerance and body weight traits,and->Is the additive genetic variance of the two traits. In calculating phenotype correlations, σ xy Is the phenotypic covariance between the high salt tolerance trait and the body weight trait, +.>And->Is the phenotypic variance.
The method is characterized by comprising the following steps of: the genetic power estimated values of the high salinity stress survival trait and the weight trait of urechis unicinctus are 0.7323 +/-0.2169 and 0.7244 +/-0.1559 respectively, which belong to the higher genetic power level, and show that the two traits have the potential of further breeding (Table 3). In addition, the survival time and the variation coefficient of the body weight are 45.09% and 59.02% (table 1) respectively, and the high variation coefficient range further shows that the breeding potential of the salt tolerance and the body weight characteristics is very high. The genetic and phenotype correlation coefficients between the high salt tolerance and the body weight traits of urechis unicinctus are 0.3914 +/-0.1597 and 0.3803 +/-0.0796 (table 3), respectively, which indicates that indirect selection can be performed between the two traits.
Table 372h tolerance survival time and weight trait genetic parameters of urechis unicinctus under semi-lethal high salinity stress conditions (genetic correlation on diagonal, phenotypic correlation under diagonal)
| Traits (3) | High salt | Weight of body |
| High salt | 0.7323±0.2169 | 0.3914±0.1597 |
| Weight of body | 0.3803±0.0796 | 0.7244±0.1559 |
Seed reserving of F1 generation parent
According to the high-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 high salt stress (Table 4). Sequencing by using comprehensive breeding values, and screening to obtain families ranked in the top 5 families as follows: 7-3, 7-2, 8-3, 4-1, 4-2 (ranked in order of ranking) can be found to contain families that were initially determined based on phenotypic data. And (5) taking the selected five families as high-salt breeding candidate families, and continuing to cultivate the families into adults according to the seed reserving rate of 3-5%.
Table 4 72h high salt tolerance trait, weight trait and weighted comprehensive breeding value of urechis unicinctus of each isotactic cell line under semi-lethal high salinity stress condition
F2 generation family construction and subculture selection
And (3) breeding individuals reserved in the five high-salt breeding candidate families screened by the F1 generation until the gonad maturation period, selecting the strong urechis unicinctus male and female individuals with large individuals and strong vitality, and carrying out pairing breeding between the same family source 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 high salt tolerance and excellent weight property as breeding indexes, and healthy male and female urechis unicinctus individuals with far relativity, excellent weight property and pink body color are selected from the finally obtained family to be industrially popularized and applied as excellent strains.
The above embodiments are only for illustrating the technical solution of the present invention, and are not limited thereto. Modifications and substitutions of some of the technical means described in the above embodiments may be made by those skilled in the art, and these modifications and substitutions fall within the scope of the present invention.
Claims (5)
1. A breeding method of a new urechis unicinctus strain tolerant to high salt is characterized by comprising the following steps:
1) Basic breeding population introduction: taking multiple tails of wild urechis unicinctus groups from natural sea areas, and selecting urechis unicinctus individuals with excellent phenotypic characters and rich renal tubes as basic breeding groups;
2) F1 generation family construction and offspring cultivation: selecting a urechis unicinctus adult with mature gonad development and full kidney tube, and establishing an F1-generation male parent half-sibling family of urechis unicinctus and a female isotactic-sibling family of urechis unicinctus by adopting a nest mating design method (one male mating and three female); culturing factory seedlings after hatching the larvae of each family of the F1 generation;
3) Calculation of high salt stress semi-lethal conditions: after the F1 generation offspring seed is cultivated to 200 days old, an acute toxicity attack experiment is adopted to explore the salinity value of the semi-lethal for 72 hours under the condition of high salt stress of urechis unicinctus; setting the high salinity gradient experimental group to be 47.5 permillage, 50 permillage, 52.5 permillage and 55 permillage, wherein the experimental seawater is obtained by dissolving sea salt in the culture seawater; a high salinity gradient experiment group and a normal sea water control group (32%o) are arranged together, and three groups are arranged in parallel; continuously attacking for 120 hours, recording death conditions of the insects every 8 hours, and calculating semi-lethal salinity by using a linear regression method according to the survival rate of 72 hours under each salinity gradient;
4) High salinity stress: stress is carried out by utilizing the explored seawater with the half-lethal high salinity value for 72 hours; in the high salinity stress process, 60 insects in each family are randomly selected, the weight of the insects is weighed, the insects are placed in independent incubators (the incubators are isolated by miniature isolating plates to ensure that each insect is in one area) for stress, all the incubators are placed in one culture pond and are uniformly cultured by using semi-lethal high salinity seawater, and the survival time of each individual is counted;
5) Salinity tolerance and body weight genetic parameter determination: carrying out genetic parameter analysis by using the survival time and the weight value of the urechis unicinctus individuals counted in the high salinity stress process; determining salt tolerance genetic force according to individual survival time under salinity stress, and estimating individual salinity tolerance breeding value and family salinity tolerance average breeding value according to the salt tolerance genetic force; determining the genetic transmission of the body weight character according to the body weight of the test individual, and estimating the body weight breeding value of the individual and the average breeding value of the family body weight according to the genetic transmission of the body weight character; the estimation of the breeding value is performed by using a BLUP method (best linear unbiased estimation method); estimating genetic and phenotypic correlations between traits based on salinity stress survival time and weight trait parameters;
for the high-salinity tolerance character, establishing a high-salinity tolerance genetic parameter estimation model: y is i ＝μ+α i +е i ；
Wherein y is i For the high salt tolerance survival time of the ith test individual, μ is the mean, α, of the high salt tolerance survival times of all test individuals i For the i-th test individual to have a high salt-tolerant survival time additive genetic effect, e.g. i High salt resistant survival time random residuals for the ith test individual;
the genetic force calculation formula of the animal model is as follows:
wherein,for high salt-tolerant survival time additive genetic variance, < >>Residual variance, which is high salt-tolerant time-to-live; weight genetic force calculation method is the same as that above;
at the same time, genetic and phenotypic correlation between high salt tolerance and body weight traits (r x，y ) The calculation is as follows:
in calculating genetic correlations, σ xy For additive genetic covariance between high salt tolerance and body weight traits,and->Additive genetic variance of two traits; in calculating phenotype correlations, σ xy Is the phenotypic covariance between the high salt tolerance trait and the body weight trait, +.>And->Is the phenotypic variance;
6) And F1, parent seed reserving: weighting the breeding values of the average salt resistance and the weight character of each family according to the proportion of 1:1 to obtain comprehensive breeding values of all families under the high-salt condition; sequencing by using comprehensive breeding values, reserving families ranked in the top 5 as high-salt breeding candidate families, and continuously breeding the selected families to adults according to a seed reserving rate of 3-5%;
7) F2 generation family construction and subculture selection: breeding high-salt breeding candidate families of F1 generation reserved seeds until the gonad maturation period, screening robust urechis unicinctus individuals with large individuals and strong vitality from the high-salt breeding candidate families, carrying out pairing breeding on the urechis unicinctus individuals with the same family sources and different family sources to construct F2 generation families, and controlling the inbreeding coefficient within 0.1; and then, continuously selecting F2 generation families by using high 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 the cultivation of the family seedlings in step 2) is divided into three stages: (1) each family was incubated in a separate incubator before insemination was carried out until transformation to an allergic benthonic life (about 25 days, 18-19.5 ℃); (2) the method comprises the steps of transferring to an indoor culture pond for cultivation in a period from the metamorphosis benthonic life to 100 days of age, and uniformly dividing the culture pond into different areas with equal area by using 100-mesh bolting silk for cultivation; (3) transferring the culture process of 100-200 days old to an outdoor pond, and dividing the pond into areas with the same area by using 20-mesh bolting silk for culture; note that each time a family individual transfer between different culture environments is performed, the culture densities of all families need to be controlled at 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 high salt stress semi-lethal condition screening process of step 3) performs a gradient stress experiment for randomly selecting individuals throughout the family system; three parallels are arranged on each salinity condition, and at least 10 insects participate in the stress process under each parallels; the 72h semi-lethal high salinity obtained by exploration is 53 per mill.
4. The method of claim 1, wherein in step 4) for high salinity stress conditions, the high salinity stress test group and the control group are treated in a single culture pond; observing survival condition every 4 hours, fishing out dead individuals, 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, 53 permillage of salinity seawater is selected for replacement when water is changed (32 permillage of salinity 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 parameters of the high salinity tolerance trait and the body weight trait in step 5) are both processed using ASReml software.
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