Breeding method of new single-cyclone nereis virens line with high salt tolerance

Abstract

The application discloses a breeding method of a new single-cycle spine eel line tolerating high salt, and comprises the following steps: (1) introducing a basic breeding population; (2) constructing a F1 generation family and cultivating seedlings; (3) calculating a high-salt stress semi-lethal condition; (4) stressing by using the high-salt stress semi-lethal condition and counting survival time data; (5) establishing a genetic evaluation model to estimate high-salt tolerance traits and body weight traits breeding values; (6) determining a comprehensive breeding value according to the two trait breeding values and screening out excellent F1 generation families for seed saving; and (7) constructing a F2 generation family and performing subculture selection by using the seed-saving high-salt breeding candidate family. By using the method, a new single-cycle spine eel line with high high-salt tolerance and growth trait advantages can be selected and bred, and the problems of low survival rate and poor economic indexes of the single-cycle spine eel in high-salt water area breeding in the prior art are solved, thereby seriously restricting the development of the industry.

Description

Technical Field

[0001] This invention belongs to the field of aquatic genetics and breeding, specifically relating to a breeding method for a new strain of *Ulva monocyclic urticaria* that is tolerant to high salt content. Background Technology

[0002] Uni-ringed thorn beetle ( Urechis unicinctus *Ulva unicinctus* is a type of annelid that lives in U-shaped caves in the intertidal zone, mainly distributed in Russia, North Korea, Japan, and the Yellow and Bohai Sea coasts of China. It is rich in essential amino acids, glycosaminoglycans, and unsaturated fatty acids, giving it high nutritional value. Furthermore, it possesses active substances such as polypeptides, fibrinolytic enzymes, and macromolecular proteins with antioxidant, thrombolytic, and in vitro antitumor effects, indicating potential medicinal value. *Ulva unicinctus* has a delicious flavor and was recognized as one of the top ten excellent aquatic germplasm resources in 2022, showing broad prospects for industrial development. In recent years, overfishing of wild *Ulva unicinctus* resources has led to a sharp decline in its yield and a significant increase in market price, reaching as high as 200 yuan / kg, highlighting the growing supply-demand imbalance. On the other hand, artificial breeding and cultivation of *Ulva unicinctus* are currently in a rapid development stage, with the main breeding method being the capture of wild populations as parent stock, without prior genetic improvement. Breeding superior varieties is one of the key factors for the sustainable and rapid development of the *Ulva unicornu* aquaculture industry. By cultivating superior varieties with strong resistance through artificial targeted breeding, the ability of *Ulva unicornu* to resist environmental stress can be enhanced, and the survival rate of aquaculture can be improved.

[0003] Salinity is a crucial environmental factor for the growth, metabolism, and reproduction of aquatic organisms, influencing enzyme activity, energy allocation, metabolic rate, and the secretion and distribution of related hormones. Therefore, adapting aquatic organisms to a wide range of salinities requires significant physiological resources. As an important physicochemical factor in the aquaculture waters of *Ulva monocyclicis*, natural phenomena such as high temperatures and droughts, seasonal rainfall, and tidal activity cause fluctuations in seawater salinity in the aquaculture area. These salinity changes affect the physiological and biochemical responses of *Ulva monocyclicis*. For example, low osmotic pressure environments easily lead to cell swelling due to water absorption, while high osmotic pressure environments affect the activity of osmotic and immunomodulatory enzymes. Existing research indicates that appropriate breeding screening methods and salinity acclimatization models can significantly improve the survival rate of aquatic organisms in high-salinity environments. Furthermore, my country possesses abundant high-salinity water resources along the coasts of the Yellow and Bohai Seas, with the area of ​​high-salinity waters along the Liaodong and Shandong Peninsulas alone reaching 1300 km². 2High-salinity waters are widely distributed, but most areas have not yet been industrialized. *Ulva unicinctus* has high economic value and a wide salinity tolerance range, making it a promising aquatic species for high-salinity aquaculture development. Screening and breeding superior new *Ulva unicinctus* varieties with strong high-salinity tolerance and fast growth rates through quantitative genetics is of great significance for expanding the development space of artificial aquaculture of *Ulva unicinctus*, promoting the high-value utilization of high-salinity resources along the Yellow and Bohai Seas in my country, and increasing the scale of the artificial aquaculture industry of *Ulva unicinctus*. Summary of the Invention

[0004] The purpose of this invention is to address the current lack of high-salt-tolerant new varieties in the *Ulva monocyclicis* aquaculture industry by providing a breeding method for a new high-salt-tolerant *Ulva monocyclicis* strain. This method can cultivate new *Ulva monocyclicis* strains with excellent growth traits and strong high-salt resistance, laying a solid foundation for the rapid, stable, and sustainable development of the *Ulva monocyclicis* industry.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A breeding method for a new high-salt-tolerant monocyclic urchin strain, characterized by the following steps:

[0007] 1) Introduction of basic breeding population: Multiple individuals of wild Ulva unicinctus population from natural sea areas were selected, and individuals with excellent phenotypic traits and abundant nephridia were selected as basic breeding population;

[0008] 2) F1 generation family construction and seedling cultivation: Adult *Ulva unicinctus* with mature gonads and full nephridia were selected, and nesting mating design (one male to three females) was used to establish paternal half-sib families and maternal full-sib families for the F1 generation. After hatching, the larvae of each F1 generation family were cultured in a factory-scale manner.

[0009] 3) Calculation of the half-lethal salinity under high salinity stress: After F1 generation seedlings reached 200 days of age, an acute challenge experiment was conducted to investigate the 72-hour half-lethal salinity value of *Ulva uniflora* under high salinity stress. High salinity gradient experimental groups were set up at 47.5‰, 50‰, 52.5‰, and 55‰. The experimental seawater was obtained by dissolving sea salt in the culture seawater. A total of three replicates were set up for each high salinity gradient experimental group and one normal seawater control group (32‰). The challenge was continued for 120 hours, with insect mortality recorded every 8 hours. The half-lethal salinity was calculated using linear regression based on the survival rate at 72 hours under each salinity gradient.

[0010] 4) High salinity stress: Seawater with a 72-hour semi-lethal high salinity value obtained from the study was used for stress. During the high salinity stress process, 60 parasites were randomly selected from each family, weighed, and placed in independent incubators (each incubator was isolated with a miniature isolation plate to ensure that each parasite was in one area) for stress. All incubators were placed in one culture tank and cultured uniformly using semi-lethal high salinity seawater. The survival time of each individual was recorded.

[0011] 5) Determination of salinity tolerance and body weight genetic parameters: Genetic parameter analysis was performed using the survival time and body weight of individuals of *Urtica uniflora* during high salinity stress. The heritability of salt tolerance was determined based on individual survival time under salinity stress, and the breeding value of individual salinity tolerance and the average breeding value of family salinity tolerance were estimated based on this heritability. The heritability of body weight was determined based on the body weight of the tested individuals, and the breeding value of individual body weight and the average breeding value of family body weight were estimated based on this heritability. The breeding value was estimated using the BLUP method (best linear unbiased estimation method). Genetic and phenotypic correlations between traits were estimated based on salinity stress survival time and body weight trait parameters.

[0012] For the trait of high salt tolerance, establish a genetic parameter estimation model for high salt tolerance: y i = μ+α i +e i

[0013] in y i Let be the high salt tolerance survival time of the i-th test individual. μ This represents the mean high-salt tolerance survival time for all tested individuals. α i The high salt tolerance survival time of the i-th test individual is an additive genetic effect. e i Let be the random residual of the high salt tolerance survival time of the i-th test individual.

[0014] The formula for calculating the heritability of the above animal models is as follows:

[0015] h 2 =

[0016] in, The additive genetic variance of high salt tolerance survival time. This represents the variance of the residuals for high-salt tolerance survival time. The method for calculating the heritability of body weight is the same as above.

[0017] Meanwhile, there are genetic and phenotypic correlations between high salt tolerance and body weight traits. r x，yThe calculation is as follows:

[0018] r x, y =

[0019] When calculating genetic correlations, The additive genetic covariance between the high salt tolerance trait and the body weight trait. and This represents the additive genetic variance of the two traits. When calculating phenotypic correlations, The phenotypic covariance between the high salt tolerance trait and the body weight trait. and For phenotypic variance;

[0020] 6) F1 generation parental breeding: The average salt tolerance trait and body weight trait of each family are weighted at a 1:1 ratio to obtain the comprehensive breeding value of all families under high salt conditions. The comprehensive breeding values ​​are used to rank the families, and the top 5 families are retained as candidate families for high salt breeding. The selected families are then raised to adulthood at a retention rate of 3-5%.

[0021] 7) F2 Generation Family Construction and Subsequent Selection: For the high salt tolerance trait, F1 generation high salt breeding candidate families were cultured to gonadal maturity. Large, vigorous, and robust *Ulva unicinctus* individuals were selected for breeding with both individuals from the same and different family lines to construct F2 generation families, with the inbreeding coefficient controlled below 0.1. Subsequently, using strong high salt tolerance and excellent body weight as selection criteria, 4-5 generations of continuous selection were conducted on the F2 generation families. From the final obtained families, healthy *Ulva unicinctus* males and females with distant kinship, large size, and pink body color were selected as superior strains for industrial promotion and application.

[0022] Preferably, the wild *Ulva unicinctus* population in the natural marine environment in step 1) is a wild population in the Qinhuangdao sea area, which is artificially caught and transported at low temperature to the breeding base for selection.

[0023] Preferably, in step 2), the family seedling cultivation process involves pairing parents using in vitro fertilization (IVF). The parents are dissected, and their renal tubes are collected in a dry, clean dissection tray. After all parents have been dissected, the renal tubes are cut into small pieces, diluted with seawater, and sprinkled into the rearing tank for fertilization and hatching. After hatching, before transitioning to a metamorphic benthic lifestyle (25 days, 18-19.5℃), the larvae are transferred to a specific area of ​​the rearing pond for further rearing. Once the larvae reach hatching size (100 days, 18-20℃), the larvae from each family are filtered and transferred to an outdoor pond for further rearing (200 days, 19-23℃). Throughout the rearing process, different families are isolated using silk screens to prevent cross-contamination. The rearing process employs a basic method of two feedings per day (spirulina solution during indoor rearing, and filtered spirulina powder during outdoor pond rearing), one water change per day (replacing half the volume of the rearing pond), and one bottom suction every 3 days. Adjustments are made as needed based on specific circumstances.

[0024] Preferably, in step 3), the screening process for the semi-lethal condition of high salinity stress involves randomly selecting individuals from all families to conduct gradient stress experiments. Three replicates are set up for each salinity gradient, with at least 10 insects participating in the stress process in each replicate. Observations are conducted every 8 hours during the study, and dead individuals are promptly removed. The semi-lethal high salinity at 72 hours was determined to be 53‰.

[0025] Preferably, the high salinity stress investigation system in step 4) is conducted in a separate culture tank, with the seawater in the culture environment at 53‰. During this period, the survival status is observed every 4 hours and dead individuals are removed in a timely manner. A small amount of food is fed once a day (the feeding is finely adjusted according to the number of surviving insects and their vitality status) and a water change is performed once a day (the water volume is half of the culture tank volume; the high salinity experimental group is replaced with 53‰ salinity seawater, and the control group is replaced with normal salinity seawater at 32‰).

[0026] Preferably, the determination of genetic parameters for high salt tolerance and weight traits in step 5) is performed using ASReml software.

[0027] Preferably, in step 6), the F1 generation parental lineages retained are the families ranked in the top 5 in terms of comprehensive breeding value as candidate families for high-salt breeding. The selected families are then raised to adulthood at a retention rate of 3-5%, and the entire breeding process is carried out in outdoor ponds.

[0028] Preferably, the family breeding method in step 7) for F2 generation family selection and subsequent generation selection is consistent with the in vitro fertilization operation for F1 generation. F2 generation families are constructed by pairing and breeding individuals from the same and different family origins, with the inbreeding coefficient controlled below 0.1. Continuous selection is conducted using strong high-salt tolerance and excellent body weight traits as breeding indicators.

[0029] In summary, this invention uses a wild *Urtica monocyclicis* population as the base population and selects families with significant potential for genetic improvement and breeding through high-salt tolerance selection. The superior families are then propagated to obtain new strains with excellent growth traits and strong high-salt tolerance. This breeding method is simple, practical, and highly operable. Through multiple generations of selection, superior *Urtica monocyclicis* varieties with excellent high-salt tolerance can be developed. Attached Figure Description

[0030] Figure 1 Survival curves of *Ulva unicornu* under different high salinity stress conditions

[0031] Figure 2 Survival curves of *Ulva unicinctus* at 72 hours under different high salinity stress conditions.

[0032] Figure 3 Weight and survival time of a paternal half-sib family of *Ulva occulta* under 72-hour half-lethal high salinity stress.

[0033] Figure 4 Weight and survival time of maternal full-sib families of *Ulva occulta* under 72-hour semi-lethal high salinity stress. Detailed Implementation

[0034] To more clearly explain the objectives and technical details of this invention, the technical solution of this invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0035] 1. Introduction of basic breeding population

[0036] Mature wild *Ulva unicinctus* parents were caught in the waters off Qinhuangdao. Individuals with good physiological condition, abundant nephridia, pink and undamaged body walls, and the ability to respond quickly to external stimuli were selected and transported at low temperature to Qingdao Dayou Haichuang Biotechnology Co., Ltd. as the basic breeding population for passage.

[0037] 2. F1 generation family construction and seedling cultivation

[0038] From the basic breeding population, parents of *Ulva unicinctus* with a weight 15-20% greater than the average weight and with well-developed and vigorous gonads were selected for in vitro fertilization and propagation. A nested mating design was used to establish 24 full-sib families (8 males × 24 females). The parents were separated by sex and cultured in a seawater environment for 12 hours to allow for recovery. After the parents regained vigor, all parents were dissected, and the nephridia obtained were cut into small pieces and placed in 50ml centrifuge tubes. The nephridia were diluted with seawater and then poured into a rearing box (length × width × height: 60cm × 50cm × 35cm) for fertilization and hatching. After hatching, the larvae were cultured for approximately 25 days (18-19.5℃) before transitioning to a benthic lifestyle and then transferred to a specific area of ​​a rearing pond for rearing until 100 days of age (18-20℃). Once the larvae reached hatching size, the larvae from each family were filtered and transferred to an outdoor pond (19-23℃) for further rearing. To avoid cross-contamination between families during the cultivation process, each family was individually housed in an incubator, separated in the culture ponds using 100-mesh silk sieves, and in the outdoor ponds using 20-mesh silk sieves. The cultivation process employed a basic method: two feedings per day (spirulina solution in indoor culture ponds and filtered spirulina powder in outdoor ponds), one water change per day (half the volume of the culture pond or pond), and bottom suction every 3 days. Adjustments were made as needed based on specific circumstances. Due to sudden environmental changes and human error during cultivation, three full-sib families were lost, resulting in 20 families subsequently experiencing salinity stress.

[0039] 3. Calculation of the half-lethal condition of high salt stress

[0040] After F1 generation seedlings were raised to 200 days old in outdoor ponds, an acute high-salt stress experiment was conducted to investigate the 72-hour median lethal salinity of *Ulva unicinctus* under high-salt stress conditions. High-salt gradient experimental groups were set up at 47.5‰, 50‰, 52.5‰, and 55‰. The experimental seawater was obtained by dissolving sea salt in the culture seawater. Four high-salt gradient experimental groups and one normal seawater control group (32‰) were set up. Each group had three replicates, with 10 insects participating in the stress (19-19.5℃) in each replicate. The challenge was continued for 120 hours, and insect mortality was recorded every 8 hours. The survival rate of insects at each time point under different high-salt gradient conditions was statistically analyzed. Figure 1 The semi-lethal salinity survival curve equation was calculated using linear regression to determine the survival rate at 72 hours under various salinity gradients. Figure 2 The half-lethal salinity value of *Ulva moniliforme* under high salinity stress was calculated to be 53‰ at 72 hours using the trend line equation.

[0041] 4. High salinity stress

[0042] Sixty individuals from each family were randomly selected, weighed, and placed in independent incubators for subsequent stress investigation. A total of 20 incubators were used for cultivation, with each incubator isolated by a miniature partition to ensure that each individual incubator was in a separate area. The individuals were subjected to stress using seawater with a 72-hour median lethal salinity obtained during the initial investigation. All incubators were placed in a single culture tank (19-20℃) and the stress was continued until all individuals died. Observations were conducted every 4 hours to record the survival time of each individual. Throughout the stress period, a small amount of food was fed daily (the amount was adjusted based on the number of surviving individuals and their activity level) and a water change was performed once daily (the water volume was half the volume of the culture tank; the high-salinity experimental group received seawater with a salinity of 53‰, while the control group received seawater with a salinity of 32‰). The phenotypic parameters of individuals and families within the entire experimental system are shown in Tables 1 and 2. It can be seen that the survival time of *Ulva unicornu* within the stress system varies considerably: Significant differences exist at the individual level, with a minimum survival time of 36 hours and a maximum survival time of 440 hours, resulting in an average survival time of 236.5933 ± 3.0796 hours per individual (Table 1); at the family level, the survival time also varies considerably, with a minimum survival time of 112.5333 ± 8.2374 hours and a maximum survival time of 381.2667 ± 6.0763 hours (Table 2). Similarly, significant differences in body weight are also observed between different families in Tables 1 and 2. Graphical analysis using survival time and body weight data from different paternal half-sib and maternal full-sib families of *Ulva unicornu* reveals that among paternal half-sib families, families 4, 7, and 8 exhibit stronger salt tolerance. Figure 3 Among maternal full-sib families: families with full siblings 4-1, 7-3, and 8-3 showed stronger salt tolerance. Figure 4 This indicates that the above families have a better ability to tolerate high salinity, so the above families were initially labeled.

[0043] Table 1. Individual survival time and weight phenotypic data of *Ulva monocranium* under 72-hour semi-lethal high salinity stress.

[0044] Properties mean Standard error Minimum value Maximum value Coefficient of variation / % body weight / g 0.8368 0.0143 0.2334 3.7334 59.02% Survival time / h 236.5933 3.0796 36 440 45.09%

[0045] Table 2. Survival time and weight phenotypic data of full-sib families of *Urtica monocyclic* under 72-hour semi-lethal high salinity stress (mean ± standard error).

[0046] Family name High salt tolerance survival time / h body weight / g 1-1 129.0667±8.5536 0.6337±0.0261 1-2 232.7333±8.8132 2.1496±0.0809 1-3 221.2000±9.7658 0.8041±0.0315 2-1 166.2000±9.5036 0.6158±0.0167 2-2 179.6000±8.9794 0.6054±0.0218 2-3 151.0667±8.7962 0.5053±0.0171 3-1 112.5333±8.2374 0.4783±0.0249 3-2 196.2000±10.4638 0.5295±0.0241 3-3 254.4667±7.7463 0.7041±0.0272 4-1 381.2667±6.0763 1.6251±0.0581 4-2 327.6000±11.8467 1.1553±0.0362 4-3 266.8667±9.6656 0.6993±0.0186 5-1 120.9333±9.7449 0.5725±0.0272 5-2 187.5333±10.9074 0.6278±0.0276 5-3 228.8667±8.7455 0.6452±0.0297 7-2 305.3333±9.4110 0.6546±0.0267 7-3 341.2667±9.3979 0.6730±0.0223 8-1 281.2667±10.1552 0.9419±0.0454 8-2 298.4667±8.4464 0.8913±0.0342 8-3 349.4000±9.6763 1.2250±0.0549

[0047] 5. Determination of genetic parameters for salinity tolerance and body weight

[0048] Genetic parameters were analyzed using the survival time and body weight of *Urtica uniflora* individuals collected during the high salinity stress process. A genetic parameter estimation model for high salinity tolerance was established. y i = μ+α i +e i

[0049] in y i Let be the high salt tolerance survival time of the i-th test individual. μ This represents the mean high-salt tolerance survival time for all tested individuals. α i The high salt tolerance survival time of the i-th test individual is an additive genetic effect. e i Let be the random residual of the high salt tolerance survival time of the i-th test individual.

[0050] The formula for calculating the heritability of this animal model is as follows:

[0051] h 2 =

[0052] in, The additive genetic variance of high salt tolerance survival time. This represents the variance of the residuals for high-salt tolerance survival time. The method for calculating the heritability of body weight is the same as above.

[0053] Meanwhile, there are genetic and phenotypic correlations between high salt tolerance and body weight traits. r x，y The calculation formula is as follows:

[0054] r x, y =

[0055] When calculating genetic correlations, The additive genetic covariance between the high salt tolerance trait and the body weight trait. and This represents the additive genetic variance of the two traits. When calculating phenotypic correlations, The phenotypic covariance between the high salt tolerance trait and the body weight trait. and This represents the phenotypic variance.

[0056] Calculations and analysis revealed that the heritability estimates for the high-salinity stress survival and weight traits in *Ulva monocyclicis* were 0.7323±0.2169 and 0.7244±0.1559, respectively, indicating a high level of heritability and suggesting potential for further selective breeding of these traits (Table 3). Furthermore, the coefficients of variation for survival time and weight were 45.09% and 59.02%, respectively (Table 1), falling within the high coefficient of variation range, further indicating significant potential for selective breeding of salt tolerance and weight traits. The genetic and phenotypic correlation coefficients between high-salt tolerance and weight traits in *Ulva monocyclicis* were 0.3914±0.1597 and 0.3803±0.0796, respectively (Table 3), indicating that indirect selection can be performed between the two traits.

[0057] Table 3. Genetic parameters of tolerance survival time and weight traits of *Ulva uniflora* under 72-hour semi-lethal high salinity stress (diagonal lines represent heritability, lines above the diagonal represent genetic correlation, lines below the diagonal represent phenotypic correlation).

[0058] Properties High salt weight High salt 0.7323±0.2169 0.3914±0.1597 weight 0.3803±0.0796 0.7244±0.1559

[0059] 6. F1 generation parental seed preservation

[0060] Based on the heritability of high salt tolerance and weight traits, the individual breeding value of *Ulva unicinctus* was estimated using the BLUP method (best linear unbiased estimation method), and then the average breeding value of families was determined based on the individual breeding values. The average breeding values ​​of salt tolerance and weight traits of each family were weighted at a 1:1 ratio to obtain the comprehensive breeding value of all families under high salt stress (Table 4). Using the comprehensive breeding values, the top 5 families were selected as follows: 7-3, 7-2, 8-3, 4-1, and 4-2 (arranged in order of ranking). These five families included those initially identified based on phenotypic data. These five selected families were designated as candidate families for high salt breeding, and were continued to be cultured to adulthood at a retention rate of 3-5%.

[0061] Table 4. High salt tolerance traits, body weight, and weighted comprehensive breeding values ​​of all full-sib families of *Ulva monocyclicis* under 72-hour semi-lethal high salinity stress.

[0062] Family name High salt tolerance breeding value Body weight breeding value Weighted average breeding value 1-1 -92.6681 0.0004 -46.3339 1-2 -122.2293 0.5898 -60.8197 1-3 -15.6458 -0.0042 -7.8250 2-1 -54.0170 -0.0160 -27.0165 2-2 -39.7424 -0.1217 -19.9320 2-3 -59.4318 -0.1660 -29.7989 3-1 -95.4402 -0.3237 -47.8820 3-2 -16.4904 -0.1686 -8.3295 3-3 26.3115 -0.1699 13.0708 4-1 72.2136 0.3299 36.2717 4-2 59.8831 0.2498 30.0665 4-3 39.2700 -0.0975 19.5862 5-1 -95.3500 -0.2292 -47.7896 5-2 -33.7743 -0.1257 -16.9500 5-3 5.9213 -0.0205 2.9504 7-2 81.6108 -0.1863 40.7123 7-3 115.8433 -0.1106 57.8664 8-1 32.3621 0.2081 16.2851 8-2 53.9538 0.2145 27.0842 8-3 75.6537 0.2455 37.9496

[0063] 7. F2 Generation Family Structure and Succession Selection

[0064] Individuals from five high-salt breeding candidate families selected in the F1 generation were cultured to gonadal maturity. Large, vigorous, and robust *Ulva unicinctus* individuals were then selected for breeding between individuals from the same and different family lines to construct the F2 generation, with the inbreeding coefficient controlled below 0.1. Subsequent selection processes involved 4-5 generations of continuous selection in the F2 generation, using strong high-salt tolerance and excellent weight traits as selection indicators. From the final families, healthy *Ulva unicinctus* individuals with distant kinship, excellent weight traits, and pink body color were selected as superior strains for industrial promotion and application.

[0065] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Those skilled in the art can modify the technical solutions described in the above embodiments or replace some of the technical means, and such modifications or substitutions will all fall within the protection scope of the present invention.

Claims

1. A breeding method of a new line of single-cyclidium Ucaudatus which is resistant to high salt, characterized in that Its method steps are: 1) Introduction of the basic breeding population: take many tails from the wild single ring stingray population in natural sea area, select single ring stingray individuals with excellent phenotypic traits and rich nephridia as the basic breeding population; 2) F1 generation family construction and seedling cultivation: select single ring stingray adults with mature gonads and full nephridia, and use nest mating design method to establish F1 generation paternal half-sib families and maternal full-sib families; After the larvae of F1 generation families are hatched, factory seedling cultivation is carried out; 3) Calculation of high salt stress semi-lethal condition: after F1 generation seedlings are cultivated to 200 days old, acute challenge experiment is used to explore the 72h semi-lethal salinity value of single ring stingray under high salt stress condition; set high salinity gradient experimental groups as 47.5‰, 50‰, 52.5‰ and 55‰, and the experimental seawater is obtained by dissolving sea salt in cultivation seawater; a normal seawater control group with salinity of 32‰ is set, and three parallels are set in each group; continue the challenge to 120h, and record the death of the worms every 8h; the semi-lethal salinity is calculated by linear regression method through the survival rate under each salinity gradient for 72h; 4) High salinity stress: use the 72h semi-lethal high salinity value seawater obtained to carry out stress; randomly select 60 worms in each family, weigh them and place them in independent incubators for stress, the incubators are isolated by micro isolation plates to ensure that each worm is in an area, and all incubators are placed in a culture tank and cultured with semi-lethal high salinity seawater, and the survival time of each individual is counted; 5) Determination of salt tolerance and body weight genetic parameters: genetic parameter analysis is carried out by using the survival time and body weight of single ring stingray individuals during high salinity stress; the salt tolerance breeding value of individuals and the average breeding value of family salt tolerance are estimated according to the salt tolerance trait heritability of individuals under salt stress; the body weight breeding value of individuals and the average breeding value of family body weight are estimated according to the body weight trait heritability; the estimation of breeding value is carried out by using BLUP method (best linear unbiased estimation method); the genetic and phenotypic correlation between traits is estimated according to the salt stress survival time and body weight trait parameters; For high salt tolerance traits, a high salinity tolerance genetic parameter estimation model is established: y i = μ+α i +е i ; wherein y i is the high salt survival time for the i-th test individual, μ is the mean high salt survival time for all test individuals, α i is the additive genetic effect for the high salt survival time of the i-th test individual, е i is the random residual for the high salt survival time of the i-th test individual. The heritability calculation formula is as follows: h 2 = wherein, is the additive genetic variance for high salt survival time, is the residual variance for high salt survival time; the heritability of body weight is calculated as above; At the same time, genetic and phenotypic correlations between high salt tolerance and body weight traits were calculated (Table 2). r x，y ) were calculated as follows: r x, y = When calculating genetic correlations, The additive genetic covariance between the high salt tolerance trait and the body weight trait. and This represents the additive genetic variance of the two traits; when calculating phenotypic correlation, The phenotypic covariance between the high salt tolerance trait and the body weight trait. and For phenotypic variance; 6) F1 generation parent seed selection: the average salt resistance trait and body weight trait breeding value of each family are weighted in the proportion of 1:1 to obtain the comprehensive breeding value of all families under high salt condition; the comprehensive breeding value is sorted, and the families ranked in the top 5 are selected as high salt breeding candidate families, and the selected families are further cultivated to adults according to the seed retention rate of 3-5%; 7) F2 generation family construction and subculture selection: the high-salt breeding candidate families of F1 generation were bred to the period of gonadal maturation, and the robust individual single ringed worms with large size and strong vitality were selected to construct F2 generation families by pairing breeding of the same family source and different family sources, and the inbreeding coefficient was controlled within 0.1; then, the high-salt tolerance and excellent body weight traits were selected as the breeding index to carry out continuous selection of 4-5 generations of F2 generation families, and the healthy single ringed worm individuals with distant genetic relationship, large size and pink body color were selected as excellent lines for industrialization and popularization.

2. The method of claim 1, wherein, The family seedling cultivation process in step 2) is divided into three stages: ① continue to culture for 25 days after fertilization and hatching, the temperature is 18-19.5℃, and each family is hatched in a separate incubator before metamorphosis and benthic life; ② indoor aquaculture pond culture from metamorphosis to 100 days old, use 100 mesh gauze to divide the aquaculture pond into different areas with equal area for breeding; ③ transfer the culture process from 100 to 200 days old to outdoor ponds, use 20 mesh gauze to divide the ponds into areas with the same area for breeding; attention should be paid to controlling the stocking density of all families to the same level when transferring individuals between different breeding environments each time; the basic method of twice feeding / day, once water changing / day, and once bottom suction / 3 days is adopted throughout the culture process, and appropriate adjustments are made according to the specific situation during the process.

3. The method of claim 1, wherein, The high-salt stress semi-lethal condition selection process in step 3) is to randomly select individuals within the entire family system to carry out gradient stress experiments; three parallel conditions are set for each salinity, and at least 10 individuals participate in the stress process under each parallel; the obtained 72h semi-lethal high salinity is 53‰.

4. The method of claim 1, wherein, In step 4), for high salinity stress conditions, the high salinity stress experiment group and the control group are placed in a separate culture tank for treatment; during the period, the survival situation is observed every 4 hours and the dead individuals are fished out, and a small amount of feeding and water changing are carried out once a day; the feeding is adjusted according to the number of surviving individuals and the vitality state, and the water changing is carried out with 53‰ salinity seawater for the experiment group and 32‰ salinity natural seawater for the control group, and the water changing amount is half of the volume of the culture tank.

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