Breeding method of alkalinity-tolerant urechis unicinctus excellent strain

Abstract

The invention discloses a breeding method for an excellent alkalinity-tolerant urechis unicinctus strain. The breeding method comprises the following steps: (1) establishing and breeding a family (preparing a breeding pool, selecting parents and hatching and breeding); (2) exploring a semi-lethal concentration condition by alkalinity concentration gradient stress; (3) constructing a semi-lethal concentration stress environment to count survival time of individuals of different families; (4) establishing a genetic evaluation model to estimate genetic parameters and breeding values of the alkalinity tolerance character and the body weight character; (5) evaluating a comprehensive breeding value and screening an F1-generation excellent family as a parent for reserving seeds; and (6) F2 generation family construction and subculture selection. The method is high in operability and breeding efficiency, the bred urechis unicinctus offspring seeds have the advantages of being high in alkalinity tolerance, excellent in growth character and high in survival rate in industrial aquaculture, and the problems that in the existing urechis unicinctus breeding industry, the breeding scale is small, the survival rate is low and the like, and industrial development is restricted can be solved.

Description

Technical Field

[0001] The invention belongs to the field of aquatic genetic breeding, and particularly relates to a method for breeding a new strain of an alkalinity-tolerant Echinops monocyclica. Background Art

[0002] Urechis unicinctus is an annelid that lives in U-shaped caves in the lower intertidal zone of muddy banks, shallow subtidal areas and coral reef areas. It is mainly distributed in Russia, North Korea, Japan and China. Urechis unicinctus has crisp meat and delicious taste. In 2022, it was identified as one of the top ten characteristic aquatic germplasm resources in China by the Ministry of Agriculture and Rural Affairs of the People's Republic of China, and has broad prospects for industrial development. Urechis unicinctus is rich in essential amino acids, glycosaminoglycans and unsaturated fatty acids for the human body, and has high nutritional value. In addition, it also has active substances such as peptides, fibrinolytic enzymes and macromolecular proteins with antioxidant, thrombolytic and in vitro anti-tumor effects, and has potential medicinal value. However, with the overfishing of wild Urechis unicinctus resources in recent years, the contradiction between market supply and demand has become increasingly prominent. On the other hand, the breeding area of ​​Urechis unicinctus is relatively small, and its breeding scale and output are greatly restricted. This requires innovation in the breeding model of the single-ringed spiny caterpillar. Broadening the breeding environment and selecting and breeding high-quality seed sources are important directions that need to be explored in the subsequent sustainable development of the industry.

[0003] Saline water is a non-marine saltwater resource distributed in land areas. Different from seawater and fresh water, it has a higher carbonate alkalinity (HCO3 - and CO3 2- ), high pH content, complex ion composition and weak buffering capacity. Most aquatic economic species cannot survive or grow in saline-alkali environments, so most saline-alkali waters have poor biological resources and low fishery productivity. According to statistics, there are currently 1947Mhm 2 Degraded land, of which saline-alkali land accounts for 970 Mhm 2 , covering at least more than 100 countries and regions, and my country is a country with large saline-alkali land, with the area of ​​saline-alkali land ranking third in the world, about 99130khm 2 , of which low-lying saline-alkali waters account for 45870khm 2, covering 19 provinces, municipalities and autonomous regions in my country, accounting for about 55% of the total area of ​​lakes in China. Therefore, the development and utilization of saline-alkali water (land) has important ecological, economic and social value for improving my country's land resources. In addition, saline-alkali waters will threaten the growth, development, reproduction and survival of aquatic organisms due to their special water quality environment, and carrying out breeding research on alkaline-resistant aquatic organisms is an effective way to utilize saline-alkali water resources. Production practice shows that "using fish to control alkali" is an effective way to develop and utilize saline-alkali water resources. At present, transplanting and domesticating marine aquaculture varieties in low-salinity and alkaline waters (carbonate alkalinity <10mM) has been basically developed and utilized and has achieved certain results, while high-salinity and alkaline waters (carbonate alkalinity >10mM, 30-50mM) have not yet been effectively developed and utilized. The reasons may be as follows: first, the salt-alkali tolerance mechanism of aquaculture varieties is unclear, and it is difficult to guide breeding from a technical level; second, there is a lack of salt-alkali tolerant varieties, and there is a lack of good varieties that can adapt to medium and high alkalinity, grow fast, and have good quality; finally, the breeding technology of salt-alkali tolerant varieties is relatively backward, the breeding method is single and extensive, and the energy and material circulation rate of breeding ponds is low. Therefore, the selection of aquaculture species with short growth period and high economic benefits has become an urgent problem to be solved in the development and utilization of saline-alkali waters in my country.

[0004] The present invention aims at the utilization of saline-alkali water areas in my country and the current status of aquaculture of Spinella monocyclica, and evaluates the genetic parameters of the alkalinity tolerance traits and body weight traits of a Spinella monocyclica population through genetic breeding means, and then screens and cultivates new excellent Spinella monocyclica varieties with fast growth rate, high meat quality and strong alkalinity tolerance. This is of great significance for efficiently utilizing saline-alkali water resources, expanding aquaculture space and meeting the growing market demand for Spinella monocyclica. Summary of the invention

[0005] The invention aims to provide a method for selecting and breeding a new strain of Spinella monocyclica with alkalinity tolerance and producing seeds in view of the current situation that there is no excellent strain with alkalinity resistance in the industrial breeding of Spinella monocyclica, so as to cultivate a new variety of Spinella monocyclica with excellent growth traits and strong alkalinity resistance.

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

[0007] A method for breeding alkalinity-tolerant Echinops monocyclic strains, characterized in that the method steps are:

[0008] 1) Family establishment and cultivation: Construct a breeding environment, prepare a certain number of culture boxes (length × width × height: 60cm × 50cm × 35cm) as early incubation boxes, use a certain concentration of potassium permanganate solution for disinfection and use brushes and other tools for mechanical cleaning. After cleaning and disinfection, inject fresh filtered seawater for standby use. Collect wild monocypris populations with mature gonads in natural waters as breeding populations, select monocypris individuals with vigorous vitality and full nephric ducts that are 15-20% heavier than the average body weight as parents, and use the nest mating design method to establish a monocypris F1 generation full-sib family according to the principle of one male and three females. After hatching in the incubator, the F1 generation full-sib family is subsequently cultivated in a specific area according to the factory seedling cultivation model;

[0009] 2) Alkalinity concentration gradient stress to explore the semi-lethal concentration conditions: An acute attack experiment was used to explore the 72h semi-lethal alkalinity value of the monocyclic stingray under alkalinity stress conditions. After the F1 generation seedlings were cultivated to 200 days of age, the alkalinity gradient experimental groups were set to 48.3995mmol / L, 56.9658mmol / L, 86.2955mmol / L, 100.1606mmol / L and a normal filtered seawater control group (tested to be 2.7127mmol / L). Each group was set up with three parallels and the attack lasted for 120h. During this period, the death of the insects was recorded every 8h. The linear regression method was used to calculate the semi-lethal alkalinity value of the monocyclic stingray population through the survival rate at 72h under each alkalinity gradient stress condition;

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

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

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

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

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

[0015]

[0016] in, is the additive genetic variance of alkalinity tolerance survival time, is the residual variance of alkalinity tolerance survival time. The method for calculating the heritability of body weight is the same as above.

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

[0018]

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

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

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

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

[0023] Preferably, in step 2), the individuals of the monocyclic stingray in the 72h half-lethal concentration screening process of alkalinity stress are randomly selected in the entire cultivation system. The alkalinity stress experimental seawater is obtained by dissolving Na2CO3 and NaHCO3 in a certain proportion in aquaculture filtered seawater, the alkalinity detection method is acid-base titration, and the indicator reagent is methyl orange-aniline blue solution. Three parallels are set for each alkalinity concentration stress experimental group and control group, and at least 10 monocyclic stingrays participate in the stress process in each parallel. The alkalinity concentration gradient of the experimental group can be set accordingly according to the tolerance of different aquatic economic species.

[0024] Preferably, in step 3), for the alkalinity stress environment construction of the whole family, the alkalinity stress experimental group and the control group are placed in a separate culture pool for treatment to ensure the consistency of experimental conditions such as temperature. During the period, the survival of the monocyclic spiny worms is observed every 4 hours and the dead individuals are fished out in time, and a small amount of feeding and a water change operation are performed every day. When feeding, specific adjustments are made according to the number of surviving insects and the vitality state. In addition, since the alkalinity is prone to change during the preparation of high-alkalinity seawater, the water is prepared on the spot when changing the water. The experimental group is replaced with the prepared 72h half-lethal alkalinity value seawater, and the control group chooses naturally filtered seawater, and the amount of water change is half of the volume of seawater in the breeding box.

[0025] Preferably, in step 4), the genetic parameter estimation of the alkalinity stress tolerance trait and the body weight trait is achieved by using ASReml-R4.0 software.

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

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

[0028] In summary, the present invention selects a wild monocyclic thorn worm population as a basic breeding population, selects a family with a large genetic improvement space and breeding potential through alkalinity tolerance breeding, and retains seeds, expands and selects the family with excellent performance, so as to obtain a new strain with excellent growth traits and strong alkalinity tolerance. The breeding method is simple and practical, and has strong operability. After multiple generations of breeding, a new monocyclic thorn worm strain with fast growth rate and excellent alkalinity tolerance can be cultivated. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Survival curves of Echinops unicyclicus under different alkalinity concentration gradient stress conditions

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

[0031] Figure 3Scatter plot of weight-survival time distribution of different individuals of Echinops monocyclicus under 72h semi-lethal alkalinity concentration stress

[0032] Figure 4 Body weight and survival time of half-sib families of different paternal lines of Echinops monocytogenes under 72h semi-lethal alkalinity stress Figure 5 Body weight and survival time of full-sib families of different maternal lines of Echinops monocytogenes under 72h semi-lethal alkalinity stress DETAILED DESCRIPTION

[0033] In order to explain the purpose and technical solution details of the present invention more clearly, the technical solution of the present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments:

[0034] 1. Family establishment and cultivation

[0035] The mature parents of wild monocyclic thorn worms in Qinhuangdao sea area were collected, and individuals with good physiological conditions and abundant nephric ducts were selected and transported to Shandong Dayi Luyu Marine Technology Co., Ltd. at low temperature as the basic breeding population. Individuals with an average body weight of 15-20% or more (the individual weight used in the present embodiment is greater than 120g) were selected in the basic breeding population to carry out in vitro fertilization and generation as parents, and 24 (8 heads ♂×24 heads ♀) full-sib families of monocyclic thorn worms were established by nest mating design. The in vitro fertilization process is as follows: first, the parents are classified according to the sexes and the recovery culture is carried out in the filtered seawater environment for 12h, and the parents are dissected after the vitality is restored and the nephric ducts are neatly placed in a dry and clean dissecting dish, and the nephric ducts are transferred to 24 different 50ml centrifuge tubes according to different parent individuals after all the parents are dissected, and the nephric ducts are cut into pieces and diluted with seawater and then sprinkled in an incubator (length×width×height: 60cm×50cm×35cm) for fertilization and hatching. During fertilization, the density of the egg cells in the incubator is ensured to be 8-10 / ml and the number of sperms around each egg cell is about 5-8. The family cultivation process is as follows: the fertilized eggs in the incubator are hatched and cultivated until the early stage of metamorphosis benthic life, and in this embodiment, they are cultivated to 25 days old (the culture water temperature during the cultivation period is: 17-18°C); before the embryos of the monocyclic schizonts are about to develop to metamorphosis benthic life, they are moved out of the incubator to a specific area of ​​the indoor culture pond for cultivation until they reach the seedling size stage in industrialized cultivation, and in this example, they are cultivated to 100 days old (the culture water temperature during the cultivation period is: 17-19°C); after reaching the seedling size, the monocyclic schizonts of each family are filtered out from the indoor culture pond and transferred to the outdoor pond for continued cultivation until they are 200 days old (the culture water temperature during the cultivation period is: 18-22°C). During the period, the incubation process in the incubator was carried out in an independent box, the indoor breeding pond breeding process was carried out by using a 100-mesh silk screen to isolate the breeding area of ​​each family, and the outdoor pond breeding process was carried out by using a 20-mesh silk screen to isolate the breeding area of ​​each family. In addition, each time the family individuals in different breeding environments were transferred, the breeding density of all families was controlled at the same level. The basic method of feeding twice a day, changing water once, and removing dirt from the bottom every three days was maintained throughout the culture process (the conditions for breeding and filtering seawater were: the dissolved oxygen content was greater than 5 mg / L, and the salinity was 31-32 ppt; the feeding conditions were: feeding a mixture of spirulina and marine red yeast (100 million / ml) at day age, and then feeding a spirulina powder solution filtered by a 300-mesh silk screen, and the standard was that the culture water became clear 3-4 hours after feeding), and appropriate adjustments were made during the process according to the specific situation. Due to the problems of family loss and seed retention and generation during the breeding process, a total of 15 families were finally used to participate in the subsequent alkalinity stress breeding.

[0036] 2. Alkalinity concentration gradient stress to explore the semi-lethal concentration conditions

[0037] After the F1 generation seedlings were cultivated to 200 days old in outdoor ponds, an acute alkalinity stress experiment was used to explore the 72h semi-lethal alkalinity value of Echinops monocylindrica under alkalinity stress conditions. Four alkalinity concentration gradient experimental groups were set up (48.3995mmol / L, 56.9658mmol / L, 86.2955mmol / L, 100.1606mmol / L, the experimental seawater was obtained by dissolving Na2CO3 and NaHCO3 in a certain proportion in aquaculture filtered seawater, the alkalinity detection method was acid-base titration, and the indicator reagent was methyl orange-aniline blue solution) and a normal seawater control group (detected to be 2.7127mmol / L), each group was set up with three parallels, and 10 Echinops monocylindrica were randomly selected in each parallel to participate in the stress. The 72h semi-lethal alkalinity stress exploration experiment was carried out in the aquaculture pool of the cultivation workshop (the aquaculture water temperature was: 17.5-19℃). The attack was continued for 120 h, during which the insect death was recorded every 8 h, and the insect survival rate of each alkalinity gradient at different time points was calculated ( Figure 1 The survival curve equation was calculated by linear regression method based on the survival rate at 72h under each alkalinity concentration gradient ( Figure 2 ), and the half-lethal alkalinity value of Echinops monocyrthospermum under alkalinity stress was calculated by trend line equation to be 82.9073mmol / L in 72h.

[0038] 3. Construct a 72-hour half-lethal alkalinity concentration stress environment to calculate the survival time of individuals from different families

[0039] 60 insects in each family were randomly selected, weighed and numbered, and placed in an independent space in an incubator (length × width × height: 28cm × 19.5cm × 16.5cm) according to the individual number for alkalinity stress exploration. The control group consisted of 60 randomly selected insects from an experimental system containing 15 full-sib families. After the same treatment, they were cultured in filtered seawater. To ensure that each insect was in one area, each incubator was separated into 30 areas with micro-isolation boards. Each family and the control group used two incubators for stress exploration. The experimental group used the 72h semi-lethal alkalinity seawater obtained in the exploration for stress, and continued to stress until all individuals died. During this period, they were observed every 4h and the survival time of each individual was counted. The control group used filtered seawater for unified culture and observed the survival status. All 32 incubators were placed in a culture pool for stress culture (the culture water temperature was: 17.5-19℃). During the whole stress process, a small amount of feeding was performed once a day (the feeding was fine-tuned according to the number of surviving insects and the state of vitality) and a water change operation was performed (the amount of water changed was half the volume of the culture box, the alkalinity experimental group replaced the seawater with an alkalinity of about 83mmol / L, and the control group replaced the normal filtered seawater). The phenotypic parameters of the entire experimental system and each family in the system are shown in Tables 1 and 2. It can be seen that the survival time of the single-ringed thorn worm in the stress system is quite different: the difference is significant at the individual level, the minimum survival time is 24h, the maximum survival time is 144h, and the average survival time of each single-ringed thorn worm is 71.32±0.93h (Table 1); the survival time of the single-ringed thorn worm at the family level is also quite different, the minimum survival time is 60.00±3.56h, and the maximum survival time is 80.90±4.42h (Table 2), indicating that the tolerance of single-ringed thorn worms under different genetic backgrounds to alkalinity stress is different. . In addition, it can be seen from Tables 1 and 2 that there are significant differences in body weight parameters between different families. Scatter plot analysis of the survival time data of the individual of the monocypris punctatus of different body weights in the entire stress system shows that the survival time of the individual has a tendency to increase with the increase of body weight ( Figure 3 ). By using the survival time and weight data of different paternal half-sib families and maternal full-sib families for graphical analysis, it can be seen that among the paternal half-sib families: half-sib family No. 1, half-sib family No. 7 and half-sib family No. 8 have stronger alkalinity tolerance ( Figure 4 ); among the maternal full-sib families: the 1-1 full-sib family, the 3-1 full-sib family, the 5-1 full-sib family, the 7-3 full-sib family, and the 8-1 full-sib family had stronger alkalinity tolerance ( Figure 5 ), indicating that the above families have better tolerance to alkalinity, and the above families were preliminarily marked.

[0040] Table 172h semi-lethal alkalinity concentration stress condition tolerance survival time and body weight phenotypic data of the individual

[0041] Characteristics Mean Standard error Minimum Maximum Coefficient of variation Weight / g 0.39 0.01 0.10 1.23 45.64％ Survival time / h 71.32 0.93 24 144 39.16％

[0042] Table 272h semi-lethal alkalinity concentration stress condition tolerance survival time and body weight phenotypic data of each full-sib family of Echinops monocyrthosiphon (mean ± standard error)

[0043]

[0044]

[0045] 4. Determination of genetic parameters of alkalinity tolerance and body weight

[0046] Genetic parameter analysis was performed using the survival time and body weight of the individuals of the monocyclic worms collected during the alkalinity stress process. i =μ+α i +е i

[0047] where y i is the alkalinity tolerance survival time of the i-th test individual, μ is the mean alkalinity tolerance survival time of all test individuals, α i is the additive genetic effect of alkalinity tolerance survival time of the i-th test individual, е i is the random residual of the alkalinity tolerance survival time of the i-th test individual.

[0048] The heritability calculation formula of this animal model is as follows:

[0049]

[0050] in, is the additive genetic variance of alkalinity tolerance survival time, is the residual variance of alkalinity tolerance survival time. The method for calculating the heritability of body weight is the same as above.

[0051] At the same time, the genetic and phenotypic correlations between alkalinity tolerance and body weight traits (r x，y ) is calculated as follows:

[0052]

[0053] When calculating genetic correlation, σ xy is the additive genetic covariance between alkalinity tolerance and body weight traits, and is the additive genetic variance of the two traits. When calculating phenotypic correlation, σ xy is the phenotypic covariance between alkalinity tolerance and body weight, and is the phenotypic variance of the two traits.

[0054] Through calculation and analysis, the estimated heritability of the survival trait of alkalinity stress in C. monocylindrica was 0.1404±0.0637, indicating that it has certain breeding potential. The estimated heritability of the weight trait of C. monocylindrica was 0.7244±0.1559, which belongs to the high heritability level, indicating that the weight trait also has the potential for further breeding (Table 3). In addition, the coefficients of variation of survival time and weight were 39.16% and 45.64% respectively (Table 1), further indicating that the breeding potential of alkalinity tolerance and weight traits of C. monocylindrica was strong. The genetic and phenotypic correlations between the two were 0.9680±0.0159 and 0.8221±0.0167 respectively (Table 3). The high correlation analysis results between the two traits also showed that the alkalinity tolerance trait can be indirectly selected through weight, which provides theoretical guidance for the comprehensive breeding of alkalinity tolerance and growth traits of C. monocylindrica.

[0055] Table 3 Genetic parameters of survival time and body weight of Echinops monocypris under 72h semi-lethal alkalinity stress (bold diagonal lines represent heritability, those above the diagonal lines represent genetic correlation, and those below the diagonal lines represent phenotypic correlation)

[0056] Characteristics Alkalinity tolerance weight Alkalinity tolerance 0.1404±0.0637 0.9680±0.0159 weight 0.8221±0.0167 0.7244±0.1559

[0057] 5. F1 generation parent seed preservation

[0058] According to the heritability of alkalinity tolerance and body weight traits, the BLUP method (best linear unbiased estimation method) was used to estimate the individual breeding values ​​of A. monocyrthosiphon, and then the average breeding value of the family was determined based on the individual breeding values. The average breeding values ​​of alkalinity tolerance and body weight traits of all families were weighted at a ratio of 1:1 to obtain the comprehensive breeding values ​​of all families under alkalinity stress (Table 4). The top five full-sib families were screened using the comprehensive breeding values: 7-3 full-sib family, 8-1 full-sib family, 7-2 full-sib family, 3-1 full-sib family, and 5-1 full-sib family (arranged in order of ranking). Except for the 1-1 full-sib family, the other families were consistent with the families initially determined based on the phenotypic data. The analysis showed that the reason may be that the alkalinity tolerance of different individuals in the 1-1 full-sib family was quite different. The five selected families were used as candidate families for alkalinity breeding and were further cultured to adulthood at a seed retention rate of 3-5%.

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

[0060] Family name Alkalinity tolerance breeding value Weight breeding value Weighted average breeding value 1-1 -10.7977 0.0004 -5.3987 1-3 -2.7666 -0.0042 -1.3854 2-1 -6.4759 -0.0160 -3.2460 2-2 -1.7538 -0.1217 -0.9378 2-3 -8.0917 -0.1660 -4.1289 3-1 5.5400 -0.3237 2.6082 3-2 -0.7219 -0.1686 -0.4453 4-2 -3.6240 0.2498 -1.6871 4-3 -4.9379 -0.0975 -2.5177 5-1 3.2090 -0.2292 1.4899 5-2 0.1676 -0.1257 0.0210 7-2 8.0152 -0.1863 3.9145 7-3 8.7606 -0.1106 4.3250 8-1 8.3842 0.2081 4.2962 8-2 1.1104 0.2145 0.6625

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

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

[0063] The above embodiments are only used to illustrate the technical solutions in the present invention. For professionals in the field, the operating methods and technical means recorded herein can be modified or replaced in other embodiments without departing from the spirit or scope of the present invention to achieve the breeding goals, but these modifications or replacements made without departing from the concept of the present invention will fall within the protection scope of the present invention.

Claims

1. A method for breeding a strain of Echinops monocyclica with alkalinity tolerance, characterized in that Quantitative genetics and family breeding methods are used together to screen and cultivate new industrial breeding varieties with excellent growth traits and strong alkalinity tolerance. The breeding method comprises the following steps: 1) Family establishment and cultivation: Construct a breeding environment, prepare a certain number of culture boxes (length × width × height: 60cm × 50cm × 35cm) as early incubation boxes, use a certain concentration of potassium permanganate solution for disinfection and use brushes and other tools for mechanical cleaning. After cleaning and disinfection, inject fresh filtered seawater for standby use. Collect wild monocypris populations with mature gonads in natural waters as breeding populations, select monocypris individuals with vigorous vitality and full nephric ducts that are 15-20% heavier than the average body weight as parents, and use the nest mating design method to establish a monocypris F1 generation full-sib family according to the principle of one male and three females. After hatching in the incubator, the F1 generation full-sib family is subsequently cultivated in a specific area according to the factory seedling cultivation model; 2) Alkalinity concentration gradient stress to explore the semi-lethal concentration conditions: An acute attack experiment was used to explore the 72h semi-lethal alkalinity value of the monocyclic stingray under alkalinity stress conditions. After the F1 generation seedlings were cultivated to 200 days of age, the alkalinity gradient experimental groups were set to 48.3995mmol / L, 56.9658mmol / L, 86.2955mmol / L, 100.1606mmol / L and a normal filtered seawater control group (tested to be 2.7127mmol / L). Each group was set up with three parallels and the attack lasted for 120h. During this period, the death of the insects was recorded every 8h. The linear regression method was used to calculate the semi-lethal alkalinity value of the monocyclic stingray population through the survival rate at 72h under each alkalinity gradient stress condition; 3) Construct a semi-lethal concentration stress environment to count the survival time of individuals from different families: Use the 72h semi-lethal alkalinity value obtained in the study to prepare seawater of corresponding concentration for aquaculture stress. 60 insects were randomly selected from each family, weighed and placed in an independent incubator for alkalinity stress culture. At the same time, 60 single-ringed spiny worms were randomly selected from the experimental system and subjected to the same treatment. They were placed in an incubator and cultured using filtered seawater as a control group. The incubators were isolated with micro-isolation plates to ensure that each insect was in a separate area. All incubators were placed in a culture pool for stress observation. For the experimental group, 72h semi-lethal alkalinity seawater was used for unified culture, and the stress continued until all individuals died, and the survival time of each individual was counted. For the control group, filtered seawater was used for unified culture, and observation was stopped after the experimental group completed the stress statistics; 4) Establish a genetic evaluation model to estimate the genetic parameters and breeding values ​​of alkalinity tolerance and body weight traits: The heritability of alkalinity tolerance traits was determined by the survival time of individuals under 72h semi-lethal alkalinity stress conditions, and the individual alkalinity tolerance breeding value and the average breeding value of family alkalinity tolerance were estimated based on the heritability of alkalinity tolerance traits. The heritability of body weight traits was determined by the body weight of the individuals of the monocyclic worms under stress conditions, and the individual body weight breeding value and the average breeding value of family weight were estimated based on the heritability of body weight traits. The best linear unbiased estimation method (BLUP method) of animal models was used to estimate genetic parameters and breeding values. Establishment of genetic parameter estimation model for alkalinity stress tolerance: i =μ+α i +е i where y i is the alkalinity tolerance survival time of the i-th test individual, μ is the mean alkalinity tolerance survival time of all test individuals, α i is the additive genetic effect of alkalinity tolerance survival time of the i-th test individual, е i is the random residual of the alkalinity tolerance survival time of the i-th test individual. The heritability calculation formula for the above animal model is as follows: in, is the additive genetic variance of alkalinity tolerance survival time, is the residual variance of alkalinity tolerance survival time. The method for calculating the heritability of body weight is the same as above. At the same time, the genetic and phenotypic correlations among the traits (r x，y ) is calculated as follows: When calculating genetic correlation, σ xy is the additive genetic covariance between alkalinity tolerance and body weight traits, and is the additive genetic variance of the two traits. When calculating phenotypic correlation, σ xy is the phenotypic covariance between alkalinity tolerance and body weight, and is the phenotypic variance; 5) Evaluate the comprehensive breeding value and select the F1 generation excellent families as parents for seed retention: weight the average breeding value of alkalinity tolerance and the average breeding value of body weight traits of each family at a ratio of 1:1 to obtain the comprehensive breeding value of each family under alkalinity stress. Use the comprehensive breeding value to sort and retain the top 5 families as candidate families for alkalinity tolerance breeding. The selected candidate families will continue to be raised to adulthood at a seed retention rate of 3-5%; 6) Construction of F2 generation families and selection of successive generations: The seed individuals of the F1 generation alkalinity tolerance breeding candidate families are cultured to the stage of gonadal maturity, and the strong and vigorous individuals of the monocyprids with weight in the top 15-20% are selected for mating and breeding with the same family source and different family sources to construct the F2 generation family, and the inbreeding coefficient is controlled within 0.

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

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

3. The method according to claim 1, characterized in that In step 2), the individuals of the monocyclic stingray used in the 72h half-lethal concentration screening process of alkalinity stress are randomly selected in the entire cultivation system. The alkalinity stress experimental seawater is obtained by dissolving Na2CO3 and NaHCO3 in a certain proportion in aquaculture filtered seawater, the alkalinity detection method is acid-base titration, and the indicator reagent is methyl orange-aniline blue solution. Three parallels are set for each alkalinity concentration stress experimental group and control group, and at least 10 monocyclic stingrays participate in the stress process in each parallel. The alkalinity concentration gradient of the experimental group can be set accordingly according to the tolerance of different aquatic economic species.

4. The method according to claim 1, characterized in that In step 3), for the alkalinity stress environment of the family, the alkalinity stress experimental group and the control group are placed in a separate culture pool for treatment to ensure the consistency of experimental conditions such as temperature. During the period, the survival situation is observed every 4 hours and the dead individuals are fished out in time. A small amount of feeding and a water change operation are performed every day. When feeding, specific adjustments are made according to the number of worm survival and vitality status. In addition, since the alkalinity is prone to change during the configuration of high-alkalinity seawater, the water is used and prepared when changing the water. The experimental group is replaced with the configured 72h half-lethal alkalinity value seawater, and the control group chooses naturally filtered seawater, and the water change amount is half of the volume of seawater in the breeding box.

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

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

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