CN113940294B - Breeding method for negative correlation characters of fishes - Google Patents
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- CN113940294B CN113940294B CN202111190868.9A CN202111190868A CN113940294B CN 113940294 B CN113940294 B CN 113940294B CN 202111190868 A CN202111190868 A CN 202111190868A CN 113940294 B CN113940294 B CN 113940294B
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Abstract
The invention discloses a breeding method of negative correlation characters of fishes, belonging to the technical field of biological breeding. The breeding method comprises the following steps: aiming at the negative correlation characters of fish growth and environmental stress resistance, fitting the relationship between the development period and the environmental stress resistance, and determining the stress resistance development sensitive period and stress resistance breeding parameters; high-concentration acute stress lethal screening is carried out, individuals with poor tolerance to environmental stress are eliminated, fish individuals cultured and retained in a conventional environment are implanted into a PIT fish chip, individuals growing fast are selected and retained in a low-concentration chronic stress environment, meanwhile, stress-resistant main-effect genes are selected as evaluation indexes for stress resistance to be detected, and individuals with abnormal gene expression level are eliminated. The breeding method of the invention gives consideration to growth and adverse-resistant negative correlation properties, realizes accurate polymerization breeding, greatly shortens breeding period and improves efficiency.
Description
Technical Field
The invention belongs to the technical field of biological breeding, and particularly relates to a breeding method for negative correlation traits of fishes.
Background
There are different types or degrees of phenotypic and genetic associations, i.e., positively and negatively associated traits, between biological traits. The value of a certain trait is correspondingly increased along with the increase of another trait, the correlation is called positive correlation, and the pair of traits is the positive correlation trait. For example, Atlantic salmon high temperature resistant strains are also highly resistant to hypoxia, high temperature resistance and hypoxia resistance may share some common signaling pathways, and there is a significant positive correlation between high temperature resistance and hypoxia resistance. In contrast to the above, when the value of a trait decreases with the increase of another trait, the relationship is called negative correlation, and the pair of traits are negative correlation traits, such as short stalk and large grain, ear grain number and thousand grain number, yield and quality, lean meat percentage and meat quality of pig, and the like, which are inherent genetic characteristics of the animal and plant. In breeding practice, two negatively correlated traits need to be combined together and bred into a variety with production expectation.
The fish negative correlation character is polymerized and bred through hybridization, colony breeding and other modes. In the crossbreeding, strains with excellent characters need to be found in time, and distant hybridization needs to overcome interspecific separation, so that the seed production efficiency is low, and the seedling yield cannot meet the production requirement. Selective breeding, which is an effective and common method for artificially deciding to reserve individuals with good traits as breeding stocks, and increasing the frequency of favorable alleles in the population, thereby increasing the genetic potential of the herd. However, for the negative correlation traits, the total number of genes of an individual is determined, and the number of genes determining the two traits is offset, namely, the two negative correlation traits do not show excellent characteristics on the same individual, only a relative balance state can be achieved, and the traits show different stage characteristics along with the development of the individual. Therefore, individuals with certain advantages and negative correlation traits are hidden in the population and are easy to lose in the breeding process, and how to accurately select a suitable selective-remaining object from a large number of individuals is a key technical difficulty in performing polymerization group breeding on the negative correlation traits.
The negative correlation between the characters of the animals and the plants is controlled by heredity as the inherent characteristics of the animals and the plants, so that the difficulty of combining the negative correlation characters is high, the common family breeding method is suitable for the characters with low heritability, and the requirement of large family is met, and the difference between families or the correlation in the family caused by the common environment is small. The group breeding is subdivided into a sequential selection method, an independent elimination method, a comprehensive index selection method and gene and marker assisted selection, and the methods have the following defects: (1) the sequential selection method has a relatively fast improvement speed for a certain trait, but takes a long time to improve a plurality of traits, cannot consider the correlation among the traits, and is easy to cause the failure; (2) the independent elimination method usually eliminates some species with excellent performance on certain characters and deficient performance on individual characters, is rarely used for selecting important economic characters and is mainly used for eliminating individuals with defects in heredity or poor compactness; (3) the comprehensive index selection method (a) is not large enough because of the breeding population, and the genetic parameter estimation has deviation; (b) the economic weighted value of each target character is difficult to be accurately determined; (c) the inbreeding degree in the breeding group is higher; (d) the selection character is not consistent with the target character (if the target character is lean meat percentage, the selection character is backfat thickness or eye muscle area) and the like, so that the theoretically expected effect cannot be achieved in the practical application; (4) gene and marker assisted selection because (a) the trait is not controlled by a single gene, the entire trait may not be controlled by the selected marker-associated gene; (b) the main molecular markers shared among the groups are few, and the application and the development of the main molecular markers are limited to a certain extent; (c) the breeding families are few or the population is small, and the molecular marker screening needs a large number of families or populations to guarantee diversity and the like, so that the effect of the molecular marker screening is not obviously superior to that of the traditional breeding.
In conclusion, the existing fish negative correlation character polymerization breeding method has respective defects, such as low efficiency, high cost, incapability of meeting production and the like, and particularly, an accurate breeding scheme is difficult to determine in group breeding and the breeding period is long.
Disclosure of Invention
The invention aims to provide a breeding method of negative correlation characters of fish aiming at typical negative correlation characters of fish growth and environmental stress resistance (saline-alkali resistance, low oxygen resistance and the like), so as to realize accurate breeding, shorten breeding period and improve efficiency.
In order to achieve the purpose, the invention adopts the technical scheme that:
the invention provides a breeding method of negative correlation traits of fishes, which comprises the following steps:
(1) aiming at the negative correlation characters of fish growth and environmental stress resistance, fitting the relationship between the development period and the environmental stress resistance, and determining the stress resistance development sensitive period and stress resistance breeding parameters;
(2) carrying out high-concentration acute stress lethal screening according to the stress-resistant breeding parameters, and eliminating individuals with poor tolerance to environmental stress;
(3) culturing the remaining fish individuals in the conventional environment, and implanting PIT fish chips when the fish individuals are in proper sizes;
(4) and selecting and reserving fast-growing individuals under the low-concentration chronic stress environment, and selecting the stress-resistant major gene as an evaluation index for detecting the stress resistance, thereby eliminating the individuals with abnormal gene expression level.
Preferably, the stress-resistant development sensitive period and the stress-resistant breeding parameters are determined in the step (1) by the following method:
(1-1)96h environmental alkali stress experiment: selecting at least 5 types of fishes according to the physical quality from small to large, setting at least 3 corresponding environmental stress gradients for each type of fishes, temporarily culturing the fishes in a control water body without environmental stress, directly putting the fishes into water bodies with different environmental stresses, setting 3 fishes in each group of 40 fishes in parallel, and carrying out an acute stress experiment for 96 hours; observing and recording death conditions every 4h after the experiment begins, keeping the state stable during the period, not feeding, sucking out excrement by using a siphon tube to keep a water body clean, and taking the condition that fish is still at the bottom of a water tank, breathing stops, and a glass rod touches no reaction as a death standard;
(1-2) fitting the cumulative mortality curve surfaces of different specifications of fishes in different time intervals under different environmental stress treatment by adopting a complementary log-log model (CLL), and respectively calculating the corresponding 96h semi-lethal concentration of each specification of fishes;
(1-3) fitting a relationship curve between a development period (body mass) and the 96h semi-lethal concentration by using a Logistic regression model according to the 96h semi-lethal concentration corresponding to the fish with each specification;
(1-4) determining the development sensitivity period of the stress resistance according to the relation curve, and setting the breeding parameters of the stress resistance.
Preferably, the stress-resistant breeding parameters comprise the age of the bred fish, the weight of the fish, the breeding stress intensity and the stress period.
Preferably, the PIT fish chip has a diameter of 2.05mm, a length of 12.05mm and an average mass of 0.094 g.
Preferably, the anti-adversity major gene comprises Heat Shock Protein (HSP), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase), 24 dehydrocholesterol reductase (DHCR24), Platelet Derived Growth Factor (PDGF), growth differentiation factor 15(GDF15), miRNA-26a, miRNA-30c and miRNA-204.
More preferably, the bulk mass does not exceed 30 g.
More preferably, the complementary log-log model fitting employs a DPS data processing system.
More preferably, the fish is selected from nile tilapia.
Compared with the prior art, the invention has the beneficial effects that: the problem that the existing fish group breeding is subjected to environmental stress screening of resistant individuals exists, such as the breeding retention rate is only considered and the correlation of individual development and resistance is ignored; or only simulating the condition of the chronic stress environment required by production without considering the difference between acute stress and chronic stress; or the breeding is monitored by lacking the application of molecular markers suitable for fishes, the breeding process of the negative correlation characters is relatively random and blind, the breeding period is long, and the like, the invention carries out statistical processing on data through a reasonable mathematical model, fits a relation curve of a development period (body mass) and 96h semi-lethal concentration, systematically considers the relation of the development period (body mass) and stress resistance, finds out a sensitive development period of the fishes enduring environmental stress so as to accurately set breeding parameters, and carries out high-concentration stress selection to retain resistance dominant individuals; meanwhile, the method is used for marking and tracking the fishes in the low-concentration chronic stress environment, screening individuals with fast growth speed, selecting a plurality of stress-resistant major genes as stress-resistant evaluation indexes for collaborative screening so as to ensure stable characters, giving consideration to growth and stress-resistant negative related characters, realizing accurate polymerization breeding, greatly shortening breeding period and improving efficiency, and having certain general applicability.
Drawings
Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:
FIG. 1 is a fitted curve of developmental stage (body mass) and 96h semilethal concentration in example 1 (independent variable is body mass, dependent variable is semilethal concentration).
FIG. 2 is a gene expression assay.
FIG. 3 is a flow chart of a breeding method for negative correlation traits of fishes in the invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention are clearly and completely described below with reference to the drawings of the embodiments of the present invention. It should be apparent that the described embodiments are only some of the embodiments of the present invention, and not all of them. All other embodiments, which can be derived by a person skilled in the art from the described embodiments of the invention without any inventive step, are within the scope of protection of the invention.
Taking the negative correlation characters of the growth and the environmental stress resistance of the tilapia nilotica as an example in the following examples, breeding the negative correlation characters of the fishes according to the method shown in the figure 3; the method comprises the following steps:
step S1, determining stress resistance breeding parameters by fitting the relationship between the development period and the stress resistance, specifically:
(1)96h environmental alkali stress experiment: selecting at least 5 types of fish with different sizes according to the body weight from small to large (the body weight can be set according to 1: 2: 3 and the like, the maximum body weight is recommended not to exceed 30g), and setting at least 3 corresponding environmental stress gradients for the fish with each size according to actual experience or pre-experiments. Temporarily culturing the fish in a control water body without environmental stress (such as a fully aerated tap water group is used as a control), directly putting the experimental fish into water bodies with different environmental stresses, setting 3 parallel fishes in each group of 40 fishes, and carrying out an acute stress experiment for 96 h. After the experiment is started, the death condition is observed and recorded every 4h, the state is kept stable during the experiment, the feeding is not carried out, excrement is sucked out by a siphon tube to keep the water body clean, and the death standard is that the experimental fish is stood still at the bottom of a water tank, the breathing is stopped, and no response is caused by touching the experimental fish by a glass rod.
(2) And fitting a CLL model to the cumulative mortality curve of the fishes with different specifications in each time interval under different environmental stresses for analyzing the time-alkalinity-mortality relationship under acute stress, respectively calculating the 96h semi-lethal concentration corresponding to each specification, and fitting the model under a DPS data processing system. Compared with linear regression, the CLL model can directly reflect death change conditions in different time intervals, system errors caused by too high or too low set concentration are remarkably reduced, the fitting degree is higher, and the obtained 96h semi-lethal concentration is more practical.
(3) A Logistic regression model was fitted to the relationship between developmental stage (body mass) and 96h semilethal concentration based on the 96h semilethal concentration for each size of fish.
(4) Determining the development sensitivity period of stress resistance according to the relation curve, and setting stress resistance breeding parameters including the age of breeding fish, the weight of the fish, the breeding stress intensity, the stress period and the like. The fish has a development sensitive period of stress resistance or adaptation to the environment along with the growth and development, if the development period is early or the physique quality is too low, a stress resistance mechanism is not formed, the breeding stress condition leads a large amount of or even all the fish to die, the reasonable population quantity (avoiding inbreeding) cannot be ensured, and the difference among individuals cannot be effectively distinguished (the stage is greatly influenced by the weight, the reserved individuals only grow faster and do not mean the absolute strong stress resistance). Generally, the resistance of a developing individual is gradually increased along with the environmental resistance of the developing individual, but the resistance is not continuously and linearly increased, the resistance is not obviously increased when a perfect defense mechanism is established and the fish age or the body mass is increased. However, too late development period or too large physique can increase breeding cost. The appropriate development sensitivity period is a reasonable period for breeding, and screening under high-concentration acute stress can be carried out according to the parameters of the period.
And step S2, carrying out high-concentration acute stress lethal screening according to the stress-resistant breeding parameters, and eliminating individuals with poor tolerance to environmental stress.
And step S3, culturing the remaining fish individuals in the conventional environment, and implanting PIT fish chips when the sizes are proper.
S4, selecting and reserving individuals with fast growth in a low-concentration chronic stress environment, selecting a stress-resistant major gene as an evaluation index for stress resistance, and eliminating individuals with abnormal gene expression level, wherein the method specifically comprises the following steps:
(1) setting a low-concentration (or low-intensity) stress environment which can not cause fish death but can cause fish stress reaction, taking blood from tail veins under the condition of ensuring the survival of the fish after feeding for 24 hours in the environment, and detecting the expression of the following major genes by a fluorescence quantitative method: heat Shock Protein (HSP), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase), 24 dehydrocholesterol reductase (DHCR24), Platelet Derived Growth Factor (PDGF), growth differentiation factor 15(GDF15), miRNA-26a, miRNA-30c, and miRNA-204. The genes comprise classical genetic and epigenetic regulation and relate to main upstream regulation pathways of fish response adaptive environmental stress processes, such as stress response, cell repair, immunity and the like. By verification, after 24 hours of environmental stress, HSP, HMG-CoAreductase, DHCR24, PDGF and GDF15 can be expressed obviously in an up-regulation mode, and miRNA-26a, miRNA-30c and miRNA-204 can be expressed obviously in a down-regulation mode, which means good stress resistance.
(2) After the fish is raised for 60 days under the low-concentration chronic stress environment, the fish is fished, and the shape data of the weight, the body length and the like of each fish are measured and recorded again. And selecting and reserving individuals with fast weight increment, wherein the selecting and reserving proportion is generally not higher than 20%, and simultaneously combining gene expression analysis results, because the concentration (or strength) of the stress environment is lower, the gene expression conditions are different, and the individuals with abnormal gene expression level are eliminated, so that the stability of the character is ensured, and the growth and stress resistance characters are considered.
Example 1
Selecting Nile tilapia with average body weight of 6g, 15g, 20g, 50g, 80g and 150g, setting 4-10 g.L -1 NaHCO 3 The alkalinity gradient was subjected to a 96h acute stress experiment, the time-alkalinity-mortality relationship under acute stress was analyzed using CLL model, and the 96h semi-lethal concentrations of different body mass of fish were calculated (table 1).
Table 1: 96h semi-lethal carbonate concentration for tilapia of different body masses
Wherein the experimental fish is at time t j (J ═ 12, 24.., J) by an agent (NaHCO) 3 ) Dose d of i (I ═ 6, 7.., I) the probability of lethality can be expressed as:
p ij ＝1－exp[－exp(τ j +βlg(d i ))]①；
in the formula: beta is a parameter to be estimated describing the dose effect; tau. j Is to time t j A time effect parameter of (a); p is a radical of ij Is dose d i Cumulative probability of death occurring over 0 to j time units;
dose d i Subject individual is allowed to remain in time interval t j -1,t j ]The mortality rate that may be suffered is called the conditional probability of death and can be expressed as:
q ij ＝1－exp[－exp(γ j +βlg(d i ))]②；
in the formula: beta is a parameter to be estimated describing the dose effect; gamma ray j To describe the time interval t j －1,t j ]The parameter to be estimated of the internal time effect, and j with some differences in the meaning of time, γ j And β is obtained by a binomial distribution maximum likelihood function approximation. And (3) for the difference between the fitting value and the measured value of the model, respectively adopting Pearson chi-square test and Hosmer-Lemoshow statistic to test the fitting degree of the model.
Logistic model was used to fit a curve relating developmental stage (body mass) to 96h semi-lethal concentration as shown in figure 1. Fitting a regression equation: the semilethal concentration of 96h is 9.3878/(1+ EXP (0.183941-0.152365 developmental stage)), and P is 0.0003. According to the fitting curve and the equation, after the weight of the nile tilapia exceeds 80g, the semilethal concentration of the nile tilapia does not change basically for the alkali resistance, 10g to 20g is the sensitive period for the formation of the alkali resistance, the factors such as the breeding accuracy, the breeding cost and the like are comprehensively considered, the fish body mass is 15g to 20g as a breeding object during acute stress, and the screening concentration range is 8 g to 9g.L -1 The breeding parameters are more suitable.
The juvenile tilapia nilotica with the body mass of about 15g is directly transferred into 9g.L from fresh water -1 NaHCO 3 In the alkalinity water body, the surviving individuals are left for the next breeding after 96 hours, the dead individuals are eliminated, and the elimination rate is about 50 percent.
The preserved nile tilapia is raised to about 100g in the conventional environment (the alkali resistance of the nile tilapia at the growth stage is not changed basically), PIT fish chips (with the diameter of 2.05mm, the length of 12.05mm and the average mass of 0.094g in the United states) are implanted, and the weight and the body length of each fish are measured and recorded.
Set at 2g.L -1 NaHCO 3 The method comprises the steps of using an alkaline water body to fish the nile tilapia 24 hours after being stressed in the environment, taking blood from tail veins, sampling the blood for detecting the expression of major genes by a fluorescence quantitative method, and then putting the nile tilapia back to recover 2g.L -1 The culture is continued in the coerced environment. The fluorescent quantitative manufacturing process comprises the following steps: extracting total RNA with Trizol, extracting with RNase free ddH 2 O dissolved, RNA concentration and A260/A280 value were measured with spectrophotometer (Onedrop, national), and sample retention with ABS value between 1.8-2.1. Taking total RNA as a template, carrying out reverse transcription according to the instruction of PrimeScript RTreagent Kit With gDNA Eraser to synthesize first strand cDNA, carrying out real-time fluorescence quantitative PCR amplification according to the instruction of SYBR Premix ExTaq, and carrying out the amplification program: pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 5s, annealing and extension at 58-62 ℃ for 30s, and 40 cycles; melting at 95 deg.C for 10s, and annealing at 65 deg.C for 5 s. Adjusting the amplification system according to the result to make the amplification efficiency of the target gene and the reference gene close to 100 percent, the amplification procedure is the same as that of the standard curve, and finally adopting 2 -ΔΔCt The method can be used for comparing and analyzing the expression quantity of different genes.
After 60 days of culture, measuring body weight and body length, calculating the weight gain rate, selecting and reserving 10% of individuals with faster growth, and then checking the expression condition of major genes of the individuals after 24 hours of stress, wherein all gene expressions in normal individuals show a significant rising trend (figure 2A, P is less than 0.05), and the expression rising condition of growth differentiation factor 15(GDF15) in abnormal individuals is lower in detected individuals, so that the abnormal individuals are eliminated (figure 2B).
Claims (4)
1. A breeding method for negative correlation traits of fishes is characterized by comprising the following steps:
(1) aiming at the negative correlation characters of fish growth and environmental stress resistance, fitting the relationship between the development period and the environmental stress resistance, and determining the stress resistance development sensitive period and stress resistance breeding parameters;
(2) carrying out high-concentration acute stress lethal screening according to the stress-resistant breeding parameters, and eliminating individuals with poor tolerance to environmental stress;
(3) culturing the remaining fish individuals in the conventional environment, and implanting PIT fish chips when the fish individuals are proper in size;
(4) selecting and reserving fast-growing individuals under the low-concentration chronic stress environment, and selecting a stress-resistant major gene as an evaluation index for stress resistance to detect, so as to eliminate individuals with abnormal gene expression level;
determining the stress resistance development sensitive period and the stress resistance breeding parameters, comprising the following steps:
(1-1)96h environmental alkali stress experiment: selecting at least 5 types of fishes according to the physical quality from small to large, setting at least 3 corresponding environmental stress gradients for each type of fishes, temporarily culturing the fishes in a control water body without environmental stress, directly putting the fishes into water bodies with different environmental stresses, setting 3 fishes in each group of 40 fishes in parallel, and carrying out an acute stress experiment for 96 hours; observing and recording death conditions every 4h after the experiment begins, keeping the state stable during the period, not feeding, sucking out excrement by using a siphon tube to keep a water body clean, and taking the condition that fish is still at the bottom of a water tank, breathing stops, and a glass rod touches no reaction as a death standard;
(1-2) fitting the accumulated mortality curve surfaces of different specifications of fishes in different time intervals under different environmental stress treatment by using a complementary logarithm model, and calculating the corresponding 96h semi-lethal concentration of each specification of fishes respectively;
(1-3) fitting a relationship curve between the development period and the 96h semi-lethal concentration by using a Logistic regression model according to the 96h semi-lethal concentration corresponding to the fish with each specification;
(1-4) determining the development sensitivity period of stress resistance according to the relation curve, and setting stress resistance breeding parameters;
the stress-resistant breeding parameters comprise breeding age, fish weight, breeding stress intensity and stress period;
fitting the complementary logarithm model by using a DPS data processing system;
the stress-resistant major gene comprises heat shock protein, 3-hydroxy-3-methylglutaryl coenzyme A reductase, 24 dehydrocholesterol reductase, platelet-derived growth factor, growth differentiation factor 15, miRNA-26a, miRNA-30c and miRNA-204.
2. The method for breeding fish with negative correlation traits as claimed in claim 1, wherein the PIT fish chip has a diameter of 2.05mm, a length of 12.05mm and an average mass of 0.094 g.
3. The method for breeding fish negative correlation traits as claimed in claim 1, wherein the body mass is not more than 30 g.
4. The method for breeding fish with negative correlation traits according to any one of claims 1 to 3, wherein the fish is selected from Nile tilapia.
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