Method for breeding low-oxygen-resistant variety of trachinotus ovatus

Abstract

The invention provides a low-oxygen-resistant variety breeding method for trachinotus ovatus, and relates to the technical field of low-oxygen-resistant variety breeding for trachinotus ovatus, and the method specifically comprises the following steps: carrying out a low-oxygen stress test on a trachinotus ovatus group, determining a dissolved oxygen value and survival time of a suffocation point, screening phenotype-resistant extreme individuals, and obtaining genotype data of the individuals; identifying a target SNP marker significantly related to the hypoxia resistance character through whole genome association analysis; performing genotyping on the backup parents by using the target SNP, calculating a hypoxia resistance evaluation index, and screening excellent parents to establish a core breeding population; filial generations are obtained through breeding, and candidate groups are screened in combination with favorable allele detection and genome estimation breeding values; and in combination with an intermittent hypoxia stress test performed on the candidate group, calculating a comprehensive growth expression index based on the body length and the body weight, and selecting a final hypoxia-resistant trachinotus ovatus variety. According to the invention, efficient and accurate hypoxia-resistant character breeding can be realized.

Description

Technical Field

[0001] This invention relates to the field of breeding technology for low-oxygen-tolerant varieties of oval pomfret, specifically to a method for breeding low-oxygen-tolerant varieties of oval pomfret. Background Technology

[0002] The golden pomfret, also known as the oval pomfret, is an important economic fish species in marine aquaculture in southern my country. In 2024, its national aquaculture production reached 305,600 tons, making it the largest aquaculture species in my country. Dissolved oxygen is an indispensable element for fish survival, and its concentration has a profound impact on their feeding behavior, feed utilization, and overall growth. When fish are under hypoxic stress, they experience slow growth, decreased immunity, and even mass mortality, resulting in severe economic losses. The golden pomfret is an active swimmer with high activity levels. Compared to other conventionally farmed fish, its oxygen consumption rate and asphyxiation point are relatively high, requiring a dissolved oxygen level of 5.0 mg / L. When dissolved oxygen falls below 3.0 mg / L, normal feeding is affected, with a minimum critical dissolved oxygen level of 2.5 mg / L. Therefore, breeding new golden pomfret varieties with strong hypoxia tolerance is an urgent need for the sustainable development of the industry. Traditional breeding methods rely on individual phenotypes for selection, but such traits are easily affected by environmental factors, resulting in low breeding efficiency, long cycles, and difficulty in accurately identifying individuals with excellent genetic potential.

[0003] Among existing breeding techniques, the population selection method for oval pomfret (publication number CN110150190A) aims to cultivate varieties with growth advantages by establishing families and combining specific aquaculture management strategies. This method mainly relies on macroscopic phenotypic data such as morphology and growth status for multi-generational selection. Although it avoids inbreeding to some extent, it still falls within the scope of traditional population selection. The breeding method for hypoxia-tolerant golden pomfret (publication number CN118633550A) aims to obtain offspring with oxygen-carrying capacity and hypoxia tolerance through intermittent hypoxia training of parents. This method only has a certain effect on first-generation seedlings and cannot identify and aggregate key genes directly related to hypoxia tolerance from a genetic perspective, thus failing to fix the hypoxia tolerance trait. Therefore, it has significant technical limitations in the efficient and precise breeding of the specific and complex physiological trait of "hypoxia tolerance." Our team previously invented a SNP molecular marker associated with the hypoxia tolerance trait of oval pomfret and its application (Patent No.: ZL202110313332.5). By performing genome-wide association analysis on oval pomfret after hypoxia treatment, we screened and obtained an SNP molecular marker associated with the hypoxia trait, which provides support for molecular marker-assisted selection of the hypoxia tolerance trait in oval pomfret.

[0004] The information disclosed in the background section is only intended to enhance the understanding of the background of this disclosure, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0005] The purpose of this invention is to provide a method for breeding low-oxygen-tolerant varieties of oval pomfret, in order to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A method for breeding low-oxygen-tolerant varieties of oval pomfret, the specific steps of which include: Step 1: Conduct a hypoxia stress tolerance test on the oval pomfret population. The dissolved oxygen value at the asphyxiation point and the survival time under hypoxia stress are used as tolerance phenotypes to screen out extreme individuals with tolerance phenotypes. Step 2: Obtain genotype data of individuals with extreme tolerance phenotypes; perform genome-wide association analysis on the genotype data and the tolerance phenotypes identified in Step 1 to screen for target SNP molecular markers that are significantly associated with hypoxia tolerance traits; Step 3: Using the selected target SNP molecular markers, the genotype of the reserve parent population of oval pomfret is classified. Based on the number and weight of favorable alleles at all target SNP loci for each reserve parent, its hypoxia tolerance evaluation index is determined. Based on the hypoxia tolerance evaluation index, superior parents are selected to form a core breeding population. Step 4: Based on the constructed core breeding population, breed to obtain offspring population, and by detecting whether each individual in the offspring population carries the favorable allele in the target SNP molecular marker, combined with the genomic estimation breeding value, screen out low-oxygen-tolerant oval pomfret varieties as candidate populations; Step 5: Conduct a second hypoxia stress test on the candidate population. The second hypoxia stress test adopts an intermittent hypoxia treatment method. After the test, the body length and weight of each individual in the candidate population are measured. The comprehensive growth performance index is determined based on the body length and weight, and the final hypoxia-tolerant oval pomfret breeding variety is selected based on the comprehensive growth performance index.

[0007] Furthermore, the asphyxiation point dissolved oxygen value refers to the dissolved oxygen concentration in the water when the test individual begins to lose balance and the ability to maintain body position during the hypoxia stress test. The lower the value, the stronger the hypoxia tolerance. Survival time under hypoxia stress refers to the time from the start of the test to the individual's death in a hypoxia stress tolerance test. The longer the time, the stronger the hypoxia tolerance. The logic behind conducting hypoxia stress tolerance tests on oval pomfret populations is as follows: A population of oval pomfret was formed by selecting several individuals. The population was placed in a controlled, closed breathing chamber for a hypoxia stress test. Nitrogen was continuously introduced while the dissolved oxygen concentration in the water was monitored, and the dissolved oxygen was reduced at a constant rate to a predetermined endpoint. During this process, the dissolved oxygen concentration at which the individual first became unbalanced and unable to maintain a normal swimming position was recorded as its asphyxiation point dissolved oxygen value. Under the stress environment of maintaining the set hypoxia concentration, the survival time of each individual from the start of the test to death was continuously observed and recorded. Finally, based on the asphyxiation point dissolved oxygen value from low to high and the survival time from long to short, extreme individuals that met the preset requirements in both indicators were selected and defined as tolerance phenotype extreme individuals. Subsequently, tail fin tissue samples were immediately collected from the tolerance phenotype extreme individuals and stored at low temperature in centrifuge tubes containing RNA protectant for later use.

[0008] Furthermore, genomic DNA was extracted and quality-checked from caudal fin tissue samples of the collected individuals with the tolerance phenotype, and a whole-genome sequencing library was constructed. Subsequently, the library was re-sequencing the whole genome using a high-throughput sequencing platform to obtain raw sequencing data. The raw sequencing data was compared with the reference genome of the oval pomfret, and single nucleotide polymorphism (SNP) sites were called based on the comparison results to finally obtain genome-wide SNP marker genotype data. Among them, the genotype of each SNP site was confirmed by filtering based on its genotype quality value. When generating the genotype quality value, the error probability of genotype calling is used as the basis, and the value is transformed by a negative logarithm function with base 10. The resulting value is the genotype quality value. Genotype data for a SNP locus are only accepted if the genotype quality value of the SNP locus is not less than 20.

[0009] Furthermore, the filtered and confirmed single nucleotide polymorphism marker genotype data were subjected to genome-wide association analysis with the tolerance phenotype data determined in step 1; In the association analysis, a mixed linear model was used for modeling. The dependent variable of the model was composed of the asphyxiation point dissolved oxygen value and survival time under hypoxia stress of all individuals participating in the hypoxia stress tolerance test, as determined in step 1, after standardization. The model includes two parts: fixed effects and random effects. The fixed effects part is used to construct a design matrix using the genotype data of each SNP locus to be tested, which is used to estimate the effect value of each SNP locus. The random effects part is used to characterize the genetic correlation between individuals using a kinship matrix, and it is assumed that the random effects follow a multivariate normal distribution with a mean of zero and a covariance equal to the product of the kinship matrix and the genetic variance. The residuals of the model are also assumed to follow a multivariate normal distribution with a mean of zero and a covariance equal to the product of the identity matrix and the residual variance, in order to characterize the phenotypic variation caused by environmental factors and random errors. For each SNP locus in the mixed linear model, a significance test was performed and the p-value was calculated. Specifically, the Wald test was used to calculate the effect size. The degree of deviation from the null hypothesis is used to generate a test statistic. This test statistic is then substituted into a chi-square distribution to calculate the original p-value for the association analysis of the SNP locus. The Bonferroni correction method is applied to correct the original p-values ​​of all tested SNP loci, and SNP loci with corrected p-values ​​less than or equal to 0.05 are selected as target SNP molecular markers significantly associated with hypoxia tolerance. The null hypothesis is... This indicates that the SNP site has no effect; When performing a significance test for each SNP locus, the test statistic is generated as follows: the estimated value of the effect value of the SNP locus and its variance are obtained by fitting the mixed linear model, and then the square of the estimated value is divided by its variance. The result is the statistic used for the significance test.

[0010] Furthermore, the specific execution process of step 3 is as follows: The remaining individuals that are homologous to the oval pomfret population described in step 1 and have not undergone hypoxia stress test are used as the oval pomfret reserve parent population. Caudal fin tissue samples are collected from the oval pomfret reserve parent population, genomic DNA is extracted, and SNP typing technology is used to perform genotyping on the screened target SNP molecular markers to determine the genotype of each reserve parent at each target SNP locus. Based on the results of genome-wide association analysis, alleles positively associated with hypoxia tolerance at each target SNP locus were identified as favorable alleles, and weights were assigned according to the p-values ​​corrected for each SNP locus. In setting the weight of each target SNP site, the weight is allocated based on the significance level of each SNP site after correction in the genome-wide association analysis. Specifically, the weight of each SNP site is determined by the proportion of the negative logarithm of the corrected p-value to the total negative logarithm of all target SNP sites, and it is ensured that the sum of the weights of all target SNP sites is one. The number of favorable alleles for each backup parent at all target SNP loci was counted. If the genotype of the backup parent at a target SNP locus was homozygous for a favorable allele, 2 favorable alleles were recorded; if the genotype was heterozygous, 1 favorable allele was recorded; and if the genotype was homozygous for an unfavorable allele, 0 favorable alleles were recorded. This was used to obtain the number of favorable alleles for each backup parent at each target SNP locus. Number of favorable alleles at each target SNP locus .

[0011] Furthermore, when determining the hypoxia tolerance evaluation index for each backup parent, the final hypoxia tolerance evaluation index is obtained by comprehensively considering the number of favorable alleles of the backup parent at all target SNP sites and the weight of their corresponding sites. All reserve parents were sorted from high to low according to their hypoxia tolerance evaluation index. The top 30% of the reserve parents were selected as superior parents. The superior parents whose male-female ratio met the preset requirements were selected to form the core breeding population.

[0012] Furthermore, the core breeding population is placed in a controlled breeding environment for artificial breeding to obtain offspring population; for each individual in the offspring population, the same SNP genotyping technology as in step 3 is used to detect its genotype at all target SNP molecular marker sites. When calculating the estimated breeding value of each offspring individual's genome, the genotype data of the individual at all target SNP loci and their corresponding allele substitution effect values ​​are integrated, and a weighted summation method is used to conduct a comprehensive genetic assessment, thereby obtaining the estimated breeding value of the genome that reflects its genetic potential for hypoxia tolerance. The estimated genomic breeding values ​​of all offspring individuals were calculated, and they were sorted from high to low according to the estimated genomic breeding values. The top 50% of individuals were selected to construct a candidate population.

[0013] Furthermore, the secondary hypoxia stress test specifically includes: Body length and weight data of each individual in the candidate population were collected, and the candidate population was placed in a controlled circulating water system for periodic intermittent hypoxia treatment. Each cycle included a 6-hour hypoxia treatment phase and an 18-hour normoxic recovery phase. The experiment continues After that, the body length and weight data of each individual in the candidate group were measured. Based on the body length and weight data, the comprehensive growth performance index of each individual was calculated, and individuals with a comprehensive growth performance index greater than or equal to the group average were selected as the final low-oxygen tolerant oval pomfret breeding varieties.

[0014] Furthermore, when calculating the comprehensive growth performance index, the individual's weight and length changes before and after the two hypoxia stress tests are comprehensively evaluated based on preset weights, thereby obtaining a comprehensive growth performance index that reflects the individual's growth performance under hypoxia stress.

[0015] Compared with the prior art, the beneficial effects of the present invention are: This invention is the first to combine hypoxia stress tolerance phenotype with whole-genome resequencing technology, accurately screening SNP molecular markers significantly associated with hypoxia tolerance through genome-wide association analysis. Based on the number and weight of favorable alleles at target SNP loci and estimated breeding values ​​from a genome perspective, a complete hypoxia tolerance evaluation system was established, achieving a leap from phenotypic selection to genotypic selection, and significantly improving the accuracy and reliability of breeding.

[0016] Traditional population breeding relies on multi-generational genotyping, which is time-consuming and inefficient. This invention, by performing genotyping of parental and offspring populations using target SNP molecular markers, can screen for individuals with high hypoxia tolerance potential in the early developmental stages, helping to shorten the breeding cycle, improve breeding efficiency, and meet the needs of large-scale, targeted breeding of superior varieties.

[0017] Based on a mixed linear model analysis of genetic variance and residual variance, environmental interference was effectively isolated, ensuring that the selected hypoxia tolerance trait possesses high genetic stability. The obtained oval pomfret cultivar exhibits stronger survival and growth advantages in hypoxia environments, helping to reduce aquaculture risks and improve economic benefits, and providing reliable technical support for stress-resistant breeding of oval pomfret and the green and healthy development of the industry.

[0018] By conducting a second intermittent hypoxia stress test on the candidate population, the accuracy of the previous genotype selection can be verified, and individuals that can maintain good growth performance under continuous and periodic hypoxia pressure can be screened out. This will ultimately ensure that the selected varieties have synergistic improvement in hypoxia tolerance and growth traits, and have stronger practical application value. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the overall method flow of the present invention; Figure 2 A parallel coordinate graph of individual indexes, genotype codes, and estimated breeding values ​​based on genomes. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments.

[0021] It should be noted that, unless otherwise defined, the technical or scientific terms used in this invention should have the ordinary meaning understood by one of ordinary skill in the art to which this invention pertains. The terms "first," "second," and similar terms used in this invention do not indicate any order, quantity, or importance, but are merely used to distinguish different components. Terms such as "comprising" or "including" mean that the element or object preceding the word encompasses the elements or objects listed following the word and their equivalents, without excluding other elements or objects. Terms such as "connected" or "linked" are not limited to physical or mechanical connections, but can include electrical connections, whether direct or indirect. Terms such as "upper," "lower," "left," and "right" are used only to indicate relative positional relationships; when the absolute position of the described object changes, the relative positional relationship may also change accordingly.

[0022] Example: Please see Figures 1-2 The present invention provides a technical solution: A method for breeding low-oxygen-tolerant varieties of oval pomfret, the specific steps of which include: Step 1: Conduct a hypoxia stress tolerance test on the oval pomfret population. The dissolved oxygen value at the asphyxiation point and the survival time under hypoxia stress are used as tolerance phenotypes to screen out extreme individuals with tolerance phenotypes. In this embodiment, the dissolved oxygen value at the asphyxiation point refers to the dissolved oxygen concentration in the water when the test individual begins to lose balance and loses the ability to maintain body position during the hypoxia stress test. The lower the value, the stronger the hypoxia tolerance. Survival time under hypoxia stress refers to the time from the start of the test to the individual's death in a hypoxia stress tolerance test. The longer the time, the stronger the hypoxia tolerance. The logic behind conducting hypoxia stress tolerance tests on oval pomfret populations is as follows: A group of healthy oval pomfret individuals with a body length of 10cm-15cm and a weight of 150g-300g were selected to form an oval pomfret population. The oval pomfret population was placed in a controlled, closed breathing chamber for a hypoxia stress test. Nitrogen gas was continuously introduced and the dissolved oxygen concentration in the water was monitored to reduce the dissolved oxygen at a constant rate to a predetermined endpoint. During this process, the dissolved oxygen concentration at which the experimental individuals first became unbalanced and unable to maintain a normal swimming position was recorded as their asphyxiation point dissolved oxygen value. Under the stress environment of maintaining the set hypoxia concentration, the survival time of each individual from the start of the test to death was continuously observed and recorded. Finally, based on the asphyxiation point dissolved oxygen value from low to high and the survival time from long to short, the top 25% of individuals in both indicators were selected and defined as extreme individuals with a tolerance phenotype. Subsequently, tail fin tissue samples were immediately collected from the extreme individuals with a tolerance phenotype and placed in centrifuge tubes containing RNA protectant for cryopreservation for later use.

[0023] Step 1 plays a crucial role in phenotypic screening and laying the foundation for genetic material in this invention. Through systematic hypoxia stress tests on an oval pomfret population, two key phenotypic indicators—dissolved oxygen level at the asphyxiation point and survival time—were accurately measured. This allowed us to screen for individuals exhibiting the best tolerance phenotype under extreme hypoxia conditions. This step not only provides a reliable phenotypic data basis for subsequent genome-wide association studies but also lays an important material and informational foundation for obtaining genetic samples with significant hypoxia tolerance potential and establishing a core breeding population. It is the primary step in achieving precise breeding from phenotype to genotype.

[0024] Step 2: Obtain genotype data of individuals with extreme tolerance phenotypes; perform genome-wide association analysis on the genotype data and the tolerance phenotypes identified in Step 1 to screen for target SNP molecular markers that are significantly associated with hypoxia tolerance traits; In this embodiment, genomic DNA was extracted and quality-checked from caudal fin tissue samples collected from individuals with the extreme tolerance phenotype, and a whole-genome sequencing library was constructed. Subsequently, the library was re-sequencing the entire genome using a high-throughput sequencing platform to obtain raw sequencing data. The raw sequencing data was compared with the reference genome of *S. ovalis*, and single nucleotide polymorphism (SNP) site calling was performed based on the comparison results to finally obtain genome-wide SNP marker genotype data. The genotype of each SNP site was confirmed by filtering based on its genotype quality value. The calculation formula is:

[0025] In the formula, This is the genotype quality score, used to measure the reliability of genotype determination. The higher the value, the higher the reliability of the genotype determination. This represents the error probability of genotype calling, used to assess the reliability of SNP genotyping. Its value is calculated internally by the sequencing quality and genotyping algorithm.

[0026] Genotype data for a SNP locus are only accepted if the genotype quality value of the SNP locus is not less than 20.

[0027] The filtered and confirmed single nucleotide polymorphism marker genotype data were compared with the tolerance phenotype data determined in step 1 to perform a genome-wide association analysis. The correlation analysis employs a mixed linear model, based on the following formula:

[0028] In the formula, The phenotypic value vector is composed of the asphyxiation point dissolved oxygen value and survival time under hypoxia stress of all individuals participating in the hypoxia stress tolerance test determined in step 1, after standardization. A fixed-effects matrix is ​​designed, with columns including the genotype of each SNP locus to be tested, to estimate the fixed effect of that SNP. This is a fixed-effects vector, containing the effect values ​​of SNP sites; Design a matrix for random effects; The random effects vector follows a multivariate normal distribution, i.e. ,in A kinship matrix; Genetic variance represents the phenotypic differences caused by genetic factors; The residual vector follows a multivariate normal distribution, i.e. ,in It is the identity matrix; The residual variance represents the phenotypic differences caused by environmental factors and random errors. in, The asphyxiation point dissolved oxygen value and survival time were standardized by Z-score and then combined. It consists of the genotype code for each SNP locus, and the genotype code includes 0, 1, and 2; For the fixed effects value of the SNP to be estimated, the restricted maximum likelihood method or generalized least squares method is used for fitting estimation; Let it be the design matrix corresponding to the random effects structure; in common cases, it is set as the identity matrix. The random effects vector has a covariance structure derived from the kinship matrix. and genetic variance Common definition, Constructed based on genome-wide SNPs using a genetic relationship matrix; and The results were obtained by using restricted maximum likelihood or similar algorithms based on joint estimation of phenotypic and genotypic data.

[0029] For each SNP locus in the mixed linear model, a significance test was performed and the p-value was calculated. Specifically, the Wald test was used to calculate the effect size. The degree of deviation from the null hypothesis is used to generate a test statistic. This test statistic is then substituted into a chi-square distribution to calculate the original p-value for the association analysis of the SNP locus. The Bonferroni correction method is applied to correct the original p-values ​​of all tested SNP loci, and SNP loci with corrected p-values ​​less than or equal to 0.05 are selected as target SNP molecular markers significantly associated with hypoxia tolerance. The null hypothesis is... This indicates that the SNP site has no effect.

[0030] The formula for calculating the test statistic is as follows:

[0031] In the formula, To test the statistic, The estimated SNP effect value is obtained by fitting the mixed linear model; for The variance is obtained by fitting the mixed linear model.

[0032] Step 2 involves decoding the genetic information of the extreme individuals with the tolerance phenotype identified in Step 1. Tissue samples from these oval pomfret, exhibiting exceptional hypoxia tolerance, are collected for whole-genome sequencing to obtain comprehensive genotypic data. This step transforms the invisible genetic trait of hypoxia tolerance into readable and analyzable molecular information, laying a solid foundation for accurately identifying key gene markers controlling hypoxia tolerance.

[0033] Step 3: Using the selected target SNP molecular markers, the genotype of the reserve parent population of oval pomfret is classified. Based on the number and weight of favorable alleles at all target SNP loci for each reserve parent, its hypoxia tolerance evaluation index is determined. Based on the hypoxia tolerance evaluation index, superior parents are selected to form a core breeding population. In this embodiment, the specific execution process of step 3 is as follows: The remaining individuals that are homologous to the oval pomfret population described in step 1 and have not undergone hypoxia stress test are used as the oval pomfret reserve parent population. Caudal fin tissue samples are collected from the oval pomfret reserve parent population, genomic DNA is extracted, and SNP typing technology is used to perform genotyping on the screened target SNP molecular markers to determine the genotype of each reserve parent at each target SNP locus. Genome-wide association analysis (GWA) results identified alleles positively correlated with hypoxia tolerance at each target SNP locus as favorable alleles. Weights were assigned based on the corrected p-value of each SNP locus, calculated using the following formula:

[0034] In the formula, For the first The weights of each target SNP site. An index for the target SNP site; For the first p-values ​​of the target SNP loci after Bonferroni correction; The number of target SNP sites is denoted by , and the sum of the weights of all target SNP sites is 1.

[0035] The purpose of calculating the weights of target SNP loci is to quantify their relative importance based on the significance of their association with the hypoxia tolerance trait. By converting the corrected p-values ​​into weights, SNP loci with more significant associations (i.e., smaller p-values) receive higher weights in subsequent evaluations. This allows for a more accurate reflection of the actual genetic contribution of different loci to the hypoxia tolerance trait in parental selection and breeding value estimation, thereby improving the precision and efficiency of breeding.

[0036] The number of favorable alleles for each backup parent at all target SNP loci was counted. If the genotype of the backup parent at a target SNP locus was homozygous for a favorable allele, 2 favorable alleles were recorded; if the genotype was heterozygous, 1 favorable allele was recorded; and if the genotype was homozygous for an unfavorable allele, 0 favorable alleles were recorded. This was used to obtain the number of favorable alleles for each backup parent at each target SNP locus. Number of favorable alleles at each target SNP locus .

[0037] The hypoxia tolerance evaluation index is determined based on the number and weight of favorable alleles at all target SNP loci for each reserve parent, using the following formula:

[0038] In the formula, As an evaluation index for the hypoxia tolerance of reserve parents, For the first The number of favorable alleles at each target SNP locus.

[0039] Dependent variable This score is used to characterize the potential hypoxia tolerance of reserve parent individuals at the genetic level. The higher the value, the more favorable alleles positively correlated with hypoxia tolerance are carried by the reserve parent, and the higher their weight, indicating a stronger potential for passing on hypoxia tolerance to offspring; conversely, the lower the value, the weaker the genetic potential for hypoxia tolerance.

[0040] The number of favorable alleles directly affects the hypoxia tolerance evaluation index because each favorable allele is considered to contribute positively to the hypoxia tolerance trait. With fixed weights, the more favorable alleles a replacement parent carries at a given locus, the greater its contribution to the dependent variable at that locus. The greater the contribution, the higher the probability. Therefore, the total number of favorable alleles at all target loci and their product with the weights together determine the probability of an individual's success. Value; the more favorable alleles, the higher the hypoxia tolerance index. Generally, the higher the value, the greater the genetic value of the parent.

[0041] All reserve parents were sorted from high to low according to their hypoxia tolerance evaluation index. The top 30% of reserve parents were selected as superior parents, and the superior parents with a female-to-male ratio of 1:1.5 were selected to form the core breeding population.

[0042] Step 3 aims to locate key gene markers directly related to hypoxia tolerance. By performing association analysis between the gene data obtained in Step 2 and the hypoxia tolerance phenotype recorded in Step 1, SNP molecular markers significantly associated with hypoxia tolerance are screened from the gene information. This step is equivalent to identifying the genes that truly play a role in hypoxia tolerance from numerous gene loci, thereby establishing a reliable link between genotype and hypoxia tolerance phenotype, allowing breeding work to move from relying on external observation to understanding the genetic essence.

[0043] Step 4: Based on the constructed core breeding population, breed to obtain offspring population, and by detecting whether each individual in the offspring population carries the favorable allele in the target SNP molecular marker, combined with the genomic estimation breeding value, screen out low-oxygen-tolerant oval pomfret varieties as candidate populations; In this embodiment, the core breeding population is placed in a controlled breeding environment for artificial breeding to obtain a progeny population. For each individual in the progeny population, the same SNP genotyping technology as in step 3 is used to detect its genotype at all target SNP molecular marker loci. Based on the genotype, the estimated genomic breeding value of each progeny individual is calculated using the following formula:

[0044] In the formula, The breeding value of the genome of the j-th offspring is estimated to predict its genetic potential for hypoxia tolerance; Indicates the first The offspring individuals in the first The genotype encoding of each target SNP locus, when the genotype is homozygous for the favorable allele, heterozygous for the unfavorable allele, or homozygous for the unfavorable allele. The values ​​are 2, 1, and 0 respectively; For the first The allele substitution effect value of each target SNP locus is equal to the estimated value of the SNP effect value. Dependent variable A comprehensive predictive value used to characterize the potential genetic advantage of offspring in the trait of hypoxia tolerance inherited from their parents. The higher the value, the more favorable alleles positively associated with hypoxia tolerance the individual carries and the stronger their genetic effect, indicating that the individual may have better survival and growth potential in hypoxic environments; conversely, the lower the value, the lower the genetic potential.

[0045] It directly impacts the estimated breeding value of the genome because it quantifies the genetic contribution of offspring individuals to the hypoxia tolerance trait at each key SNP locus. When An increase in the value indicates an increase in the positive genetic contribution to hypoxia tolerance at that locus. Therefore, assuming the allele substitution effect remains constant, The higher the value, the greater its positive contribution to the estimated breeding value of the genome, thus leading to a corresponding increase in the estimated breeding value of the genome.

[0046] The formula's formal rationality lies in its construction of an additive genetic effect model by multiplying and summing the genotype codes of each key SNP locus with their corresponding allele substitution effect values. This form intuitively reflects the cumulative contribution of multiple gene loci to the hypoxia tolerance trait, consistent with the fundamental principle of polygenic control of traits in quantitative genetics—that is, an individual's total breeding value can be approximately represented as the linear superposition of the genetic effects of each locus. Therefore, the formula is reasonable both mathematically and genetically.

[0047] Table 1: Statistics of Genomic Estimated Breeding Values

[0048] It should be noted that the individual index, SNP1 genotype code, SNP2 genotype code, SNP3 genotype code, SNP4 genotype code, and SNP5 genotype code mentioned in Table 1 correspond to... , , , , , Furthermore, in this embodiment, the first... Allelic substitution effect value of each target SNP locus , No. Allelic substitution effect value of each target SNP locus , No. Allelic substitution effect value of each target SNP locus , No. Allelic substitution effect value of each target SNP locus , No. Allelic substitution effect value of each target SNP locus .

[0049] Analysis of the data in Table 1 shows significant differences in the genetic potential for hypoxia tolerance among individuals. Some individuals carried homozygous favorable alleles at multiple target SNP loci, exhibiting high breeding values ​​and demonstrating excellent genetic potential for hypoxia tolerance; while others carried more unfavorable alleles, resulting in relatively lower breeding values. Overall, the data validates the feasibility of using target SNP molecular markers and effect values ​​to estimate breeding values ​​based on genome sequencing. This method effectively distinguishes the genetic advantages of individuals and provides a reliable basis for the precise breeding of hypoxia-tolerant varieties.

[0050] The estimated genomic breeding values ​​of all offspring individuals were calculated, and they were sorted from high to low according to the estimated genomic breeding values. The top 50% of individuals were selected to construct a candidate population.

[0051] The core objective of step 4 is to select the best among the best and establish a breeding population with high hypoxia tolerance potential. Using the key gene markers identified in the previous steps, genotypic screening is performed on the reserve parents to assess the hypoxia tolerance genetic potential of each parent, and a core breeding population is established accordingly. This step is equivalent to selecting the best candidates for subsequent breeding, ensuring that the most superior hypoxia tolerance genes are concentratedly passed on to the next generation, laying the parental foundation for cultivating new varieties with truly strong hypoxia tolerance.

[0052] Step 5: Conduct a second hypoxia stress test on the candidate population. The second hypoxia stress test adopts an intermittent hypoxia treatment method. After the test, the body length and weight of each individual in the candidate population are measured. The comprehensive growth performance index is determined based on the body length and weight. The final hypoxia-tolerant oval pomfret breeding variety is selected based on the comprehensive growth performance index. In this embodiment, the secondary hypoxia stress test specifically includes: Body length and weight data were collected for each individual in the candidate population. The candidate population was then placed in a controlled circulating water system for periodic intermittent hypoxia treatment. Each cycle included a 6-hour hypoxia treatment phase and an 18-hour normoxic recovery phase. During the hypoxia phase, the dissolved oxygen concentration in the water was controlled at [value missing]. Dissolved oxygen concentration during the normoxic phase is controlled at ; The experiment continues After that, the body length and weight data of each individual in the candidate population were measured. Based on the body length and weight data, the comprehensive growth performance index of each individual was calculated, and individuals with a comprehensive growth performance index greater than or equal to the population mean were selected as the final low-oxygen tolerant oval pomfret breeding varieties; in this embodiment, Set to 30 days; The formula for calculating the comprehensive growth performance index is as follows:

[0053] In the formula, The comprehensive growth performance index, This refers to the individual's body weight data prior to the second hypoxia stress test. The data pertains to the individual's body weight after undergoing a second hypoxia stress test. The data represents the individual's body length prior to the second hypoxia stress test. Body length data for individuals after undergoing a second hypoxia stress test; and Preset weights for body weight and body length, respectively. , .

[0054] In this embodiment, the following settings are provided: , The reason is that, in actual fish farming production, body weight is a more direct and crucial indicator for measuring growth performance and economic benefits. Body weight gain directly reflects muscle accumulation and overall weight gain in the fish, and is closely related to yield and farming profits. While body length is also a growth indicator, its correlation with final yield is not as significant as that of body weight, and body length growth may not always be accompanied by a synchronous increase in body weight. Therefore, giving body weight a higher weight allows for a more accurate assessment of the actual growth performance and commercial value of fish under hypoxic stress, making breeding results more aligned with industry needs.

[0055] Dependent variable This value is used to characterize the overall growth performance of oval pomfret individuals after undergoing two hypoxic stress tests. A higher value indicates a greater increase in body weight and length during the two hypoxic stress tests, meaning its growth is less affected by the hypoxic environment and it has stronger hypoxia tolerance; conversely, a lower value indicates a lower tolerance. A smaller value indicates that the individual's growth was severely inhibited during the second hypoxia stress test and that its hypoxia tolerance was weak.

[0056] when or A larger increase indicates that the oval pomfret experienced a significant increase in weight or body length during the second hypoxic stress test; this suggests that it was still able to effectively ingest, digest, and accumulate energy, maintaining normal growth and metabolism. An elevated value indicates that the individual has a strong tolerance to hypoxia and is less affected by stress; conversely, if the increase is small or even negative, it indicates that hypoxia has severely inhibited its feeding and metabolism, leading to growth retardation or emaciation. The value subsequently decreased, indicating that the individual had poor adaptation to low oxygen and significant growth impairment. This means that... and , They show a positive correlation.

[0057] The formula's formal rationality lies in its summation of the sum of the relative growth rates of body weight and body length multiplied by preset weights, thus comprehensively reflecting the growth performance of oval pomfret individuals under two hypoxic stress experiments. This design considers both body weight and body length, two key growth indicators, and emphasizes the contribution of body weight changes to overall growth performance through weight allocation. This aligns with the practical need in aquaculture to prioritize weight gain, making the evaluation results more scientific and practical.

[0058] Step 5 serves to verify and comprehensively evaluate the results of the previous breeding selection. By conducting intermittent hypoxic stress experiments on the candidate population, which closely resemble actual aquaculture conditions, and comprehensively examining their growth performance under hypoxic environments, individuals that not only have excellent genotypes but also maintain good growth performance under actual hypoxic pressure are selected. This ensures that the selected oval pomfret variety not only has excellent genes but also truly possesses hypoxia tolerance and growth advantages in actual production.

[0059] The above formulas are all dimensionless calculations. The formulas are derived from software simulations based on a large amount of collected data to obtain the most recent real-world results. The preset parameters in the formulas are set by those skilled in the art according to the actual situation.

[0060] The above embodiments can be implemented, in whole or in part, by software, hardware, firmware, or any other combination thereof. When implemented in software, the above embodiments can be implemented, in whole or in part, as a computer program product. Those skilled in the art will recognize that the units and algorithm steps of the various examples described in conjunction with the embodiments disclosed herein can be implemented by electronic hardware, or a combination of computer software and electronic hardware. Whether these functions are implemented in hardware or software depends on the specific application and design constraints of the technical solution.

[0061] The units described as separate components may or may not be physically separate. The components shown as units may or may not be physical units; they may be located in one place or distributed across multiple network units. Some or all of the units can be selected to achieve the purpose of this embodiment according to actual needs.

[0062] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application.

Claims

1. A method for breeding low-oxygen-tolerant varieties of oval pomfret, characterized in that, The specific steps include: Step 1: Conduct a hypoxia stress tolerance test on the oval pomfret population. The dissolved oxygen value at the asphyxiation point and the survival time under hypoxia stress are used as tolerance phenotypes to screen out extreme individuals with tolerance phenotypes. Step 2: Obtain genotype data of individuals with extreme tolerance phenotypes; perform genome-wide association analysis on the genotype data and the tolerance phenotypes identified in Step 1 to screen for target SNP molecular markers that are significantly associated with hypoxia tolerance traits; Step 3: Using the selected target SNP molecular markers, the genotype of the reserve parent population of oval pomfret is classified. Based on the number and weight of favorable alleles at all target SNP loci for each reserve parent, its hypoxia tolerance evaluation index is determined. Based on the hypoxia tolerance evaluation index, superior parents are selected to form a core breeding population. Step 4: Based on the constructed core breeding population, breed to obtain offspring population, and by detecting whether each individual in the offspring population carries the favorable allele in the target SNP molecular marker, combined with the genomic estimation breeding value, screen out low-oxygen-tolerant oval pomfret varieties as candidate populations; Step 5: Conduct a second hypoxia stress test on the candidate population. The second hypoxia stress test adopts an intermittent hypoxia treatment method. After the test, the body length and weight of each individual in the candidate population are measured. The comprehensive growth performance index is determined based on the body length and weight, and the final hypoxia-tolerant oval pomfret breeding variety is selected based on the comprehensive growth performance index.

2. The method for breeding low-oxygen-tolerant varieties of oval pomfret according to claim 1, characterized in that: The asphyxiation point dissolved oxygen value refers to the dissolved oxygen concentration in the water when the test individual begins to lose balance and loses the ability to maintain body position during the hypoxia stress test. The lower the value, the stronger the hypoxia tolerance. Survival time under hypoxia stress refers to the time from the start of the test to the individual's death in a hypoxia stress tolerance test. The longer the time, the stronger the hypoxia tolerance. The logic behind conducting hypoxia stress tolerance tests on oval pomfret populations is as follows: A population of oval pomfret was formed by selecting several individuals. The population was placed in a controlled, closed breathing chamber for a hypoxia stress test. Nitrogen was continuously introduced while the dissolved oxygen concentration in the water was monitored, and the dissolved oxygen was reduced at a constant rate to a predetermined endpoint. During this process, the dissolved oxygen concentration at which the individual first became unbalanced and unable to maintain a normal swimming position was recorded as its asphyxiation point dissolved oxygen value. Under the stress environment of maintaining the set hypoxia concentration, the survival time of each individual from the start of the test to death was continuously observed and recorded. Finally, based on the asphyxiation point dissolved oxygen value from low to high and the survival time from long to short, extreme individuals that met the preset requirements in both indicators were selected and defined as tolerance phenotype extreme individuals. Subsequently, tail fin tissue samples were immediately collected from the tolerance phenotype extreme individuals and stored at low temperature in centrifuge tubes containing RNA protectant for later use.

3. The method for breeding low-oxygen-tolerant varieties of oval pomfret according to claim 2, characterized in that: Genomic DNA was extracted and quality-checked from caudal fin tissue samples of the collected extreme individuals with the tolerance phenotype, and a whole-genome sequencing library was constructed. Subsequently, the library was re-sequencing the whole genome using a high-throughput sequencing platform to obtain the raw sequencing data. The raw sequencing data was compared with the reference genome of the oval pomfret, and single nucleotide polymorphism (SNP) sites were called based on the comparison results to obtain genome-wide SNP marker genotype data. The genotype of each SNP site was confirmed by filtering based on its genotype quality value. When generating the genotype quality value, the error probability of genotype calling is used as the basis, and the value is transformed by a negative logarithm function with base 10. The resulting value is the genotype quality value. Genotype data for a SNP locus are only accepted if the genotype quality value of the SNP locus is not less than 20.

4. The method for breeding low-oxygen-tolerant varieties of oval pomfret according to claim 3, characterized in that: The filtered and confirmed single nucleotide polymorphism marker genotype data were compared with the tolerance phenotype data determined in step 1 to perform a genome-wide association analysis. In the association analysis, a mixed linear model was used for modeling. The dependent variable of the model was composed of the asphyxiation point dissolved oxygen value and survival time under hypoxia stress of all individuals participating in the hypoxia stress tolerance test, as determined in step 1, after standardization. The model includes two parts: fixed effects and random effects. The fixed effects part is used to construct a design matrix using the genotype data of each SNP locus to be tested, which is used to estimate the effect value of each SNP locus. The random effects part is used to characterize the genetic correlation between individuals using a kinship matrix, and it is assumed that the random effects follow a multivariate normal distribution with a mean of zero and a covariance equal to the product of the kinship matrix and the genetic variance. The residuals of the model are also assumed to follow a multivariate normal distribution with a mean of zero and a covariance equal to the product of the identity matrix and the residual variance, in order to characterize the phenotypic variation caused by environmental factors and random errors. For each SNP locus in the mixed linear model, a significance test was performed and the p-value was calculated. Specifically, the Wald test was used to calculate the effect size. The degree of deviation from the null hypothesis is used to generate a test statistic; Substituting the test statistic into the chi-square distribution, the original p-value for the association analysis of this SNP locus was calculated. The Bonferroni correction method was applied to correct the original p-values ​​of all tested SNP loci, and SNP loci with corrected p-values ​​less than or equal to 0.05 were selected as target SNP molecular markers significantly associated with hypoxia tolerance. The null hypothesis is... This indicates that the SNP site has no effect; When performing a significance test for each SNP locus, the test statistic is generated as follows: the estimated value of the effect value of the SNP locus and its variance are obtained by fitting the mixed linear model, and then the square of the estimated value is divided by its variance. The result is the statistic used for the significance test.

5. The method for breeding low-oxygen-tolerant varieties of oval pomfret according to claim 1, characterized in that: The specific execution process of step 3 is as follows: The remaining individuals that are homologous to the oval pomfret population described in step 1 and have not undergone hypoxia stress test are used as the oval pomfret reserve parent population. Caudal fin tissue samples are collected from the oval pomfret reserve parent population, genomic DNA is extracted, and SNP typing technology is used to perform genotyping on the screened target SNP molecular markers to determine the genotype of each reserve parent at each target SNP locus. Based on the results of genome-wide association analysis, alleles positively associated with hypoxia tolerance at each target SNP locus were identified as favorable alleles, and weights were assigned according to the p-values ​​corrected for each SNP locus. In setting the weight of each target SNP site, the weight is allocated based on the significance level of each SNP site after correction in the genome-wide association analysis. Specifically, the weight of each SNP site is determined by the proportion of the negative logarithm of the corrected p-value to the total negative logarithm of all target SNP sites, and it is ensured that the sum of the weights of all target SNP sites is one. The number of favorable alleles for each backup parent at all target SNP loci was counted. If the genotype of the backup parent at a target SNP locus was homozygous for a favorable allele, 2 favorable alleles were recorded; if the genotype was heterozygous, 1 favorable allele was recorded; and if the genotype was homozygous for an unfavorable allele, 0 favorable alleles were recorded. This was used to obtain the number of favorable alleles for each backup parent at each target SNP locus. Number of favorable alleles at each target SNP locus .

6. The method for breeding low-oxygen-tolerant varieties of oval pomfret according to claim 5, characterized in that: When determining the hypoxia tolerance evaluation index for each backup parent, the final hypoxia tolerance evaluation index is obtained by comprehensively considering the number of favorable alleles of the backup parent at all target SNP sites and the weight of their corresponding sites. All reserve parents were sorted from high to low according to their hypoxia tolerance evaluation index. The top 30% of the reserve parents were selected as superior parents. The superior parents with the male-female ratio that met the preset requirements were selected to form the core breeding population.

7. A method for breeding low-oxygen-tolerant varieties of oval pomfret according to claim 6, characterized in that: The core breeding population is placed in a controlled breeding environment for artificial breeding to obtain offspring population; for each individual in the offspring population, the same SNP genotyping technology as in step 3 is used to detect its genotype at all target SNP molecular marker sites. When calculating the estimated breeding value of each offspring individual's genome, the genotype data of the individual at all target SNP loci and their corresponding allele substitution effect values ​​are integrated, and a weighted summation method is used to conduct a comprehensive genetic assessment, thereby obtaining the estimated breeding value of genome that reflects its genetic potential for hypoxia tolerance. The estimated genomic breeding values ​​of all offspring individuals were calculated, and they were sorted from high to low according to the estimated genomic breeding values. The top 50% of individuals were selected to construct a candidate population.

8. The method for breeding low-oxygen-tolerant varieties of oval pomfret according to claim 1, characterized in that: The secondary hypoxia stress test specifically includes: Body length and weight data of each individual in the candidate population were collected, and the candidate population was placed in a controlled circulating water system for periodic intermittent hypoxia treatment. Each cycle included a 6-hour hypoxia treatment phase and an 18-hour normoxic recovery phase. The experiment continues After that, the body length and weight data of each individual in the candidate group were measured. Based on the body length and weight data, the comprehensive growth performance index of each individual was calculated, and individuals with a comprehensive growth performance index greater than or equal to the group average were selected as the final low-oxygen tolerant oval pomfret breeding varieties.

9. A method for breeding low-oxygen-tolerant varieties of oval pomfret according to claim 8, characterized in that: When calculating the comprehensive growth performance index, the data on the individual's weight change and body length change before and after the two hypoxia stress tests are combined with preset weights for comprehensive evaluation, thereby obtaining the comprehensive growth performance index that reflects the individual's growth performance under hypoxia stress.

Images