A method for breeding a few interosseous bone strain of cyprinus carpio based on whole genome selection

Abstract

The application belongs to the technical field of aquatic genetic breeding, and particularly relates to a breeding method of a few intermuscular bones strain of a carp based on whole genome selection. The reference population is established by determining the number of intermuscular bones of the carp, the candidate population is established, then the reference population and the candidate population are resequenced, and high-quality SNP typing results are obtained; the genetic parameters of the target traits are estimated according to the high-quality SNP typing results, the genomic breeding value (GEBV) of the candidate population is further estimated, the candidate individuals are selected as the parent fish according to the GEBV value, and the breeding of the few-spine F1 generation seedlings is carried out. The purpose of breeding the few-spine fine strain is achieved by only calculating the GEBV value through gene sequencing without killing the fish. The application develops a set of genome selection scheme suitable for the improvement of the number of intermuscular bones of the carp, the operation is clear, and a new molecular marker assisted breeding technology is provided for the breeding of the few-spine fine strain of the carp.

Description

Technical Field

[0001] This invention belongs to the field of aquatic genetic breeding technology, specifically relating to a breeding method for carp strains with few intermuscular bones based on whole-genome selection. Background Technology

[0002] carp( Cyprinus carpio ) belongs to the order Cypriniformes ( Cypriniformes Cyprinidaceae ( Cyprinidae Cyprinids are currently the most widely farmed and important economic fish species internationally, cultivated in over 100 countries worldwide. Intermuscular bones are membranous bones located in the intermuscular septa on either side of the vertebrae, and are present in most cyprinid fish. However, the presence of intermuscular bones causes inconvenience in consuming fish meat and limits the deep processing of fish products. Therefore, selecting fish with a higher number of intermuscular bones has become an important goal in the breeding of new fish varieties.

[0003] The exploration of methods to reduce the number of intermuscular bones began in 1967. Some researchers bred fish with fewer bones through hybridization. For example, Wu et al. bred a new hybrid culter (sharphead bream ♀ × topmouth bream ♂) using distant hybridization, resulting in a 5.7% reduction in intermuscular bones compared to the paternal parent. With the development of gene editing technology, some scholars have focused on obtaining boneless fish through gene editing. For example, Zhong et al. knocked out the sp7a gene in carp, but bone defects appeared. Gao Zexia et al. knocked out the scxa gene in zebrafish, resulting in a 70% reduction in the number of intermuscular bones in the mutant, but also problems such as rib development defects appeared. This team also knocked out the runx2b gene in blunthead bream, resulting in a reduction of more than 30% in the number of intermuscular bones in the mutant blunthead bream. Kuang Youyi et al. bred crucian carp with fewer or no bones by knocking out the bmp6 gene. Although gene editing technology has significantly reduced the number of intermuscular bones at the individual level in fish, no new boneless / few-boned strains / species have been reported at the population level. In addition, genetically modified fish are subject to strict restrictions in aquaculture, and their industrialization prospects are unclear.

[0004] Selective breeding can effectively improve target traits at the population level. Assessments of the heritability of intermuscular bone quantity showed moderate heritability for vascular arch ossicle quantity, further demonstrating that selective breeding can be used to cultivate this trait. However, for traits difficult to measure in vivo, conventional breeding relies on selection of siblings, which is inefficient and time-consuming. Marker-assisted selection depends on the accurate identification of major genes or trait-related molecular markers, but significant progress has not yet been made in elucidating the genetic mechanisms of intermuscular bone quantity. Genome-wide selection technology offers the possibility of breeding complex traits, enabling significant genetic progress in the short term for intermuscular bone quantity. Summary of the Invention

[0005] The purpose of this invention is to provide a method for breeding carp with fewer intermuscular bones based on whole-genome selection technology, thereby achieving the breeding of carp with a reduced number of intermuscular bones at the population level.

[0006] The technical solution of this invention is implemented as follows:

[0007] A method for breeding carp with few intermuscular bones based on genome-wide selection technology, comprising the following steps:

[0008] (1) Determination of the number of intermuscular bones in carp and establishment of a reference population.

[0009] At least 500 healthy carp with no deformities and a weight greater than 100g were selected from a wide range of sources as the test population for the quantitative trait of intermuscular bones. They were electronically tagged, and their fin rays were cut off and preserved in anhydrous ethanol.

[0010] The specific steps for determining the quantity of intermuscular bone in the test group are as follows:

[0011] Live or frozen whole fish are tightly wrapped in at least three layers of newspaper and steamed at high temperature until the flesh can be completely separated. After the fish cools down, an electronic tag is scanned to record the fish's identification information. The skin tissue is then removed on a dissecting tray, and the muscles are carefully removed. From tail to head, the medullary arch bones and vascular arch bones are removed sequentially and arranged neatly according to their position in the fish. They are then pasted onto black cardstock. After all intermuscular bones have been removed, the cardstock is laminated to fix them, thus creating an intermuscular bone specimen. The total number of intermuscular bones in an individual and the number of intermuscular bones in each location are used as phenotypic data for subsequent analysis.

[0012] The phenotypic distribution of the measured phenotypic population was fitted with a normal distribution, and genotyped individuals were selected to establish a reference population.

[0013] (2) Establishment of the candidate population:

[0014] Candidate populations refer to individuals that have not undergone intermuscular bone quantitative phenotyping or do not have an intermuscular bone quantitative phenotypic phenotype. A subset of individuals whose intermuscular bone quantitative phenotypic phenotype is unknown are selected from the population, or parent fish used for breeding in the cultivation of superior varieties are selected as candidate populations.

[0015] (3) Perform whole-genome resequencing on the reference population and candidate population to obtain high-quality SNP genotyping results.

[0016] Genomic DNA was extracted from the reference and candidate populations, and after library construction, low-depth resequencing of the genome was performed. SNP genotyping of the population was carried out using software such as GATK. The original genotyping data was cleaned and quality controlled, missing sites were filled, and finally a high-quality SNP genotyping dataset that can be used for genome selection was generated.

[0017] (4) Estimate the genetic parameters of the target trait

[0018] ① Use the high-quality SNP genotyping dataset of the reference population obtained in step (3) to perform haplotype genome-wide association analysis, obtain the significance P-value of each haplotype, sort them from low to high according to the P-value, select the tag SNPs of the top 50, 100, 500, 1000, 5000, 10000, 50000 and all haplotypes respectively, and use ASREML software to build a genome-wide selection model. Calculate the model prediction accuracy of each SNP subset by using the 5-fold cross-validation method; the number of tags in the corresponding SNP subset when the model accuracy is the highest is the optimal tag density.

[0019] ② Based on the high-quality SNP genotyping dataset from step (3), construct a kinship matrix A or a genome relationship matrix G that includes all individuals in the reference population and the candidate population. Estimate the variance components using the average information-constrained maximum likelihood method, thereby calculating the heritability of traits and the genetic correlation between traits.

[0020] (5) Estimate the genomic breeding value (GEBV) of the candidate population.

[0021] At the optimal marker density obtained in step (4), the GEBV of the candidate population is estimated based on the G matrix and heritability from step (4), and the individuals are sorted from smallest to largest. The GEBV of each individual in the candidate population is calculated. The accuracy of the GEBV results of the reference population is evaluated through cross-validation. The GEBV of each individual in the candidate population is further calculated. If the candidate population already has offspring, the correlation between the phenotype of the target trait in the offspring and the GEBV of the individuals in the candidate population is analyzed to verify the accuracy of genomic selection. If the candidate population completes performance testing, it can be placed into the reference population to estimate the GEBV of the new candidate population, and this process is repeated.

[0022] (6) Select candidate individuals as parent fish based on GEBV value and breed them into first-generation fry with fewer thorns.

[0023] Verification has shown a positive correlation between the number of intermuscular bones and GEBV in carp; individuals with fewer intermuscular bones also have lower GEBV. Therefore, individuals can be selected for breeding based on their GEBV. GEBV is sorted from lowest to highest, and with a certain selection intensity (top 10%), individuals with the highest GEBV are selected as parent fish to establish family lines for the breeding of first-generation fry with fewer spines.

[0024] The present invention has the following beneficial effects:

[0025] This invention belongs to the field of aquatic genetics and breeding technology, specifically relating to a method for breeding carp strains with fewer intermuscular bones based on whole-genome selection. This application establishes a reference population and a candidate population by measuring the number of intermuscular bones in carp. Then, whole-genome resequencing is performed on both the reference and candidate populations to obtain high-quality SNP genotyping results. Based on the high-quality SNP genotyping results, the genetic parameters of the target trait are estimated, and the genomic breeding value (GEBV) of the candidate population is further estimated. Candidate individuals are selected as broodstock based on the GEBV value for breeding F1 fry with fewer bones. This method achieves the goal of breeding superior strains with fewer bones without killing the fish, simply by calculating the GEBV value through gene sequencing of candidate individuals, effectively solving the problem of individual selection being impossible in intermuscular bone selection. This invention develops a genomic selection scheme suitable for improving the number of intermuscular bones in carp, providing a new molecular marker-assisted breeding technology for breeding superior strains of carp with fewer bones. The F1 fry with fewer bones obtained by this invention have significantly fewer intermuscular bones than the control group, indicating that the average number of intermuscular bones in the genomically selected breeding lines is reduced by 11%-7% compared to the control group. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 This is a distribution diagram of body weight and the number of intermuscular bones; where imb.num is the total number of intermuscular bones; wt is body weight; ep is the number of ossicles in the vascular arch; and en is the number of ossicles in the myeloid arch.

[0028] Figure 2 To improve the accuracy of GEBV estimation for the intermuscular bone quantity trait in Yellow River carp populations under different marker densities.

[0029] Figure 3 For reference, a scatter plot of total intermuscular bone count and GEBV.

[0030] Figure 4 This is a genomic phylogenetic diagram of the reference population and the candidate population.

[0031] Figure 5 Scatter plot of GEBV among candidate families. Detailed Implementation

[0032] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0033] Example 1

[0034] The embodiments of the present invention include the following steps:

[0035] (1) Determination of the number of intermuscular bones in carp and establishment of a reference population.

[0036] In April 2021, parent fish were selected from the core breeding group of Yellow River carp to construct 68 Yellow River carp families. Fifty individuals from each family were collected and cultured together. In November 2021, 500 six-month-old juvenile fish with an average weight of 319g were randomly selected from this mixed group, and their fins were cut off and preserved in anhydrous ethanol.

[0037] The specific steps for determining the quantity of intermuscular bone in the test group are as follows:

[0038] Live or frozen whole fish are tightly wrapped in at least three layers of newspaper and steamed at high temperature until the flesh can be completely separated. After the fish has cooled, the skin tissue is removed on a dissecting tray, and the muscles are carefully removed. From tail to head, the medullary arch bones and vascular arch bones are removed sequentially and arranged neatly according to their position in the fish. They are then pasted onto black cardstock. After all the intermuscular bones have been removed, the cardstock is laminated to fix them, thus creating an intermuscular bone specimen. The total number of intermuscular bones in an individual and the number of intermuscular bones in each location are used as phenotypic data for subsequent analysis. Figure 1 The distribution of intermuscular bone number in different parts of the phenotypic population is presented. Individuals were selected from this distribution, and 299 individuals were ultimately chosen as the reference population.

[0039] (2) Establishment of candidate groups.

[0040] In December 2021, 119 two-year-old Yellow River carp that were about to reach sexual maturity were selected from the core breeding group of Yellow River carp as a candidate group for intensive breeding. These parent fish are the candidate parents for the first generation with fewer thorns.

[0041] (3) Perform whole-genome resequencing on the reference population and candidate population to obtain high-quality SNP genotyping results.

[0042] Genomic DNA was extracted from the reference and candidate populations, and after library construction, 5× depth genome resequencing was performed, achieving an individual genome coverage of 82.91%. The raw sequencing data were aligned with the carp genome (GCA_018340385.1). SNP genotyping was performed on 418 fish from both the reference and candidate populations using GATK software, generating VCF files. The raw genotyping data was cleaned and quality-controlled using Plink software, with the following quality control conditions: SNP detection rate > 0.98, individual SNP detection rate > 0.95, minimum allele frequency of SNPs > 0.05, and Hardy-Weinberg equilibrium. Deletion genotypes were filled using Beagle software. LD filtering conditions were a 100kb sliding window, a 2bp step size, and r 2 A value greater than 0.2 was used to generate a high-quality SNP genotyping dataset that can be used for genome selection, containing 533,878 SNP markers, with the SNPs evenly distributed across 50 chromosomes.

[0043] (4) Estimation of genetic parameters

[0044] A kinship G-matrix was constructed using the genotypes of all SNP markers, and the genetic variance was calculated. The heritability of total intermuscular bone count, ossicles in the medullary arch, and ossicles in the vascular arch were 0.38, 0.45, and 0.29, respectively. The genetic and phenotypic correlations among traits are shown in Table 1. There was no significant genetic correlation between total intermuscular bone count and body weight, indicating that selection for intermuscular bone count does not have a significant impact on body weight.

[0045] Table 1. Heritability of traits (diagonal), genetic correlations between traits (lower triangle), and phenotypic correlations between traits (upper triangle).

[0046]

[0047] (5) Evaluation of the effect of genomic selection by different gradient SNP densities

[0048] Due to the massive number of SNPs obtained from resequencing, the computational burden was too high. Therefore, low-density SNPs were further screened for breeding value estimation. First, the accuracy of breeding value estimation under different SNP densities was evaluated to select the optimal SNP density. The accuracy evaluation method employed 5-fold cross-validation, where the reference population was randomly divided into 5 groups, with 4 groups forming the training group and the remaining group serving as the validation group. The prediction accuracy was calculated as the Pearson correlation coefficient between the GEBV and phenotypic value of the training group divided by the square root of heritability. This was repeated 50 times, and the mean was used as the final prediction accuracy. SNP density gradients were set to 50, 100, 500, 1000, 5000, 10k, 50k, 100k, and 500k. It was found that with the current reference population size, using 100k SNP markers achieved the highest prediction accuracy, with prediction accuracies of 0.32, 0.28, and 0.38 for the total number of intermuscular bones, ossicles in the medullary arch, and ossicles in the vascular arch, respectively.

[0049] (6) Calculate the GEBV of candidate individuals and breed to produce a generation of less spurs.

[0050] It has been verified that the number of intermuscular bones is positively correlated with GEBV, and individuals with fewer intermuscular bones also have lower GEBV. Figure 3 The candidate population can be selected for propagation based on GEBV. 100k SNP markers covering the entire genome were randomly selected to construct a genotypic relationship G matrix between the candidate and reference populations. Figure 4 ), estimating the GEBV (Gross Birth Value) of 119 individuals in the candidate population. Figure 5 The GEBV of the candidate populations were sorted from smallest to largest. Combined with pedigree selection, 12 male fish from the top 10% of the candidate populations with the highest GEBV were selected as the sires of the GS breeding line, and the remaining parent fish were used as the control group parent fish.

[0051] In May 2022, pedigrees were constructed, and the first generation (F1) was produced. In December 2022, the number of intermuscular bones in this batch of pedigrees was dissected and measured. The number of intermuscular bones in all GS-selected pedigrees was less than the population mean. The correlation coefficient between the number of intermuscular bones in the offspring and the paternal GEBV was 0.7. The average total number of intermuscular bones in the selected pedigrees was 90.83, while the average total number of intermuscular bones in the control group was 98.81, representing a decrease of 8.08% in the total number of intermuscular bones in the selected pedigrees compared to the control group.

[0052] The above implementation details sufficiently demonstrate the feasibility and efficiency of the breeding method for *Carassius acutus* strains based on genome-wide selection provided by this invention. The breeding process provided by this invention can be directly applied to the breeding of *Carassius acutus*. Compared to traditional breeding methods, which cannot perform individual selection for traits that are difficult to measure directly in vivo, this invention can directly predict the breeding value of candidate individuals, improving breeding efficiency. Simultaneously, it does not alter the fish's genome, making it safe and reliable. It allows for simultaneous breeding and promotion, possessing high industrial value.

[0053] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for breeding carp strains with few intermuscular bones based on whole-genome selection, characterized in that, The steps are as follows: (1) Select no less than 500 carp as the carp population for determining the quantitative traits of intermuscular bones, inject PIT electronic tags, cut off the fin rays and preserve them in anhydrous ethanol, then prepare intermuscular bone specimens of the carp population, determine the total number of intermuscular bones in the intermuscular bone specimens and the number of intermuscular bones in each part as phenotypes, analyze the carp population with the measured phenotypes, and establish a reference population. (2) Select individual fish with unknown intermuscular bone quantitative traits as candidate groups and inject them with PIT electronic tags, and cut off the fin rays and preserve them in anhydrous ethanol. (3) After DNA was extracted from the fin samples of the reference population in step (1) and the candidate population in step (2), low-depth genome resequencing was performed. Then, the DNA samples were genotyped. After the sequencing data were analyzed by population SNP genotyping, the obtained raw genotyping data was further cleaned and quality controlled. Then, the missing genotypes were filled in using Beagle software to obtain a high-quality SNP genotyping dataset. (4) Use the high-quality SNP genotyping dataset of the reference population obtained in step (3) to perform haplotype genome-wide association analysis, obtain the significance P value of each haplotype, screen tag SNPs to establish a genome-wide selection model, and calculate the model prediction accuracy under each SNP subset to obtain the optimal marker density. (5) Based on the high-quality SNP genotyping dataset from step (3), construct a kinship matrix A or a genome relationship matrix G that includes all individuals in the reference population and the candidate population. Estimate the variance components using the average information-constrained maximum likelihood method, thereby calculating the heritability of traits and the genetic correlation between traits. (6) Under the optimal marker density obtained in step (4), estimate the GEBV value of each individual in the candidate population based on the G matrix and heritability obtained in step (4), and sort them from smallest to largest; (7) Select individuals with low GEBV values ​​as parent fish according to a certain selection intensity, breed them, and build families. The individuals in these families are the first generation of less spiny fish. The first generation of less spiny fish is bred according to normal carp breeding methods. A carp strain with less intermuscular bone was obtained through continuous selective breeding. The method for preparing intermuscular bone specimens of carp in step (1) is as follows: After wrapping the live or frozen whole fish tightly, steam it at high temperature until the fish meat can be completely peeled off. After the fish cools down, remove the skin tissue on the dissecting tray, carefully remove the muscle, and take out the small bone of the medullary arch and the small bone of the vascular arch from tail to head. Arrange them neatly according to their position in the fish body and paste them on black cardboard in turn. After all the intermuscular bones are removed, fix the cardboard with plastic to make intermuscular bone specimens. The analysis refers to fitting the phenotypic distribution of the measured phenotypic population to a normal distribution, selecting genotyped individuals, and establishing a reference population; The quality control conditions in step (3) are: SNP detection rate greater than 0.98, individual SNP detection rate greater than 0.95, minimum allele frequency of SNP greater than 0.05, Hardy-Weinberg equilibrium; LD filtering conditions are a sliding window of 100kb, a step size of 2bp, and r 2 Greater than 0.2; In step (4), the tag SNP selection refers to selecting the top 50, 100, 500, 1000, 5000, 10000, 50000, and all haplotypes of the tag SNPs ranked by significance P-value. In step (7), a certain selection intensity refers to the 10% of individuals with the lowest GEBV ranking.

2. The breeding method for carp strains with few intermuscular bones based on genome-wide selection according to claim 1, characterized in that, The live or frozen whole fish is wrapped in at least three layers of newspaper.

3. The method for breeding carp strains with few intermuscular bones based on genome-wide selection according to claim 2, characterized in that, In step (5), the GEBV of each individual in the candidate population is calculated using the 5-fold cross-validation method.

4. The breeding method for carp strains with few intermuscular bones based on genome-wide selection according to claim 3, characterized in that, The optimal label density refers to the number of labels in the SNP subset corresponding to the highest model prediction accuracy.

5. The application of the breeding method according to any one of claims 1-4 in the breeding of carp strains with less intermuscular bone.

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