A liquid-phase chip for disease-resistant genomic selection breeding of turbot and its application

Abstract

The present invention relates to the technical field of aquaculture genetic breeding, and particularly to a liquid-phase chip for genome selection breeding of turbot disease resistance and its application. The liquid-phase chip for genome selection breeding of turbot disease resistance contains 500 SNP loci, and the nucleotide sequences of the SNP loci are shown in SEQ ID No.1 to 250. The core loci in the liquid-phase chip have the characteristics of extremely low SNP locus number, low computational amount, low cost and high accuracy. In the first-line production, the core loci in the liquid-phase chip are easy to operate and calculate, and can accurately and effectively perform genotyping (the detection rate in different populations reaches 95%), and the heritable information of the turbot's resistance to Edwardsiella tarda can be basically obtained (the heritability can reach 80% of that using 2.3M markers covering the whole genome); the core loci in the liquid-phase chip can stably and efficiently implement genome selection breeding for turbot disease resistance.

Description

Technical Field

[0001] The present invention relates to the technical field of aquaculture genetics and breeding, and particularly to a liquid-phase chip for genome selection breeding of disease-resistant turbot and its application. Background Art

[0002] Aquaculture has made important contributions to meeting the fish needs of urban and rural residents in China, increasing the supply of high-quality animal protein, improving the national nutritional and health level, and ensuring food security in China. Since its introduction into China in 1992, turbot has been massively cultured in coastal areas such as Shandong, Hebei, Liaoning, Jiangsu, Zhejiang and Fujian, and has rapidly developed into one of the important pillar industries of seawater fish culture, and has become one of the nine research objects of the national seawater fish industry technology system. However, with the rapid development of intensive turbot culture, problems of germplasm degradation and diseases have gradually emerged. For example, in recent years, diseases with high mortality rates mainly characterized by enteritis, ascites and acute hemorrhagic symptoms of turbot caused by infection with Edwardsiella tarda have occurred in Shandong, Hebei and Liaoning. Since turbot of different months of age are highly susceptible to Edwardsiella tarda and there is no obvious seasonality, Edwardsiella tarda disease has become one of the main bottlenecks for the sustainable development of the turbot aquaculture industry. At present, during the high-incidence period of turbot diseases, methods such as disinfecting the aquaculture environment, controlling water temperature, injecting vaccines and using a large amount of antibiotics are often used. However, these methods are costly and cumbersome to operate and are difficult to apply on a large scale. Therefore, cultivating disease-resistant fry is an urgent need for the green and sustainable development of the turbot industry.

[0003] For fish disease-resistant traits, which are often determined by multiple genes, the accuracy of using traditional breeding methods (population selection, family selection and hybridization) is relatively low. Therefore, the breeding progress of disease-resistant fish varieties is slow. Genome selection breeding technology is a method for estimating breeding values based on SNP markers that may be linked to traits on the whole genome. This method has high accuracy, does not require the measurement of the phenotypes of candidate populations, can effectively reduce the detection of breeding generations, and is suitable for the breeding of disease-resistant fish varieties. This method has been successfully applied to fish such as Japanese flounder, half-smooth tongue sole, and large yellow croaker.

[0004] The accuracy of genomic selection breeding is mainly affected by the construction of the reference population and the accuracy and detection rate of SNP genotyping in the candidate population. Whole-genome SNP genotyping is the core technical means for analyzing the association between gene genetic variation and traits within the whole genome. The cGBS liquid chip genotyping technology is based on targeted capture sequencing of next-generation sequencing, which can achieve high-depth resequencing of target SNP sites, thereby accurately detecting genotypes and applying them to genomic selection breeding. Compared with solid-phase chip technology, the cGBS liquid chip technology has the characteristics of low cost, low requirements for detection platforms, additivity of information, and broad-spectrum application. This technology has been widely used in agricultural breeding, disease diagnosis and other fields, and has achieved remarkable results (such as species like pigs, corn, half-smooth tongue sole, etc.). However, in the field of turbot breeding, there is currently no report on a gene chip for turbot breeding against Edwardsiella tarda at home and abroad. Therefore, it is urgent to develop a low-cost, high-throughput and highly flexible whole-genome SNP chip and disease-resistant genomic selection breeding technology to meet the needs of cultivating disease-resistant high-quality turbot. Summary of the Invention

[0005] In order to solve the above problems, the present invention provides a liquid chip for turbot disease-resistant genomic selection breeding and its application, making up for the deficiencies of traditional breeding methods. And based on the MLE-rank model, 500 core SNP sites with high genetic effects in the liquid chip are selected to reduce the computational difficulty in actual production, thereby improving the usability of the chip. To promote the process of breeding disease-resistant high-quality turbot and achieve rapid progress in turbot selection breeding technology.

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

[0007] The present invention provides a liquid chip for turbot disease-resistant genomic selection breeding, and the liquid chip for turbot disease-resistant genomic selection breeding contains 500 SNP sites, and the nucleotide sequences of the SNP sites are as shown in SEQ ID No.1 to 250.

[0008] The present invention also provides the application of the liquid chip for turbot disease-resistant genomic selection breeding described in the above technical solution in detecting turbot DNA.

[0009] The present invention also provides the application of the liquid chip for turbot disease-resistant genomic selection breeding described in the above technical solution in turbot genomic selection.

[0010] The present invention also provides the application of the liquid chip for turbot disease-resistant genomic selection breeding described in the above technical solution in the evaluation of the heritability of turbot traits against Vibrio harveyi.

[0011] The present invention also provides the application of the liquid chip for turbot disease-resistant genomic selection breeding described in the above technical solution in turbot genotyping.

[0012] The present invention also provides the application of the turbot disease-resistant genomic selection breeding liquid chip described in the above technical solution in turbot breeding.

[0013] The beneficial effects of the present invention are as follows:

[0014] 1. The core loci in the turbot disease-resistant genomic selection breeding liquid chip provided by the present invention have the characteristics of extremely low SNP locus number, low computational complexity, low cost and high accuracy. It is easy to operate and calculate in the first-line production;

[0015] 2. The core loci in the turbot disease-resistant genomic selection breeding liquid chip provided by the present invention can accurately and effectively perform genotyping (the detection rate reaches 95% in different populations), and can basically obtain the heritable information of the turbot's resistance to Edwardsiella tarda (the heritability can reach 80% of that using 2.3M markers covering the whole genome);

[0016] 3. The core loci in the turbot disease-resistant genomic selection breeding liquid chip provided by the present invention can stably and efficiently implement turbot disease-resistant genomic selection. Description of the Drawings

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments.

[0018] Figure 1 For the comparison of prediction accuracy based on four different SNP combinations;

[0019] Figure 2 For the comparison of the average survival time of the parents of the high GEBV families and the average survival time of the low GEBV families. Detailed Embodiments

[0020] Term Explanation:

[0021] SNP: Single Nucleotide Polymorphism, which represents DNA sequence polymorphism caused by single nucleotide variation at the genomic level.

[0022] Reference population: In genomic selection, a population with phenotypes and genotypes. The phenotypes are obtained through manual measurement, and the genotypes are obtained through genomic sequencing. This population is used to train the model.

[0023] Candidate population: In genomic selection, a population with only genotypes but no phenotypes, usually the alternative individuals in the breeding process. After measuring the genotypes, the breeding values of these individuals can be estimated in combination with the reference population, and the individuals can be preferentially selected and bred based on the high and low breeding values.

[0024] GBLUP: Genomic Best Linear Unbiased Prediction, an abbreviation for Genomic Best Linear Unbiased Prediction, is a method for estimating individual breeding values by constructing a genomic relationship matrix G matrix using high-density molecular markers covering the genome.

[0025] GEBV: Genomic Estimated Breeding Value, an abbreviation for Genomic Estimated Breeding Value, is the breeding value estimated at the genomic level through genomic selection methods.

[0026] The present invention provides a liquid-phase chip for genomic selection breeding of turbot for disease resistance. The liquid-phase chip for genomic selection breeding of turbot for disease resistance contains 500 SNP loci. The nucleotide sequences of the SNP loci are the 36th and 107th bases of any one of the sequences SEQ ID NO: 1–SEQ ID NO: 250, which are represented by the degenerate base y. Specifically as follows:

[0027] The present invention does not particularly limit the preparation method of the liquid-phase chip for genomic selection breeding of turbot for disease resistance, and conventional methods can be adopted.

[0028] The present invention also provides the application of the liquid-phase chip for genomic selection breeding of turbot for disease resistance in detecting turbot DNA.

[0029] The present invention also provides the application of the liquid-phase chip for genomic selection breeding of turbot for disease resistance in genomic selection of turbot.

[0030] The present invention also provides the application of the liquid-phase chip for genomic selection breeding of turbot for disease resistance in evaluating the heritability of the trait of turbot against Vibrio harveyi slow disease.

[0031] The present invention also provides the application of the liquid-phase chip for genomic selection breeding of turbot for disease resistance in genotyping of turbot.

[0032] The present invention also provides the application of the liquid-phase chip for genomic selection breeding of turbot for disease resistance in breeding of turbot.

[0033] In order to further illustrate the present invention, the following examples are used to describe the present invention in detail, but they should not be construed as limiting the protection scope of the present invention.

[0034] Example 1

[0035] Obtaining of the core SNP loci of the chip and chip preparation.

[0036] 1. Establishment of a reference population of turbot against Vibrio edwardsiella tarda and collection of phenotypes

[0037] The reference population of turbot resistant to Edwardsiella tarda disease is derived from turbot families that have been selectively bred by the Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences for many years. Through artificial infection experiments with Edwardsiella tarda by intraperitoneal injection, our research group has carried out the breeding work of disease-resistant improved turbot varieties.

[0038] In the reference population described in the present invention, artificial infection experiments with Edwardsiella tarda were carried out on 2400 turbot from 40 families in 2022. Fin rays of the tested fish were collected and the death time and individual data were recorded. According to the equal proportion of each family and its death time, 14 - 21 individual samples were randomly selected, and a total of 699 samples were used to form the reference population. The genotypes of the reference population were obtained by whole-genome resequencing based on the BGI T7 sequencer.

[0039] 2. Obtaining and quality control of SNPs

[0040] Genomic DNA of 699 individuals in the reference population was extracted. Using the qualified DNA, a paired-end DNA library for second-generation sequencing was constructed, and sequencing and data output were completed based on the BGI T7 sequencing platform. The clean reads after quality control were aligned with the turbot reference genome (BioProjects: PRJNA792617) using the BWA software, and duplicate sequences were removed. Then, basic information statistics and map alignment statistics of the data were carried out. Finally, based on the alignment results between the sequences generated by sequencing and the genomic reference sequences, SNPs were called using the GATK software, and a total of 5,233,470 SNPs were obtained.

[0041] The obtained original reference population vcf file was processed using the VCFtools software. First, sites with a sequencing depth less than 15 were removed. Then, filtering was carried out according to the following criteria: removing sites with a missing rate > 0.05, sites with a minor allele frequency (MAF) < 0.05, and removing sites that did not conform to the Hardy-Weinberg equilibrium (P value threshold was set to 0.00001). A total of 2.3M turbot SNP markers were obtained. Finally, the Beagle v5.1 software was used for imputation.

[0042] 3. Extracting SNP site information

[0043] All quality-controlled SNP loci (Geno2) were extracted from the reference population (turbot.Ref) using Plink v1.9 software and stored as "geno2.bin"; and 500 evenly distributed SNP loci (Geno3) were extracted using the plink v1.9–bp-space command according to the physical positions of the SNPs and stored as "geno3.bin". A genotype file "geno4.bin" containing 500 SNP loci (Geno4) was randomly selected in R. Based on "geno2.bin" and the reference population phenotype file "pheno.turbot.Ref.dat", the relative importance of each SNP locus was calculated using the MLE-rank model. Using the top 5000 SNP loci obtained by MLE-rank, based on the principle of uniform filling of the whole genome, Changsha Huazhi Biotechnology Co., Ltd. selected 31,803 SNPs in total to synthesize 31,671 capture probes according to the requirements of cGPS chip development, capturing 31,671 intervals to construct a liquid-phase chip system. Design a liquid-phase chip for turbot disease-resistant breeding.

[0044] For the MLE-rank model, the analysis model is:

[0045]

[0046] where y n represents the phenotypic value of the nth sample, and x nj is the genotype (0 / 1 / 2 represents AA / Aa / aa) of the jth SNP on the nth sample. N is the total population of samples obtained, which is 699 in this example. p is the proportion of individuals defined as disease-resistant in the samples. The vec j obtained by the MLE-rank model represents the effect of the jth SNP locus on the phenotype.

[0047] For each SNP in the above cGPS chip, they were sorted from largest to smallest according to vec, and the top 500 were taken as the loci described in the present invention (SEQ ID No.1 to 250) and denoted as geno1.bin.

[0048] Example 2

[0049] The core SNP loci of the chip were used to evaluate the accuracy of genomic selection breeding.

[0050] 1. Construction of genotype association matrix (Gmatrix)

[0051] The specific content can be obtained, and the specific extraction method is in "3. Extract SNP site information" in Example 1. Among them, geno1.bin is the information of 500 SNP sites extracted by MLE-rank described in the present invention. geno2.bin is the information of all SNP sites. geno3.bin is the information of 500 SNP sites evenly distributed. geno4.bin is the information of 500 SNP sites randomly distributed. For the genotype files such as "geno1.bin", "geno2.bin", "geno3.bin", "geno4.bin" in Example 1, G matrices ("Gmatrix1.bin", "Gmatrix2.bin", "Gmatrix3.bin", "Gmatrix4.bin") are constructed respectively. The construction of the G matrix is implemented using the A.mat function in the "sommer" package of the R language.

[0052] 2. Calculation of GBLUP prediction accuracy

[0053] Using ASReml-R for the genotype relationship matrix files "Gmatrix_geno1.bin", "Gmatrix_geno2.bin", "Gmatrix_geno3.bin", "Gmatrix_geno4.bin" constructed from four different SNP sets, and the phenotypes of the reference population. Using the GBLUP model for 5-fold cross-validation 5 times (a total of 25 times), retaining the phenotypes of the training set, and estimating the GEBV of the validation set with the training set as the reference population. And the prediction accuracy of different SNP combinations is evaluated by calculating the area under the ROC curve (AUC value) between the GEBVs of the validation set and the phenotypic values of the validation set. The results are shown in Table 1 and Figure 1 .

[0054] The results are organized into Table 1

[0055] Table 1 Prediction accuracy (AUC) of different SNP sets

[0056]

[0057]

[0058] From the cross-validation results, it can be seen that the prediction accuracy using the 500 core SNPs (Geno1) of the chip described in the present invention is 0.75, which is significantly higher than that using the high-density (2.3M) SNP sites covering the whole genome (Geno2; prediction accuracy is 0.65), 500 randomly selected SNP sites (Geno3, prediction accuracy is 0.61), and 500 evenly selected SNP sites (Geno4, prediction accuracy is 0.63). The results show that the core SNP sites of the chip described in the present invention have good genotyping effects.

[0059] Example 3

[0060] Based on the 500 SNPs described in the present invention, the GEBVs and narrow-sense heritability of the reference population of turbot were evaluated.

[0061] 1. Evaluation of family GEBVs

[0062] Using the 500 core SNPs of the chip described in the present invention, the GEBV of 699 turbot in the reference population was evaluated by the GBLUP method (implemented by asreml4-R). The family GEBV was defined as the average of the GEBVs of the fish within the family. The GEBVs and survival time of the 40 families that make up the reference population are shown in Table 2 and Figure 2 .

[0063] Table 2 Family average survival time and family GEBV

[0064]

[0065]

[0066] The results showed that the Person correlation coefficient between the family GEBV and the survival time after infection was 0.885, showing a strong positive correlation.

[0067] 2. Evaluation of narrow-sense heritability

[0068] Using the core loci of the chip (Gene1) in Example 1 and the SNP combination covering the whole genome (Gene2). Based on the 699 turbot reference population, the heritability of the trait of resistance to Edwardsiella tarda in turbot was evaluated by the GBLUP method (implemented by asreml4-R). The results showed that the heritability obtained using Gene1 (0.18 ± 0.053) was close to the heritability obtained using Gene2 (0.22 ± 0.056), indicating that the core loci of the chip described in the present invention can obtain 80% of the stably heritable information.

[0069] The results showed that the core SNP loci of the chip described in Example 3 of the present invention can meet the computational accuracy requirements of genomic selection and can also obtain most of the genetic information, ensuring the smooth progress of breeding work.

[0070] Example 4

[0071] Detection rate evaluation of 500 core loci of the chip

[0072] Experimental materials: Sample 1 (a total of 40 randomly selected turbot collected from Haiyang, Yantai, China), Sample 2 (a total of 56 turbot from the half-sib families of the reference population), Sample 3 (a total of 16 turbot from the reference population)

[0073] Strategy: Detect the sites of the present invention in Sample 1 and Sample 2 using the liquid-phase chip in Example 1, and count the detection of different SNP sites in the samples.

[0074] Experimental results: The detection rates of samples from different sources can all reach over 95%, indicating that the SNP sites of the present invention have a high detection rate for turbot from different sources and can meet the needs of turbot disease-resistant breeding.

[0075] Table 3 Site detection rate

[0076]

[0077]

[0078] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A liquid phase chip for disease resistance genomic selection breeding of turbot, characterized in that: The turbot disease resistance genomic selective breeding liquid phase chip contains nucleic acid for detecting 500 SNP sites, the nucleotide sequence of the nucleic acid is shown in SEQ ID No.1-250, and the SNP site is the 36th and 107th bases of any sequence in the sequence SEQ ID No.1-SEQ ID No.250, which are indicated by the degenerate base y; The disease to be resisted is Edwardsiella tarda.

2. Use of the turbot disease resistance genomic selection breeding liquid phase chip according to claim 1 in the evaluation of the heritability of turbot resistance to Edwardsiella tarda disease.

3. The turbot disease resistance genomic selection breeding liquid phase chip according to claim 1 is used in the breeding of turbot resistant to Edwardsiella tarda.

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