SNP molecular marker of scophthalmus maximus against edwardsiella tarda and application thereof

Abstract

This invention discloses a SNP molecular marker for resistance to Edwardsiella tarda in turbot and its application, belonging to the fields of molecular breeding and biotechnology. The nucleotide sequence of the SNP molecular marker is shown in SEQ ID No. 1, and the polymorphic site of the SNP molecular marker is A / C. This invention also provides the application of the SNP molecular marker in screening turbot with resistance to Edwardsiella tarda; turbot with the CC genotype are individuals with resistance to Edwardsiella tarda. Using the SNP molecular marker provided by this invention, DNA-level selection can be performed in the early stages of fish fry development, unaffected by environmental factors, significantly improving the accuracy and efficiency of breeding and shortening the breeding cycle. The molecular marker was significantly validated in an independent validation population through linear regression analysis, demonstrating stable and reliable genotype-phenotype association, excluding false positives, and showing good market application prospects.

Description

Technical Field

[0001] This invention belongs to the field of molecular breeding and biotechnology, specifically relating to an SNP molecular marker for turbot resistance to Edwardsiella tarda and its application. Background Technology

[0002] turbot( Scophthalmus maximus Turbot is a cold-water marine fish native to European waters and an important marine aquaculture species introduced to my country. After years of development, my country's turbot industry has gradually built an integrated technology system of "breeding, propagation, and promotion," forming a complete industrial chain from breeding planning, broodstock cultivation, seedling propagation to adult fish raising.

[0003] However, while the industry is developing steadily, the aquaculture sector faces challenges from bacterial diseases, particularly Edwardsiella tarda (…). Edwardsiella tarda The infection poses a serious challenge. This pathogen can cause ascites, skin ulcers, and septicemia in turbot, and is highly contagious with a high mortality rate, causing huge economic losses to fish farmers. In addition, the domestic industry still relies to some extent on limited germplasm resources introduced in the early stages, and long-term inbreeding may lead to germplasm degradation, resulting in a continuous decline in disease resistance and survival rate.

[0004] Traditional breeding techniques, centered on large-scale family selection and best linear unbiased prediction (BLUP) genetic assessment, have made significant progress in the genetic improvement of turbot, successfully selecting strains with advantages in growth rate and other aspects. However, for traits such as disease resistance, which are difficult to measure precisely, generally have low heritability, and are easily affected by environmental factors, the progress of traditional breeding methods is relatively slow and inefficient. A key reason for this is that obtaining disease resistance phenotypic data (such as survival time after challenge) is costly and time-consuming, and the accuracy and reliability of selection based solely on phenotypic and pedigree information still need improvement.

[0005] Molecular marker-assisted breeding (MAS) offers an effective solution to this bottleneck problem. Currently, research on molecular markers for the specific trait of Edwardsiella tarda resistance in turbot is lacking, particularly regarding key SNP loci that have been systematically discovered through genome-wide association studies (GWAS) and rigorously validated in independent populations. This absence of core markers directly limits the early and precise selection of disease-resistant turbot germplasm, hindering the breeding process of highly resistant strains.

[0006] Therefore, it is of great significance to first screen out the molecular markers that have been rigorously verified and are highly correlated with the trait of Edwardsiella tarda in turbot, and to establish an efficient application system for them. This is crucial for breaking through the bottleneck of disease resistance breeding in turbot, cultivating new strains with high survival rates, and ensuring the healthy and sustainable development of the industry. Summary of the Invention

[0007] In view of the current status of the prior art, the present application aims to provide a SNP molecular marker for Scophthalmus maximus against Edwardsiella tarda and application thereof, so that individuals of Scophthalmus maximus against Edwardsiella tarda can be quickly screened by using the SNP molecular marker provided by the present application, thereby being used for efficient assisted breeding of Scophthalmus maximus against Edwardsiella tarda.

[0008] To achieve the above-mentioned application purposes, the present application adopts the following technical solutions:

[0009] The present application provides a SNP molecular marker for Scophthalmus maximus against Edwardsiella tarda, wherein the nucleotide sequence of the SNP molecular marker is shown as SEQ ID No. 1.

[0010] Further, the 201st base of the SNP molecular marker is A or C. The 201st base of the SNP molecular marker is located at the physical position of 13,943,695 of the chromosome NC_061520.1 of the Scophthalmus maximus genome.

[0011] Further, the genotype CC of the 201st base of the SNP molecular marker is the genotype of the trait of being against Edwardsiella tarda.

[0012] The present application also provides amplification primers of the SNP molecular marker, wherein the nucleotide sequences of the amplification primers are shown as SEQ ID No. 2 and SEQ ID No. 3.

[0013] The present application also provides application of the SNP molecular marker in preparing a preparation for screening Scophthalmus maximus against Edwardsiella tarda.

[0014] Further, the application comprises the following steps:

[0015] (1) Extracting the genomic DNA of the to-be-tested Scophthalmus maximus individual;

[0016] (2) Using the genomic DNA of the to-be-tested Scophthalmus maximus individual as a template, performing PCR amplification by using the amplification primers of the SNP molecular marker, and performing sequencing on the PCR product to detect the genotype of the 201st base of the SNP molecular marker;

[0017] (3) According to the genotyping result, screening the Scophthalmus maximus individual with the CC genotype as the individual with the trait of being against Edwardsiella tarda.

[0018] Further, compared with the individual with the disadvantageous genotype (AA type), the individual carrying the C allele (i.e., the individual with the AC or CC genotype) has a longer survival time after the attack.

[0019] Furthermore, in step (2), the PCR reaction system is 25 μL: containing 12.5 μL of 2× Taq PCRMaster Mix, forward and reverse primers with a final concentration of 0.4 μM, 50-100 ng of genomic DNA template, and made up to 25 μL with sterile double-distilled water.

[0020] Furthermore, the PCR reaction program in step (2) is as follows: pre-denaturation at 95°C for 5 minutes; 35 cycles of amplification, each cycle including denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds and extension at 72°C for 1 minute; and final extension at 72°C for 10 minutes.

[0021] This invention also provides the application of the SNP molecular marker or the amplification primers in the selection of turbot strains resistant to Edwardsiella tarda.

[0022] Furthermore, during the breeding process, genomic DNA was extracted from the male and female turbot parents, and the genotype of the 201st base of the SNP molecular marker was detected. Based on the genotyping results:

[0023] (1) Both the father and mother parents, who are both of the CC genotype, are retained for seed saving and propagation;

[0024] (2) If the father and mother are not both of the CC genotype, then the father and mother are selected to be of the CC genotype and the mother is of the AC genotype respectively for mating; or the father and mother are both of the AC genotype for mating, and individuals with the CC genotype are further screened from the offspring.

[0025] Compared with the prior art, the present invention has the following advantages:

[0026] 1. Early selection and high accuracy: Using the SNP molecular markers provided by this invention, DNA-level selection can be carried out in the early stage of fish fry (such as when fin tissue is taken), without being affected by environmental factors, which significantly improves the accuracy and efficiency of selection and shortens the breeding cycle.

[0027] 2. Rigorous verification: The SNP molecular markers provided by this invention were discovered in the experimental population through GWAS analysis and were significantly verified in an independent validation population through linear regression analysis. The genotype-phenotype association is stable and reliable, and false positives are excluded.

[0028] 3. Broad application prospects: The SNP molecular markers provided by this invention can be combined with the existing BLUP genetic assessment system to construct the MA-BLUP model, realize genomic selection, and significantly improve the genetic progress of turbot in terms of disease resistance and growth rate. It has broad and promising market application prospects. Attached Figure Description

[0029] Figure 1 This is a Manhattan plot of the SNP molecular markers described in this invention.

[0030] Figure 2 This is a box plot showing the post-infection survival time of different genotypes in the SNP molecular marker verification population described in this invention. Detailed Implementation

[0031] The present invention will be further described below with reference to embodiments, but these are not intended to limit the scope of the invention.

[0032] Example 1: Screening and Validation of SNP Molecular Markers

[0033] 1. Experimental population and phenotype collection

[0034] The turbot population used in this invention originated from the Marine Flounder Genetic Breeding Center of the Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences. Fifty full-sib families were constructed through artificial mating, and each family was reared in a 500-liter fiberglass tank. Approximately two months after hatching, about six juveniles from each family were randomly selected and transferred to a new fiberglass tank (1.5 meters in radius and 0.6 meters in height). In November, approximately five months after hatching, the population underwent Edwardsiella tarda resistance testing.

[0035] In the challenge experiment, each turbot was injected intramuscularly with 100 μL to obtain a concentration of 4.61 × 10⁻⁶. 6 CFU / mL of Edwardsiella tarda bacterial suspension was administered. Infected fish were returned to their original water temperature environment (16±0.5℃), and feeding amounts were adjusted based on actual feeding behavior. Mortality was recorded every 2-3 hours from the onset of mortality. The weight of dead fish was measured, and fin samples were collected and stored at -80℃ for genomic DNA extraction. After all individuals had died, the survival time post-infection was used as phenotypic data, recorded to the nearest 0.5 days. Individuals were sorted in descending order of survival time, and every other individual was selected for genome resequencing, ultimately yielding approximately 311 individuals for resequencing and subsequent analysis.

[0036] 2. Whole genome resequencing and SNP genotyping

[0037] Genomic DNA was extracted from fin rays using the standard phenol-chloroform method. DNA concentration and quality were assessed using a Qubit quantitative PCR analyzer and 1% agarose gel electrophoresis. Double-digestion restriction site-associated sequencing libraries were constructed and paired-end sequencing was performed on an Illumina HiSeq 2500 platform. Sequencing data from different individuals were extracted from the library using indexing and barcoding, and low-quality reads were filtered using Stacks 2.0. High-quality, clean reads were aligned to the turbot reference genome (GCF_022379125.1) using BWA's mem algorithm. After processing the sequenced BAM files, SNP genotyping was performed using the Populations parameter in Stacks software.

[0038] The genotyping SNP set was filtered using the following parameters: Geno 0.1, MAF 0.05, and Mind 0.2. SNPs with excessively high deletion rates (>10%), rare variants with excessively low allele frequencies (<5%), and individuals with severe genotypic deletions (>20%) were removed, ultimately yielding 2,816,337 high-quality SNPs for analysis.

[0039] 3. Genome-wide association study (GWAS)

[0040] Using the FarmCPU function in the R package rMVP, a GWAS analysis was performed using a mixed linear model (MLM).

[0041] y = Xβ + Sγ + Zu + ε

[0042] Where y is the phenotypic observation (survival time); X is the fixed-effects design matrix (including the first three principal components used to correct for population stratification and body weight); β is the fixed-effects vector (i.e., the coefficients of the principal components); S is the genotype vector of the SNP to be detected; γ is the effect size of the SNP to be detected; Z is the random-effects design matrix; u is the random-effects vector (following a multivariate normal distribution, whose variance-covariance structure is defined by the kinship matrix); and ε is the random residual vector. Since sex has no significant effect on growth rate in juvenile turbot, sex was not included as a covariate in this model. The significance threshold for SNP loci was set to P < 1 × 10⁻⁶. −6 .

[0043] 4. Screening for trait-associated loci of resistance to Edwardsiella tarda

[0044] Based on GWAS analysis, a locus NC_061520.1-13943695, located at position 13943695 on linkage group NC_061520.1 of the genome, was identified as significantly associated with post-infection survival time. Its significance in genome-wide association with post-infection survival time was demonstrated. PThe value is 4.27E-07. The CC genotype at this locus is the dominant genotype. The nucleotide sequence of this SNP molecular marker associated with the trait of resistance to Edwardsiella tarda is shown in SEQ ID No. 1 (the nucleotide sequence shown in SEQ ID No. 1 has a C base at position 201, which is a variable base site). The Manhattan plot analyzed by FarmCPU is shown below. Figure 1 As shown.

[0045] Example 2: Validation and application of the association between SNP molecular markers and resistance to Edwardsiella tarda.

[0046] 1. Construction and trait testing of the validation population

[0047] Ten full-sib families were artificially constructed through mating. Two months after hatching, ten individuals from each family were randomly selected and mixed-cultured to form a validation population. When the individuals in the validation population reached 50g, 200 individuals were randomly selected for phenotypic testing against Edwardsiella tarda, following the procedure in Example 1 (1). After the test, fin tissue samples were taken from all individuals tested for the phenotypic traits, fixed in 95% ethanol, and stored at -20℃.

[0048] 2. Verify the SNP typing of the population

[0049] Genotyping of the NC_061520.1-13943695 loci in the validation population was performed using first-generation sequencing. PCR reactions were performed using forward primer F: 5'-ACAATGCTGAAGAAAGAGAT-3' (SEQ ID No. 2) and reverse primer R: 5'-ACAATGCTGAAGAAAGAGAT-3' (SEQ ID No. 3).

[0050] The PCR reaction system was 25 μL, containing 12.5 μL of 2× Taq PCR Master Mix (DNA polymerase, dNTPs, Mg²⁺ and reaction buffer), forward and reverse primers with a final concentration of 0.4 μM, 50-100 ng of genomic DNA template, and sterile double-distilled water to bring the total volume to the limit.

[0051] The PCR reaction program was as follows: pre-denaturation at 95°C for 5 minutes; followed by 35 cycles of amplification, each cycle consisting of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 1 minute; and a final extension at 72°C for 10 minutes. The PCR products were purified and directly used for first-generation sequencing to complete SNP genotyping.

[0052] 3. Validation of the association between infection and survival time at the NC_061520.1-13943695 site.

[0053] At the NC_061520.1-13943695 locus, three genotypes (AA, AC, and CC) were detected in the aforementioned 200 individuals. Individuals were divided into three groups based on their genotypes, with 86, 80, and 38 individuals for each genotype, respectively. The CC genotype had the fewest homozygous individuals. The mean survival time after infection was compared among the three groups. Figure 2 One-way ANOVA showed that there were significant differences in post-infection survival time among the three groups. P The values ​​were all 2E-16 (Table 1). Therefore, the NC_061520.1-13943695 sites in the SNP molecular markers screened by the above method also showed a significant association with post-infection survival time in the validation population.

[0054] Table 1. Post-infection survival time of individuals with different genotypes at the NC_061520.1-13943695 locus.

[0055]

[0056] As demonstrated above, the SNP molecular marker obtained by this invention can be used to assist in screening for parents resistant to Edwardsiella tarda. By using the CC genotype at the NC_061520.1-13943695 locus of this marker to select parents, the growth rate of the breeding population can be improved.

[0057] Example 3

[0058] This invention provides the application of the aforementioned SNP molecular marker in screening turbot for resistance to Edwardsiella tarda, specifically including the following steps:

[0059] 1. Extract genomic DNA from individual turbot individuals to be tested;

[0060] 2. Using the genomic DNA of the turbot individuals to be tested as a template, PCR amplification was performed using the forward primer F: 5'-ACAATGCTGAAGAAAGAGAT-3' (SEQ ID No. 2) and the reverse primer R: 5'-ACAATGCTGAAGAAAGAGAT-3' (SEQ ID No. 3).

[0061] The PCR reaction system was 25 μL, containing 12.5 μL of 2× Taq PCR Master Mix (DNA polymerase, dNTPs, Mg²⁺ and reaction buffer), forward and reverse primers with a final concentration of 0.4 μM, 50-100 ng of genomic DNA template, and sterile double-distilled water to bring the total volume to the limit.

[0062] The PCR reaction program was as follows: pre-denaturation at 95°C for 5 minutes; followed by 35 cycles of amplification, each cycle consisting of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 1 minute; and a final extension at 72°C for 10 minutes. The PCR products were purified and directly used for first-generation sequencing to complete SNP genotyping.

[0063] 3. Sequencing the PCR products and detecting the genotype of the 201st base of the SNP molecular marker. Based on the genotyping results, turbot with the CC genotype are selected as individuals with resistance to Edwardsiella tarda.

[0064] Example 4

[0065] This invention provides the application of the aforementioned SNP molecular marker in the selection of turbot strains resistant to Edwardsiella tarda. During the selection process, genomic DNA is extracted from the paternal and maternal turbot parents, and the genotype at the NC_061520.1-13943695 locus of the SNP molecular marker is detected. Based on the genotyping results:

[0066] (1) Both the father and mother parents, who are both of the CC genotype, are retained for seed saving and propagation;

[0067] (2) If the father and mother are not both of the CC genotype, then the father and mother are selected to be of the CC genotype and the mother is of the AC genotype respectively for mating; or the father and mother are both of the AC genotype for mating, and individuals with the CC genotype are further screened from the offspring.

[0068] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. The application of SNP molecular markers in the preparation of formulations for screening turbot against Edwardsiella tarda, characterized in that, The nucleotide sequence of the SNP molecular marker is shown in SEQ ID No. 1; the 201st base of the SNP molecular marker is A or C; the genotype CC of the 201st base of the SNP molecular marker is the genotype of resistance to Edwardsiella tarda.

2. The application according to claim 1, characterized in that, The application includes the following steps: (1) Extract genomic DNA from individual turbot individuals to be tested; (2) Using the genomic DNA of the turbot individual to be tested as a template, PCR amplification was performed using the amplification primers of the SNP molecular marker, and the PCR product was sequenced to detect the genotype of the 201st base of the SNP molecular marker; the nucleotide sequence of the amplification primers is shown in SEQ ID No.2 and SEQ ID No.3; (3) Based on the genotyping results, turbot with the CC genotype were screened as individuals with resistance to Edwardsiella tarda.

3. The application according to claim 2, characterized in that, In step (2), the PCR reaction system is 25 μL, containing 12.5 μL of 2× Taq PCR Master Mix, forward and reverse primers with a final concentration of 0.4 μM, 50-100 ng of genomic DNA template, and is made up to 25 μL with sterile double-distilled water.

4. The application according to claim 2, characterized in that, The PCR reaction program in step (2) is as follows: pre-denaturation at 95°C for 5 minutes; 35 cycles of amplification, each cycle including denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds and extension at 72°C for 1 minute; and final extension at 72°C for 10 minutes.

5. The application of SNP molecular markers in the breeding of turbot strains resistant to Edwardsiella tarda, characterized in that, The nucleotide sequence of the SNP molecular marker is shown in SEQ ID No. 1; the 201st base of the SNP molecular marker is A or C; the genotype CC of the 201st base of the SNP molecular marker is the genotype of resistance to Edwardsiella tarda.

6. The application according to claim 5, characterized in that, During the breeding process, genomic DNA was extracted from the male and female turbot parents, and the genotype of the 201st base of the SNP molecular marker was detected. Based on the genotyping results: (1) Both the father and mother parents, who are both of the CC genotype, are retained for seed saving and propagation; (2) If the father and mother are not both of the CC genotype, then the father and mother are selected to be of the CC genotype and the mother is of the AC genotype respectively for mating; or the father and mother are both of the AC genotype for mating, and individuals with the CC genotype are further screened from the offspring.

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