SNP (Single Nucleotide Polymorphism) molecular marker for resisting Edwardsiella tarda of scophthalmus maximus and application of SNP molecular marker

Abstract

The invention discloses a scophthalmus maximus anti-edwardsiella tarda SNP molecular marker and application thereof, and belongs to the technical field of molecular breeding and biology. The nucleotide sequence of the SNP molecular marker is shown as SEQ ID No.1, and the polymorphic site of the SNP molecular marker is A / C. The invention further provides application of the SNP molecular marker in screening of turbots with the Edwardsiella tarda resistant character, and the CC genotype turbots are individuals with the Edwardsiella tarda resistant character. By utilizing the SNP molecular marker provided by the invention, DNA level selection can be carried out in the early stage of fish fries, interference of environmental factors is avoided, the accuracy and efficiency of breeding are remarkably improved, and the breeding period is shortened. The molecular marker is remarkably verified in an independent verification group through linear regression analysis, genotype-phenotype association is stable and reliable, false positive is eliminated, and the molecular marker has a good market application prospect.

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: 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.

[0009] 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 Scophthalmus maximus genome chromosome NC_061520.1.

[0010] Further, the genotype CC of the 201st base of the SNP molecular marker is the genotype of the trait of being against Edwardsiella tarda.

[0011] 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.

[0012] The present application also provides application of the SNP molecular marker in preparing a preparation for screening Scophthalmus maximus against Edwardsiella tarda.

[0013] Further, the application comprises the following steps: (1) extracting genomic DNA of a to-be-tested Scophthalmus maximus individual; (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; (3) according to the genotyping result, screening Scophthalmus maximus individuals with CC genotype as individuals with the trait of being against Edwardsiella tarda.

[0014] Further, compared with individuals with a disadvantageous genotype (AA type), individuals carrying C alleles (i.e., AC or CC genotype) have a longer survival time after challenge.

[0015] Further, the PCR reaction system in step (2) is 25 μL, containing 12.5 μL of 2x Taq PCR Master Mix, 0.4 μM of the forward primer and the reverse primer, 50-100 ng of the genomic DNA template, and sterile double distilled water to make up to 25 μL.

[0016] Further, the PCR reaction procedure in step (2) is as follows: 95°C pre-denaturation for 5 minutes; 35 cycles of amplification, each cycle including 95°C denaturation for 30 seconds, 55°C annealing for 30 seconds and 72°C extension for 1 minute; and finally 72°C terminal extension for 10 minutes.

[0017] The application further provides the SNP molecular marker or the amplification primer in the application of breeding Scophthalmus maximus resistant to Edwardsiella tarda.

[0018] Further, in the breeding process, the genomic DNA of the male and female parents of Scophthalmus maximus is extracted, the genotype of the SNP molecular marker at the 201st base is detected, and according to the typing result: (1) the male and female parents with the CC genotype are reserved for breeding and passing; (2) if the male and female parents are not both of the CC genotype, the male and female parents with the CC genotype and the AC genotype are selected for mating; or the male and female parents with the AC genotype are selected for mating, and individuals with the CC genotype are continuously screened in the offspring.

[0019] Compared with the prior art, the application has the following beneficial effects: 1. Early breeding and high accuracy: the SNP molecular marker provided by the application can be used for DNA level selection at an early stage (such as fin strip tissue) of fry, is not disturbed by environmental factors, significantly improves the accuracy and efficiency of breeding, and shortens the breeding cycle.

[0020] 2. Strict verification: the SNP molecular marker provided by the application is found in an experimental population through GWAS analysis, and is significantly verified through linear regression analysis in an independent verification population, the genotype-phenotype correlation is stable and reliable, and false positives are excluded.

[0021] 3. Wide application prospect: the SNP molecular marker provided by the application can be combined with the existing BLUP genetic evaluation system to construct a MA-BLUP model, realize genomic selection, and greatly improve the genetic progress of Scophthalmus maximus in disease resistance and growth rate. It has a wide and good market application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is the Manhattan plot of the SNP molecular marker provided by the application.

[0023] Figure 2 Box plot of survival time after infection in different genotypes of the SNP molecular marker validation population of the present application. DETAILED DESCRIPTION

[0024] The present application is further illustrated by the following examples, which are not intended to limit the scope of the present application.

[0025] Example 1: Screening and validation of SNP molecular markers 1. Experimental population and phenotype collection The Scophthalmus maximus population used in the present application was from the Marine Scophthalmidae Genetic Breeding Center of the Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences. 50 full-sib families were constructed by artificial mating, and each full-sib family was raised in a 500-liter glass steel tank. That is, about two months after hatching, about 6 juvenile fish were randomly selected from each family and transferred to a new glass steel tank (radius 1.5 meters, height 0.6 meters). In November, about five months after hatching, the population was tested for Edwardsiella tarda resistance.

[0026] In the challenge test, each Scophthalmus maximus was injected intramuscularly with 100 μL of Edwardsiella tarda bacterial solution with a concentration of 4.61 × 10 6 CFU / mL. The infected fish were returned to their original water temperature environment (16±0.5℃), and the feeding amount was adjusted according to the actual feeding situation. From the time of death, the death situation was recorded every 2-3 hours. The body weight of the dead fish was weighed, and the fin bar sample was collected and stored at -80℃ for genomic DNA extraction. After all individuals died, the survival time after infection was used as the phenotype data, and the numerical value was recorded to 0.5 days. The individuals were arranged in descending order of survival time, and every other individual was selected for genomic resequencing, and finally about 311 individuals were obtained for resequencing and subsequent analysis.

[0027] 2. Whole genome resequencing and SNP typing Genomic DNA was extracted from the fin bar using the standard phenol chloroform method. The concentration and quality of the DNA were detected by Qubit fluorescence quantitative analyzer and 1% agarose gel electrophoresis. Double enzyme digestion restriction site associated sequencing library was constructed for double-end sequencing on the Illumina HiSeq 2500 platform. After extracting the sequencing data of different individuals from the library by indexing and barcoding, low-quality reads were filtered using Stacks 2.0. The high-quality clean reads were aligned to the Scophthalmus maximus reference genome (GCF_022379125.1) using the mem algorithm of BWA. After processing the sorted BAM file, SNP typing was performed by the Populations parameter of the Stacks software.

[0028] The set of typing SNPs was filtered by the following parameters --Geno 0.1 --MAF 0.05 --Mind 0.2. SNPs with high missing rate (>10%), rare variants with low allele frequency (<5%) and individuals with high genotype missing rate (>20%) were removed, and finally 2816337 high-quality SNPs were obtained for analysis.

[0029] 3. Genome-wide association study (GWAS) GWAS analysis was performed using the FarmCPU function in the R package rMVP using a mixed linear model (MLM): y = Xβ + Sγ + Zu + ε Where y is the phenotype observation (survival time); X is the fixed effect design matrix (including the first three principal components for correcting population stratification and body weight); β is the fixed effect vector (i.e. the coefficient of the principal components); S: is the genotype vector of the SNPs to be detected; γ is the effect value of the SNPs to be detected; Z is the random effect design matrix; u is the random effect vector (subject to multivariate normal distribution, whose variance-covariance structure is defined by the kinship matrix); ε is the random residual vector. Since the gender of the Scophthalmus maximus juvenile stage has no significant effect on the growth rate, the model does not include gender as a covariate. The significance threshold for SNP sites is set to P < 1 x 10 −6 .

[0030] 4. Screening of anti-E. tarda trait-associated SNPs According to the results of the GWAS analysis, a SNP site NC_061520.1-13943695 significantly associated with survival time after infection was screened out, which is located at position 13943695 on chromosome NC_061520.1 linkage group, and its significance value with survival time after infection at the whole genome level is 4.27E-07. The CC genotype of the site is the dominant genotype, and the nucleotide sequence of the SNP molecular marker related to the anti-E. tarda trait is shown in SEQ ID No. 1 (the 201st base in the nucleotide sequence shown in SEQ ID No. 1 is C, which is a variable base site). The Manhattan plot of the FarmCPU analysis is shown in P . Figure 1

[0031] Example 2: Verification and application of SNP molecular marker and anti-E. tarda association 1. Construction of verification population and trait test ​Ten full-sib families were constructed by artificial mating, and two months after hatching, 10 individuals from each family were randomly selected to form a verification population. When the individuals in the verification population grew to 50 g, 200 individuals were randomly selected for testing of the phenotype of Edwardsiella tarda resistance, and the testing process was as described in step (1) of Example 1. After the test, fin tissue samples were taken from all the individuals tested for traits, fixed in 95% ethanol, and stored at -20°C.

[0032] 2. SNP typing of the verification population The NC_061520.1-13943695 locus of the verification population was typed by first-generation sequencing. The PCR reaction 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) for amplification.

[0033] The PCR reaction system was 25 μL, containing 12.5 μL of 2x Taq PCR Master Mix (DNA polymerase, dNTPs, Mg² + and reaction buffer), 0.4 μM of the forward and reverse primers, 50-100 ng of genomic DNA template, and sterile double-distilled water to a total volume.

[0034] The PCR reaction program was as follows: 95°C pre-denaturation for 5 minutes, followed by 35 cycles of amplification, each cycle including 95°C denaturation for 30 seconds, 55°C annealing for 30 seconds, and 72°C extension for 1 minute; and finally 72°C terminal extension for 10 minutes. The PCR product was purified and directly used for first-generation sequencing to complete SNP typing.

[0035] 3. Verification of the correlation between the NC_061520.1-13943695 locus and post-infection survival time Among the 200 individuals described above, the NC_061520.1-13943695 locus detected AA, AC, and CC genotypes. According to different genotypes, the individuals were divided into 3 groups, and the number of individuals of the 3 genotypes was 86, 80, and 38, respectively, with the least number of CC homozygotes. The mean values of post-infection survival time of the 3 groups were compared Figure 2 ). One-way ANOVA showed that there was a significant difference in post-infection survival time among the 3 groups, P with a value of 2E-16 (Table 1). Therefore, the NC_061520.1-13943695 locus in the SNP molecular marker screened by the above method also showed significant correlation with post-infection survival time in the verification population.

[0036] Table 1 Survival time after infection of individuals with different genotypes at NC_061520.1-13943695 locus

[0037] As demonstrated above, the SNP molecular marker obtained in the present application can be used to assist in screening parent against Edwardsiella tarda, and the CC genotype at NC_061520.1-13943695 locus in the marker can be used to select parent, so as to improve the growth rate of breeding population.

[0038] Example 3 The present application provides the use of the SNP molecular marker in screening Scophthalmus maximus against Edwardsiella tarda, and the specific use includes the following steps: 1. Extracting the genomic DNA of the Scophthalmus maximus to be tested; 2. Using the genomic DNA of the Scophthalmus maximus to be tested as a template, and using the forward primer F: 5'-ACAATGCTGAAGAAAGAGAT-3' (SEQ ID No. 2) and the reverse primer R: 5'-ACAATGCTGAAGAAAGAGAT-3' (SEQ ID No. 3) to perform PCR amplification.

[0039] The PCR reaction system is 25 μL, containing 12.5 μL of 2x Taq PCR Master Mix (DNA polymerase, dNTPs, Mg² + and reaction buffer), 0.4 μM of the forward and reverse primers, 50-100 ng of the genomic DNA template, and sterile double distilled water to make up the total volume.

[0040] The PCR reaction program is as follows: 95°C pre-denaturation for 5 minutes; followed by 35 cycles of amplification, each cycle including 95°C denaturation for 30 seconds, 55°C annealing for 30 seconds and 72°C extension for 1 minute; and finally 72°C terminal extension for 10 minutes. The PCR product is directly used for first-generation sequencing after purification to complete SNP typing.

[0041] 3. Sequencing the PCR product to detect the genotype of the 201st base of the SNP molecular marker, and screening the Scophthalmus maximus with CC genotype according to the genotyping results, which is the individual with the trait of resistance to Edwardsiella tarda.

[0042] Example 4 The application provides application of the SNP molecular marker in breeding of scophthalmus maximus strains resistant to Edwardsiella tarda, in the breeding process, genomic DNA of male and female scophthalmus maximus is extracted, genotypes of the SNP molecular marker NC_061520.1-13943695 site are detected, and according to the typing results: (1) the male and the female which are both CC genotypes are reserved for seed saving and generation; (2) if the male and the female are not both CC genotypes, the male and the female which are CC genotypes and AC genotypes are selected for mating, or the male and the female which are both AC genotypes are selected for mating, and individuals with CC genotypes are continuously screened in the offspring.

[0043] The above examples are only used to illustrate the technical solutions of the present application, but not limit it; although the present application has been described in detail with reference to the foregoing examples, the technical solutions recorded in the foregoing examples can still be modified or some technical features can be replaced by equivalents for ordinary skilled in the art; and the modification or replacement does not make the essence of the corresponding technical solution deviate from the spirit and scope of the technical solutions claimed by the present application.

Claims

1. A SNP molecular marker for resistance to Edwardsiella tarda in turbot, characterized in that, The nucleotide sequence of the SNP molecular marker is shown in SEQ ID No.

1.

2. The SNP molecular marker according to claim 1, characterized in that, The 201st base of the SNP molecular marker is either A or C.

3. The SNP molecular marker according to claim 1, characterized in that, The genotype CC at the 201st base of the SNP molecular marker is the genotype for resistance to Edwardsiella tarda.

4. The amplification primers for the SNP molecular marker according to claim 1, characterized in that, The nucleotide sequences of the amplification primers are shown in SEQ ID No. 2 and SEQ ID No.

3.

5. The use of the SNP molecular marker according to any one of claims 1-3 in the preparation of formulations for screening turbot against Edwardsiella tarda.

6. The application according to claim 5, 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. (3) Based on the genotyping results, turbot with the CC genotype were screened to identify individuals with resistance to Edwardsiella tarda.

7. The application according to claim 6, 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.

8. The application according to claim 6, 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.

9. The application of the SNP molecular marker of claim 1 or the amplification primer of claim 4 in the selection of turbot strains resistant to Edwardsiella tarda.

10. The application according to claim 9, 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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