SNP site associated with trachinotus ovatus and streptococcus iniae disease resistance character and application

Abstract

The invention relates to an SNP site associated with trachinotus ovatus and streptococcus iniae disease resistance characters and application, and belongs to the technical field of molecular biological breeding, the SNP site is located at the 14078454 site on the No.7 chromosome, the polymorphism site is C / T, and the genotype of the site with disease resistance is CC. The invention also provides application of the SNP site in identification of streptococcus iniae resistant trachinotus ovatus, and the application method comprises the following steps: extracting DNA (deoxyribonucleic acid) of the trachinotus ovatus to be identified, and selecting individuals with CC genotype at the SNP site through sequencing. And screening the genotype of which the SNP site is CC as a breeding parent. The method is used for cultivating a new trachinotus ovatus variety resistant to streptococcus iniae.

Description

[0001] This patent claims priority to patent number 2024104412485, filed on April 12, 2024, titled "SNP sites and applications associated with resistance to dolphin streptococcal disease in an oval pomfret." Technical Field

[0002] The invention belongs to the technical field of molecular biological breeding, and specifically discloses a SNP site associated with the resistance trait of dolphin streptococcus disease in ovate pomfret and its application. Background Art

[0003] Trachinotus ovatus, commonly known as golden pomfret, belongs to the order Perciformes, family Carangidae, and genus Trachinotus. It grows fast, has few intramuscular spines, and boasts delicious meat. Streptococcus iniae causes mortality rates in ovatus of up to 30% to 50%, severely impacting the ovatus industry. Currently, the primary treatment for streptococcal infections is antibiotics. However, long-term use and overuse of these drugs has led to resistance to many drugs in streptococci. Research on streptococcal vaccines remains largely experimental, and their potential application remains uncertain due to factors such as increased aquaculture costs and difficulties in widespread application. The most fundamental strategy for addressing streptococcal infections is to breed new strains or varieties that are resistant to streptococci. This is the most effective approach to combating the disease. However, little research has been conducted domestically or internationally on streptococcal resistance in ovatus.

[0004] Traditional breeding methods have drawbacks such as long cycles and low efficiency. Molecular breeding techniques can conduct targeted breeding of organisms at the molecular level, significantly shortening the breeding cycle. Genome-wide association study (GWAS) is a newly emerging breeding method that primarily identifies the association between phenotype and genotype. GWAS is based on linkage disequilibrium (LD) at the population level. By detecting SNPs across the genomes of multiple individuals, a suitable model is used to statistically analyze the association between each genetic marker and the target trait (expressed as a P value). Based on the P value, molecular markers closely associated with the target trait are selected and the SNPs and related key genes that influence the trait are identified. Summary of the Invention

[0005] To address existing technical challenges, the present invention provides a single-nucleotide polymorphism (SNP) site associated with resistance to Streptococcus iniae in the ovate pomfret and its application. This invention uses genome-wide association analysis to identify SNPs associated with resistance to Streptococcus iniae across the entire genome of the pomfret. Using this large-scale screening approach combined with multi-population validation, SNPs associated with Streptococcus resistance are identified and applied to the breeding of new strains or varieties resistant to Streptococcus.

[0006] The present invention is achieved through the following technical solutions:

[0007] One of the technical solutions of the present invention is to identify a single nucleotide polymorphism (SNP) associated with resistance to Streptococcus iniae based on the genome of the scad croaker (NCBI: GCA_900607315.1) provided by NCBI. This SNP is located at position 14078454 on chromosome 7 and has a C / T polymorphism, with the CC genotype being significantly associated with disease resistance.

[0008] The second technical solution of the present invention is to provide the application of the SNP site in identifying ovate pomfret resistant to indiatric Streptococcus infection. The application method is to extract DNA from the ovate pomfret to be identified and select individuals with CC genotype at the SNP site through sequencing.

[0009] The third technical solution of the present invention is to provide the application of the SNP site in breeding a new variety of oval pomfret resistant to dolphin streptococcus disease, and the application method is to screen the genotype of the SNP site CC as the breeding parent.

[0010] The fourth technical solution of the present invention is to provide an application of the gene at the SNP site associated with the resistance trait of the oval pomfret and Streptococcus iniae. The application is used to identify the oval pomfret with resistance to Streptococcus iniae. The application method is to extract DNA from the oval pomfret to be identified and select individuals with the CC genotype at the SNP site through sequencing.

[0011] The fifth technical solution of the present invention is to provide an application of the gene at the SNP site associated with the resistance trait of the oval pomfret and Streptococcus iniae, wherein the application is used to breed a new variety of oval pomfret resistant to Streptococcus iniae disease, and the application method is to screen individuals with the CC genotype of the SNP site as breeding parents for breeding the next generation.

[0012] The beneficial effects of the present invention compared with the prior art are as follows:

[0013] The present invention provides a genome-wide association analysis method for the association between the ovate pomfret and the resistance trait of Streptococcus iniae. The method uses established susceptible and resistant populations of ovate pomfret as experimental subjects for the genome-wide association analysis to divide the resistance trait. Fins are used to extract genomic DNA for resequencing to conduct genome-wide association analysis between the ovate pomfret and the resistance trait of Streptococcus iniae. A single polymorphism (SNP) site associated with the resistance trait of Streptococcus iniae is screened out, thereby providing a scientific method and theoretical support for the future breeding of high-quality disease-resistant new varieties (lines) of ovate pomfret. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 For DNA quality testing;

[0015] Figure 2 The density distribution of SNPs associated with resistance to S. iniae screened across the entire genome;

[0016] Figure 3 Manhattan plot of genome-wide association analysis of SNPs significantly associated with resistance to S. iniae in the oval pomfret;

[0017] Figure 4 The linkage disequilibrium (LD) between SNPs loci significantly associated with S. iniae resistance in ovata pomfret;

[0018] Figure 5 Principal component analysis of samples of scad pomfret to screen for SNPs associated with resistance to S. iniae;

[0019] Figure 6 QQ plot of the oval pomfret samples screened for SNPs associated with resistance to Streptococcus iniae. DETAILED DESCRIPTION

[0020] The technical solution of the present invention is further studied below through examples, but the protection scope of the present invention is not limited in any form by the examples.

[0021] Example 1

[0022] 1 Experimental Materials and Methods

[0023] 1.1 Screening of susceptible and resistant populations of ovate pomfret

[0024] In this experiment, 300 healthy oval pomfret (average weight 150±10g) were selected and placed in a pond (4*4*1.2m 3) for one week, with continuous ventilation and water changes every other day. Intraperitoneal injection of S. iniae was performed at a dose of 1 μL / g, and 60 fish were injected with the same dose of PBS buffer as a control group. All individuals were marked as 0 h before the challenge, and then the water temperature was kept constant, with continuous aeration and regular water changes. Symptoms were observed regularly and the time of death was recorded. Based on the time of death, the 60 ovate pomfret that died first and showed obvious symptoms of S. iniae infection were used as the susceptible group. 15 days after the challenge, 60 individuals were randomly selected from the surviving individuals as the resistant group.

[0025] 1.2 Sample collection and processing

[0026] Dorsal fin tissue and muscle tissue of susceptible and resistant groups of oval pomfret were preserved in anhydrous ethanol and RNAwait respectively, transported to the laboratory, and stored in a -80℃ refrigerator for future use.

[0027] 1.3 Extraction and detection of genomic DNA

[0028] The genomic DNA was extracted using the phenol-chloroform extraction method. The extracted DNA was tested for concentration, purity, and integrity, and qualified samples were stored at -80°C.

[0029] 1.4 Whole-genome resequencing and typing

[0030] GBS simplified genome sequencing technology and Illumina HiSeq sequencing platform were used to perform paired-end PE150 sequencing on the genomic DNA of the oval pomfret described in step 3; the oval pomfret genome was used as the reference sequence for alignment, and SAMtools software was used to detect population variation and obtain a VCF file storing SNPs genotyping data; Plink and VCFtools software were used to filter genotypes based on the criteria of sample detection rate > 0.95, SNP detection rate > 0.90, minimum allele frequency ≥ 0.05, Hardy-Weinberg equilibrium > 0.0001, deletion rate and heterozygosity rate < 20%, and glimpse software was used to fill in genotypes in the haplotype library of this research group, finally obtaining high-quality SNPs polymorphic sites for subsequent analysis.

[0031] 1.5 Genome-wide association analysis

[0032] A genome-wide association analysis was conducted between the ovate pomfret and S. iniae resistance using GEMMA's mixed linear model (MLM) on the quality-controlled and imputed sequencing data to identify SNPs associated with S. iniae resistance. A QQ plot (Quantile-Quantile Plot) was also created to assess bias and sample population stratification in the association analysis.

[0033] 1.6 Gene annotation of significant SNPs

[0034] According to the genomic annotation file and the NCBI reference genome sequence, the SNPs identified in GWAS were annotated using the ANNOVAR program. By calculating the LD at the genomic level of Trachinotus ovatus, the genomic regions within 50 kb upstream and downstream of the relevant SNPs where the candidate genes were located were determined. The candidate genes in the candidate regions were annotated using the NCBI BLAST program.

[0035] 1.7 Verification of significant SNPs

[0036] After the above steps were performed on 60 Trachinotus ovatus in the verification population to obtain all their SNP loci, the SNP allele frequencies at position 14078454 on chromosome 7 of resistant and susceptible Trachinotus ovatus were extracted using the grep command in the linux server. These data were summarized in an Excel table to calculate the corresponding allele and genotype frequencies. Then, the chi-square test was performed on these frequencies using SPSS software to verify the significant differences between the resistant group and the susceptible group.

[0037] 2 Result analysis

[0038] 2.1 Phenotypic records of the Trachinotus ovatus challenge test

[0039] After the challenge, the phenotypic characteristics of the susceptible population of Trachinotus ovatus were as follows: black body color, protruding eyes, turbidity, orbital congestion, cerebral congestion, bleeding at the base of the fin rays, and congestion and swelling of the internal organs after dissection.

[0040] 2.2 Genomic DNA detection

[0041] All extracted DNA samples need to be detected by concentration and agarose gel electrophoresis. The specific requirements are as follows: for the purity detection of DNA, 1.6 < OD260 / OD280 < 2.0 and 1.8 < OD260 / OD230 < 2.1; the results of agarose gel electrophoresis need to have clean and pollution-free sample loading wells, clear main bands, and no trailing. All the extracted DNA needs to meet these two results before being sent for testing, otherwise it will be discarded or re-extracted. Finally, all 120 samples sent for testing met the requirements (see some DNA agarose gel electrophoresis diagrams in Figure 1 )

[0042] 2.3 Quality control of sequencing results

[0043] Quality control was carried out according to the following standards: sample detection rate > 0.95, SNP detection rate > 0.90, minimum allele frequency ≥ 0.05, Hardy-Weinberg equilibrium > 0.0001, and the deletion rate and heterozygosity rate < 20% for genotype filtering.

[0044] 2.4 Analysis of SNPs results

[0045] After the sequencing results are quality controlled, the number of extracted SNPs and their distribution on the chromosomes are as follows: Figure 2 The results show that chromosome 1 has the most SNPs, while chromosome 24 has the least. The marker density ranges from 1.77 kb / SNP to 3.58 kb / SNP, with an average density of 2.23 SNPs / kb across the entire genome.

[0046] 2.5 Genome-wide association analysis

[0047] The present invention uses a mixed linear model (MLM) of GEMMA to perform genome-wide association analysis on the resistance traits of ovate pomfret and Streptococcus iniae, and screens related SNPs sites within the whole genome.

[0048] 2.5.1 Screening of significant SNPs

[0049] The present invention uses the whole genome association analysis method to screen 128 SNPs significantly associated with the resistance trait of Streptococcus iniae in the oval pomfret. Finally, a SNP site with a polymorphic position C / T at position 14078454 on chromosome 7 (Oval pomfret genome NCBI: GCA_900607315.1) was selected. The Manhattan plots and linkage disequilibrium plots corresponding to all SNPs are shown in Figure 3 and Figure 4 .

[0050] Table 1 SNPs significantly associated with S. iniae resistance in Genome-wide association analysis of ovate pomfret

[0051]

[0052] 2.6 Population structure analysis

[0053] All the traits studied have significant SNPs sites, and their principal component analysis (PCA) plots and QQ plots are shown in Figure 2. Figure 5 Figure 6 As shown. Principal component analysis showed that there was no obvious clustering within the group, indicating no obvious stratification. In the QQ diagram, the horizontal axis represents the expected value and the vertical axis represents the observed value. The thin line in the figure represents the 45° line, which is the predicted threshold. The gray area is the 95% confidence interval of the scattered points on the figure. The farther the SNPs are from the solid line, the better the association strength. Figure 6 As can be seen, most of the loci in the lower left corner of the graph are on the diagonal line, indicating that the model selection is reasonable. The loci in the upper right corner that exceed the diagonal line and the confidence interval are highly significant with the target trait. In summary, this shows that there is no population stratification in the experimental population.

[0054] 2.7 Annotation of SNPs with significant association at the whole genome level

[0055] Through genome-wide association analysis, the significant SNPs obtained were used as the basis for screening candidate genes. The candidate genes screened are shown in Table 2.

[0056] Table 2 Candidate genes associated with resistance to S. iniae

[0057]

[0058] 2.8 Significant SNP Verification

[0059] In the validation population analysis, the SNP at position 14078454 on chromosome 7 exhibited a C / T polymorphism. The allele frequencies in the susceptible population were C: 0.833333 and T: 0.166667, while in the resistant group, the C frequency was 1 and the T frequency was 0. Chi-square tests showed significant differences (P < 0.05) in both genotype frequency (P = 0.005) and allele frequency (P = 0.02) between the resistant and susceptible groups, confirming the significance of the selected SNP in the validation population. The results indicate that the CC homozygous genotype at this site is associated with enhanced resistance to S. iniae.

[0060] Table 3 SNP significance test of validation population

[0061]

[0062] 3 Conclusion

[0063] In the aquaculture industry of ovate pomfret, huge losses are caused by iniae streptococcus every year, so it is very important to breed new varieties (lines) of ovate pomfret that are resistant to iniae streptococcus. The present invention provides a theoretical reference for the future breeding of ovate pomfret that is resistant to iniae streptococcus.

[0064] 3.1 Quality requirements for genomic DNA of ovate pomfret

[0065] In this example, the extracted DNA passed strict testing, and its integrity, concentration, and purity all met the standards.

[0066] 3.2 Genome-wide association analysis

[0067] In this study, genome-wide association analysis (GWAS) was used to analyze the phenotypes and genotypes of S. iniae in susceptible and resistant populations. Sequencing data was first subjected to quality control to extract all single nucleotide polymorphisms (SNPs). 128 SNPs were identified as associated with S. iniae resistance. A SNP located at position 14,078,454 on chromosome 7, with a C / T polymorphism, was selected. Validation in a validation population confirmed that this SNP is useful for identifying S. iniae resistance in scads, and that homozygous C / C at this position indicates strong resistance in scads.

[0068] Based on the above examples, the present invention discloses a SNP locus associated with resistance to Streptococcus iniae in the ovate pomfret. Through genome-wide association analysis of samples with both susceptible and resistant traits, a SNP located at position 14,078,454 on chromosome 7 was identified, providing theoretical and practical support for the future breeding of new ovate pomfret varieties (lines) resistant to Streptococcus iniae.

Claims

1. A SNP site associated with resistance to Streptococcus dolphinum disease in ovate pomfret, characterized in that: The SNP site is located at position 14078454 on chromosome 7, the polymorphism is C / T, and the genotype of the site with disease resistance is CC.

2. Use of the SNP site according to claim 1 in identifying Streptococcus iniae-resistant scads, characterized in that: The application method comprises extracting DNA of the oval pomfret to be identified, and selecting individuals with CC genotype at the SNP site by sequencing.

3. Use of the SNP site according to claim 1 in breeding a new variety of pomfret resistant to Streptococcus iniae, characterized in that: The application method is to screen the genotype of the SNP site being CC as the breeding parent.

4. The application of the gene containing the SNP site associated with the resistance trait of Streptococcus iniae in the ovate pomfret, characterized in that: The SNP site is as shown in claim 1, and the application is for identifying oval pomfret with resistance to dolphin streptococcal disease. The application method is to extract DNA from the oval pomfret to be identified, and select individuals with CC genotype at the SNP site through sequencing.

5. The application of the gene containing the SNP site associated with the resistance trait of Streptococcus iniae in Pomfret ovata, characterized in that: The application is used for breeding new varieties of oval pomfret resistant to dolphin streptococcal disease, and the application method is to screen individuals with CC genotype at the SNP site as breeding parents for breeding the next generation.

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