Haplotype of rachycentron canadum body weight associated SNP (Single Nucleotide Polymorphism) site, primer group and application thereof

Abstract

The invention relates to haplotypes of rachycentron canadum body weight related SNP sites, a primer group and application thereof, and belongs to the technical field of molecular markers, seven SNP sites related to rachycentron canadum body weight are screened out, primers for amplifying the SNP sites are provided, the SNP sites or the primers can assist in breeding of new varieties with body weight traits, and the breeding efficiency of rachycentron canadum body weight related SNP sites is improved. The method can greatly shorten the breeding period, reduce the dependence on phenotypic characters, improve the selective breeding efficiency, and have good application prospects.

Description

Technical Field

[0001] This invention belongs to the field of molecular marker technology, specifically, it relates to a haplotype, primer set and application of a body weight-related SNP site in cobia. Background Technology

[0002] With drastic global climate change, marine pollution, and increasing overfishing, the quality of wild cobia germplasm resources is facing an unprecedented decline. Under current aquaculture practices, farmed cobia rely primarily on wild-caught stock for reproduction. However, the widespread disordered mating during farming, coupled with a lack of rigorous selection and control of germplasm resources, has directly led to severe degradation in important aquaculture traits such as growth rate, disease resistance, and adaptability to water temperature changes. This degradation not only affects the profitability of cobia farming but also significantly hinders the large-scale and efficient development of the cobia factory farming industry. Therefore, cultivating superior cobia breeds with growth advantages is of paramount importance, as it not only helps improve economic efficiency but also enhances the market competitiveness of the cobia farming industry.

[0003] Currently, while there are some reports on growth traits in cobia, there are no reports on the application of haplotypes of associated SNP loci in breeding for the key economic trait of cobia weight. In similar research areas, some studies focus on growth traits; however, these studies mostly employ traditional breeding methods, which suffer from low precision and efficiency. For example, traditional breeding methods often only allow selection based on phenotypes, making it difficult to delve into the genetic level for precise genetic improvement. Regarding molecular markers, while commonly used microsatellite markers have some applications in genetic diversity research, they have significant limitations. Microsatellite markers can only provide a rough identification based on fragment size during electrophoresis, and cannot precisely locate SNP loci related to weight, nor can they accurately determine whether cobia with growth advantages are homozygous or whether this advantage is heritable. In contrast, the emerging SNP marker technology in recent years has provided a new opportunity for precise breeding of cobia. In related studies, researchers have identified a large number of SNP loci in the cobia genome using high-throughput sequencing technology and screened out some loci associated with traits such as growth. For the weight trait of cobia, haplotype analysis using SNP markers can more accurately locate genes or gene regions related to weight, and deeply analyze their genetic mechanisms, thus providing key technical support for the improvement of cobia's weight. Summary of the Invention

[0004] To address the current technical challenges faced by cobia, this invention provides haplotypes, primer sets, and applications of SNP sites associated with cobia weight, overcoming various limitations and enabling strict monitoring of cobia weight. It can be used for molecular-assisted breeding of new cobia strains with high weight.

[0005] This invention is achieved through the following technical solution: Haplotypes of SNP sites associated with body weight in cobia, wherein the SNP site includes at least one of the following: The SNP site chr19_4747364 is located on chromosome 19, with a physical location of 4747364 and alleles of G or A. The nucleotide sequence of the 100 bp before and after this marker is shown in SEQ ID NO.1. The SNP site chr19_5847505 is located on chromosome 19, with a physical location of 5847505 and alleles of T or C. The nucleotide sequence of the 100 bp before and after this marker is shown in SEQ ID NO.4. The SNP site chr19_6518522 is located on chromosome 19, with a physical location of 6518522 and alleles of T or C. The nucleotide sequence of the 100 bp before and after this marker is shown in SEQ ID NO.7. The SNP site chr19_6063646 is located on chromosome 19, with a physical location of 6063646 and alleles of A or T. The nucleotide sequence of the 100 bp before and after this marker is shown in SEQ ID NO.10. The SNP site chr19_5062858 is located on chromosome 19, with a physical location of 5062858 and alleles of A or G. The nucleotide sequence of the 100 bp before and after this marker is shown in SEQ ID NO.13. The SNP site chr19_5753438 is located on chromosome 19, with a physical location of 5753438 and alleles of A or C. The nucleotide sequence of the 100 bp before and after this marker is shown in SEQ ID NO.16.

[0006] The present invention also provides a primer set for SNP markers of the weight trait of cobia, wherein the nucleotide sequence of the primer set is SEQ ID NO2-3, SEQ ID NO5-6, SEQ ID NO.8-9, SEQ ID NO.11-12, SEQ ID NO.14-15, SEQ ID NO.17-18 or SEQ ID NO.20-21.

[0007] The present invention also provides the application of the haplotype or primer set in the selection of body weight traits in cobia. The method is to select or discard individuals with different body weight traits caused by a single base change, which is used for molecular-assisted breeding of new fast-growing cobia strains.

[0008] Furthermore, the application method involves using the primers to amplify cobia DNA fragments and selecting individuals with SNP loci genotypes that have a positive effect on weight trait as breeding parents.

[0009] The advantages of this invention compared to existing technologies are as follows: This invention utilizes genome-wide association analysis to screen for body length in cobia, obtaining SNP molecular markers significantly associated with the trait, including at least one of the seven SNP loci, which can be applied to molecular-assisted breeding of cobia. By using the breeding method disclosed in this invention for genomic-level screening, the breeding cycle can be significantly shortened, dependence on phenotypic traits reduced, and selection breeding efficiency improved, showing promising application prospects. Attached Figure Description

[0010] Figure 1 This is a frequency distribution map of body weight. Figure 2 The Manhattan plot uses chromosomes as the horizontal axis and the vertical axis to represent the -log of each SNP. 10 (p) value. Detailed Implementation

[0011] To better understand the technical content of this invention, specific embodiments and accompanying drawings are described below. Unless otherwise specified, the experimental methods in the following examples are conventional methods. The instruments, reagents, and kits used in the experiments are all commercially available.

[0012] Example 1 I. Materials and Methods The cobia used in the experiment came from Yangjiang Jinyuan Marine Biological Research Co., Ltd. 167 fish were randomly selected from the fish population and transferred to a new pond for temporary rearing. They were not fed for one day. During the experiment, the operators strictly followed the ethical guidelines for animal experiments, measured phenotypic traits, including body length, total length, body height, and weight, and performed statistical analysis on the phenotypic traits. At the same time, the tail fins of the cobia were taken and preserved in dry ice for later use.

[0013] II. Organizing and Analyzing Phenotypic Traits Statistical analysis was performed on the collected and organized phenotypic data, including minimum, maximum, mean, standard deviation, and coefficient of variation. The results are shown in Table 1. The normal distribution of the cobia body length measurement data was verified, and the results are as follows: Figure 1 ; Table 1. Statistical analysis of growth performance phenotypic data of cobia. ; Note: Body length, caudal fin length, and total length are measured in cm; body weight is measured in g, and fullness is measured in g / cm³.

[0014] III. Acquisition of Genomic Data Genomic DNA was extracted from the caudal fin samples of cobia and sent to MegiGene for whole-genome resequencing and variant detection. After the DNBSeq T7™ sequencing data was processed, quality control was performed to filter out low-quality data and obtain high-quality data. The clean data was aligned to the reference genome sequence using BWA-MEME software to determine the sequence location. The BAM file was corrected using the Best Practices workflow of GATK software to obtain the vcf file of the population genotype. Ultimately, we obtained 1,129.45 G reads, with a sequencing Q30 of 97.17%, a GC content of 40.60%, and 3,143,741 SNPs.

[0015] IV. SNP Quality Control and Filling First, using vcftools v0.1.16, filter parameters were set to ensure a minimum allele count of 2, a maximum deletion rate of less than 0.95, a minimum allele frequency of greater than 0.05, a measurement quality of greater than 30 for each SNP, and a minimum mean depth of 10. After screening, 980,781 SNPs meeting the parameters were retained. Then, Beagle v5.5 genotyping software was used to perform genotyping on the SNP data to resolve genotype deletion issues. Finally, SNP quality control was performed, and Plink v1.9.0-b.7.7 was used to convert the genotype vcf file to binary format.

[0016] V. Genome-wide association analysis A kinship matrix was constructed using GEMMA v0.98.5. The constructed kinship matrix and phenotypic traits were then used in conjunction with a mixed linear model for genome-wide association analysis. The model is as follows: y = Xβ + Zu + e, where y is the phenotypic vector, Xβ is the population structure effect, Zu is the marker effect to be tested, and e is the residual effect. In the small polygenic effect, K is the marker-inferred kinship matrix. The results are as follows... Figure 2 The Manhattan diagram shown is a genome-wide association analysis of body length in cobia, with a total of 7 SNP loci. The results are shown in Table 2-4. Table 2. SNP locus information for body length in cobia ; Table 3. Screening of SNPs and their corresponding genotypes and phenotypic traits ; ; Table 4. Molecular marker site information ; ;

[0017] Note: The underlined part is the target fragment amplified by the corresponding primer, and the shaded bases are the SNP sites.

[0018] VI. Validation in different groups Different batches of adult fish were selected for verification. First, phenotypic data were statistically analyzed, including minimum, maximum, mean, standard deviation, and coefficient of variation. The results are shown in Table 5. Screening analysis was then performed on the seven SNP loci mentioned above. At the chr19_4747364 locus, AA levels were increased by 57.5% and 41.4% relative to GG and GA, respectively. CC at the chr19_5847505 site was increased by 61.4% and 41.1% relative to TT and TC, respectively. CC at the chr19_6518522 site was increased by 37.7% and 17.6% relative to TT and TC, respectively. At the chr19_6063646 locus, TT was increased by 62.7% relative to AA and 40.2% relative to TA. At the chr19_5062858 locus, GG showed a 60.8% and 40.7% increase relative to AA and GA, respectively. At the chr19_5753438 locus, AA was increased by 62.7% relative to CC and 44.6% relative to CA. At the chr19_12436450 locus, AA was increased by 67.4% relative to TT and 36.2% relative to AT. See Table 6 for specific loci and phenotypic data.

[0019] Table 5. Phenotypic data of the validation group. ; Note: Body length is measured in cm; weight is measured in g; and fullness is measured in g / cm³.

[0020] Table 6. Loci and Phenotypes .

[0021] VII. The specific combined effects of mutations are shown in Table 7; Table 7. Phenotypic data for different mutation combinations ; ; Note: The SNP sites of GG, TT, TT, AA, AA, CC, TT from left to right are chr19_4747364, chr19_5847505, chr19_6518522, chr19_6063646, chr19_5062858, chr19_5753438, and chr19_1243645.

[0022] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, are covered within the scope of protection of the present invention.

Claims

1. A haplotype of a body weight-associated SNP locus in cobia, characterized in that, The SNP site includes at least one of the following: The SNP site chr19_4747364 is located on chromosome 19, with a physical location of 4747364 and alleles of G or A. The nucleotide sequence of the 100 bp before and after this marker is shown in SEQ ID NO.

1. The SNP site chr19_5847505 is located on chromosome 19, with a physical location of 5847505 and alleles of T or C. The nucleotide sequence of the 100 bp before and after this marker is shown in SEQ ID NO.

4. The SNP site chr19_6518522 is located on chromosome 19, with a physical location of 6518522 and alleles of T or C. The nucleotide sequence of the 100 bp before and after this marker is shown in SEQ ID NO.

7. The SNP site chr19_6063646 is located on chromosome 19, with a physical location of 6063646 and alleles of A or T. The nucleotide sequence of the 100 bp before and after this marker is shown in SEQ ID NO.

10. The SNP site chr19_5062858 is located on chromosome 19, with a physical location of 5062858 and alleles of A or G. The nucleotide sequence of the 100 bp before and after this marker is shown in SEQ ID NO.

13. The SNP site chr19_5753438 is located on chromosome 19, with a physical location of 5753438 and alleles of A or C. The nucleotide sequence of the 100 bp before and after this marker is shown in SEQ ID NO.

16. The SNP site chr19_12436450 is located on chromosome 19, with a physical location of 12436450 and alleles of T or A. The nucleotide sequence of the 100 bp before and after this marker is shown in SEQ ID NO.

19.

2. The application of the haplotype of the cobia weight-related SNP locus as described in claim 1 in the breeding of cobia weight traits, characterized in that, The application involves selecting or discarding individuals with different weight traits due to single base changes, for molecular-assisted breeding of new fast-growing strains of cobia.

3. A primer set for SNP markers of weight traits in cobia, characterized in that, The nucleotide sequences of the primer set are SEQ ID NO2-3, SEQ ID NO5-6, SEQ ID NO.8-9, SEQ ID NO.11-12, SEQ ID NO.14-15, SEQ ID NO.17-18 or SEQ ID NO.20-21, and the primer set amplifies the corresponding SNP sites in the haplotype of the body weight-related SNP sites of cobia as described in claim 1.

4. The application of the primer set described in claim 3 in the breeding of cobia weight traits.

5. The application according to claim 4, characterized in that, The application involves using the primer set to amplify cobia DNA fragments and selecting individuals with a positive effect of the SNP locus genotype on weight trait as breeding parents.

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