Haplotype of low temperature related snp site of cobia and primer set and application thereof

Abstract

The application relates to a haplotype of a low-temperature-resistant SNP site of Larimichthys crocea, a primer set and application thereof, and belongs to the technical field of molecular markers. Seven SNP sites significantly related to body length are identified through whole genome association analysis. Single base change in the sites can significantly affect the low-temperature resistance. The application also provides a primer set for amplifying the SNP sites. The advantage haplotype constructed based on the SNP sites can be used for molecular assisted breeding of a new strain of Larimichthys crocea with low-temperature resistance, significantly shortens the breeding cycle, improves the selection accuracy, and overcomes the limitations of traditional breeding on phenotype. The application provides an efficient molecular tool for Larimichthys crocea germplasm improvement, and has an important application prospect.

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 low-temperature-resistance-associated 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, although there are some reports on the growth traits of cobia, there are no reports on the application of haplotypes of related SNP loci in breeding for the key economic trait of low temperature tolerance in cobia. Summary of the Invention

[0004] To address the current technical problems faced by cobia, this invention provides haplotypes of SNP sites associated with low-temperature tolerance in cobia, primer sets, and their application in the breeding of low-temperature tolerance traits. This overcomes various limitations, strictly monitors the low-temperature tolerance trait of cobia, and can be used for molecular-assisted breeding of new low-temperature tolerant strains of cobia.

[0005] This invention is achieved through the following technical solution:

[0006] Haplotypes of low-temperature tolerance-associated SNP sites in cobia, wherein the SNP sites include at least one of the following:

[0007] chr6_15616256 is located on chromosome 6, with a physical location of 15616256 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.

[0008] chr6_15926088 is located on chromosome 6, with a physical location of 15926088 and alleles of C or A. The nucleotide sequence of the 100 bp before and after this marker is shown in SEQ ID NO.4.

[0009] chr6_21059880 is located on chromosome 6, with a physical location of 21059880 and alleles of A or T. The nucleotide sequence of the 100 bp before and after this marker is shown in SEQ ID NO.7.

[0010] chr6_18380688 is located on chromosome 6, with a physical location of 18380688 and alleles of T or G. The nucleotide sequence of the 100 bp before and after this marker is shown in SEQ ID NO.10.

[0011] chr6_20945793 is located on chromosome 6, with a physical location of 20945793 and alleles of T or C. The nucleotide sequence of the 100 bp before and after this marker is shown in SEQ ID NO.13.

[0012] chr6_16770206 is located on chromosome 6, with a physical location of 16770206 and alleles of T or C. The nucleotide sequence of the 100 bp before and after this marker is shown in SEQ ID NO.16.

[0013] chr6_14494167 is located on chromosome 6, with a physical location of 14494167 and alleles of A or G. The nucleotide sequence of the 100 bp before and after this marker is shown in SEQ ID NO.19.

[0014] The present invention also provides a set of SNP marker primers for identifying the low-temperature tolerance trait of cobia, wherein the nucleotide sequences of the primer set are SEQ ID NO2-3, SEQ ID NO.5-6, 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.

[0015] The present invention also provides the application of the haplotype or primer set in the breeding of low-temperature tolerance traits in cobia. The application method is to select or discard individuals with different low-temperature tolerance traits in cobia by selecting or discarding a single base change, which is used for molecular-assisted breeding of new low-temperature tolerant strains of cobia.

[0016] Furthermore, the application method involves using the primers to amplify cobia DNA fragments, and selecting individuals with SNP genotypes that have a positive effect on low-temperature tolerance as breeding parents.

[0017] The advantages of this invention compared to existing technologies are as follows: This invention utilizes genome-wide association analysis to screen for the low-temperature tolerance trait 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 can be reduced, and selection breeding efficiency can be improved, showing promising application prospects. Attached Figure Description

[0018] Figure 1 This is a frequency distribution diagram for low-temperature resistance.

[0019] Figure 2 The Manhattan plot uses chromosomes as the horizontal axis and the vertical axis to show the -log of each SNP. 10 (p) value. Detailed Implementation

[0020] 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.

[0021] Example 1

[0022] I. Materials and Methods

[0023] 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, weight, and accumulated temperature under shock, 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.

[0024] II. Organizing and Analyzing Phenotypic Traits

[0025] In the low-temperature experiment, the initial temperature was 30 degrees Celsius, and the water temperature was measured every ten minutes to record the accumulated temperature during shock in the cobia. The collected and processed phenotypic data were statistically analyzed, including the minimum, maximum, mean, standard deviation, and coefficient of variation. The results are shown in Table 1. The low-temperature tolerance data of the cobia were validated for normality, and the results are as follows: Figure 1 ;

[0026] Table 1. Statistical analysis of growth performance phenotypic data of cobia.

[0027] .

[0028] III. Acquisition of Genomic Data

[0029] 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.

[0030] IV. SNP Quality Control and Filling

[0031] 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.

[0032] V. Genome-wide association analysis

[0033] 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:

[0034] 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 the genome-wide association analysis of cold tolerance in cobia, which includes 7 SNP loci. The results are shown in Table 2-4.

[0035] Table 2. Low-temperature tolerance SNP information in cobia

[0036] ;

[0037] Table 3. Screening of SNPs and their corresponding genotypes and phenotypic traits

[0038] ;

[0039] Table 4. Molecular marker site information

[0040] ;

[0041] ;

[0042] Note: The underlined part is the target fragment amplified by the corresponding primer, and the shaded bases are the SNP sites.

[0043] VI. Validation in different groups

[0044] 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 six SNP loci mentioned above.

[0045] At chr6_15616256, GG was increased by 13.45% and 42.90% relative to GA and AA, respectively; at chr6_15926088, CA was increased by 19.2% relative to CC; at chr6_21059880, TT was increased by 52.7% and 60.6% relative to AT and AA, respectively; and at chr6_18380688, TG was increased by 13.2% relative to TT.

[0046] CC at chr6_20945793 increased by 76.6% and 83.3% relative to TC and TT, respectively; TC at chr6_16770206 increased by 17.6% relative to TT; AG at chr6_14494167 increased by 50.9% relative to AA. See Table 6 for details on loci and phenotypic data.

[0047] Table 5. Validation group phenotypic data

[0048] ;

[0049] Table 6. Loci and Phenotypes

[0050] .

[0051] 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 molecular marker for low-temperature tolerance in cobia, characterized in that, The molecular marker is at least one of the following: The nucleotide sequence of the molecular marker is shown in SEQ ID NO.

1. At position 100 of the molecular marker, there is an SNP site associated with the low-temperature tolerance of cobia, and the allele of this site is G or A. The nucleotide sequence of the molecular marker is shown in SEQ ID NO.

4. At position 100 of the molecular marker, there is an SNP site associated with the cold tolerance of cobia, and the allele of this site is C or A. The nucleotide sequence of the molecular marker is shown in SEQ ID NO.

7. At position 100 of the molecular marker, there is an SNP site associated with the cold tolerance of cobia, and the allele of this site is A or T. The nucleotide sequence of the molecular marker is shown in SEQ ID NO.

10. At position 99 of the molecular marker, there is an SNP site associated with the cold tolerance of cobia, and the allele of this site is T or G. The nucleotide sequence of the molecular marker is shown in SEQ ID NO.

13. At position 100 of the molecular marker, there is an SNP site associated with the cold tolerance of cobia, and the allele of this site is T or C. The nucleotide sequence of the molecular marker is shown in SEQ ID NO.

16. At position 100 of the molecular marker, there is an SNP site associated with the cold tolerance of cobia, and the allele of this site is T or C. The nucleotide sequence of the molecular marker is shown in SEQ ID NO.

19. At position 100 of the molecular marker, there is an SNP site associated with the cold tolerance of cobia, and the allele of this site is A or G.

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