Haplotype, primer combination of body length associated snp site of cichla temminckii and application thereof

Abstract

The application relates to a haplotype of a SNP site of a body length of a cobia, a primer combination and application thereof, and belongs to the technical field of molecular markers. Six SNP sites significantly related to the body length are identified through whole genome correlation analysis, and a single base change in the sites can significantly affect the body length. The application also provides a primer group for amplifying the SNP sites. The superior haplotype constructed based on the SNP sites can be used for molecular assisted breeding of a new strain of the cobia in terms of the body length, significantly shortens a breeding cycle, improves selection accuracy, and overcomes the limitation of phenotype dependence in traditional breeding. The application provides an efficient molecular tool for cobia 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 of a body length-associated SNP site in cobia, primer combinations, and their applications. 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 the growth traits of cobia, there are no reports on the application of haplotypes of associated SNP loci in breeding for the key economic trait of body length. 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 body length, 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. By combining SNP marker haplotype analysis with the body length trait of cobia, we can more accurately locate genes or gene regions related to body length and deeply analyze their genetic mechanisms, thus providing key technical support for the improvement of body length in cobia. 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 body length. These methods overcome various limitations, allow for strict monitoring of cobia body length, and can be used for molecular-assisted breeding of new cobia strains with high body length.

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

[0006] Haplotypes of SNP sites associated with body length in cobia, wherein the SNP sites include at least one of the following:

[0007] The SNP site chr14_2882510 is located on chromosome 14, with a physical location of 2882510 and alleles of T or C. The nucleotide sequence of the 100 bp before and after this marker is shown in SEQ ID NO.1.

[0008] The SNP site chr14_2996332 is located on chromosome 14, with a physical location of 2996332 and alleles of A or C. The nucleotide sequence of the 100 bp before and after this marker is shown in SEQ ID NO.4.

[0009] The SNP site chr14_1491846 is located on chromosome 14, with a physical location of 1491846 and alleles of A or C. The nucleotide sequence of the 100 bp before and after this marker is shown in SEQ ID NO.7.

[0010] The SNP site chr14_1791709 is located on chromosome 14, with a physical location of 1791709 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.

[0011] The SNP site chr14_683410 is located on chromosome 14, with a physical location of 683410 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] The SNP site chr7_3748958 is located on chromosome 7, with a physical location of 3748958 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] The present invention also provides a set of SNP marker primers for identifying the body length 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 or SEQ ID NO.17-18.

[0014] The present invention also provides the application of the haplotype or primer set in the selection of body length traits in cobia. The application method is to select or discard individuals with different body length traits in cobia by selecting or discarding a single base change, which is used for molecular-assisted breeding of new fast-growing cobia strains.

[0015] 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 body length traits as breeding parents.

[0016] 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 six 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

[0017] Figure 1 This is a frequency distribution diagram of body length;

[0018] 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

[0019] 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.

[0020] Example 1

[0021] I. Materials and Methods

[0022] 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.

[0023] II. Organizing and Analyzing Phenotypic Traits

[0024] 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 .

[0025] Table 1. Statistical analysis of growth performance phenotypic data of cobia.

[0026] ;

[0027] 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.

[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 2The Manhattan diagram shown is a genome-wide association study of body length in cobia. There are three SNP loci in the first four positions, as shown in Table 2-4.

[0035] Table 2. SNP locus information for body length in cobia

[0036] ;

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

[0038] ;

[0039] ;

[0040] Table 4. Molecular marker site information

[0041] ;

[0042] ;

[0043] Note: The underlined part is the target fragment amplified by the corresponding primer, and the shaded bases are the SNP sites.

[0044] VI. Verification was conducted in different schools of Sergeant fish.

[0045] 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.

[0046] CC and CT at the chr14_2882510 locus were increased by 9.6% and 12.9% respectively compared to TT.

[0047] CC and CA at the chr14_2996332 site were increased by 10.2% and 15.6% respectively compared to AA.

[0048] CC and CA at the chr14_1491846 site were increased by 10.4% and 10.9% respectively compared to AA.

[0049] At the chr14_271210 locus, TT and TA were increased by 10.4% and 13.2% respectively relative to AA.

[0050] CC and CT at the chr14_683410 locus were increased by 10.4% and 13.2% respectively compared to TT.

[0051] CC and CT at the chr7_3748958 locus were increased by 10.4% and 10.9% respectively compared to TT.

[0052] See Table 6 for specific locus and phenotypic data;

[0053] Table 5. Phenotypic data of the validation group.

[0054] ;

[0055] Note: Body length is measured in cm; weight is measured in g; and fullness is measured in g / cm³.

[0056] Table 6. Locus and Phenotypic Data

[0057] ;

[0058] VII. The combined effects of multiple mutants are shown in Table 7;

[0059] Table 7. Effects of different mutant combinations on phenotypic data

[0060] ;

[0061] Note: The SNP tags corresponding to TT, AA, AA, AA, TT, and TC are chr14_2882510, chr14_2996332, chr14_1491846, chr14_271210, chr14_683410, and chr7_3748958, respectively.

[0062] As can be seen from Table 7, multiple positive SNP mutations may produce synergistic or antagonistic effects.

[0063] 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. Molecular markers associated with body length in cobia, characterized in that, The molecular marker may be any 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 body length of the cobia, and the allele of this site is T or C. 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 body length of the cobia, and the allele of this site is A or C. 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 body length of the cobia, and the allele of this site is A or C. The nucleotide sequence of the molecular marker is shown in SEQ ID NO.

10. At position 100 of the molecular marker, there is an SNP site associated with the body length of the cobia, and the allele of this site is A or T. 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 body length of the 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 body length of the cobia, and the allele of this site is T or C.

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