Haplotype, primer set of snp site related with fatness of cobia and application thereof

Abstract

The application relates to a haplotype of a SNP site related to the fullness degree of Larimichthys crocea, a primer set and application thereof, and belongs to the technical field of molecular markers. Seven SNP sites related to the fullness degree of Larimichthys crocea are screened, and primers for amplifying the SNP sites are provided. The SNP sites or the primers can assist the breeding of new varieties with the fullness degree, can greatly shorten the breeding period, reduce the dependence on phenotypic traits, improve the selection and breeding efficiency, and have a good 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 SNP site associated with the fatness of cobia. Background Technology

[0002] With drastic global climate change, marine pollution, and increasingly severe overfishing, the quality of wild cobia germplasm resources is facing an unprecedented decline. Under current aquaculture practices, farmed cobia rely primarily on wild 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 industrial aquaculture industry. Therefore, cultivating superior cobia breeds with growth advantages and excellent plumpness is of paramount importance. This will not only help improve the economic benefits of aquaculture but also enhance 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 cobia condition. In related 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 the size of electrophoretic fragments, and cannot precisely locate SNP loci associated with condition, nor can they accurately determine whether cobia with growth advantages are homozygous or whether this advantage is heritable. 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's conditionability. These methods overcome various limitations, allow for strict monitoring of cobia's conditionability, and can be used for molecular-assisted breeding of new cobia strains with high conditionability.

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

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

[0007] The SNP site chr7_6908611 is located on chromosome 7, with a physical location of 6908611 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 chr7_24273367 is located on chromosome 7, with a physical location of 24273367 and an allele of G or T. The nucleotide sequence of the 100 bp before and after this marker is shown in SEQ ID NO.4.

[0009] The SNP site chr21_13711537 is located on chromosome 21, with a physical location of 13711537 and an allele of C or T. The nucleotide sequence of the 100 bp before and after this marker is shown in SEQ ID NO.7.

[0010] The SNP site chr21_7207269 is located on chromosome 21, with a physical location of 7207269 and an allele of G 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 chr21_1718482 is located on chromosome 21, with a physical location of 1718482 and alleles of A 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_988975 is located on chromosome 7, with a physical location of 988975 and an allele of C or T. The nucleotide sequence of the 100 bp before and after this marker is shown in SEQ ID NO.16.

[0013] The SNP site chr21_17165459 is located on chromosome 21, with a physical location of 17165459 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 primer set for identifying the SNP markers of cobia fatness, wherein the primer set is 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 of the SNP site associated with the body condition of the cobia or the primer set thereon in the breeding of the body condition trait of cobia. The application is characterized in that the application is to select or discard individuals with different body condition traits of cobia by selecting or discarding a single base change, for molecular-assisted breeding of new fast-growing cobia strains.

[0016] Furthermore, the method of application involves amplifying cobia DNA fragments using the primers, and selecting individuals with SNP loci genotypes that have a positive effect on condition factor as breeding parents.

[0017] The beneficial effects of this invention compared with the prior art are as follows: This invention uses genome-wide association analysis to screen for the condition of cobia and found that seven SNP molecular markers are significantly associated with condition, which can greatly shorten the breeding cycle, reduce dependence on phenotypic traits, improve the efficiency of selective breeding, and has good application prospects. Attached Figure Description

[0018] Figure 1 This is a frequency distribution diagram of body fat percentage;

[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, and weight, calculated body condition, and performed statistical analysis on the phenotypic traits. At the same time, the cobia's tail fin was taken and preserved in dry ice for later use.

[0024] II. Organizing and Analyzing Phenotypic Traits

[0025] 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 body condition measurement data of cobia was verified, and the results are as follows: Figure 1 ;

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

[0027] ;

[0028] 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.

[0029] III. Acquisition of Genomic Data

[0030] 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.

[0031] IV. SNP Quality Control and Filling

[0032] 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.

[0033] V. Genome-wide association analysis

[0034] 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:

[0035] y = Xβ + Zu + e

[0036] 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. Results are as follows... Figure 2(Manhattan) is a Manhattan diagram obtained from a genome-wide association analysis of the fatness of cobia. There are a total of 7 SNP sites, and the results are shown in Table 2.

[0037] Table 2. SNP locus information for the condition of cobia.

[0038] .

[0039] VI. The genotypes and phenotypic traits corresponding to the SNPs are shown in Tables 3 and 4;

[0040] Table 3. Genotypes and phenotypic traits corresponding to SNPs

[0041] ;

[0042] ;

[0043] Table 4. Molecular marker site information

[0044] ;

[0045] ;

[0046] Note: Underlined sites represent amplified product fragments of the SNP sites, and single shaded sites represent the SNP sites.

[0047] VII. Validation in different groups

[0048] 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.

[0049] The TC at the chr19_4747364 site was 23.65% higher than the CC.

[0050] The TT at the chr19_5847505 locus was increased by 8.19% and 5.54% relative to GG and GT, respectively; the TT at the chr19_6518522 locus was increased by 6.67% and 3.74% relative to CC and CT, respectively.

[0051] The TT level at the chr19_6063646 locus was increased by 0.77% and 0.55% relative to GG and GT, respectively.

[0052] CC at the chr19_5062858 locus was increased by 5.18% relative to AA and 5.08% relative to AC.

[0053] The TT at the chr19_5753438 locus was increased by 7.34% relative to CC and 5.92% relative to CT.

[0054] At the chr19_12436450 locus, GG was increased by 2.67% relative to AA and 2.25% relative to AG.

[0055] See Table 6 for specific loci and phenotypic data.

[0056] Table 5. Validation group phenotypic data

[0057] ;

[0058] Note: Body length is measured in cm; weight is measured in g; and fullness is measured in g / cm³.

[0059] Table 6. Loci and Phenotypes

[0060] ;

[0061] .

[0062] 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 associated with the condition of P. argentee, characterized in that, The molecular marker is at least one of the following: a nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1, and a SNP site associated with the flesh fullness of C. ichthys is at position 98 of the molecular marker, and the allelic gene of the site is T or C; a nucleotide sequence of the molecular marker is shown as SEQ ID NO. 4, and a SNP site associated with the flesh fullness of C. ichthys is at position 100 of the molecular marker, and the allelic gene of the site is G or T; a nucleotide sequence of the molecular marker is shown as SEQ ID NO. 7, and a SNP site associated with the flesh fullness of C. ichthys is at position 100 of the molecular marker, and the allelic gene of the site is C or T; a nucleotide sequence of the molecular marker is shown as SEQ ID NO. 10, and a SNP site associated with the flesh fullness of C. ichthys is at position 100 of the molecular marker, and the allelic gene of the site is G or T; a nucleotide sequence of the molecular marker is shown as SEQ ID NO. 13, and a SNP site associated with the flesh fullness of C. ichthys is at position 100 of the molecular marker, and the allelic gene of the site is A or C; a nucleotide sequence of the molecular marker is shown as SEQ ID NO. 16, and a SNP site associated with the flesh fullness of C. ichthys is at position 100 of the molecular marker, and the allelic gene of the site is C or T; a nucleotide sequence of the molecular marker is shown as SEQ ID NO. 19, and a SNP site associated with the flesh fullness of C. ichthys is at position 100 of the molecular marker, and the allelic gene of the site is A or G.

Images