Haplotype of cobia fatness-degree-associated SNP site, primer group and application of haplotype and primer group of cobia fatness-degree-associated SNP site

Abstract

The invention relates to haplotypes of cobia fatness degree related SNP sites, a primer group and application of the haplotypes and the primer group, and belongs to the technical field of molecular markers, seven SNP sites related to cobia fatness degree are screened, primers for amplifying the SNP sites are provided, breeding of new varieties with fatness degree characters can be assisted by utilizing the SNP sites or the primers, and breeding of cobia fatness degree characters is facilitated. 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] The application belongs to the technical field of molecular markers, and particularly relates to a haplotype of a SNP site related to the fatness of Cobia, a primer set and application thereof. BACKGROUND

[0002] With the severe changes in global climate, the pollution of marine ecological environment and the increasingly serious overfishing phenomenon, the quality of wild germplasm resources of Cobia is facing an unprecedented decline crisis. Under the current breeding mode, the breeding population of Cobia mainly relies on wild original species to breed offspring. However, due to the existence of disordered mating phenomenon in the breeding process, and the lack of strict screening and control measures for germplasm resources, serious degradation phenomenon of important breeding traits such as growth rate, disease resistance and temperature adaptation ability of the breeding Cobia has occurred. This degradation phenomenon not only affects the breeding efficiency of Cobia, but also greatly restricts the scale and efficient development of the industrialization of Cobia breeding. Therefore, it is of great significance to breed Cobia strains with growth advantage and excellent fatness, which not only helps to improve the economic efficiency of breeding, but also enhances the market competitiveness of the Cobia breeding industry.

[0003] Nowadays, although there have been some reports on the growth traits of Cobia, there is no report on the application of haplotype of SNP site related to fatness in breeding. In the similar research field, some studies focus on growth traits, however, these studies mostly use traditional breeding methods, which have the problems of low precision and low efficiency. For example, the traditional breeding method can only select according to the phenotype, and it is difficult to carry out precise genetic improvement at the gene level. In the aspect of molecular markers, although the commonly used microsatellite markers have certain application in genetic diversity research, they have obvious limitations. Microsatellite markers can only be roughly identified by electrophoretic fragment size, and cannot accurately locate the SNP site related to fatness, nor can they accurately determine whether the Cobia with growth advantage is homozygous and whether the advantage is heritable. SUMMARY

[0004] In order to solve the technical problems faced by Cobia at present, the application provides a haplotype of SNP site related to the fatness of Cobia, a primer set and application thereof, which overcomes the limitation of multiple factors, strictly monitors the fatness of Cobia, and can be used for molecular assisted breeding of new strains with high fatness of Cobia.

[0005] The application is realized by the following technical solutions: The haplotype of SNP site related to the fatness of Cobia comprises at least one of the following: The SNP site chr7_6908611 is located on chromosome 7, the physical position is 6908611, the allele is T or C, and the nucleotide sequence of 100bp before and after the marker is shown as SEQ ID NO. 1. The SNP site chr7_24273367 is located on chromosome 7, the physical position is 24273367, the allele is G or T, and the nucleotide sequence of 100bp before and after the marker is shown as SEQ ID NO. 4. The SNP site chr21_13711537 is located on chromosome 21, the physical position is 13711537, the allele is C or T, and the nucleotide sequence of 100bp before and after the marker is shown as SEQ ID NO. 7. The SNP site chr21_7207269 is located on chromosome 21, the physical position is 7207269, the allele is G or T, and the nucleotide sequence of 100bp before and after the marker is shown as SEQ ID NO. 10. The SNP site chr21_1718482 is located on chromosome 21, the physical position is 1718482, the allele is A or C, and the nucleotide sequence of 100bp before and after the marker is shown as SEQ ID NO. 13. The SNP site chr7_988975 is located on chromosome 7, the physical position is 988975, the allele is C or T, and the nucleotide sequence of 100bp before and after the marker is shown as SEQ ID NO. 16. The SNP site chr21_17165459 is located on chromosome 21, the physical position is 17165459, the allele is A or G, and the nucleotide sequence of 100bp before and after the marker is shown as SEQ ID NO. 19.

[0006] The application also provides a primer set for identifying the SNP marker of the P. carcharias plumpness, wherein the primer set is SEQ ID NO. 2-3, 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.

[0007] The application also provides the haplotype of the SNP site related to the P. carcharias plumpness or the primer set, and the application in the selection of the P. carcharias plumpness trait, characterized in that the application is to select or discard individual base changes to produce different individuals in the P. carcharias plumpness trait, and is used for molecular assisted breeding of a new strain of P. carcharias with fast growth.

[0008] 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.

[0009] 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

[0010] Figure 1 This is a frequency distribution diagram of body fat percentage; 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, 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.

[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 body condition measurement data of cobia 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. 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. Table 2. SNP locus information for the condition of cobia. .

[0017] VI. The genotypes and phenotypic traits corresponding to the SNPs are shown in Tables 3 and 4; Table 3. Genotypes and phenotypic traits corresponding to SNPs ; ; Table 4. Molecular marker site information ; ; Note: Underlined sites represent amplified product fragments of the SNP sites, and single shaded sites represent the SNP sites.

[0018] VII. 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 six SNP loci mentioned above. The TC at the chr19_4747364 site was 23.65% higher than the CC. 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. The TT level at the chr19_6063646 locus was increased by 0.77% and 0.55% relative to GG and GT, respectively. CC at the chr19_5062858 locus was increased by 5.18% relative to AA and 5.08% relative to AC. The TT at the chr19_5753438 locus was increased by 7.34% relative to CC and 5.92% relative to CT. At the chr19_12436450 locus, GG was increased by 2.67% relative to AA and 2.25% relative to AG. See Table 6 for specific loci and phenotypic data.

[0019] Table 5. Validation group phenotypic data ; 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] 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 SNP locus associated with the fatness of cobia, characterized in that, The SNP site includes at least one of the following: 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. 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. SNP locus chr21_13711537 is located on chromosome 21, with a physical location of 13711537 and alleles of C or T. 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.

7. 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.

10. 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.

13. 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.

16.

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

3. A primer set for identifying the SNP markers of fatness in cobia, characterized in that, 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, and the primer set amplifies the corresponding SNP sites in the haplotype of the fatness-associated SNP site of cobia as described in claim 1.

4. Application of the primer set of claim 3 in the breeding of the conditionality trait of cobia.

5. The application according to claim 4, characterized in that, The primer set amplifies the DNA fragment of cobia, and individuals with a positive effect of the SNP locus genotype on conditionability are selected as breeding parents.

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