Haplotype, primer set of weight-related snp site of cichla temminckii and application thereof

Abstract

The application relates to a haplotype of a SNP site related to body weight of Larimichthys crocea, a primer set and application thereof, and belongs to the technical field of molecular markers. Seven SNP sites related to the body weight of the 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 body weight, can greatly shorten a breeding period, reduce the dependence on phenotypic traits, improve the selection and breeding efficiency, and have a good application prospect.

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 body weight of Cobia, a primer set and application thereof. BACKGROUND

[0002] With the dramatic 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, it directly causes serious degradation of important breeding traits such as growth rate, disease resistance and the ability to adapt to water temperature changes of the farmed Cobia. This degradation 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 varieties with growth advantage, 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 research of growth traits of Cobia, there is no report on the application of haplotype of SNP site to breeding for the key economic trait of body weight of Cobia. 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 body weight, nor can they accurately determine whether the Cobia with growth advantage is homozygous and whether the advantage is heritable. In contrast, the SNP marker technology emerging in recent years provides a new opportunity for the precise breeding of Cobia. In related research, through high-throughput sequencing technology, researchers have identified a large number of SNP sites in the genome of Cobia, and screened out some sites related to growth traits. For the body weight trait of Cobia, combined with haplotype analysis of SNP markers, it can more accurately locate the genes or gene regions related to body weight, and deeply analyze its genetic mechanism, thereby providing key technical support for the body weight improvement of Cobia. SUMMARY

[0004] In order to solve the technical problems faced by the Cobia at present, the application provides a haplotype of a Cobia weight-related SNP site, a primer group and application thereof, overcomes multiple factor restrictions, strictly monitors the weight of the Cobia, and can be used for molecular-assisted breeding of a new strain with high Cobia weight.

[0005] The application is realized through the following technical solutions:

[0006] The haplotype of the Cobia weight-related SNP site comprises at least one of the following:

[0007] The SNP site chr19_4747364 is located on chromosome 19, the physical position is 4747364, the allele is G or A, and the nucleotide sequence of 100 bp before and after the marker is shown as SEQ ID NO. 1.

[0008] The SNP site chr19_5847505 is located on chromosome 19, the physical position is 5847505, the allele is T or C, and the nucleotide sequence of 100 bp before and after the marker is shown as SEQ ID NO. 4.

[0009] The SNP site chr19_6518522 is located on chromosome 19, the physical position is 6518522, the allele is T or C, and the nucleotide sequence of 100 bp before and after the marker is shown as SEQ ID NO. 7.

[0010] The SNP site chr19_6063646 is located on chromosome 19, the physical position is 6063646, the allele is A or T, and the nucleotide sequence of 100 bp before and after the marker is shown as SEQ ID NO. 10.

[0011] The SNP site chr19_5062858 is located on chromosome 19, the physical position is 5062858, the allele is A or G, and the nucleotide sequence of 100 bp before and after the marker is shown as SEQ ID NO. 13.

[0012] The SNP site chr19_5753438 is located on chromosome 19, the physical position is 5753438, the allele is A or C, and the nucleotide sequence of 100 bp before and after the marker is shown as SEQ ID NO. 16.

[0013] The application also provides a primer set of the SNP marker of the body weight trait of Larimichthys crocea, and the nucleotide sequences of the primer set are 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.

[0014] The application also provides the application of the haplotype or the primer set in the selection of the body weight trait of Larimichthys crocea, and the method is to select or discard the individual with the difference in the body weight trait of Larimichthys crocea caused by the single base change, and is used for the molecular assisted breeding of the new strain of Larimichthys crocea with fast growth.

[0015] Further, the application method is to amplify the DNA fragment of Larimichthys crocea by using the primer, and select the individual with the positive effect of the SNP site genotype and the body weight trait as the breeding parent.

[0016] Compared with the prior art, the application has the beneficial effects that the application screens the body length of Larimichthys crocea by using the whole genome correlation analysis, obtains the SNP molecular marker with the significant correlation with the trait, includes at least one of the 7 SNP sites, and can be applied to the molecular assisted breeding of Larimichthys crocea. The breeding method disclosed in the application can greatly shorten the breeding period, reduce the dependence on the phenotype trait, improve the selection and breeding efficiency, and has a good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 is a body weight frequency distribution chart;

[0018] Figure 2 is a Manhattan plot with the chromosome as the horizontal axis and the -log 10 (p) value of each SNP as the vertical axis. DETAILED DESCRIPTION

[0019] In order to better understand the technical content of the application, the following specific embodiments and drawings are used for specific introduction, and the experimental methods in the following experimental examples are all conventional methods without special instructions. The instruments, reagents and kits used in the experiments can be obtained by market purchase.

[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 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.

[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. Validation in different groups

[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 seven SNP loci mentioned above.

[0046] At the chr19_4747364 locus, AA levels were increased by 57.5% and 41.4% relative to GG and GA, respectively.

[0047] CC at the chr19_5847505 site was increased by 61.4% and 41.1% relative to TT and TC, respectively.

[0048] CC at the chr19_6518522 site was increased by 37.7% and 17.6% relative to TT and TC, respectively.

[0049] At the chr19_6063646 locus, TT was increased by 62.7% relative to AA and 40.2% relative to TA.

[0050] At the chr19_5062858 locus, GG showed a 60.8% and 40.7% increase relative to AA and GA, respectively.

[0051] At the chr19_5753438 locus, AA was increased by 62.7% relative to CC and 44.6% relative to CA.

[0052] At the chr19_12436450 locus, AA was increased by 67.4% relative to TT and 36.2% relative to AT.

[0053] See Table 6 for specific loci and phenotypic data.

[0054] Table 5. Phenotypic data of the validation group.

[0055] ;

[0056] Note: Body length is measured in cm; weight is measured in g; and fullness is measured in g / cm³.

[0057] Table 6. Loci and Phenotypes

[0058] .

[0059] VII. The specific combined effects of mutations are shown in Table 7;

[0060] Table 7. Phenotypic data for different mutation combinations

[0061] ;

[0062] ;

[0063] 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.

[0064] 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 body weight of 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 body weight of the 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 body weight 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.

7. At position 100 of the molecular marker, there is an SNP site associated with the body weight 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.

10. At position 100 of the molecular marker, there is an SNP site associated with the body weight 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.

13. At position 101 of the molecular marker, there is an SNP site associated with the body weight of cobia, and the allele of this site is A or G. 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 weight 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.

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

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