Haplotype of rachycentron canadum body length associated SNP (Single Nucleotide Polymorphism) site, primer combination and application thereof

Abstract

The invention relates to haplotypes of rachycentron canadum body length associated SNP sites, a primer combination and application of the haplotypes and the primer combination, and belongs to the technical field of molecular markers. Six SNP sites remarkably associated with the body length are identified through whole genome association analysis, and the body length can be remarkably influenced by the change of a single basic group in the sites. The invention also provides a primer group for amplifying the SNP site. The dominant haplotype constructed on the basis of the SNP site can be used for molecular assisted breeding of a new cobia body length strain, the breeding period is remarkably shortened, the selection accuracy is improved, and the limitation that traditional breeding depends on phenotypes is overcome. The invention provides an efficient molecular tool for germplasm improvement of cobia, and has an important application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular markers, and particularly relates to a haplotype of a body length associated SNP site of Cobia, a primer combination and application thereof. BACKGROUND

[0002] With the dramatic changes in global climate, 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 temperature adaptation ability of farmed Cobia. This degradation not only affects the breeding efficiency of Cobia, but also greatly restricts the scale and efficient development of Cobia factory farming industry. 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 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 associated SNP site to breeding for the key economic trait of body length 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 length, 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 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 length trait of Cobia, combined with haplotype analysis of SNP markers, it can more accurately locate the genes or gene regions related to body length, and deeply analyze its genetic mechanism, thereby providing key technical support for the body length 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 body length related SNP site, a primer group and application thereof, overcomes multiple factor restrictions, strictly monitors the body length of the Cobia, and can be used for molecular assisted breeding of a new strain with high Cobia body length.

[0005] The application is realized by the following technical solutions: The haplotype of the Cobia body length related SNP site comprises at least one of the following: The SNP site chr14_2882510 is located on chromosome 14, the physical position is 2882510, the allele is T or C, and the nucleotide sequence of 100 bp before and after the marker is shown as SEQ ID NO. 1; The SNP site chr14_2996332 is located on chromosome 14, the physical position is 2996332, the allele is A or C, and the nucleotide sequence of 100 bp before and after the marker is shown as SEQ ID NO. 4; The SNP site chr14_1491846 is located on chromosome 14, the physical position is 1491846, the allele is A or C, and the nucleotide sequence of 100 bp before and after the marker is shown as SEQ ID NO. 7; The SNP site chr14_1791709 is located on chromosome 14, the physical position is 1791709, 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; The SNP site chr14_683410 is located on chromosome 14, the physical position is 683410, the allele is T or C, and the nucleotide sequence of 100 bp before and after the marker is shown as SEQ ID NO. 13; The SNP site chr7_3748958 is located on chromosome 7, the physical position is 3748958, the allele is T or C, and the nucleotide sequence of 100 bp before and after the marker is shown as SEQ ID NO. 16.

[0006] The application further provides a Cobia body length related SNP marker primer group, and the nucleotide sequences of the primer group 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 or SEQ ID NO. 17-18.

[0007] The application also provides the application of the haplotype or the primer set in the selection of the body length trait of C. undecacanthus, and the method is to select or discard individual base changes to produce different individuals in the body length trait of C. undecacanthus, and is used for molecular assisted breeding of a new strain of C. undecacanthus with fast growth.

[0008] Further, the application method is to amplify the DNA fragments of C. undecacanthus by using the primer, and select individuals with a positive effect of the SNP site genotype and the body length trait as breeding parents.

[0009] Compared with the prior art, the application has the beneficial effects that the application screens the body length of C. undecacanthus by using whole genome association analysis, obtains SNP molecular markers significantly associated with the trait, includes at least one of the 6 SNP sites, and can be applied to molecular assisted breeding of C. undecacanthus. The selection method disclosed in the application can greatly shorten the breeding period, reduce the dependence on phenotypic traits, improve the selection and breeding efficiency, and has a good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 is a body length frequency distribution graph; Figure 2 is a Manhattan graph, and the -log 10 (p) value of each SNP is displayed as the vertical axis. DETAILED DESCRIPTION

[0011] In order to better understand the technical content of the application, the following specific embodiments and drawings are specifically introduced, and the experimental methods in the following experimental examples are all conventional methods unless otherwise specified. The instruments, reagents and kits used in the experiments can be obtained by market purchase.

[0012] Example 1 I. Materials and methods The experimental C. undecacanthus was obtained from Yangjiang Jin Yuan Marine Biological Scientific Research Co., Ltd., and 167 tails were randomly taken from the fish population and transferred into a new pool for temporary culture. The operator strictly abided by the animal experiment ethics specification during the experimental process, measured the phenotypic traits including body length, total length, body height and body weight, and statistically analyzed the phenotypic traits, and meanwhile, the tail fins of C. undecacanthus were taken and stored in dry ice for standby.

[0013] II. Arrangement and analysis of phenotypic traits The collected and arranged phenotypic data were statistically analyzed, including minimum value, maximum value, mean value, standard deviation, coefficient of variation, and the results are shown in Table 1. The body length measurement data of C. undecacanthus were verified by normal distribution, and the results are shown in Figure 1 .

[0014] Table 1. Statistical analysis of growth performance phenotypic data of C. undecacanthus ; Note: The unit of body length, tail fin length and total length is cm; the unit of body weight is g, and the unit of condition factor is g / cm³.

[0015] III. Obtaining of genomic data The genomic DNA of the obtained L. japonicus tail fin sample was extracted and sent to Meiji Bio for whole genome resequencing and variation detection. After the DNBSeq T7TMTM sequencing data were obtained, quality control was performed on the obtained data, low-quality data was filtered out, and high-quality data was obtained. The Clean Data was aligned to the reference genome sequence using BWA-MEME software to obtain the sequence position attribution. The BAM file was corrected using the Best Practices process of GATK software to obtain the vcf file of the population genotype. Finally, 1,129.45G reads data were obtained, the sequencing Q30 was 97.17%, the GC content was 40.60%, and 3,143,741 SNPs were obtained.

[0016] IV. SNP quality control and filling First, vcftools v0.1.16 was used to set filtering parameters to ensure that the minimum allele was 2, the maximum missing rate was less than 0.95, the minimum allele frequency was greater than 0.05, the measurement quality of each SNP was required to be greater than 30, and the minimum average depth was 10. After screening, 980,781 SNPs that met the parameters were retained, and then beagle v5.5 genotype filling software was used to fill the SNP data with genotypes, solve the genotype missing question, and then perform SNP quality control. The genotype vcf file was converted to binary format using Plink v1.9.0-b.7.7.

[0017] V. Whole genome association analysis GEMMA v0.98.5 was used to establish the kinship matrix, and the established kinship matrix and phenotype traits were used to perform whole genome association analysis using a mixed linear model, and the model was as follows: y = Xβ + Zu + e, wherein y is the phenotype vector, Xβ is the population structure effect, Zu is the marker effect to be tested, and e is the residual effect. K in the microgene effect is the kinship matrix inferred by the marker. The results are shown in FIG. 2-1 (Manhattan), which is a Manhattan plot obtained by whole genome association analysis of the body length of L. japonicus. There are three SNP sites, and the results are shown in Tables 2-4. Figure 2

[0018] Table 2 SNP site information of L. japonicus body length ; ​Table 3. Genotype and phenotype traits of SNP screening ; ; Table 4. Information of molecular marker sites ; ; Note: The underlined part is the target fragment amplified by the primer, and the shaded base is the SNP site.

[0019] Six. Verification in different groups of P. olivaceus Different batches of adult fish were selected for verification. First, the phenotype data was statistically analyzed, including minimum value, maximum value, mean value, standard deviation, coefficient of variation, and the results are shown in Table 5. The above six SNP sites were screened and analyzed; The CC and CT of chr14_2882510 site improved 9.6% and 12.9% compared with TT; The CC and CA of chr14_2996332 site improved 10.2% and 15.6% compared with AA; The CC and CA of chr14_1491846 site improved 10.4% and 10.9% compared with AA; The TT and TA of chr14_271210 site improved 10.4% and 13.2% compared with AA; The CC and CT of chr14_683410 site improved 10.4% and 13.2% compared with TT; The CC and CT of chr7_3748958 site improved 10.4% and 10.9% compared with TT; Specifically, see Table 6 site and phenotype data; Table 5. Phenotype data of verification group ; Note: The unit of body length is cm; the unit of body weight is g, and the unit of fatness is g / cm³.

[0020] Table 6. Site and phenotype data ;

[0021] Seven. Combination effect of multiple mutants is shown in Table 7; Table 7. Effect of different mutant combinations on phenotype data ; Note: TT, AA, AA, AA, TT, TC correspond to SNP markers of chr14_2882510, chr14_2996332, chr14_1491846, chr14_271210, chr14_683410, chr7_3748958 respectively.

[0022] As can be seen from Table 7, the SNP superposition mutations of multiple positive effects can produce synergistic effects or antagonistic effects.

[0023] The above description is merely preferred specific embodiments of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can make equivalent replacements or changes within the technical scope disclosed by the present application according to the technical solutions and inventive concepts of the present application, which are all covered within the protection scope of the present application.

Claims

1. A haplotype of a SNP site associated with body length in cobia, characterized in that, The SNP site includes at least one of the following: 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. 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. 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. 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. 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. 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.

2. The application of the haplotype described in claim 1 in the breeding of cobia body length traits, characterized in that, The application involves selecting or discarding individuals with different body length traits due to single base changes, for molecular-assisted breeding of new fast-growing strains of cobia.

3. A primer set for identifying SNP markers related to the body length trait of cobia, characterized in that, 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, and the primer set amplifies the SNP sites described in the haplotype of the body length-associated SNP sites of claim 1.

4. The application of the primer set described in claim 3 in the breeding of cobia body length traits.

5. The application according to claim 4, characterized in that, The application 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.

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