SNP (Single Nucleotide Polymorphism) molecular marker related to pig body length character and application

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) molecular marker related to a pig body length character and application, and aims to solve the technical problem that gene research on the pig body length character is lacked at present. The molecular marker related to the pig body length character is detected and screened on the basis of a pig whole genome association analysis strategy by utilizing a pig whole genome sequence, and the molecular marker is based on A / G conversion type single nucleotide polymorphism variation at 97652204bp on a No.7 chromosome of an international pig genome 11.1 version reference sequence. According to the present invention, the genotype of the 97652204 site on the chromosome 7 of the pig population is detected, the SNP site (chr7: 97652204) GG genotype individual has the longer body length than the AA / AG genotype individual, and the body length of the pig can be significantly improved by selecting the favorable genotype individual for breeding and eliminating the unfavorable genotype individual so as to achieve the purpose of accelerating the genetic progress of the body length character of the pig;

Description

Technical Field

[0001] The present invention relates to the technical field of pig molecular marker screening, and specifically to a SNP molecular marker related to pig body length traits and its application. Background Art

[0002] Pig body length is one of the important economic traits in pig production, and it is closely related to the pig's body size and meat production. Since the body length trait is easier to evaluate through external observation, people have often used traditional breeding methods based on phenotypic and pedigree data to evaluate the pig's body length trait. This method has improved the pig's body length trait to a certain extent, but body length is a quantitative trait and is genetically regulated by multiple genes. Studies have shown that the body length trait has above medium heritability and has significant phenotypic and genetic correlations with other important economic traits. Therefore, understanding the genetic mechanism behind this trait is of great significance in the genetic improvement of pig body length.

[0003] With the advancement of high-throughput sequencing technology, whole genome resequencing technology is increasingly being used in the genetic improvement of important economic traits in pigs. By using commercial chips to genotype SNPs on the pig genome, combined with genome-wide association analysis (GWAS) technology, marker sites closely related to pig body length variation can be identified. High-density resequencing data is then used to finely locate body length-related variation sites, which can more clearly and accurately locate key mutation sites that affect pig body length. By improving the body length traits of core group pigs, the advantages of the improvement can be passed on to future generations of commercial pigs to a greater extent, thereby accelerating the genetic progress of pig body length, improving pig meat production, and improving the economic benefits of the pig industry.

[0004] The information disclosed in this background technology section is only used to deepen the understanding of the background technology of the present disclosure, and should not be regarded as acknowledging or suggesting in any form that the information constitutes the prior art known to those skilled in the art. Summary of the invention

[0005] The purpose of the present invention is to provide a SNP molecular marker related to pig body length traits and apply it to genotype identification, genetic breeding, etc., aiming to solve the current technical problem of lack of genetic research on pig body length related traits.

[0006] By using the pig whole genome resequencing (WGS), based on the pig genome-wide association study (GWAS) strategy, molecular markers related to pig body length were detected and screened. The SNP molecular marker is based on the A / G mutation at the 97652204th base of chromosome 7 of the international pig genome version 11.1 reference sequence; the application of this SNP molecular marker in molecular marker-assisted selection and genomic selection can promote the genetic improvement process of pigs and improve the economic benefits of breeding.

[0007] The first aspect disclosed in the present application relates to a SNP molecular marker related to the body length trait of pigs. Based on the international pig genome version 11.1 reference sequence, there is an A / G conversion type single nucleotide polymorphism variation at the 97652204th base of chromosome 7, which is recorded as the mutation site g.97652204.

[0008] In a second aspect disclosed in the present application, the SNP molecular marker is used in the preparation of reagents for selecting / identifying pig breeds / lines related to body length traits.

[0009] According to a third aspect disclosed in the present application, the SNP molecular marker is applied in the selection / identification of breeds / lines related to pig body length traits.

[0010] The fourth aspect disclosed in the present application relates to a method for selecting / identifying related pig body length traits using the SNP molecular marker, comprising the following steps: (1) Extracting whole genome DNA from pig ear tissue samples; (2) scanning the whole genome DNA using pig whole genome resequencing; (3) Obtain resequencing data and, based on the international pig genome version 11.1 reference sequence, check the genotype of site 97652204 on chromosome 7, determine the A / G polymorphism of this site, and select / identify the pig body length trait based on the characteristic that individuals with the GG genotype have longer body length than individuals with the AA / AG genotype.

[0011] The fifth aspect disclosed in the present application provides a primer pair for identifying the above-mentioned SNP molecular marker, comprising primers primer-F and primer-R, whose nucleotide sequences are as follows: Upstream primer primer-F: 5′-TCATTTTTTTGGCCATGCCC-3′; Downstream primer primer-R: 5'-GGGAAAGTAGAGTGGTGGT-3'.

[0012] The sixth aspect of the present application is directed to a breeding method for increasing the body length of pigs, comprising the following steps: (1) Based on the international pig genome version 11.1 reference sequence, the genotype of chromosome 7 at locus 97652204 of the pig population was determined; (2) Select individual sows with the GG genotype at the 97652204th locus and eliminate those with the AG or AA genotype at the same locus, so as to increase the frequency of the allele G at this locus from generation to generation, thereby increasing the body length of the offspring pigs.

[0013] In some embodiments of the present disclosure, the breeding pigs are Duroc pigs, Landrace pigs, Large White pigs and their synthetic lines.

[0014] One or more technical solutions provided in the embodiments of the present application have at least any of the following technical effects or advantages: The present application studies and determines the SNP molecular markers that affect the pig body length trait. Based on the international pig genome version 11.1 reference sequence, a single nucleotide polymorphism variation of the A / G conversion type exists at the 97652204th base of chromosome 7, and its effect on the pig body length trait is verified. By optimizing the dominant allele of the molecular marker, a breeding method for improving the pig body length is provided, and finally a molecular marker-assisted selection breeding approach for rapid improvement of the pig body length trait is established, which greatly improves the breeding process of Duroc pigs, Landrace pigs, Large White pigs and their synthetic lines, shortens the time for improvement of Duroc, Landrace pigs and Large White pigs, adapts to the needs of the breeding pig market, and effectively improves the economic benefits of breeding pigs. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the Manhattan plot of the genome-wide association analysis (GWAS) of Duroc, Landrace and Large White pigs on chromosome 7 for the pig body length trait in one embodiment of the present application; wherein: the horizontal axis represents the chromosome number of the pig; the vertical axis represents -log10 (P value).

[0016] Figure 2 This is a genome-wide association analysis (GWAS) QQ graph in one embodiment of the present application.

[0017] Figure 3 This is a diagram showing the body length analysis results of pigs with different genotypes in one embodiment of the present application.

[0018] Figure 4 This is a diagram of the sequencing results of the product after PCR amplification using primers primer-F and primer-R in one embodiment of the present application; in the figure, M is the mutation site (the mutated base in brackets is an allele mutation), and the first position of the sequence is bold, indicating the designed primer sequence position. DETAILED DESCRIPTION

[0019] The specific implementation modes of the present invention are described below in conjunction with examples. However, the following examples are only used to illustrate the present invention in detail and are not intended to limit the scope of the present invention in any way.

[0020] Unless otherwise specified, the instruments and equipment involved in the following examples are all conventional instruments and equipment; the reagents and raw materials involved are all commercially available conventional products unless otherwise specified; the test methods involved are all conventional methods unless otherwise specified.

[0021] Example 1: The process of measuring body length 1. Source of experimental animals In this case, 278 purebred pigs from Henan Muyuan Breeding Farm were used as the experimental group. The pigs were allowed to eat and drink freely, and the entire feeding method and feeding conditions were kept consistent, all of which were conventional methods.

[0022] 2. Sample collection and phenotypic recording The ear tissues of the above pigs were collected, stored in 75% ethanol, and kept in a -20°C refrigerator. The live length of each pig was measured and various measurement information was recorded.

[0023] Example 2: SNP molecular marker detection and identification process 1. Extraction and detection of genomic DNA The DNA extraction method of ear tissue samples of Duroc pigs, Landrace pigs and Large White pigs refers to the phenol-chloroform method for extracting whole genome DNA; the quality and concentration of DNA of purebred Duroc pigs, Landrace pigs and Large White pigs were tested by Nanodrop-ND1000 spectrophotometer; the A260 / 280 ratio was 1.8-2.0, and the A260 / 230 ratio was 1.7-1.9, which was considered qualified. Finally, the qualified DNA samples were uniformly diluted to 50 ng / μl.

[0024] 2. Whole genome genotyping of pigs The whole genome resequencing of 278 purebred Duroc, Landrace and Large White pigs was performed using the DNBSEQ-T7 platform, with an average sequencing depth of 10X. The bcftools software was used to perform SNP and indel calling on the sequencing data after alignment with the reference genome; PLINK v1.90 was used to perform quality control on the obtained genotype data, eliminating individuals with a detection rate of <90%, a minimum allele frequency (MAF) of <10%, and excluding individuals with a detection rate of <80%.

[0025] 3. Genome-wide association analysis In order to eliminate the population stratification effect, this example uses linear mixed model single point regression analysis combined with GEMMA software for GWAS analysis. The analysis model uses the similarity of the genome between individuals to correct the stratification effect. The Bonferroin method is used to determine the significance threshold of the association between SNP and indel and body length traits. The significant threshold at the genome level is 0.05 / N, and the significant threshold at the chromosome level is 1 / N, where N is the number of SNPs and indels used for analysis. In this example, the chromosome level threshold is selected. The GWAS analysis results are shown in Figure 2. Figure 1 As shown. Figure 1 It can be seen that in Duroc, Landrace and Large White pigs, there is a site on chromosome 7 that significantly affects body length. The strongest associated SNP is chr7:97652204A>G (P=6.653859E-9), which corresponds to the A>G mutation at 97652204bp on chromosome 7 of the International Pig Reference Genome Version 11.1.

[0026] 4. Correlation analysis between different genotypes and lean meat percentage phenotype of breeding pigs As shown in Table 1, the molecular marker SNP site chr7:97652204A>G is extremely significantly correlated with the body length trait (P=6.653859E-9), indicating that this molecular marker significantly affects the body length of Duroc, Landrace and Large White pigs. Through auxiliary selection of the SNP site of pigs, the body length of the group can be improved, thereby accelerating the breeding process of breeding pigs. In addition, according to Table 1, Table 2, Figure 1 and Figure 3 It can also be seen that the AA type of SNP site chr7:97652204 is shorter than the AG and GG types, indicating that homozygous AA is the most disadvantageous to the body length of the breeding pig. Body length is an important indicator to measure the growth performance of breeding pigs. Long body length indicates good growth performance of pigs. Therefore, in the process of breeding, it is necessary to gradually eliminate the breeding pigs of the corresponding SNP site AA and AG types, and retain the GG type breeding pigs to increase the dominant allele frequency of the site generation by generation.

[0027] Table 1 Correlation between molecular marker SNP site chr7:97652204 A>G and body length .

[0028] Table 2 Statistics of molecular marker SNP locus chr7: 97652204 A>G in three varieties .

[0029] 5. Amplification and sequencing of target DNA sequence (1) The target fragment containing the SNP site significantly associated with the body length performance of Duroc, Landrace and Large White pigs is a 272 bp nucleotide sequence in chromosome 7. The upstream and downstream primers for sequence amplification are primer-F and primer-R. The nucleic acid sequence is as follows: Upstream primer primer-F: 5′-TCATTTTTTTGGCCATGCC-3′; Downstream primer primer-R: 5'-GGGAAAGTAGAGTGGTGGT-3'.

[0030] (2) PCR amplification system and condition settings Prepare a 10 μL reaction system, including 1 μL DNA template, 0.3 μL upstream primer, 0.3 μL downstream primer, 5 μL PCR mix, and 3.4 μL ddH2O.

[0031] The PCR reaction program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s, for a total of 30 cycles; and the final extension was at 72°C for 10 min.

[0032] (3) DNA sequencing identification: Sequencing gene fragments to measure both positive and negative reactions; the measured sequence is compared with the Ensembl genome sequence to obtain the mutation of the corresponding SNP site. The sequencing results are as follows: Figure 4 shown.

[0033] The present invention provides a new molecular marker for molecular marker-assisted selection of pigs by detecting SNP base mutation sites and preliminarily conducting an association analysis between the genotype and the body length trait of the pig. As shown in Table 1, the average body length of each GG-type breeding pig is about 9 cm and 6 cm longer than that of the AA-type and AG-type breeding pigs, respectively, with significant differences. Through molecular marker-assisted selection, the advantages obtained by the improvement can be passed on to the offspring commercial pig population to a large extent, thereby accelerating the genetic progress of pig body length, improving the meat production capacity of pigs, and improving the economic benefits of the pig farming industry.

[0034] Although some preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present application.

[0035] Obviously, those skilled in the art can make various changes and modifications to the present application without departing from the spirit and scope of the inventive concept. Thus, if these modifications and variations to the present application fall within the scope of the claims of the present application and their equivalents, the present application is also intended to include these modifications and variations.

Claims

1. A SNP molecular marker associated with pig body length traits, characterized in that: Based on the international pig genome version 11.1 reference sequence, the single nucleotide polymorphism variation of the A / G transition type at 97652204bp on chromosome 7.

2. Use of the SNP molecular marker described in claim 1 in the preparation of reagents for breeding / identifying breeds / strains related to pig body length traits.

3. Use of the SNP molecular marker described in claim 1 in the selection / identification of breeds / strains related to pig body length traits.

4. A method for selecting / identifying pig body length traits using the SNP molecular markers of claim 1, characterized in that: The following steps are involved: (1) Extracting whole genome DNA from pig ear tissue samples; (2) scanning the whole genome DNA using pig whole genome resequencing; (3) Obtain resequencing data and, based on the international pig genome version 11.1 reference sequence, check the genotype of site 97652204 on chromosome 7, determine the A / G polymorphism of this site, and select / identify the pig body length trait based on the characteristic that individuals with the GG genotype have longer body length than individuals with the AA / AG genotype.

5. A primer pair for identifying the SNP molecular marker according to claim 1, characterized in that: Contains primers primer-F and primer-R, and their nucleotide sequences are as follows: Upstream primer primer-F: 5′-TCATTTTTTTGGCCATGCCC-3′; Downstream primer primer-R: 5'-GGGAAAGTAGAGTGGTGGT-3'.

6. A breeding method for increasing the body length of pigs, characterized in that: The steps include: (1) Based on the international pig genome version 11.1 reference sequence, the genotype of chromosome 7 at locus 97652204 of the pig population was determined; (2) Select individual sows with the GG genotype at the 97652204th locus and eliminate those with the AG or AA genotype at the same locus, so as to increase the frequency of the allele G at this locus from generation to generation, thereby increasing the body length of the offspring pigs.

7. The breeding method according to claim 6, characterized in that: The breeding pigs are Duroc pigs, Landrace pigs, Large White pigs or their synthetic lines.

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