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

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) molecular marker influencing a pig body length character and application, and aims to solve the technical problem of slow breeding caused by lack of gene research on the pig body length-related character at present. According to the application, a pig whole genome sequence is utilized, based on pig whole genome association analysis strategy detection, the SNP molecular marker influencing the pig body length character is found, and the molecular marker is based on G / A mutation conversion type single nucleotide polymorphism variation at 16624558bp on chromosome 17 of an international pig genome 11.1 version reference sequence. According to the present invention, the genotype of the 16624558 site on the No.17 chromosome of the pig population is detected, it can be known that the AA genotype individual of the SNP site (chr17: 16624558) has the longer body length than the GG / GA genotype individual, and the pig body length 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 pig body length character.

Description

Technical Field

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

[0002] Pig body length is a key indicator for measuring pig body shape and evaluating its production performance. It reflects the genetic diversity of pigs and is closely related to production performance indicators such as growth rate, feed conversion efficiency and lean meat rate. For a long time, people have improved pig production performance through traditional phenotypic selection of pig body length. This method has improved the pig body length trait to a certain extent, but body length is a quantitative trait and is genetically regulated by multiple genes. Simple phenotypic selection methods often result in limited selection effects and slow results.

[0003] 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

[0004] The purpose of the present invention is to provide a SNP molecular marker that affects the pig body length trait 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, which leads to slow breeding progress.

[0005] Using the pig whole genome resequencing (WGS), based on the pig genome-wide association study (GWAS) strategy, we detected and discovered the SNP molecular marker that affects the pig body length trait. The SNP molecular marker is a G / A mutation at the 16624558th base of chromosome 17 based on the international pig genome version 11.1 reference sequence. Applying this SNP molecular marker to molecular marker-assisted selection and genomic selection can promote the genetic improvement of pigs and improve the economic benefits of breeding.

[0006] The first aspect disclosed in the present application relates to a SNP molecular marker that affects the body length trait of pigs. Based on the international pig genome version 11.1 reference sequence, a single nucleotide polymorphism variation of the G / A conversion type exists at the 16624558th base of chromosome 17, which is recorded as the mutation site g.16624558.

[0007] 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.

[0008] 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.

[0009] 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 16624558 on chromosome 17, determine the G / A polymorphism of this site, and select / identify the pig body length trait based on the characteristic that individuals with the AA genotype have longer body length than individuals with the GA or GG genotype.

[0010] The fifth aspect disclosed in the present application relates to 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′-AACTGTCACAACTTGGAGG-3′; Downstream primer primer-R: 5'-TGTTTTTCCTCAGGTGTCA-3'.

[0011] The sixth aspect of the present application is directed to a method for selecting a breed / strain to increase 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 17 at locus 16624558 of the pig population was determined; (2) Select individual sows with the AA genotype at the 16624558th locus and eliminate those with the GA or GG genotype at the same locus, so as to increase the frequency of allele A at the locus from generation to generation, thereby increasing the body length of the offspring pigs.

[0012] In some embodiments of the present disclosure, the breeding pigs are Duroc pigs, Landrace pigs, Large White pigs and their synthetic lines.

[0013] 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 identifies the SNP molecular marker that affects the pig body length trait. Based on the international pig genome version 11.1 reference sequence, a single nucleotide polymorphism variation of the G / A conversion type exists at the base position 16624558 of chromosome 17, and its effect on the pig body length trait is verified. By optimizing the dominant allele of the molecular marker, a breed / strain breeding method for improving the pig body length is provided, and finally a molecular marker-assisted selection breeding technology 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

[0014] Figure 1 This is the Manhattan plot of the genome-wide association analysis (GWAS) of Duroc, Landrace and Large White pigs on chromosome 17 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).

[0015] Figure 2 This is a genome-wide association analysis (GWAS) QQ graph in one embodiment of the present application.

[0016] Figure 3 This is a diagram showing the body length analysis results of pigs with different genotypes in one embodiment of the present application.

[0017] Figure 4 This is a diagram of the sequencing results of the products 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 position of the designed primer sequence. DETAILED DESCRIPTION

[0018] 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.

[0019] Unless otherwise specified, the instruments and equipment involved in the following examples are all conventional instruments and equipment; unless otherwise specified, the biological reagents and raw materials involved are all commercially available conventional products; unless otherwise specified, the experimental methods involved are all conventional methods.

[0020] Example 1: The process of determining the body length of pigs 1. Source of experimental animals The experimental group used in this case is a group of 278 purebred pigs from Henan Muyuan Breeding Farm, including 55 Duroc pigs, 185 Landrace pigs and 38 Large White pigs. The pigs were allowed to eat and drink freely, and the entire feeding method and feeding conditions were always consistent, which was a conventional method.

[0021] 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.

[0022] 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.

[0023] 2. Whole genome genotyping of pigs The whole genome of 278 purebred pigs was resequenced using the DNBSEQ-T7 platform, and the average sequencing depth reached 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, excluding individuals with a detection rate of <90%, a minimum allele frequency (MAF) of <10%, and a detection rate of <80%.

[0024] 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 1It can be seen that in Duroc pigs, Landrace pigs and Large White pigs, there is a site on chromosome 17 that significantly affects body length. The strongest associated SNP is chr17:16624558G>A (P=1.077647E-13), which corresponds to the G / A mutation at 16624558bp on chromosome 17 of the International Pig Reference Genome Version 11.1.

[0025] 4. Correlation analysis between different genotypes and lean meat percentage phenotype of breeding pigs According to Table 1, the molecular marker SNP site chr17:16624558G>A is extremely significantly correlated with the body length trait (P=1.077647E-13), indicating that this molecular marker significantly affects the body length of Duroc pigs, Landrace pigs 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 Tables 1, 2, Figure 1 and Figure 3 It can also be seen that the body length of the GG type of SNP site chr17:16624558 is smaller than that of the GA and AA types, indicating that the homozygous GG 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 GG and GA types of the corresponding SNP sites, and retain the AA type breeding pigs to increase the dominant allele frequency of the site generation by generation.

[0026] Table 1 Correlation between SNP site chr17:16624558G>A and body length .

[0027] Table 2 Statistics of SNP locus chr17:16624558G>A in three varieties .

[0028] 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 316 bp nucleotide sequence in chromosome 17. 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′-AACTGTCACAACTTGGAGG-3′; Downstream primer primer-R: 5'-TGTTTTTCCTCAGGTGTCA-3'.

[0029] (2) PCR amplification system and condition settings A 10 μL system was prepared, including 1 μL of DNA sample, 0.3 μL of upstream primer, 0.3 μL of downstream primer, 5 μL of PCR mix, and 3.4 μL of ddH2O. 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.

[0030] (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.

[0031] This application detects SNP base mutation sites and preliminarily conducts an application of association analysis between its genotype and pig body length traits, providing a new molecular marker for molecular marker-assisted selection of pigs. As shown in Table 1, the average body length of each AA-type breeding pig is 9 cm longer than that of the GG-type breeding pig, which is a significant difference. Through molecular marker-assisted selection, the breeding process of Duroc pigs, Landrace pigs, Large White pigs and their synthetic lines has been greatly improved, the time for improvement of Duroc, Landrace pigs and Large White pigs has been shortened, the needs of the breeding pig market have been met, the economic benefits of breeding pigs have been effectively improved, and the core competitiveness of breeding enterprises has been improved.

[0032] 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 invention.

[0033] 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 affecting pig body length traits, characterized in that: Based on the international porcine genome version 11.1 reference sequence, a single nucleotide polymorphism variation of the G / A mutation conversion type exists at 16624558bp on chromosome 17.

2. Use of the SNP molecular marker described in claim 1 in the preparation of reagents for selecting / identifying pig breeds / strains related to 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 16624558 on chromosome 17, determine the G / A polymorphism of this site, and select / identify the pig body length trait based on the characteristic that individuals with the AA genotype have longer body length than individuals with the GA or GG 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′-AACTGTCACAACTTGGAGG-3′; Downstream primer primer-R: 5'-TGTTTTTCCTCAGGTGTCA-3'.

6. A method for selecting a breed / strain 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 17 at locus 16624558 of the pig population was determined; (2) Select individual sows with the AA genotype at the 16624558th locus and eliminate those with the GA or GG genotype at the same locus, so as to increase the frequency of allele A at the 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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