Snps molecular markers related to pig body length traits and application

Abstract

The application discloses a SNP molecular marker related to a pig body length character and application, and aims to solve the technical problem of lacking gene research on the pig body length character at present. The application detects and screens a molecular marker related to the pig body length character by using a pig whole genome sequence and based on a pig whole genome correlation analysis strategy. The molecular marker is based on a single nucleotide polymorphism variation of A / G conversion type at 97652204bp of a chromosome 7 in an international pig genome 11.1 version reference sequence. The genotype of the 97652204 site of the chromosome 7 of a pig population is detected, and it is known that the GG genotype individual of the SNP site (chr7: 97652204) has a longer body length than the AA / AG genotype individual. By selecting the advantageous genotype individual for breeding and eliminating the disadvantageous genotype individual, the pig body length can be obviously improved, so that the purpose of accelerating the genetic progress of the pig body length character is achieved.

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 most important economic traits in pig production, closely related to the pig's body size and meat production. Because body length is relatively easy to evaluate through external observation, traditional breeding methods for pig body length have often been used based on phenotypic and pedigree data. This approach has improved pig body length to a certain extent, but body length is a quantitative trait and is genetically regulated by multiple genes. Studies have shown that body length has above-moderate heritability and is significantly phenotypic and genetically correlated with other important economic traits. Therefore, understanding the genetic mechanisms behind this trait is of great significance in the genetic improvement of pig body length.

[0003] With advances in high-throughput sequencing technology, whole-genome resequencing is increasingly being used to improve the genetics of important economic traits in pigs. By using commercial microarrays to genotype SNPs on the pig genome, combined with genome-wide association analysis (GWAS) techniques, marker loci closely associated with pig body length variation can be identified. High-density resequencing data can then be used to fine-tune the location of body length-related variants, enabling more specific and precise identification of key mutations affecting pig body length. By improving the body length trait of core breeding pigs, the improved advantages can be largely passed on to future generations of commercial pigs, thereby accelerating the genetic progression of pig body length, improving pig meat production, and increasing the economic benefits of the pig farming 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 an admission or any form of suggestion 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 in genotype identification, genetic breeding, etc., aiming to solve the current technical problem of lack of genetic research on pig body length-related traits.

[0006] Using the whole genome resequencing (WGS) of pigs, based on the genome-wide association study (GWAS) strategy of pigs, molecular markers related to pig body length were detected and screened. The SNP molecular marker is an A / G mutation at base 97652204 on chromosome 7 based on 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 pig body length trait. 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] In 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 pig body length traits using the SNP molecular marker, comprising the following steps:

[0011] (1) Extraction of whole genome DNA from pig ear tissue samples;

[0012] (2) scanning the whole genome DNA using pig whole genome resequencing;

[0013] (3) Obtain resequencing data and check the genotype of site 97652204 on chromosome 7 based on the international pig genome version 11.1 reference sequence to determine the A / G polymorphism of the site. 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.

[0014] 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, the nucleotide sequences of which are as follows:

[0015] Upstream primer primer-F: 5′-TCATTTTTTTGGCCATGCCC-3′;

[0016] Downstream primer primer-R: 5'-GGGAAAGTAGAGTGGTGGT-3'.

[0017] The sixth aspect disclosed in the present application is a breeding method for increasing the body length of pigs, comprising the following steps:

[0018] (1) Based on the International Porcine Genome Version 11.1 reference sequence, the genotype of chromosome 7 at position 97652204 of the pig population was determined;

[0019] (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 generation by generation, thereby increasing the body length of the offspring pigs.

[0020] In some embodiments of the present disclosure, the breeding pigs are Duroc pigs, Landrace pigs, Large White pigs and their synthetic lines.

[0021] One or more technical solutions provided in the embodiments of this application have at least any of the following technical effects or advantages:

[0022] This 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, an A / G conversion type single nucleotide polymorphism variation exists at base 97652204 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 improvement time of Duroc pigs, 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

[0023] Figure 1 This is the Manhattan plot of the genome-wide association study (GWAS) on chromosome 7 of Duroc, Landrace and Large White pigs 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).

[0024] Figure 2 This is a genome-wide association study (GWAS) QQ plot in one embodiment of the present application.

[0025] Figure 3 This is a diagram analyzing the body length results of pigs with different genotypes in one embodiment of the present application.

[0026] Figure 4This is a diagram showing 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 the allele mutation), and the first position in the sequence is bold, indicating the position of the designed primer sequence. DETAILED DESCRIPTION

[0027] The specific embodiments of the present invention are described below with reference to the 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.

[0028] 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.

[0029] Example 1: Influence on the measurement process of body length

[0030] 1. Source of experimental animals

[0031] In this case, a group of 278 purebred pigs from the Henan Muyuan breeding farm served as the experimental group. The pigs had free access to food and water, and the entire feeding method and rearing conditions remained consistent, using conventional methods.

[0032] 2. Sample Collection and Phenotyping

[0033] 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.

[0034] Example 2: SNP molecular marker detection and identification process

[0035] 1. Extraction and Detection of Genomic DNA

[0036] DNA was extracted from ear tissue samples of Duroc, Landrace, and Large White pigs using the standard phenol-chloroform method for whole-genome DNA extraction. DNA quality and concentration were determined using a Nanodrop-ND1000 spectrophotometer. DNA samples were considered acceptable if their A260 / 280 ratios were between 1.8 and 2.0, and their A260 / 230 ratios were between 1.7 and 1.9. Qualified DNA samples were uniformly diluted to 50 ng / μL.

[0037] 2. Whole-genome genotyping of pigs

[0038] Whole-genome resequencing of 278 purebred Duroc, Landrace, and Large White pigs was performed using the DNBSEQ-T7 platform, reaching an average sequencing depth of 10X. Sequencing data aligned to the reference genome were subjected to SNP and indel calling using bcftools software. Genotyping data were quality-controlled using PLINK v1.90, eliminating individuals with a call rate <90%, a minimal allele frequency (MAF) <10%, and a call rate <80%.

[0039] 3. Genome-wide Association Analysis

[0040] 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 SNPs and indels and body length traits. The genome level significance threshold is 0.05 / N, and the chromosome level significance threshold 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 Porcine Reference Genome version 11.1.

[0041] 4. Correlation analysis between different genotypes and lean meat percentage phenotypes of breeding pigs

[0042] 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 pig SNP site, 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 Furthermore, the AA type at SNP chr7:97652204 is shorter than the AG and GG types, indicating that homozygous AA is most detrimental to the body length of breeding pigs. Body length is an important indicator of pig growth performance, with longer body length indicating better growth performance. Therefore, during breeding, it is necessary to gradually eliminate breeding pigs with AA and AG types at the corresponding SNP, while retaining GG types to increase the frequency of the dominant allele at the locus with each generation.

[0043] Table 1 Correlation between molecular marker SNP site chr7:97652204 A>G and body length

[0044] .

[0045] Table 2 Statistics of molecular marker SNP locus chr7: 97652204 A>G in three varieties

[0046] .

[0047] 5. Amplification and sequencing of target DNA sequences

[0048] (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, and their nucleic acid sequences are as follows:

[0049] Upstream primer primer-F: 5′-TCATTTTTTTGGCCATGCC-3′;

[0050] Downstream primer primer-R: 5'-GGGAAAGTAGAGTGGTGGT-3'.

[0051] (2) PCR amplification system and condition settings

[0052] 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.

[0053] 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 final extension at 72°C for 10 min.

[0054] (3) DNA sequencing identification: Sequencing gene fragments to test 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.

[0055] The present invention applies the detection of SNP base mutation sites and the preliminary application of the association analysis between their genotype and the body length trait of pigs, providing a new molecular marker for molecular marker-assisted selection of pigs. As shown in Table 1, the average body length of each GG type pig is about 9 cm and 6 cm longer than that of AA and AG type pigs, respectively, which is a significant difference. Through molecular marker-assisted selection, the advantages obtained from the improvement can be passed on to the next generation of commercial pig populations 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.

[0056] Although some preferred embodiments of the present invention have been described, those skilled in the art may make additional 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 this application.

[0057] Obviously, those skilled in the art may make various changes and modifications to this application without departing from the spirit and scope of the inventive concept. Thus, if such changes and modifications fall within the scope of the claims of this application and their equivalents, this application is intended to include such changes and modifications.

Claims

1. Use of a primer pair for identifying SNP molecular markers in the preparation of a reagent for selecting / identifying breeds / lines related to pig body length traits, characterized in that: The SNP molecular marker is based on the international porcine genome version 11.1 reference sequence, and is a single nucleotide polymorphism variation of the A / G conversion type at 97652204bp on chromosome 7; the pig is a Duroc pig.

2. Use of a primer pair for identifying SNP molecular markers in the selection / identification of breeds / lines related to pig body length traits, characterized in that: The SNP molecular marker is based on the international porcine genome version 11.1 reference sequence, and is a single nucleotide polymorphism variation of the A / G conversion type at 97652204bp on chromosome 7; the pig is a Duroc pig.

3. The use according to claim 1 or 2, wherein the primer pair comprises primers primer-F and primer-R, and the nucleotide sequences thereof are as follows: Upstream primer primer-F: 5′-TCATTTTTTTGGCCATGCCC-3′; Downstream primer primer-R: 5'-GGGAAAGTAGAGTGGTGGT-3'.

4. A method for selecting / identifying pig body length traits using SNP molecular markers, characterized in that: The SNP molecular marker is based on the international porcine genome version 11.1 reference sequence, and is a single nucleotide polymorphism variation of the A / G transition type at 97652204 bp on chromosome 7; the pig is a Duroc pig; and the method comprises the following steps: (1) Extraction of whole genome DNA from pig ear tissue samples; (2) scanning the whole genome DNA using pig whole genome resequencing; (3) Obtain resequencing data and check the genotype of site 97652204 on chromosome 7 based on the international pig genome version 11.1 reference sequence to determine the A / G polymorphism of the site. 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 breeding method for increasing the body length of pigs, characterized in that: The steps include: (1) Based on the International Porcine Genome Version 11.1 reference sequence, the genotype of chromosome 7 at position 97652204 of the pig population was determined; (2) Select the breeding pigs with the GG genotype at the 97652204th locus and eliminate the breeding pigs with the AG or AA genotype at the same locus, so as to increase the frequency of the allele G at this locus generation by generation, thereby increasing the body length of the offspring pigs; The breeding pig is Duroc pig.

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