SNP (Single Nucleotide Polymorphism) molecular marker influencing pig rib number and application

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) molecular marker influencing a pig rib number character and application, and aims to solve the technical problems that a traditional breeding mode is low in efficiency and accuracy, and currently, related genes of the pig rib number character are lack of research. The SNP molecular marker influencing the pig rib number character is detected based on a pig whole genome association analysis strategy by utilizing a pig whole genome sequence, and the molecular marker is based on the fact that Cgt exists at 97578184bp on a No.7 chromosome of a reference sequence of an international pig genome version 11.1; a mutation conversion type of single nucleotide polymorphism variation. Compared with AA and AC genotype individuals, the CC genotype individual at the SNP site has more rib number, by selecting favorable genotype individuals for breeding and eliminating unfavorable genotype individuals, the rib number and meat production performance of pigs can be remarkably improved, and further more social and economic benefits are generated.

Description

Technical Field

[0001] The present invention relates to the technical field of pig molecular marker screening, and specifically to SNP molecular markers affecting the number of pig ribs and their application. Background Art

[0002] Spare ribs are the most expensive part of the pig carcass in the Chinese pork market. They are the most popular high-quality pork part among consumers. They are mainly composed of ribs and attached meat. The number of pig ribs is one of the important economic traits. Its number will affect the number of carcass ribs and meat production. As the number of ribs increases, the number of carcass ribs and meat production of pigs also increase accordingly. Therefore, breeding for the number of ribs has great economic value.

[0003] The number of ribs is usually measured after slaughter. The traditional breeding method is mainly based on the comprehensive evaluation of the number of ribs and other appearances. This method that relies on phenotypic selection has many limitations, such as susceptibility to environmental influences, low selection efficiency, and incomplete genetic information. These factors limit the speed and accuracy of the breeding process. Therefore, it is very important to establish a molecular marker-assisted selection breeding technology that can quickly identify, select or improve the pig rib number trait.

[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 that affects the number of pig ribs. The molecular marker that affects the number of pig ribs is detected and discovered by using pig whole genome resequencing (WGS) based on the pig whole genome association analysis strategy (GWAS), and the SNP molecular marker is applied to molecular marker-assisted selection and genomic selection, aiming to solve the technical problems of low efficiency and accuracy of traditional breeding methods and the current lack of research on genes related to the pig rib number trait.

[0006] The first aspect disclosed in the present application relates to a SNP molecular marker that affects the number of pig ribs. Based on the international pig genome version 11.1 reference sequence, there is a C / A conversion type single nucleotide polymorphism variation at the 97578184th base of chromosome 7, which is recorded as mutation site g.97578184.

[0007] In a second aspect disclosed in the present application, the SNP molecular marker is applied in the selection / identification of varieties / lines related to the trait of pig rib number.

[0008] In a third 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 rib number traits.

[0009] The fourth aspect disclosed in the present application relates to a method for selecting / identifying the number of traits related to pig ribs, 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 97578184 on chromosome 7, determine the C / A polymorphism of this site, and select / identify the pig rib number trait based on the fact that individuals with the CC genotype have more rib number characteristics than individuals with the AA / AC 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′-ACCTCTTGGTTCCTAGTTG-3′; Downstream primer primer-R: 5'-ATGCCAGATCCTTAACCCA-3'.

[0011] The sixth aspect of the present application is directed to a breeding method for increasing the number of pig ribs, comprising the following steps: (1) Based on the international pig genome version 11.1 reference sequence, the genotype of chromosome 7 at locus 97578184 of the pig population was determined; (2) Select individual pigs with CC genotype at the 97578184th locus and eliminate those with AC or AA genotype at the same locus, so as to increase the frequency of allele C at the locus from generation to generation, thereby increasing the number of ribs in the offspring.

[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 rib number trait. Based on the international pig genome version 11.1 reference sequence, the genotype of the 97578184th site on chromosome 7 has a single nucleotide polymorphism variation of C / A, and verifies its effect on the pig rib number trait. By optimizing the dominant allele of the molecular marker, the selection or identification of the pig high rib number trait is achieved, and finally a molecular marker-assisted selection breeding technology approach for rapid improvement of the pig rib number 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 a Manhattan plot of the genome-wide association analysis (GWAS) of Duroc, Landrace and Large White pigs on chromosome 7 for the pig rib number 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 QQ plot of the genome-wide association study (GWAS) in one embodiment of the present application.

[0016] Figure 3 This is a result analysis diagram of the rib number of pigs with different genotypes in an 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; 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.

[0020] Example 1: Determination process of the number of pig ribs 1. Source of experimental animals In this case, a group of 277 purebred pigs from Henan Muyuan Breeding Farm was used as the experimental group. The pigs were allowed to eat and drink freely, and the entire feeding method and breeding conditions remained consistent, all of which were conventional methods.

[0021] 2. Sample collection and phenotypic recording Ear tissues of pigs in the above groups were collected, stored in 75% ethanol, and kept in a -20°C refrigerator. The number of ribs 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 method for extracting DNA from ear tissues of Duroc pigs, Landrace pigs and Large White pigs refers to the labeled phenol-chloroform method to extract whole-genomic DNA; the quality and concentration of DNA from purebred Duroc pigs, Landrace pigs and Large White pigs were tested using a Nanodrop-ND1000 spectrophotometer; the A260 / 280 ratio was 1.8-2.0 and the A260 / 230 ratio was 1.7-1.9, which were considered qualified. Finally, the qualified DNA samples were uniformly diluted to 50 ng / μl.

[0023] 2. Whole genome genotyping of pigs The whole genome resequencing of 277 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 detect SNP and indel variations in 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%.

[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 the rib number trait. 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 the number of ribs. The strongest associated SNP is chr7:97578184C>A (P=2.481971E-8), which corresponds to the C>A mutation at 97578184bp on chromosome 7 of the International Pig Reference Genome Version 11.1.

[0025] 4. Correlation analysis between different genotypes and rib number phenotypes of breeding pigs As shown in Table 1, the molecular marker SNP site chr7:97578184C>A is extremely significantly correlated with the rib number trait (P=6.653859E-9), indicating that this molecular marker significantly affects the rib number of Duroc, Landrace and Large White pigs. Through auxiliary selection of the SNP site of pigs, the rib number of this group can be increased, 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 AA type of SNP site chr7:97578184 has fewer ribs than the AC and CC types, indicating that homozygous AA is the most disadvantageous to the number of ribs of breeding pigs. The number of ribs is an important indicator for measuring the growth performance of breeding pigs. More ribs indicate good growth performance and body shape of pigs. Therefore, in the process of breeding, it is necessary to gradually eliminate breeding pigs with AA and AC types of corresponding SNP sites, and retain CC type breeding pigs to increase the frequency of the dominant allele of the site generation by generation.

[0026] Table 1 Correlation between molecular marker SNP locus chr7:97578184C>A and rib number .

[0027] Table 2 Statistics of molecular marker SNP locus chr7:97578184C>A in three varieties .

[0028] 5. Amplification and sequencing of target DNA sequences (1) The target fragment containing the SNP site significantly associated with the number of ribs in Duroc, Landrace and Large White pigs is a 320 bp nucleotide sequence in chromosome 7. The upstream and downstream primers for sequence amplification are primer-F and primer-R. The nucleic acid sequences are as follows: Upstream primer primer-F: 5′-ACCTCTTGGTTCCTAGTTG-3′; Downstream primer primer-R: 5'-ATGCCAGATCCTTAACCCA-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] The present invention provides a new molecular marker for molecular marker-assisted selection of pigs by detecting SNP base mutation sites and preliminarily analyzing the association between the genotype and the rib number trait of pigs. As shown in Table 1, the average CC-type breeding pig has about 0.6 more ribs than the AA-type breeding pig, which is a significant difference. Through molecular marker-assisted selection, the advantages obtained by improvement can be passed on to the offspring commercial pig population to a large extent, thereby accelerating the progress of genetic research on the number of pig ribs and improving the economic benefits of the pig farming industry.

[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 the number of pig ribs, characterized in that: Based on the international pig genome version 11.1 reference sequence, the single nucleotide polymorphism variation of the C / A transition type at 97578184bp on chromosome 7.

2. Use of the SNP molecular marker described in claim 1 in the selection / identification of varieties / lines related to pig rib number traits.

3. Use of the SNP molecular marker according to claim 1 in the preparation of reagents for breeding / identifying varieties / lines related to pig rib number traits.

4. A method for selecting / identifying the number of traits related to pig ribs, 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 97578184 on chromosome 7, determine the C / A polymorphism of this site, and select / identify the pig rib number trait based on the fact that individuals with the CC genotype have more rib number characteristics than individuals with the AA / AC 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′-ACCTCTTGGTTCCTAGTTG-3′; Downstream primer primer-R: 5'-ATGCCAGATCCTTAACCCA-3'.

6. A breeding method for increasing the number of pig ribs, 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 97578184 of the pig population was determined; (2) Select individual pigs with CC genotype at the 97578184th locus and eliminate those with AC or AA genotype at the same locus, so as to increase the frequency of allele C at the locus from generation to generation, thereby increasing the number of ribs in the offspring.

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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