SNP molecular marker affecting pig rib number and application

Abstract

The application discloses a SNP molecular marker affecting a pig rib number character and application, and aims to solve the technical problems of low efficiency and accuracy of a traditional breeding method and lack of research on pig rib number character related genes at present. The application detects a SNP molecular marker affecting a pig rib number character by using a whole genome sequence of a pig and based on a whole genome correlation analysis strategy of the pig. The molecular marker is based on a single nucleotide polymorphism variation of a C>A mutation conversion type at 97578184bp on a chromosome 7 of an international pig genome 11.1 version reference sequence. The individual with a CC genotype of the SNP site has more rib numbers than the individuals with AA and AC genotypes. By selecting the individual with the advantageous genotype for breeding and eliminating the individual with the disadvantageous genotype, the rib number and meat production performance of the pig can be significantly improved, and more social and economic benefits can be generated.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pig molecular marker screening, and particularly relates to a SNP molecular marker affecting the number of pig ribs and application thereof. BACKGROUND

[0002] Ribs are the highest-priced pig carcass part in the Chinese pork market, and are the most popular high-quality pork part among consumers, mainly composed of ribs and attached meat. The number of pig ribs, as one of the important economic traits, affects the rib degree of the carcass and the meat yield. With the increase of the number of ribs, the rib degree of the carcass and the meat yield of the pig also increase accordingly, so breeding the number of ribs has great economic value.

[0003] The number of ribs is usually determined after slaughter, and the traditional breeding method mainly comprehensively evaluates the number of ribs and the like appearance. This method depending on phenotype selection has many limitations, such as being easily affected by the environment, low selection efficiency, incomplete genetic information, and the like, which limit the speed and accuracy of the breeding process. Therefore, it is crucial to establish a molecular marker-assisted selection breeding technology capable of rapidly identifying, breeding or improving the number of pig ribs.

[0004] The information disclosed in this BACKGROUND section is only for the purpose of enhancing the understanding of the background of the present disclosure and should not be taken as admitting that such information is prior art known to those of ordinary skill in the art. SUMMARY

[0005] The present application aims to provide a SNP molecular marker affecting the number of pig ribs. The SNP molecular marker affecting the number of pig ribs is detected and found by using pig whole genome sequencing (WGS) and based on pig genome-wide association study (GWAS) detection, and the SNP molecular marker is applied to molecular marker-assisted selection and genome selection, aiming to solve the technical problems of low efficiency and accuracy of the traditional breeding method and the lack of research on the genes related to the number of pig ribs.

[0006] The first aspect of the present application discloses a SNP molecular marker affecting the number of pig ribs, which is based on the international pig genome 11.1 version reference sequence, and a C / A conversion type single nucleotide polymorphism variation exists at the 97578184th base of chromosome 7, which is denoted as mutation site g.97578184.

[0007] The second aspect of the present application applies the SNP molecular marker to breeding / identifying breeds / lines related to the number of pig ribs.

[0008] The third aspect of the present application discloses application of the SNP molecular marker in preparation of reagents for selecting / identifying a pig breed / line related to the rib number trait.

[0009] The fourth aspect of the present application relates to a method for selecting / identifying a pig rib number trait, comprising the following steps:

[0010] (1) extracting whole genome DNA from an ear tissue sample of a pig;

[0011] (2) scanning the whole genome DNA by using pig whole genome resequencing;

[0012] (3) obtaining resequencing data, checking the genotype of the 97578184th site on chromosome 7 based on the international pig genome 11.1 version reference sequence, judging the C / A polymorphism of the site, and selecting / identifying the pig rib number trait based on the fact that the individual with CC genotype has more rib number characteristics than the individual with AA / AC genotype.

[0013] The fifth aspect of the present application relates to a primer pair for identifying the above-mentioned SNP molecular marker, comprising primer primer-F and primer primer-R, and the nucleotide sequences are as follows:

[0014] the upstream primer primer-F: 5'-ACCTCTTGGTTCCTAGTTG-3';

[0015] the downstream primer primer-R: 5'-ATGCCAGATCCTTAACCCA-3'.

[0016] The sixth aspect of the present application relates to a breeding method for increasing the rib number of live pigs, comprising the following steps:

[0017] (1) detecting and determining the genotype of the 97578184th site on chromosome 7 of a pig population based on the international pig genome 11.1 version reference sequence;

[0018] (2) selecting a breeding pig individual with CC genotype at the 97578184th site, and eliminating a breeding pig individual with AC or AA genotype at the site, so as to increase the frequency of the allele C of the site from generation to generation, thereby increasing the rib number of the offspring pigs.

[0019] In some embodiments of the present application, the breeding pig is a Duroc pig, a Landrace pig, a Large White pig, or a synthetic line thereof.

[0020] The one or more technical solutions provided in the embodiments of the present application have at least any of the following technical effects or advantages:

[0021] The application determines the SNP molecular marker affecting the rib number trait of pigs, based on the genotype of the 97578184th site on chromosome 7 of the international pig genome 11.1 version reference sequence, the single nucleotide polymorphism variation of C / A exists, the effect of the rib number trait of pigs is verified, the superior allele of the molecular marker is selected, the selection or identification of the high rib number trait of pigs is realized, and finally the molecular marker assisted selection breeding technology approach for rapid improvement of the rib number trait of pigs 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, Landrace and Large White pigs, adapts to the demand of the pig market, and effectively improves the economic benefit of pig breeding. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 It is a Manhattan plot of genome-wide association analysis (GWAS) of Duroc pigs, Landrace pigs and Large White pigs on chromosome 7 about the rib number trait of pigs in an embodiment of the application; wherein: the horizontal coordinate represents the chromosome number of the pig; the vertical coordinate represents -log10 (P value).

[0023] Figure 2 It is a QQ plot of genome-wide association analysis (GWAS) in an embodiment of the application.

[0024] Figure 3 It is a rib number result analysis plot of pigs with different genotypes in an embodiment of the application.

[0025] Figure 4 It is a sequencing result plot of the product amplified by PCR with primers primer-F and primer-R in an embodiment of the application; in the plot, M is the mutation site (the mutation base in the brackets is the allele mutation), which is added at the first position of the sequence in bold, indicating the design primer sequence position. DETAILED DESCRIPTION

[0026] The specific embodiments of the application will be described below in conjunction with the embodiments, but the following embodiments are only used to illustrate the application in detail, and do not limit the scope of the application in any way.

[0027] In the following examples, the instruments and equipment involved are conventional unless otherwise specified; the reagents and raw materials involved are conventional commercial products unless otherwise specified; the test methods involved are conventional unless otherwise specified.

[0028] Example 1: Determination process of rib number of pigs

[0029] 1. Source of test animals

[0030] In this example, 277 purebred pigs in Henan Muyuan Pig Farm were used as the test population. The pigs were free to eat and drink water, and the entire feeding method and feeding conditions were consistent and were all conventional methods.

[0031] 2. Sample collection and phenotype recording

[0032] The ear tissue of the pigs in the above population was collected and stored in 75% ethanol at -20°C in the refrigerator. The number of ribs of each pig was measured and the determination information was recorded.

[0033] Example Two: SNP molecular marker detection and identification process

[0034] 1. Extraction and detection of genomic DNA

[0035] The extraction method of ear tissue DNA of Duroc pigs, Landrace pigs and Large White pigs was referred to the phenol-chloroform method for extracting whole genomic DNA. The quality and concentration of the DNA of the purebred Duroc pig, Landrace pig and Large White pig population were detected 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 determined to be qualified. Finally, the qualified DNA samples were diluted to 50 nanograms per microliter.

[0036] 2. Whole genome genotyping of pigs

[0037] The whole genome resequencing of 277 purebred Duroc pigs, Landrace pigs and Large White pigs was performed using the DNBSEQ-T7 platform, and the average depth of sequencing reached 10X. The SNP and indel variation detection was performed on the sequencing data aligned with the reference genome using bcftools software. The genotype data obtained was quality controlled using PLINK v1.90, and individuals with detection rate <90%, minimum allele frequency (MAF) <10%, and exclusion rate <80% were excluded.

[0038] 3. Whole genome association analysis

[0039] In order to eliminate the population stratification effect, linear mixed model single point regression analysis was used in this example, combined with GEMMA software for GWAS analysis. The similarity of the genomes between individuals was used to correct the stratification effect in the analysis model. The Bonferroin method was used to determine the significance threshold of the association degree of SNP and indel with rib number traits, and the genome level significant threshold was 0.05 / N, and the chromosome level significant threshold was 1 / N, wherein N was the number of SNPs and indels used for analysis. The chromosome level threshold was selected in this example. The GWAS analysis results are shown in Figure 1 . From Figure 1It can be seen that in the Duroc, Landrace and Large White pigs, there is a locus on chromosome 7 that significantly affects the rib number, and the most strongly associated SNP is chr7:97578184C>A (P=2.481971E-8), that is, the C>A mutation at position 97578184 on chromosome 7 of the international pig reference genome version 11.1.

[0040] 4. Association analysis of different genotypes with the rib number phenotype of breeding pigs

[0041] As shown in Table 1, the SNP locus chr7:97578184C>A of the molecular marker is extremely significantly related to the rib number (P=6.653859E-9), indicating that the molecular marker significantly affects the rib number of Duroc, Landrace and Large White pigs, and the rib number of the population can be improved by assisted selection of the SNP locus of pigs, 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 the SNP locus chr7:97578184 has fewer rib numbers than the AC and CC types, indicating that the homozygote AA is the most unfavorable for the rib number of breeding pigs. The rib number is an important indicator for measuring the growth performance of breeding pigs, and more rib numbers indicate better growth performance and body size of pigs. Therefore, in the breeding process, breeding pigs with the SNP locus of AA and AC type need to be gradually eliminated, and breeding pigs with the SNP locus of CC type need to be retained, so as to gradually increase the frequency of the advantageous allele of the locus from generation to generation.

[0042] Table 1 Correlation of the SNP locus chr7:97578184C>A of the molecular marker with the rib number

[0043] .

[0044] Table 2 Statistics of the SNP locus chr7:97578184C>A of the molecular marker in three breeds

[0045] .

[0046] 5. Amplification and sequencing of the target DNA sequence

[0047] (1) The target fragment containing the SNP locus significantly related to the rib number of Duroc, Landrace and Large White pigs is a 320bp nucleotide sequence in chromosome 7, and the upstream and downstream primers for sequence amplification are primer-F and primer-R, and the nucleic acid sequences are as follows:

[0048] The upstream primer primer-F is 5'-ACCTCTTGGTTCCTAGTTG-3';

[0049] Downstream primer primer-R: 5'-ATGCCAGATCCTTAACCCA-3'.

[0050] (2) PCR amplification system and condition setting

[0051] A 10 μL system was configured, wherein DNA sample 1 μL, upstream primer 0.3 μL, downstream primer 0.3 μL, PCR mix 5 μL, ddH2O 3.4 μL, and the PCR reaction program was as follows: 95 ℃ pre-denaturation for 5 min; 95 ℃ denaturation for 30 s, 60 ℃ annealing for 30 s, 72 ℃ extension for 30 s, for a total of 30 cycles; and finally extension for 72 ℃ for 10 min.

[0052] (3) DNA sequence identification: sequence the gene fragments in two reactions; compare the measured sequence with the Ensembl genome sequence, obtain the mutation of the corresponding SNP site, and the sequencing result is shown in Figure 4 .

[0053] The application detects the SNP base mutation site, preliminarily performs correlation analysis between genotypes and rib bone number traits of pigs, and provides a new molecular marker for molecular marker assisted selection of pigs. As shown in Table 1, on average, each CC type boar has about 0.6 more rib bones than each AA type boar, and the difference is significant. Through molecular marker assisted selection, the advantages of improvement can be greatly passed on to the offspring commercial pig population, so as to accelerate the genetic research progress of the rib bone number of pigs and improve the economic benefit of the pig industry.

[0054] Although some preferred embodiments of the present application have been described, those skilled in the art can make further changes and modifications to these embodiments once they know the basic inventive concept. Therefore, the appended claims are intended to be interpreted as including all changes and modifications falling within the scope of the present application.

[0055] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the inventive concept. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and equivalent technologies, the present application also intends to include these modifications and variations.

Claims

1. The application of primer pairs for identifying SNP molecular markers in the breeding / identification of breeds / strains related to the rib count trait in pigs, characterized in that, The SNP molecular marker is a single nucleotide polymorphism of the C / A switching type at 97578184 bp on chromosome 7, based on the international pig genome version 11.1 reference sequence; the pig is Duroc, Landrace, Large White or their synthetic lines.

2. The application of primer pairs for identifying SNP molecular markers in the preparation of reagents for breeding / identifying breeds / strains related to the rib count trait in pigs, characterized in that, The SNP molecular marker is a single nucleotide polymorphism of the C / A switching type at 97578184 bp on chromosome 7, based on the international pig genome version 11.1 reference sequence; the pig is Duroc, Landrace, Large White or their synthetic lines.

3. The application according to claim 1 or 2, characterized in that, The primer pair comprises primers primer-F and primer-R, whose nucleotide sequences are as follows: Upstream primer-F: 5'-ACCTCTTGGTTCCTAGTTG-3'; Downstream primer primer-R: 5'-ATGCCAGATCCTTAACCCA-3'.

4. A method for selecting / identifying traits related to the number of ribs in pigs using SNP molecular markers, characterized in that, The SNP molecular marker is a single nucleotide polymorphism of the C / A switching type at position 97578184 bp on chromosome 7, based on the international pig genome version 11.1 reference sequence; the method includes the following steps: (1) Whole genome DNA was extracted from pig ear tissue samples; (2) The whole genome DNA was scanned using porcine whole genome resequencing; (3) Obtain resequencing data, and based on the international pig genome version 11.1 reference sequence, check the genotype of the 97578184th site on chromosome 7, determine the C / A polymorphism of the site, and select / identify the pig rib number trait based on the fact that individuals with CC genotype have more ribs than individuals with AA / AC genotype. The pigs mentioned are Duroc pigs, Landrace pigs, Large White pigs, or their synthetic lines.

5. A breeding method for increasing the number of ribs in pigs, characterized in that, Includes the following steps: (1) Based on the international pig genome version 11.1 reference sequence, the genotype of the pig population at the 97578184th locus on chromosome 7 was detected and determined; (2) Select breeding pig individuals with the CC genotype at the 97578184th locus and eliminate breeding pig individuals with the AC or AA genotype at the same locus to increase the frequency of the C allele at the locus generation by generation, thereby increasing the number of ribs in the offspring pigs. The pigs mentioned are Duroc pigs, Landrace pigs, Large White pigs, or their synthetic lines.

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