SNP molecular marker affecting pig body length trait and application

Abstract

The application discloses a SNP molecular marker affecting pig body length traits and application, and aims to solve the technical problem of slow breeding caused by the lack of gene research on pig body length related traits at present. The application detects and finds a SNP molecular marker affecting pig body length traits based on pig whole genome association analysis strategy by using pig whole genome sequence. The molecular marker is based on the single nucleotide polymorphism variation of G / A mutation conversion type at the position of 16624558bp on the chromosome 17 of the international pig genome 11.1 version reference sequence. The genotype of the position 16624558 on the chromosome 17 of the pig population is detected, and it is known that the AA genotype individual of the SNP site (chr17:16624558) has a longer body length than the GG / GA genotype individual. By selecting the individual with the beneficial genotype for breeding and eliminating the individual with the adverse genotype, the body length of the pig can be significantly improved, so that the purpose of accelerating the genetic progress of the pig body length traits is achieved.

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 pig body length and application thereof. BACKGROUND

[0002] Pig body length is a key index for measuring pig body size and evaluating its production performance, and it reflects the genetic diversity of pigs and is closely related to production performance indexes such as growth rate, feed conversion efficiency and lean meat rate. For a long time, people have improved the production performance of pigs by traditional phenotype selection on pig body length. This method has improved the pig body length to some extent, but the pig body length is a quantitative trait, which is controlled by multiple genes in the genetic aspect. The simple phenotype selection method often causes the problems of limited selection effect and slow effect.

[0003] 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

[0004] The purpose of the present application is to provide a SNP molecular marker affecting pig body length, and apply it in genotype identification, genetic breeding and the like, so as to solve the technical problem that the current pig body length related traits lack gene research and further lead to slow breeding progress.

[0005] The SNP molecular marker affecting pig body length is detected and found by using pig whole genome resequencing (WGS) and based on pig genome-wide association study (GWAS) detection. The SNP molecular marker is based on the G / A mutation at the 16624558th base on the 17th chromosome of the international pig genome 11.1 version reference sequence. The SNP molecular marker is applied to molecular marker assisted selection and genome selection, which can promote the genetic improvement process of pigs to improve the economic benefit of breeding.

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

[0007] The second aspect of the present application applies the SNP molecular marker in the preparation of reagents for selecting / identifying pig breeds / lines related to body length.

[0008] The third aspect of the present application discloses application of the SNP molecular marker in breeding / identifying a breed / line related to the body length trait of a pig.

[0009] The fourth aspect of the present application discloses a method for selecting / identifying a pig related to the body length 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 whole genome resequencing of a pig;

[0012] (3) obtaining resequencing data, checking the genotype of the 16624558th site on chromosome 17 based on the international pig genome 11.1 version reference sequence, judging the G / A polymorphism of the site, and selecting / identifying the body length trait of a pig based on the characteristic that the AA genotype individual has a longer body length than the GA or GG genotype individual.

[0013] The fifth aspect of the present application discloses a primer pair for identifying the 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'-AACTGTCACAACTTGGAGG-3';

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

[0016] The sixth aspect of the present application discloses a method for breeding a breed / line with improved body length of a pig, comprising the following steps:

[0017] (1) detecting and determining the genotype of the 16624558th site on chromosome 17 of a pig population based on the international pig genome 11.1 version reference sequence;

[0018] (2) selecting a breeding pig individual with AA genotype at the 16624558th site, and eliminating a breeding pig individual with GA or GG genotype at the site, so as to improve the frequency of the allele A at the site from generation to generation, thereby improving the body length of the offspring pig.

[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 body length trait of pigs, based on the international pig genome 11.1 version reference sequence, a single nucleotide polymorphism variation of G / A conversion type at the base at position 16624558 of chromosome 17, verifies the effect of the body length trait of pigs, provides a method for improving the body length of pigs by selecting the advantage allele of the molecular marker, and finally establishes a molecular marker assisted selection breeding technology for rapidly improving the body length trait of pigs, greatly improves the breeding process of Duroc pigs, Landrace pigs, Large White pigs and their synthetic lines, shortens the time for improving 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 whole genome association analysis (GWAS) of Duroc pigs, Landrace pigs and Large White pigs on chromosome 17 about the body length trait of pigs in an embodiment of the application; wherein: the abscissa represents the chromosome number of the pig; the ordinate represents -log10 (P value).

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

[0024] Figure 3 It is a body length 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 are conventional unless otherwise specified; the biological reagents and raw materials are commercially available unless otherwise specified; the test methods are conventional unless otherwise specified.

[0028] Example 1: Determination process of pig body length

[0029] 1. Source of test animals

[0030] The experimental population used in this study consisted of 278 purebred pigs from the Henan Muyuan breeding farm, including 55 Duroc pigs, 185 Landrace pigs, and 38 Large White pigs. The pigs had free access to feed and water, and the feeding regimen and housing conditions remained consistent, following conventional methods.

[0031] 2. Sample Collection and Phenotyping

[0032] 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.

[0033] Example 2: SNP molecular marker detection and identification process

[0034] 1. Extraction and Detection of Genomic DNA

[0035] 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.

[0036] 2. Whole-genome genotyping of pigs

[0037] The whole genomes of 278 purebred pigs were resequenced 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%.

[0038] 3. Genome-wide Association Analysis

[0039] 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 1It can be seen that in the Duroc pigs, Landrace pigs and Large White pigs, there is a locus on chromosome 17 which significantly affects the body length, and the most strongly associated SNP is chr17:16624558G>A (P=1.077647E-13), that is, the G / A mutation at the 16624558bp on the chromosome 17 of the international pig reference genome version 11.1.

[0040] 4. Association analysis of different genotypes with the lean meat percentage phenotype of breeding pigs

[0041] According to Table 1, the SNP locus chr17:16624558G>A of the molecular marker is extremely significantly related to the body length (P=1.077647E-13), which indicates that the molecular marker significantly affects the body length of the Duroc pigs, Landrace pigs and Large White pigs, and the body length of the population can be improved through the assisted selection of the SNP locus of the pigs, so as to accelerate the breeding process of the breeding pigs. In addition, according to Table 1, Table 2, Figure 1 and Figure 3 It can also be seen that the body length of the GG type of the SNP locus chr17:16624558 is smaller than that of the GA and AA types, which indicates that the homozygote GG is the most unfavorable for the body length of the breeding pigs. The body length is an important indicator for measuring the growth performance of the breeding pigs, and a long body length indicates a good growth performance of the pigs. Therefore, in the breeding process, the breeding pigs of the GG type and the GA type of the corresponding SNP locus need to be gradually eliminated, and the breeding pigs of the AA type are retained, so as to gradually improve the frequency of the advantageous allele of the locus from generation to generation.

[0042] Table 1 Correlation of SNP locus chr17:16624558G>A with body length

[0043] .

[0044] Table 2 Statistics of SNP locus chr17:16624558G>A in three breeds

[0045] .

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

[0047] (1) The target fragment containing the SNP locus which is significantly related to the body length performance of the Duroc, Landrace and Large White pigs is a 316bp nucleotide sequence in chromosome 17, 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'-AACTGTCACAACTTGGAGG-3';

[0049] Downstream primer primer-R: 5'-TGTTTTTCCTCAGGTGTCA-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 at 72℃ for 10 min.

[0052] (3) DNA sequence identification: the gene fragment was sequenced for positive and negative reactions; the measured sequence was compared with the Ensembl genome sequence, the mutation of the corresponding SNP site was obtained, and the sequencing result was as shown in Figure 4 .

[0053] The application provides a new molecular marker for molecular marker assisted selection of pigs by detecting SNP base mutation sites and preliminarily performing correlation analysis between genotypes and pig body length traits. As shown in Table 1, the average body length of an AA type breeding pig is 9 cm longer than that of a GG type breeding pig, and the difference is significant. Through molecular marker assisted selection, the breeding process of Duroc pigs, Landrace pigs, Large White pigs and their synthetic lines is greatly improved, the time for improving Duroc pigs, Landrace pigs and Large White pigs is shortened, the demand of the breeding pig market is met, the economic benefits of breeding pigs are effectively improved, and the core competitiveness of breeding enterprises is further improved.

[0054] Although some preferred embodiments of the present application have been described, those skilled in the art, once they know the basic creative concept, can make additional changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and 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 thereof, the present application also intends to include these modifications and variations.

Claims

1. Use of a primer pair for identifying SNP molecular markers in the preparation of a reagent for selecting / identifying pig breeds / lines related to body length traits, characterized in that: The SNP molecular marker is based on the international porcine genome version 11.1 reference sequence, and has a single nucleotide polymorphism variation of the G / A mutation conversion type at the 16624558bp site on chromosome 17; the pig is a Duroc pig or its synthetic line, or a Landrace pig or its synthetic line, or a Large White pig or its synthetic line.

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 has a single nucleotide polymorphism variation of the G / A mutation conversion type at the 16624558bp site on chromosome 17; the pig is a Duroc pig or its synthetic line, or a Landrace pig or its synthetic line, or a Large White pig or its synthetic line.

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′-AACTGTCACAACTTGGAGG-3′; Downstream primer primer-R: 5'-TGTTTTTCCTCAGGTGTCA-3'.

4. A method for selecting / identifying pig body length traits using SNP molecular markers, characterized in that: The SNP molecular marker is a single nucleotide polymorphism variation of the G / A mutation conversion type at the 16624558bp position on chromosome 17 based on the international porcine genome version 11.1 reference sequence; the pig is a Duroc pig or a synthetic line thereof, or a Landrace pig or a synthetic line thereof, or a Large White pig or a synthetic line thereof; 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, based on the international pig genome version 11.1 reference sequence, check the genotype of site 16624558 on chromosome 17 to determine the G / A polymorphism of this site. 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 method for selecting and breeding pig breeds / lines for increasing pig body length, characterized in that: The steps include: (1) Based on the International Porcine Genome Version 11.1 reference sequence, the genotype of chromosome 17 at position 16624558 of the pig population was determined; (2) Select individual sows with the AA genotype at locus 16624558 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 generation by generation, thereby increasing the body length of offspring pigs; The breeding pig is a Duroc pig or a synthetic line thereof, or a Landrace pig or a synthetic line thereof, or a Large White pig or a synthetic line thereof.

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