A SNP molecular marker located on pig chromosome 5 and associated with pig lean meat rate and its application

Abstract

The present invention relates to the technical field of pig molecular marker screening, and more particularly to a single-nucleotide polymorphism (SNP) molecular marker located on chromosome 5 and associated with pig lean meat percentage, and its application. This SNP molecular marker corresponds to a T>C mutation at bp 64,865,623 and an A>G mutation at bp 64,865,624 on chromosome 5 of the International Porcine Genome Version 11.1 reference sequence. These two adjacent sites are the highest points in the GWAS and are highly linked to surrounding SNP sites. Application of this marker in genotyping, genetic breeding, and other areas can address current technical issues, such as the lack of genetic traits associated with pig lean meat percentage.

Description

Technical Field

[0001] The present invention relates to the technical field of pig molecular marker screening, and in particular to a SNP molecular marker located on pig chromosome 5 and associated with pig lean meat rate and its application. Background Art

[0002] As modern consumers pursue a healthier diet, they increasingly prefer lean pork, making breeding high-quality, lean pigs a key breeding goal. For a long time, genetic improvement of lean meat percentage has primarily relied on traditional phenotypic selection methods. While this approach has yielded some improvements, lean meat percentage is a quantitative trait regulated by multiple genes. Therefore, improving lean meat percentage through traditional selection methods is time-consuming and ineffective.

[0003] Genome-wide association study (GWAS) is a statistical method based on single nucleotide polymorphisms (SNPs) and genomic data that can identify marker loci closely associated with phenotypic variation. Using GWAS to identify molecular markers associated with lean meat percentage in pigs can effectively accelerate the genetic improvement and breeding of lean pigs, helping to increase the economic benefits of pig farming operations.

[0004] Duroc, Landrace, and Large White pigs have rapidly captured the global hog market, becoming the most popular commercial pig breed due to their exceptional growth rates and high lean meat percentage. Therefore, by improving the lean meat percentage traits of core herds of Duroc, Landrace, and Large White pigs, these improved traits can be largely passed on to future generations of commercial pigs. This effectively increases the lean meat percentage of commercial pigs, meeting consumer demand for lean meat while also improving the economic benefits of pig farming companies. Summary of the Invention

[0005] In order to overcome the deficiencies and shortcomings of the existing technology, the primary purpose of the present invention is to provide a SNP molecular marker located on pig chromosome 5 and related to the lean meat rate of pigs, which can be applied in genotype identification, genetic breeding, etc., to solve the current technical problem of the lack of genetic traits related to the lean meat rate of pigs.

[0006] Another object of the present invention is to provide an application of the above-mentioned SNP molecular marker.

[0007] Another object of the present invention is to provide a primer pair and a kit for identifying the above-mentioned SNP molecular markers.

[0008] The fourth object of the present invention is to provide the application of the above primer pair and kit.

[0009] A fifth object of the present invention is to provide a method for genetic improvement of pigs.

[0010] The purpose of the present invention is achieved through the following technical solutions:

[0011] A single-nucleotide polymorphism (SNP) molecular marker associated with lean meat percentage on porcine chromosome 5, corresponding to the T / C mutation at position 64865623 (denoted as mutation site g.64865623) or the A / G mutation at position 64865624 (denoted as mutation site g.64865624) on chromosome 5 in the International Porcine Genome Version 11.1 reference sequence. The polymorphism of the bases at this site affects the lean meat percentage trait of pigs.

[0012] The nucleotide sequence of the SNP molecular marker is preferably as shown in SEQ ID NO.1 or SEQ ID NO.2, wherein M in the sequence is T / C or A / G; the SNP site of the SNP molecular marker is the nucleotide mutation T174-C174 at position 174 of the sequence annotation of SEQ ID NO:1 (a single base mutation at position 174 of the sequence fragment) or the nucleotide mutation A175-G175 at position 175 of the sequence annotation of SEQ ID NO:2 (a single base mutation at position 175 of the sequence fragment);

[0013] Application of the SNP molecular marker in identifying pig lean meat percentage traits, improving pig lean meat percentage or pig genetic breeding;

[0014] Application of the SNP molecular marker in screening pig strains related to lean meat percentage;

[0015] The pigs are preferably Duroc, Landrace, Large White and their synthetic strains;

[0016] A method for identifying the lean meat percentage trait of pigs comprises the following steps:

[0017] (1) Extraction of whole genome DNA from pig ear tissue samples;

[0018] (2) scanning the whole genome DNA using pig whole genome resequencing;

[0019] (3) Obtain resequencing data, based on the International Porcine Genome Version 11.1 reference sequence, determine the T / C polymorphism of the site based on the genotype of the 64865623rd site on chromosome 5; or determine the A / G polymorphism of the site based on the genotype of the 64865624th site on chromosome 5;

[0020] The pigs are preferably Duroc, Landrace, Large White and their synthetic strains;

[0021] A primer pair for identifying the above-mentioned SNP molecular marker comprises primers primer-F and primer-R, the nucleotide sequences of which are as follows:

[0022] Upstream primer primer-F: 5′-AGTAGAGCTGATTTACAATGT-3′;

[0023] Downstream primer primer-R: 5′-TTAACAAATCCGACTAGGAAC-3′;

[0024] A kit for identifying the above-mentioned SNP molecular marker, comprising the above-mentioned primer pair;

[0025] Application of the primer pair or kit in identifying the lean meat percentage trait of pigs;

[0026] Application of the primer pair or kit in improving the lean meat rate of pigs;

[0027] Application of the primer pair or kit in breeding pig strains related to lean meat percentage;

[0028] Application of the primer pair or kit in pig genetic breeding;

[0029] A method for genetic improvement of pigs, comprising the following steps:

[0030] Determine the SNP molecular markers on chromosome 5 of the breeding pigs in the breeding pig core group that are related to the lean meat rate of the pigs, and make selections based on the molecular markers: select the breeding pigs with the TT or TC genotype at the 64865623bp position on chromosome 5 of the International Porcine Reference Genome 11.1 version, and eliminate the breeding pigs with the CC genotype; or select the breeding pigs with the AA or AG genotype at the 64865624bp position on chromosome 5, and eliminate the breeding pigs with the GG genotype, so as to increase the frequency of the allele T or A at the mutation site from generation to generation, thereby improving the lean meat rate of the offspring pigs;

[0031] The pigs are preferably Duroc, Landrace, Large White and their synthetic strains;

[0032] The present invention has the following advantages and effects compared to the prior art:

[0033] (1) The present invention utilizes pig whole genome resequencing (WGS) and genome-wide association study (GWAS) strategy to study and identify SNP molecular markers that affect pig lean meat rate traits. The SNP molecular markers correspond to the T>C mutation at 64865623bp and the A>G mutation at 64865624bp on chromosome 5 of the international pig genome version 11.1 reference sequence. These two adjacent sites are the highest points in GWAS and are highly linked to the surrounding SNP sites. The present invention verifies their effects on the pig lean meat rate trait and ultimately establishes a molecular marker-assisted selection breeding technology for rapid improvement of the pig lean meat rate trait, which greatly improves the breeding process of Duroc, Landrace, Large White and their synthetic lines, adapts to the needs of the breeding pig market, and improves the core competitiveness of enterprises.

[0034] (2) In practical applications, selecting the TT genotype at the SNP locus (chr5:64865623) or the AA genotype at (chr5:64865624) can increase lean meat percentage by 3.59% compared to the CC genotype or the GG genotype. By selecting individuals with favorable genotypes for breeding and eliminating individuals with unfavorable genotypes, the lean meat percentage of pigs can be significantly increased, thereby achieving the goal of increasing the genetic progress of the lean meat percentage trait in pigs.

[0035] (3) The present invention provides a method for pig breeding by optimizing the dominant allele of the molecular marker, which can accelerate the genetic progress of Duroc, Landrace and Large White pigs, shorten the time for improvement of Duroc, Landrace and Large White pigs, and thus effectively improve the economic benefits of breeding pigs. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the Manhattan plot of the genome-wide association study (GWAS) on chromosome 5 of Duroc, Landrace and Large White pigs for the lean meat percentage trait of breeding pigs; wherein: the horizontal axis represents the chromosome number of the pig; the vertical axis represents -log10 (P value).

[0037] Figure 2 It is the genome-wide association study (GWAS) QQ plot.

[0038] Figure 3 This is an analysis chart of the lean meat rate of pigs with different genotypes at the chr5:64865623 locus.

[0039] Figure 4 This is an analysis chart of the lean meat rate of pigs with different genotypes at the chr5:64865624 locus.

[0040] Figure 5 It is the Manhattan plot of Fst analysis results. DETAILED DESCRIPTION

[0041] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0042] Example 1 specifically explains the determination process of the lean meat rate in the present invention

[0043] 1. Source of experimental animals

[0044] The experimental population used in this example was a group of 74 purebred pigs from a pig farm in Henan Province (including 15 purebred Duroc pigs, 44 purebred Landrace pigs, and 15 purebred Large White pigs, for a total of 74 pigs). The pigs had free access to food and water, and the entire feeding method and housing conditions were consistent throughout, following conventional methods.

[0045] 2. Sample Collection and Phenotypic Recording

[0046] Ear tissue samples were collected from each of the above pigs, placed in 75% alcohol by volume, and stored in a refrigerator at -20°C. The skin, bones, meat, and fat were carefully slaughtered, divided, weighed, and the measured information (weight) and lean meat rate (200±10 days of age) were recorded. Lean meat rate = meat / (skin+bone+meat+fat).

[0047] Example 2 Detailed explanation of the invention process for obtaining the gene markers of the present invention

[0048] 1. DNA Extraction from Ear Tissue

[0049] 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. 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 diluted to 50 ng / μL.

[0050] 2. Pig whole genome genotype detection

[0051] The whole genomes of the 74 purebred Duroc, Landrace, and Large White pigs described in Example 1 were resequenced using the DNBSEQ-T7 platform to an average sequencing depth of 10×. SNP calling was performed on the sequencing data after alignment to the reference genome using bcftools software. Genotype data were quality-controlled using PLINK v1.90, eliminating individuals with a call rate <90%, a minimum allele frequency (MAF) <10%, and a call rate <80%. The remaining 12,749,772 SNP markers and 74 samples were used for subsequent data analysis.

[0052] 3. Genome-wide association (GWAS) analysis

[0053] To eliminate the population stratification effect, the present invention uses linear mixed model single-point regression analysis combined with GEMMA software for GWAS analysis. The stratification effect is corrected in the analysis model using the similarity of the genomes between individuals. The Bonferroin method is used to determine the significance threshold for the association between SNPs and lean meat percentage. The genomic level significance threshold is 0.05 / 12749772 (the number of effective SNPs), that is, the genomic level significance threshold is 3.92164E-9, and the chromosome level significance threshold is 1 / 12749772 (the number of effective SNPs), that is, the chromosome level significance threshold is 7.84328E-8. The chromosome level threshold was selected in this study.

[0054] GWAS analysis results are as follows Figure 1 As shown. Figure 1 It can be seen that in Duroc, Landrace and Large White pigs, there are sites on chromosome 5 that significantly affect lean meat percentage. The strongest associated SNPs are chr5:64865623T>C (P=2.93917E-9) and chr5:64865624A>G (P=2.93917E-9), which correspond to the T>C mutation at 64865623bp on chromosome 5 of the International Porcine Reference Genome Version 11.1 and the A>G mutation at 64865624bp on chromosome 5.

[0055] 4. Correlation analysis between different genotypes and lean meat percentage phenotypes of breeding pigs

[0056] According to Table 1, the SNP sites of the molecular marker chr5:64865623T>C and chr5:64865624A>G are extremely significantly correlated with the lean meat rate trait (P=1.6263E-7), indicating that this molecular marker significantly affects the lean meat rate of Duroc, Landrace and Large White pigs. Through auxiliary selection of the SNP sites of pigs, the lean meat rate of the group can be improved, thereby accelerating the breeding process of lean meat pigs.

[0057] In addition, according to Table 1, Table 2, Table 3, Figure 3 and Figure 4 Furthermore, the CC genotype at SNP site chr5:64865623 has a lower lean meat percentage than the TT and TC genotypes, and the GG genotype at SNP site chr5:64865624 has a lower lean meat percentage than the AG and AA genotypes. This indicates that homozygous CC and homozygous GG genotypes are most detrimental to lean meat percentage in breeding pigs. Lean meat percentage is an important indicator of growth performance in breeding pigs, and a high lean meat percentage indicates good growth performance. Therefore, pigs with CC and GG genotypes have the worst growth performance. During breeding, we need to gradually eliminate pigs with CC and TC genotypes, GG and AG genotypes at the corresponding SNP sites, and retain pigs with TT and AA genotypes to increase the frequency of the dominant allele at each locus with each generation.

[0058] 5.Fst analysis

[0059] From the 74 resequenced individuals, 203 ± 2-day-old Landrace pigs were selected. Five individuals with high lean meat percentage and five individuals with low lean meat percentage were divided into two subgroups. The genetic differentiation index (Fst) was calculated using VCFtools software using a sliding window method with the following parameters: a sliding window size (--fst-window-size) of 100 Kb and a sliding window step size (--fst-window-step) of 20 Kb. Windows were sorted by Fst value from highest to lowest, and the top 1% of windows were selected as candidate regions.

[0060] The results of Fst analysis showed that ( Figure 5 ), a total of 1195 candidate regions were obtained in the top 1%, and chr5:64865623 and chr5:64865624 were also located in multiple candidate regions of chromosome 5. This analysis further demonstrated that these two sites were highly correlated with the lean meat percentage trait of pigs.

[0061] Table 1 Correlation between SNP sites chr5:64865623T>C and chr5:64865624A>G and lean meat percentage at 200±10 days of age

[0062]

[0063] Table 2 Statistics of molecular marker SNP site chr5:64865623T>C in three varieties

[0064]

[0065]

[0066] Table 3 Statistics of molecular marker SNP site chr5:64865624A>G in three varieties

[0067]

[0068] Example 2 Detailed explanation of the invention process of detecting SNP markers

[0069] (1) The target fragment containing the SNP site significantly associated with the lean meat performance of Duroc, Landrace and Large White pigs is a 318 bp nucleotide sequence on chromosome 5. The upstream and downstream primers for sequence amplification are primer-F and primer-R, and their nucleic acid sequences are as follows:

[0070] Upstream primer primer-F: 5′-AGTAGAGCTGATTTACAATGT-3′;

[0071] Downstream primer primer-R: 5′-TTAACAAATCCGACTAGGAAC-3′;

[0072] (2) PCR amplification system and condition settings

[0073] 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 final extension at 72°C for 10 min.

[0074] (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 shown below:

[0075] For chr5:64865623:

[0076]

[0077]

[0078] For chr5:64865624:

[0079] Note: The M marked in the sequence is the mutation site, which is underlined (the mutated base in brackets is the allele mutation), and the bold at the beginning and end of the sequence is the primer sequence binding position.

[0080] Example 3 Analysis of the effects of molecular markers on SNP sites chr5:64865623T>C and chr5:64865624A>G

[0081] The present invention provides a SNP molecular marker that can significantly improve the lean meat rate of pigs. The SNP molecular marker is used for marker-assisted selection. If the SNP is applied to molecular marker-assisted selection and genomic selection, the genetic improvement process of pigs can be promoted to improve the economic benefits of breeding.

[0082] If all CC-type individuals with this molecular marker are bred to TT-type individuals, or all GG-type individuals are bred to AA-type individuals, the lean meat percentage can be increased from 61.15% to 64.74%. In a large-scale pig farm with 10,000 pigs, assuming each pig has a market weight of 100 kilograms, 35,900 kilograms of lean meat can be added.

[0083] In the individual SNP molecular marker, by optimizing the dominant allele (T) or (A) of the SNP in the pig population, it is ultimately possible to improve the production performance of pigs and thus increase the profits of the enterprise.

[0084] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A SNP molecular marker located on chromosome 5 of pigs and associated with pig lean meat rate, characterized in that The SNP site corresponds to the T / C mutation at position 64865623 on chromosome 5 or the A / G mutation at position 64865624 on chromosome 5 in the International Porcine Genome Version 11.1 reference sequence. The polymorphism of the bases at this site affects the lean meat percentage trait of pigs. The pigs are Duroc, Landrace, Large White and their synthetic lines.

2. Use of the SNP molecular marker according to claim 1 in identifying the lean meat percentage trait of pigs, improving the lean meat percentage of pigs, or in pig genetic breeding, characterized in that: The CC genotype of the SNP molecular marker has a lower lean meat rate than the TT genotype and the TC genotype, and the GG genotype has a lower lean meat rate than the AG genotype and the AA genotype; The pigs are Duroc, Landrace, Large White and their synthetic lines.

3. Use of the SNP molecular marker according to claim 1 in screening pig strains related to lean meat percentage, characterized in that: The CC genotype of the SNP molecular marker has a lower lean meat rate than the TT genotype and the TC genotype, and the GG genotype has a lower lean meat rate than the AG genotype and the AA genotype; The pigs are Duroc, Landrace, Large White and their synthetic lines.

4. A method for identifying the lean meat percentage of pigs, characterized in that The following steps are included: (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, based on the International Porcine Genome Version 11.1 reference sequence, and determine the T / C polymorphism of the site based on the genotype of the 64865623rd site on chromosome 5; or determine the A / G polymorphism of the site based on the genotype of the 64865624th site on chromosome 5; The pigs are Duroc, Landrace, Large White and their synthetic lines.

5. A primer pair for identifying the SNP molecular marker according to claim 1 for use in identifying the lean meat percentage trait of pigs, improving the lean meat percentage of pigs, or in pig genetic breeding, characterized in that: The primer pair comprises primers primer-F and primer-R, and the nucleotide sequences thereof are as follows: Upstream primer primer-F: 5′-AGTAGAGCTGATTTACAATGT-3′; Downstream primer primer-R: 5′-TTAACAAATCCGACTAGGAAC-3′; The CC genotype of the SNP molecular marker has a lower lean meat rate than the TT genotype and the TC genotype, and the GG genotype has a lower lean meat rate than the AG genotype and the AA genotype; The pigs are Duroc, Landrace, Large White and their synthetic lines.

6. A kit for identifying the SNP molecular marker according to claim 1 for use in identifying the lean meat percentage trait of pigs, improving the lean meat percentage of pigs, or in pig genetic breeding, characterized in that: The kit contains primers primer-F and primer-R, the nucleotide sequences of which are as follows: Upstream primer primer-F: 5′-AGTAGAGCTGATTTACAATGT-3′; Downstream primer primer-R: 5′-TTAACAAATCCGACTAGGAAC-3′; The CC genotype of the SNP molecular marker has a lower lean meat rate than the TT genotype and the TC genotype, and the GG genotype has a lower lean meat rate than the AG genotype and the AA genotype; The pigs are Duroc, Landrace, Large White and their synthetic lines.

7. A use of a primer pair for identifying the SNP molecular marker according to claim 1 in breeding pig strains associated with lean meat percentage, characterized in that: The primer pair comprises primers primer-F and primer-R, and the nucleotide sequences thereof are as follows: Upstream primer primer-F: 5′-AGTAGAGCTGATTTACAATGT-3′; Downstream primer primer-R: 5′-TTAACAAATCCGACTAGGAAC-3′; The CC genotype of the SNP molecular marker has a lower lean meat rate than the TT genotype and the TC genotype, and the GG genotype has a lower lean meat rate than the AG genotype and the AA genotype; The pigs are Duroc, Landrace, Large White and their synthetic lines.

8. A kit for identifying the SNP molecular marker according to claim 1 for use in breeding pig strains related to lean meat percentage, characterized in that: The kit contains primers primer-F and primer-R, the nucleotide sequences of which are as follows: Upstream primer primer-F: 5′-AGTAGAGCTGATTTACAATGT-3′; Downstream primer primer-R: 5′-TTAACAAATCCGACTAGGAAC-3′; The CC genotype of the SNP molecular marker has a lower lean meat rate than the TT genotype and the TC genotype, and the GG genotype has a lower lean meat rate than the AG genotype and the AA genotype; The pigs are Duroc, Landrace, Large White and their synthetic lines.

9. A method for genetic improvement of pigs, characterized in that The following steps are included: Determine the SNP molecular markers on chromosome 5 of the breeding pigs in the breeding pig core group that are related to the lean meat rate of the pigs, and make selections based on the molecular markers: select the breeding pigs with the TT or TC genotype at the 64865623bp position on chromosome 5 of the International Porcine Reference Genome 11.1 version, and eliminate the breeding pigs with the CC genotype; or select the breeding pigs with the AA or AG genotype at the 64865624bp position on chromosome 5, and eliminate the breeding pigs with the GG genotype, so as to increase the frequency of the allele T or A at the mutation site from generation to generation, thereby improving the lean meat rate of the offspring pigs; The pigs are Duroc, Landrace, Large White and their synthetic lines.

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