SNP (Single Nucleotide Polymorphism) molecular marker located on pig chromosome 10 and related to total lean meat percentage and application of SNP molecular marker

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) molecular marker positioned on a pig chromosome 10 and related to a total lean meat percentage. An SNP site of the SNP molecular marker corresponds to Tgt at a position of 11840666bp of a chromosome 10 of an international pig reference genome Ensembl Sscrofa11.1 version; g mutation; the nucleotide sequence of the SNP molecular marker is SEQ ID NO.1, wherein M in the sequence is T or G. Researches show that the polymorphism of the basic group at the site leads to the difference of the total lean meat percentage. Accordingly, the inventor establishes a corresponding pig genetic improvement method, and by optimizing the dominant alleles of the SNP, the frequency of the dominant alleles can be increased generation by generation, the total lean meat percentage of pigs can be increased, the progress of pig genetic improvement can be accelerated, and the economic benefit of breeding pig breeding can be effectively improved. In addition, the inventor also researches and designs a primer pair for amplifying the nucleotide sequence of the SNP molecular marker, and by virtue of the primer pair, the slaughter traits can be quickly and accurately bred, and the breeding process is accelerated.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biotechnology and molecular marker technology, and in particular relates to a SNP molecular marker located on pig chromosome 10 and associated with total lean meat rate and its application. Background Art

[0002] In recent years, with the continuous rise in residents' income levels, the market demand for high-quality pork has increased significantly. Lean meat, as a core category in the pig product consumer market, accounts for the largest share and enjoys particularly strong demand. To ensure a stable supply of lean meat products, the pig industry continues to strengthen breeding efforts, achieving significant results in improving the reproductive capacity and growth rate of pig herds, effectively driving the growth of pig production. To continuously improve lean meat production efficiency, while increasing production, it is also necessary to continuously increase the lean meat percentage of the pig herd. Therefore, the selection and breeding of high-lean meat percentage commercial pigs is a key focus of commercial pig breeding, and total lean meat percentage is a key breeding goal.

[0003] The estimation of total lean meat percentage is usually done indirectly by measuring the loin eye area, loin eye depth and backfat thickness of live pigs, which makes it difficult to accurately select for this trait during the breeding process. In addition, total lean meat percentage is a typical quantitative trait, which is controlled by micro-effect polygenes. In addition, breeding work is usually only carried out in the core group, without involving the production performance of its subsequent commercial pigs. Therefore, it is difficult to improve the total lean meat percentage of commercial pigs using traditional breeding methods. However, by using molecular biology methods, molecular markers that directly affect the total lean meat percentage are discovered, and individuals with dominant alleles are screened in the parent and grandparent generations for breeding, thereby improving the total lean meat percentage of the commercial pig herd, thereby improving production efficiency.

[0004] Currently, there is a lack of molecular markers related to total lean meat percentage that have clear functions, significant effects, and can be directly used in breeding. Candidate genes identified through candidate gene approaches and QTL mapping are often genes with known biological functions and have large confidence intervals, which limits the application of molecular markers in breeding. With the large-scale application of high-density single nucleotide polymorphism (SNP) microarrays, the cost of whole-genome testing in pigs has continued to decrease, laying the foundation for large-scale genetic analysis of quantitative traits. Genome-wide association studies (GWAS) can statistically analyze SNPs across the genome with observable traits at the population level, thereby selecting molecular markers most likely to influence the trait. Using loci that can improve total lean meat percentage in pig herds for marker-assisted selection can significantly improve the genetic progress of total lean meat percentage, thereby continuously enhancing the economic benefits of commercial pigs. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a SNP molecular marker located on pig chromosome 10 and associated with the total lean meat rate and its application.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] The SNP molecular marker associated with total lean meat percentage is located on chromosome 10 of the pig. Its SNP site corresponds to the T>G mutation at position 11840666bp on chromosome 10 of the international pig reference genome Ensembl Sscrofa11.1 version (hereinafter referred to as the g.247 T>G molecular marker).

[0008] The nucleotide sequence of the SNP molecular marker is SEQ ID NO. 1, wherein M in the sequence is T or G.

[0009] The position of M in SEQ ID NO: 1 is marked as position 247, that is, its SNP site is the nucleotide mutation T247-G247 marked as position 247 in SEQ ID NO: 1.

[0010] The primer pair used to amplify the nucleotide sequence of the above-mentioned SNP molecular marker includes primer P001-F and primer P002-R, and the base sequences thereof are SEQ ID NO.2 and SEQ ID NO.3.

[0011] Application of the above-mentioned SNP molecular markers or primer pairs in pig assisted breeding.

[0012] Pig assisted breeding is the genetic improvement of pigs to increase their overall lean meat content.

[0013] The genetic improvement method for pigs is to detect the above-mentioned SNP molecular markers, eliminate individuals with TG genotype and TT genotype at the SNP site, retain individuals with GG genotype at the SNP site as breeding pigs, and increase the frequency of the dominant allele G at the site from generation to generation, thereby increasing the total lean meat rate of offspring pigs.

[0014] The pigs are Du Changda three-way commercial pigs.

[0015] In response to the current genetic improvement of slaughter traits in pig assisted breeding, the inventors discovered a SNP molecular marker related to total lean meat percentage located on chromosome 10 of the pig. The SNP site corresponds to the T>G mutation at position 11840666bp on chromosome 10 of the international pig reference genome EnsemblSscrofa11.1; the nucleotide sequence of the SNP molecular marker is SEQ ID NO.1, where M in the sequence is T or G. Studies have shown that the polymorphism of the base at this site leads to differences in total lean meat percentage. By detecting the base mutation site and conducting a preliminary association analysis between its genotype and the total lean meat percentage trait of the pig, the inventors verified its effect on the total lean meat percentage of the pig. The inventors ultimately established an efficient and accurate molecular marker-assisted breeding technology and applied it to the genetic improvement of improving the total lean meat percentage of pigs, thereby improving the total lean meat percentage of offspring pigs, thereby improving the production performance of offspring pigs and increasing the economic profits of the enterprise. Based on this, the inventors have developed a method for genetic improvement of pigs. By optimizing the dominant allele of this SNP, they are able to increase the frequency of the dominant allele with each generation, improve the overall lean meat percentage of pigs, accelerate the progress of genetic improvement, and effectively enhance the economic benefits of pig breeding. Furthermore, the inventors have also researched and designed a primer pair for amplifying the nucleotide sequence of the SNP molecular marker. This primer pair allows for rapid and accurate selection for slaughter traits, accelerating the breeding process. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the Manhattan plot of the genome-wide association analysis (GWAS) of Sanyuan commercial pigs on chromosome 10 regarding total lean meat rate. In the figure: the horizontal axis represents the position of the SNP site on chromosome 10; the vertical axis represents the -logP value.

[0017] Figure 2 This is an analysis chart of the phenotypic ratio of total lean meat rate of pigs with different genotypes.

[0018] Figure 3 This is the sequencing result diagram of the target DNA sequence. DETAILED DESCRIPTION

[0019] Example 1 Screening of molecular markers and analysis of their association with total lean meat percentage

[0020] (1) Experimental animals

[0021] The experimental pig group used in the present invention is 388 Sanyuan commercial pigs from a branch of Guangxi Yangxiang Group Co., Ltd. The pigs were allowed to eat and drink freely, and the entire feeding method and feeding conditions were kept consistent, which was a conventional method.

[0022] (2) Sample collection

[0023] The above three commercial pig ear tissues or muscle tissues were collected and immersed in a 75% ethanol solution by volume, and stored in a -20°C refrigerator for later use.

[0024] (3) Phenotypic data collection

[0025] The phenotypic trait data of the present invention were obtained from the slaughterhouse of a branch of Guangxi Yangxiang Group Co., Ltd. The slaughter traits of three-way commercial pigs, including the total lean meat rate, were measured using the AutoFom III ultrasonic image analysis system.

[0026] (4) Pig genome-wide 80K SNP typing

[0027] Ear or muscle tissue samples were collected from the 388 commercial pigs mentioned above. Whole-genome DNA was extracted using the standard phenol-chloroform method. DNA sample quality was assessed using a Nanodrop 2000 / 2000C nucleic acid and protein analyzer, and DNA concentration and purity parameters (OD260 / 280 and OD260 / 230) were determined for each sample. DNA samples that met the test criteria were uniformly diluted to a working concentration of 50 ng / μL using sterile deionized water. Six μL of the DNA solution was mixed with 2 μL of loading buffer and loaded onto a 1% mass-to-volume agarose gel for electrophoresis (150 V, 25 min). DNA band clarity and integrity were assessed using a UV spectrophotometer and gel imaging equipment, and photographed.

[0028] DNA samples were sent to Wuhan Yingzi Gene Technology Co., Ltd. for genotyping of the whole-genome pig 80K functional locus chip according to the company's standard process. PLINK (V1.9) was used to perform quality control on the 80K chip scanning typing data of all samples, and individuals with a detection rate of less than 90%, a SNP detection rate of less than 90%, a minimum allele frequency of less than 0.05, and a Hardy The Weinberg equilibrium significance level is higher than Finally, valid genotype data of 124,265 SNPs were obtained.

[0029] (5) Genome-wide association study (GWAS)

[0030] A genome-wide association analysis of total lean meat percentage was performed using a linear mixed model in the GCTA software. The Bonferrini method was used to determine the significance threshold for the association between SNPs and total lean meat percentage. The significance threshold was 1 divided by the number of effective SNP sites, i.e., the significance level threshold was 8.05×10 -6 , which is 1 / 124265 (the number of effective SNPs). The analysis framework is based on the following model:

[0031]

[0032] Where: y is the phenotypic value vector; b is the covariate coefficient vector; α is the additive SNP effect vector; g is the random polygenic effect vector; e is the residual effect. X and Z are the corresponding matrices. , , , I is the identity matrix, and G is the kinship matrix calculated from all autosomal SNPs.

[0033] GWAS analysis results are as follows Figure 1 As shown. Figure 1 It can be seen that there is a site on chromosome 10 of Sanyuan commercial pigs that significantly affects the total lean meat rate. The significantly associated SNP is the 247th nucleotide g in SEQ NO.1. 247 T>G (P=5.31×10 -06 ).

[0034] (6) Correlation analysis between different genotypes and total lean meat percentage phenotype

[0035] According to Table 1 and Figure 2 It can be seen that the molecular marker SNP site g.247 T>G is extremely significantly correlated with the total lean meat rate trait (P=5.31×10 -06 ), where GG pigs carrying this molecular marker had a higher total lean meat percentage than TG and TT pigs. This result indicates that G is a favorable allele for this molecular marker, while T is an unfavorable allele. Therefore, during breeding, it is necessary to gradually eliminate TT and TG genotype pigs and retain GG genotype pigs to increase the frequency of the G allele at this locus generation by generation, thereby improving the total lean meat percentage of pigs.

[0036]

[0037] Example 2 Target DNA sequence amplification and sequencing

[0038] (1) Primer design

[0039] The DNA sequence of SEQ ID NO: 1 on porcine chromosome 10 was downloaded from the Ensembl website (http: / / asia.ensembl.org / index.html). Primers were designed using the primer design software Primer Premier 6.0. The DNA sequences of the designed primers are shown below:

[0040] P001-F: 5'-CAGGATGTGTGCGGTTTTCG-3',

[0041] P002-R: 5'-GGCAGAAGCTCGTACCCG-3';

[0042] (2) PCR amplification

[0043] To prepare a 10 μL system:

[0044] DNA sample 1.0 μL, upstream primer 0.3 μL, downstream primer 0.3 μL, PCR mix 5 μL, ddH2O 3.4 μL.

[0045] Configure the amplification conditions:

[0046] Stage 1: pre-denaturation at 95°C for 5 min;

[0047] The second stage consisted of 34 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 45 s;

[0048] The third stage was a final extension at 72°C for 5 min and then stored at 4°C.

[0049] (3) DNA sequence determination

[0050] DNA sequencing identification: Sequencing was performed at Shenzhen BGI Genomics Co., Ltd., and the gene fragments were tested for both positive and negative reactions. The measured sequence was compared with the NCBI genome sequence to obtain the mutation of the corresponding SNP site. The sequencing results are as follows Figure 3 As shown, the marked M is the mutation site, which is underlined in red (the mutated base in brackets is the allele mutation), and the bold underlines at the beginning and end of the sequence are the positions of the designed primer sequences.

[0051] Example 3 Analysis of the Effect of Molecular Marker SNP Site g.247 T>G

[0052] The present invention can significantly improve the total lean meat percentage of pigs by using a SNP molecular marker. Using the molecular marker for marker-assisted selection can accelerate the breeding process of the pig total lean meat percentage trait.

[0053] The effect of the dominant allele GG at the SNP locus g.247 (T>G) was significantly higher than the average phenotypes of TG and TT, by 2.69% and 3.82%, respectively. By gradually eliminating individuals with TG and TT phenotypes from the breeding pig population through marker-assisted selection, the gene frequency of the dominant allele G can be continuously increased, thereby increasing the overall lean meat percentage of the population and thus increasing the company's profits.

Claims

1. A SNP molecular marker located on chromosome 10 of pigs and associated with total lean meat percentage, characterized in that Its SNP site corresponds to the T>G mutation at position 11840666bp on chromosome 10 of the international pig reference genome Ensembl Sscrofa11.1 version.

2. The SNP molecular marker according to claim 1, wherein: The nucleotide sequence of the SNP molecular marker is SEQ ID NO. 1, wherein M in the sequence is T or G.

3. The SNP molecular marker according to claim 1, wherein: The M is marked at position 247 in the SEQ ID NO: 1 sequence.

4. A primer pair for amplifying the nucleotide sequence of the SNP molecular marker according to claim 2, characterized in that The primer comprises primer P001-F and primer P002-R, and the base sequences thereof are SEQ ID NO.2 and SEQ ID NO.

3.

5. Use of the SNP molecular marker according to claim 1 or 2 or the primer pair according to claim 4 in pig assisted breeding.

6. The use according to claim 5, characterized in that: The pig assisted breeding is a genetic improvement that increases the total lean meat rate of pigs.

7. A method for genetic improvement of pigs, characterized by: Detect the SNP molecular marker according to claim 1 or 2, eliminate individuals with TG genotype and TT genotype at the SNP site, retain individuals with GG genotype at the SNP site as breeding pigs, and increase the frequency of the dominant allele G at the site generation by generation, thereby increasing the total lean meat rate of offspring pigs.

8. The method for genetic improvement of pigs according to claim 7, characterized in that: The pig is a Duchangda three-way commercial pig.

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