SNP molecular markers associated with lean meat percentage on pig chromosome 1 and their applications

Abstract

This invention discloses a SNP molecular marker located on pig chromosome 1 that is associated with lean meat percentage and its application. The SNP molecular marker is located at position 49 from the 5' end of the nucleotide sequence shown in SEQ ID NO:1, corresponding to position 161507722 bp on chromosome 1 of the International Swine Reference Genome Version 11.1, where an A>T mutation exists. The SNP molecular marker provided by this invention is significantly associated with the lean meat percentage trait in pigs. By identifying the genotype of this SNP molecular marker, the lean meat percentage trait can be identified. Furthermore, by selecting pigs with the AA genotype of the SNP molecular marker, the pig breeding process can be accelerated, achieving genetic improvement of pigs.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology and relates to an SNP molecular marker related to lean meat percentage and its application. The SNP molecular marker is located on pig chromosome 1. Background Technology

[0002] Lean Meat Percentage (LMP) refers to the percentage of lean meat mass in the total carcass weight, and is a key indicator for evaluating the quality grade of pig carcasses. With rising living standards, consumers are increasingly emphasizing the quality of pork. In most parts of my country, pork has traditionally been the main component of the overall meat consumption structure; therefore, there is significant demand and economic value for breeding high-quality lean-type pigs. High fat content in pork not only harms consumer health but also causes substantial economic losses to the livestock industry. Therefore, improving lean meat percentage and yield has become a key focus for livestock enterprises.

[0003] Lean meat percentage is a complex quantitative trait with a relatively complex genetic structure. Phenotypic selection alone is insufficient to achieve the desired results. It is necessary to identify genetic markers related to growth traits to improve this type of trait. Summary of the Invention

[0004] The purpose of this invention is to provide a SNP molecular marker located on chromosome 1 of pigs that is associated with lean meat percentage and its application.

[0005] According to one aspect of the present invention, a SNP molecular marker associated with lean meat percentage is provided on chromosome 1 of a pig. The site is located at position 49 from the 5' end of the nucleotide sequence shown in SEQ ID NO:1, corresponding to position 161507722 bp on chromosome 1 of the International Swine Reference Genome Version 11.1. This site contains an A>T mutation (a single base mutation, named: NC010443.5:g.161507722A>T), and the nucleotide type is A or T. The genotype of the SNP molecular marker is AA, TA, or TT.

[0006] The SNP molecular marker provided by this invention is significantly correlated with the lean meat percentage trait in pigs. Specifically, pigs with the AA genotype have a significantly higher lean meat percentage than pigs with the TA and TT genotypes. By identifying the single nucleotide polymorphism (SNP) of this SNP molecular marker and / or the genotype of this SNP molecular marker, the lean meat percentage trait in pigs can be identified. Furthermore, by selecting pigs with the AA genotype of the SNP molecular marker, the pig breeding process can be accelerated, achieving genetic improvement in pigs.

[0007] Therefore, the SNP molecular markers related to lean meat percentage on chromosome 1 of pigs provided by this invention can be applied to:

[0008] (1) Identify the lean meat percentage trait of pigs;

[0009] (2) Prepare products for identifying the lean meat percentage trait of pigs;

[0010] (3) Pig genetic improvement: pig genetic improvement is achieved by selecting pigs with the SNP molecular marker genotype AA.

[0011] (4) Prepare a product for assisting in the genetic improvement of pigs, which is based on the identification of the genotype of pig SNP molecular markers to assist in the genetic improvement of pigs.

[0012] In some implementations, the pigs are preferably American Duroc strains or their synthetic lines.

[0013] According to another aspect of the present invention, a primer pair is provided that can specifically amplify an amplified fragment containing a single nucleotide polymorphism at position 49 from the 5' end of the nucleotide sequence shown in SEQ ID NO:1, wherein the nucleotide sequence of the upstream primer is shown in SEQ ID NO:2 and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:3.

[0014] The primer pair provided by this invention can specifically amplify fragments containing the SNP molecular marker associated with lean meat percentage located on pig chromosome 1 provided by this invention. This can be used to identify whether the 49th nucleotide from the 5' end in the nucleotide sequence shown in SEQ ID NO:1 is A or T. Therefore, the primer pair provided by this invention can be used to identify the genotype of the SNP molecular marker associated with lean meat percentage located on pig chromosome 1 and / or to prepare products for identifying the genotype associated with lean meat percentage located on pig chromosome 1.

[0015] According to a third aspect of the present invention, a kit is provided comprising primer pairs with nucleotide sequences as shown in SEQ ID NO:2 and SEQ ID NO:3.

[0016] The applications of the primer pairs and kits provided by this invention include, but are not limited to:

[0017] (1) Identify the genotype of SNP molecular markers associated with lean meat percentage located on chromosome 1 of pigs;

[0018] (2) Prepare products to identify the genotypes of SNP molecular markers located on chromosome 1 of pigs that are associated with lean meat percentage;

[0019] (3) Identify the lean meat percentage trait of pigs. The identification of the lean meat percentage trait of pigs is achieved by identifying the genotype of the SNP molecular markers related to lean meat percentage located on chromosome 1 of pigs.

[0020] (4) Prepare a product for identifying the lean meat percentage trait of pigs. This product is based on identifying the genotype of the SNP molecular markers related to lean meat percentage located on chromosome 1 of pigs to identify the lean meat percentage trait of pigs.

[0021] (5) Pig genetic improvement, based on the selection of pigs with the genotype AA, which is a molecular marker of SNP related to lean meat percentage on chromosome 1 of pigs;

[0022] (6) Prepare a product for assisting in the genetic improvement of pigs, which is based on the identification of the genotype of SNP molecular markers located on chromosome 1 of pigs that are associated with lean meat percentage to assist in the genetic improvement of pigs.

[0023] In some embodiments, the kit provided by the present invention may further include: dNTPs, DNA polymerase, and Mg. 2+ The components of a standard PCR reaction system, including PCR reaction buffer, can be directly referenced or adopted from the relevant components of commercially available PCR amplification kits.

[0024] According to a fourth aspect of the present invention, a method for genetic improvement of pigs is provided, comprising the following steps:

[0025] (1) Determine the genotype of the SNP molecular markers on chromosome 1 of pigs that are associated with lean meat percentage;

[0026] (2) Select individuals with the SNP molecular marker genotype AA and eliminate individuals with the genotypes TA and TT, and increase the frequency of the allele A at the SNP molecular marker locus generation by generation; thereby improving the lean meat rate of offspring pigs and increasing the meat yield of offspring pigs.

[0027] In some implementations, step (1), determining the genotype of the SNP molecular marker on chromosome 1 of the pig that is associated with lean meat percentage, may include the following steps:

[0028] Whole-genome DNA was extracted from pigs and PCR amplification was performed using primer pairs with nucleotide sequences as shown in SEQ ID NO:2 and SEQ ID NO:3. The amplification products were sequenced, and the genotype of the SNP molecular markers related to lean meat percentage located on chromosome 1 of pigs was determined based on the sequencing results.

[0029] In some implementations, the pigs are American Duroc strains and their synthetic lines.

[0030] Compared with the prior art, the beneficial effects of the present invention include:

[0031] (1) This invention provides a SNP molecular marker located on the nucleotide sequence of chromosome 1 of pigs that is related to the lean meat percentage of pigs, and verifies its effect on the lean meat percentage trait of pigs. This helps to establish a molecular marker-assisted selection breeding technology for rapid improvement of the lean meat percentage trait of pigs, improve the breeding process of Duroc and its synthetic lines, so as to meet the needs of the breeding pig market, increase the price of breeding pigs, and reduce breeding costs.

[0032] (2) This invention provides a primer pair that can be used to identify SNP molecular markers located on chromosome 1 of pigs that are related to lean meat percentage. Through this primer pair, an efficient and accurate molecular marker-assisted breeding technology can be established to quickly and accurately select traits and accelerate the breeding process. Attached Figure Description

[0033] Figure 1 This is a Manhattan plot of genome-wide association analysis (GWAS) on lean meat percentage in American Duroc pigs on chromosome 1; where: the horizontal axis represents the distribution on chromosome 1 of pigs; the vertical axis represents the statistical significance - log P value;

[0034] Figure 2 This is a graph showing the phenotypic ratios of lean meat percentage in pigs of different genotypes. Detailed Implementation

[0035] The present invention will be further described in detail below with reference to the embodiments. The embodiments are for illustrative purposes only and do not limit the invention in any way. Unless otherwise specified, the raw materials and reagents used in the embodiments are conventional products that can be obtained commercially; experimental methods that do not specify specific conditions in the embodiments are generally performed under conventional conditions in the art or according to the conditions recommended by the manufacturer.

[0036] Example 1: Identification and Validation of SNPs Related to Lean Meat Percentage

[0037] (1) Experimental pig herd

[0038] The experimental pig population used in this invention consisted of 3,760 purebred American Duroc pigs from the pig farming experimental department of Wens Foodstuff Group Co., Ltd. This herd is the core group of the pig farming division, and the herd's pedigree is meticulously recorded. During the rearing process, the pigs had free access to feed and water, and the feeding methods and conditions remained consistent throughout, following conventional practices.

[0039] (2) Method for measuring lean meat percentage

[0040] This invention estimates lean meat percentage (LMP) based on back fat thickness (BF) and eye muscle depth (LMD), and the specific method is as follows:

[0041] When the live weight of pigs reaches 100±5kg, the backfat thickness and eye muscle depth between the 10th and 11th ribs are measured using an Aloka 500V SSD B-type ultrasound machine (Corometrics Medical Systems, USA). The lean meat percentage is then estimated using the following formula:

[0042] LMP(%)=61.21920-0.77665*BF+0.15239*LMD

[0043] (3) Extraction of porcine genomic DNA

[0044] Ear tissue or tail tissue from piglets was collected from the above 3,760 American Duroc breeding pigs, soaked in a 75% ethanol solution, and stored at -20°C for later use.

[0045] Whole-genome DNA was extracted from pigs using the standard phenol-chloroform method. The concentration and OD ratio (OD260 / 280, OD260 / 230) of each sample were accurately determined using a NanoDrop 2000 / 2000C nucleic acid and protein analyzer. DNA samples that passed the NanoDrop 2000 / 2000C nucleic acid and protein analyzer test were diluted to approximately 50 ng / μL. 6 μL of the extracted DNA sample was then mixed with 2 μL of loading buffer and loaded onto a 1% (w / v) agarose gel. Electrophoresis was performed at 150 V for 25 min. The DNA integrity was observed and photographed using a UV spectrophotometer and gel imaging device.

[0046] (4) Detection of 50K SNP genotypes in the whole pig genome

[0047] DNA samples were sent to Neogene Biotech (Shanghai) Co., Ltd., where genotyping of the pig whole genome using a 50K SNP chip (Illumina, USA) was performed on the Illumina Beadstration platform according to the company's standard procedures.

[0048] The 50K chip scan genotyping data of all samples were quality controlled using PLINK v1.9 software. Individuals with a detection rate below 90%, a genotype deletion rate greater than 0.1, a family Mendelian error rate greater than 0.1, a minimum allele frequency less than 0.05, and a Hardy-Weinberg equilibrium significance level less than 10 were excluded. -6 SNP.

[0049] Then, genotyping was performed using the SWIM database (https: / / swim.scau.pigselection.com / swim / ). This resulted in valid genotype data for 561,032 SNPs.

[0050] (5) Genome-wide association analysis (GWAS)

[0051] To eliminate population stratification effects, this invention employs a linear mixture model in GEMMA software for GWAS analysis. Kinship is incorporated into the model to correct for population structure, and the stratification effect is corrected using genomic similarity among individuals. Because the Bonferroni correction method is too stringent, and this invention involves numerous hypotheses requiring testing, the significance level after correction would be significantly reduced, leading to many results failing the significance test. Therefore, this invention selects a higher significance level of 1e-5 (1×10⁻⁵). -5 () is used as a threshold to avoid overly strict calibration, thus making it easier to find significant results.

[0052] GWAS analysis results are as follows Figure 1 As shown.

[0053] from Figure 1 It was found that there are SNP sites on chromosome 1 of the American Duroc line that significantly affect lean meat percentage, with the strongest association being NC 010443.5:g.161507722A>T (P=5.69×10). -7 The nucleotide 49 from the 5' end in SEQ NO.1 corresponds to the A>T mutation at 161507722bp on chromosome 1 of the International Pig Reference Genome Version 11.1.

[0054] (6) Analyze the association between different genotypes and lean meat percentage phenotype to verify the effect of SNP loci on lean meat percentage trait.

[0055] The results are shown in Table 1. Figure 2 As shown in Table 1. Figure 2 It can be known that:

[0056] (1) The SNP site NC 010443.5:g.161507722A>T of the molecular marker was highly significantly correlated with the lean meat percentage trait (P<0.001), indicating that this molecular marker significantly affects the lean meat percentage trait of pigs. The breeding process of the lean meat percentage trait of pigs can be accelerated by assisted selection of this SNP site.

[0057] (2) The lean meat percentage of pigs with the SNP molecular marker genotype AA was significantly higher than that of pigs with the TA and TT genotypes. The TA genotype had a higher lean meat percentage than the TT genotype, indicating that allele T is detrimental to the lean meat percentage of breeding pigs. Therefore, AA-type breeding pigs should be prioritized. Lean meat percentage is an important indicator for measuring carcass quality grade, and improving the lean meat percentage of pigs is beneficial to increasing economic benefits. Therefore, gradually retaining AA-type breeding pigs in breeding to increase the frequency of allele A at this locus through each generation can significantly improve the lean meat percentage trait of breeding pigs, bringing greater economic benefits to enterprises.

[0058] Table 1. Correlation analysis between SNP locus NC 010443.5:g.161507722A>T and lean meat percentage

[0059]

[0060] (7) Effect analysis

[0061] This invention provides a SNP molecular marker that is significantly associated with the lean meat percentage trait in Duroc pigs. Using this SNP molecular marker for marker-assisted selection can accelerate the lean meat percentage breeding process in Duroc pigs. Through marker-assisted selection, if all TT-type individuals with molecular markers affecting the lean meat percentage trait in pigs are selected to become AA-type individuals, the lean meat percentage trait in Duroc pigs will increase by 0.34%.

[0062] Example 2: Methods for genetic improvement of pigs

[0063] The target fragment containing SNP sites that are significantly associated with the lean meat percentage trait of American Duroc is a 128bp nucleotide sequence from chromosome 1, the specific sequence of which is shown in SEQ ID NO:1, and the primer pairs for its PCR amplification are shown in SEQ ID NO:2 and SEQ ID NO:3.

[0064] SEQ ID NO:1

[0065]

[0066] The W marked in the sequence is the mutation site, which is A or T, indicating an allele mutation; the bolded beginning and end of the sequence indicate the primer binding position.

[0067] Upstream primer-F: 5'-TCATCTGCCAGTAGACATCT-3' (SEQ ID NO: 2);

[0068] Downstream primer primer-R: 5'-ATGACTCAGCAATTCCACTT-3' (SEQ ID NO: 3).

[0069] The genetic improvement methods for pigs include the following steps:

[0070] S1. Determine the genotypes of SNP molecular markers associated with lean meat percentage in pigs.

[0071] (1) Collect ear tissue from pigs or tail tissue from piglets, extract whole genome DNA from pigs using the standard phenol-chloroform method, and then perform quality testing and concentration determination on the extracted DNA.

[0072] (2) PCR amplification

[0073] Prepare a 10 μL mixture, including: 1 μL DNA sample, 0.3 μL upstream primer, 0.3 μL downstream primer, 5 μL PCR mix, and 3.4 μL ddH2O. The PCR mix can be commercially available 2×TaqPCR Star Mix (Dye) (manufacturer: GenStar, catalog number: A012-101), which includes dNTPs, DNA polymerase, and Mg2+. 2+ Components of conventional PCR reaction systems, such as PCR reaction buffer.

[0074] PCR reaction program: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 45 s, for a total of 35 cycles, and a final extension at 72℃ for 5 min.

[0075] (3) DNA sequence sequencing identification

[0076] The PCR amplification products were sequenced, and gene fragments were measured in both forward and reverse reactions. Based on the sequencing results, the genotype of the pig SNP locus NC 010443.5:g.161507722A>T was determined.

[0077] S2. Select pigs with the genotype AA at the SNP locus as parents for breeding, and increase the frequency of the allele A at this locus generation by generation.

[0078] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. The application of products that detect SNP molecular markers located on pig chromosome 1 that are associated with lean meat percentage, characterized in that, The SNP molecular marker is located at position 161,507,722 bp on chromosome 1 of the International Swine Reference Genome Version 11.

1. The nucleotide type at this site is A or T, and the genotype of the SNP molecular marker is AA, TA, or TT. The single nucleotide polymorphism of the SNP molecular marker site affects the lean meat percentage trait. Specifically, the lean meat percentage of pigs with the SNP molecular marker genotype AA is greater than that of pigs with the genotypes TA and TT. The pigs in question are American Duroc strains and their synthetic lines; The applications include: (1) Identify the lean meat percentage trait of pigs; (2) Prepare products for identifying the lean meat percentage trait of pigs; (3) Pig genetic improvement, based on the selection of pigs with the genotype AA, which is a molecular marker of SNP related to lean meat percentage on chromosome 1 of pigs; (4) Prepare a product for assisting in the genetic improvement of pigs, the product being based on the identification of the genotype of the SNP molecular markers on chromosome 1 of pigs that are associated with lean meat percentage to assist in the genetic improvement of pigs.

2. The application of primer pairs, characterized in that, The primer pair specifically amplifies a fragment containing the single nucleotide polymorphism at position 49, starting from the 5' end, of the nucleotide sequence shown in SEQ ID NO:1; wherein... The nucleotide sequence shown in SEQ ID NO:1 has an A or T at position 49 from the 5' end. The corresponding SNP molecular marker on chromosome 1 of pigs that is related to lean meat percentage has a genotype of AA, TA or TT. Pigs with genotype AA have a higher lean meat percentage than pigs with genotype TA and genotype TT. In the primer pair, the nucleotide sequence of the upstream primer is shown in SEQ ID NO:2, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:3; The applications include: (1) Identify the genotype of SNP molecular markers associated with lean meat percentage located on chromosome 1 of pigs; (2) Prepare products to identify the genotypes of SNP molecular markers located on chromosome 1 of pigs that are associated with lean meat percentage; (3) Identify the lean meat percentage trait of pigs by identifying the genotype of the SNP molecular markers related to lean meat percentage located on chromosome 1 of pigs. (4) Prepare a product for identifying the lean meat percentage trait of pigs, wherein the product is based on identifying the genotype of the SNP molecular markers related to lean meat percentage located on chromosome 1 of pigs to achieve the identification of the lean meat percentage trait of pigs; (5) Pig genetic improvement, based on the selection of pigs with the genotype AA, which is a molecular marker of SNP related to lean meat percentage on chromosome 1 of pigs; (6) Prepare a product for assisting in the genetic improvement of pigs, said product being based on the identification of genotypes of SNP molecular markers located on chromosome 1 of pigs that are associated with lean meat percentage to assist in the genetic improvement of pigs.

3. The application of the reagent kit, characterized in that, Its composition includes a primer pair, wherein the nucleotide sequence of the upstream primer is shown in SEQ ID NO:2 and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:3; The applications include: (1) Identify the genotype of SNP molecular markers associated with lean meat percentage located on chromosome 1 of pigs; (2) Prepare products to identify the genotypes of SNP molecular markers located on chromosome 1 of pigs that are associated with lean meat percentage; (3) Identify the lean meat percentage trait of pigs by identifying the genotype of the SNP molecular markers related to lean meat percentage located on chromosome 1 of pigs. (4) Prepare a product for identifying the lean meat percentage trait of pigs, wherein the product is based on identifying the genotype of the SNP molecular markers related to lean meat percentage located on chromosome 1 of pigs to achieve the identification of the lean meat percentage trait of pigs; (5) Pig genetic improvement, based on the selection of pigs with the genotype AA, which is a molecular marker of SNP related to lean meat percentage on chromosome 1 of pigs; (6) Prepare a product for assisting in the genetic improvement of pigs, said product being based on the identification of the genotype of a SNP molecular marker on chromosome 1 of pigs that is associated with lean meat percentage to assist in the genetic improvement of pigs.

4. A method for genetic improvement of pigs, characterized in that, Includes the following steps: (1) Determine the genotype of the SNP molecular markers on chromosome 1 of pigs that are associated with lean meat percentage; (2) Select individuals with the SNP molecular marker genotype AA and eliminate individuals with the genotypes TA and TT, and increase the frequency of the allele A at this SNP molecular marker locus generation by generation; The SNP molecular marker associated with lean meat percentage on chromosome 1 of pig is located at position 49 from the 5' end of the nucleotide sequence shown in SEQ ID NO:1, corresponding to position 161507722 bp on chromosome 1 of International Swine Reference Genome Version 11.

1. The nucleotide type of this site is A or T, and the genotype is AA, TA or TT. The pigs in question are American Duroc strains and their synthetic lines.

5. The method for genetic improvement of pigs according to claim 4, characterized in that, In step (1), the method for determining the genotype of the SNP molecular markers related to lean meat percentage located on chromosome 1 of pigs includes the following steps: Whole-genome DNA was extracted from pigs and PCR amplification was performed using primer pairs with nucleotide sequences as shown in SEQ ID NO:2 and SEQ ID NO:

3. The amplification products were sequenced, and the genotype of the SNP molecular markers related to lean meat percentage located on chromosome 1 of pigs was determined based on the sequencing results.

Images