A molecular marker associated with pig eye muscle area

Abstract

The present application discloses a technical field of pig eye muscle area, specifically a molecular marker associated with pig eye muscle area. The molecular marker includes a nucleotide sequence formed by a single nucleotide polymorphism A / G at position 3593429 of chromosome 10 of the International Porcine Reference Genome 11.1. The SNP marker provided in the present application is significantly correlated with the eye muscle area of ​​Large White pig muscle. By identifying the SNP marker, Large White pig strains with large eye muscle area can be screened. The resulting Large White pig strains with large eye muscle area have important economic benefits, edible value, and social value.

Description

Technical Field

[0001] The present application relates to the technical field of pig eye muscle area, and in particular to a molecular marker associated with pig eye muscle area. Background Art

[0002] Loin muscle area (LMA) refers to the cross-sectional area of ​​the longissimus dorsi muscle between the penultimate and second thoracic vertebrae. It is of great significance in the research of livestock, including pigs, cattle, and sheep. In pigs, loin muscle area is the cross-sectional area of ​​the longissimus dorsi muscle at the last rib, measured using a planimeter. The formula for calculating loin muscle area is generally: loin height (cm) × loin width (cm) × 0.7. Loin muscle area is a key indicator of pig growth performance and carcass meat production. It is directly correlated with lean meat percentage and backfat depth, traits crucial to the pig industry. Lean pigs occupy a significant share of the pork market, and carcass lean meat percentage is a crucial indicator of carcass quality. Studies have shown a strong correlation between lean meat percentage and loin muscle area. Estimating lean meat percentage using loin muscle area can significantly reduce costs, making loin muscle area a crucial indicator in the production and breeding of lean pigs.

[0003] In breeding practice, measuring loin eye area plays a crucial role in selecting pigs with superior genetic traits for breeding, thereby improving overall meat quality and performance. By measuring loin eye area, farmers can select pigs with larger loin eye areas for breeding, thereby increasing pig production efficiency. Therefore, loin eye area is not only an important meat quality indicator, but also a key parameter for genetic improvement and breeding in the pig industry. Summary of the Invention

[0004] The inventors of the present application used genome-wide association analysis (GWAS) to screen and obtain a molecular marker associated with the eye muscle area of ​​pigs. The marker was located at the single nucleotide polymorphism site A / G at position 3593429 of pig chromosome 10 (Ensembl database number rs81425149). Among them, the eye muscle area of ​​individuals with the AA genotype at this site was higher than that of other individuals, indicating that the A-type nucleotide at this site can serve as an allele that is beneficial to the eye muscle area of ​​Large White pigs.

[0005] To this end, the embodiments of the present application disclose at least the following technical solutions:

[0006] In a first aspect, the embodiments disclose molecular markers associated with pig eye muscle area, including a nucleotide sequence formed by a single nucleotide polymorphism A / G at position 3593429 of chromosome 10 of the International Pig Reference Genome 11.1.

[0007] In a second aspect, the embodiment discloses a primer pair for detecting the molecular marker described in the first aspect, and the nucleotide sequences of the primer pair are shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0008] In a third aspect, the embodiment discloses a kit comprising the primer pair described in the second aspect and reagents required for PCR amplification.

[0009] In a fourth aspect, an embodiment discloses a method for detecting the molecular markers described in the first aspect, the method comprising the following steps: extracting the genomic DNA of the pig to be tested; performing PCR amplification on the genomic DNA of the pig to be tested to obtain a PCR amplification product; sequencing the PCR amplification product to obtain a sequencing result; based on the sequencing result, interpreting the polymorphism of the 3593429nt site on chromosome 10 of the International Porcine Reference Genome 11.1 version.

[0010] In a fifth aspect, the embodiments disclose molecular markers associated with pig eye muscle area, including nucleotide sequences shown as SEQ ID NO:3 and SEQ ID NO:4.

[0011] In the sixth aspect, an embodiment discloses a method for breeding pig breeds with large eye muscle area, the method comprising the following steps: detecting the genotype of the 3593429th nucleotide site of chromosome 10 of the International Pig Reference Genome 11.1 version, and selecting the AA type individual at the 3593429th nucleotide site as a breeding pig.

[0012] In a seventh aspect, an embodiment discloses a method for breeding a pig breed with large eye muscle area, comprising: if a nucleotide sequence as shown in SEQ ID NO: 3 is detected in the pig genomic DNA, the corresponding homozygous genotype individual is used as a breeding pig.

[0013] In an eighth aspect, the embodiments disclose the use of a single nucleotide polymorphism (SNP) site or primer pair associated with intramuscular fat content in Large White pigs for screening pig lines with large loin eye area. The SNP site is located at nucleotide position 3593429 on chromosome 10 of the International Porcine Genome Version 11.1 reference sequence and has an A / G polymorphism. The nucleotide sequences of the primer pair are shown in SEQ ID NO: 1 and SEQ ID NO: 2. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Manhattan plot of the pig eye muscle area trait provided in the example. In the figure, the black circle points to the molecular marker located on chromosome 10 of the Large White pig obtained by screening.

[0015] Figure 2This is a 2% agarose gel electrophoresis analysis of the amplified products of individual pig genomic DNA provided in the examples. M represents a 1000bp marker molecular weight standard, and 1 and 2 represent DNA products amplified from Large White pig genomic DNA at SNP mutation sites G and A, respectively. The 1000bp marker molecular weight bands, from top to bottom, are 1000bp, 700bp, 500bp, 400bp, 300bp, 200bp, and 100bp.

[0016] Figure 3 The sequencing peaks of the amplified pig genomic DNA products provided in the examples were aligned with the relevant pig gene fragments in GenBank. The top figure shows the sequence alignment result of the pig chromosome 10 at position 3593429, which is G, and the bottom figure shows the sequence alignment result of the pig chromosome 10 at position 3593429, which is A. DETAILED DESCRIPTION

[0017] In order to make the purpose, technical solutions and advantages of this application more clearly understood, the present application is further described in detail below with reference to the following examples. It should be understood that the specific examples described herein are merely for the purpose of explaining this application and are not intended to limit this application. Reagents not described in detail in this application are all conventional reagents and can be obtained from commercial channels; methods not specifically described in detail are all conventional experimental methods and can be obtained from the prior art.

[0018] Pig eye loin area is influenced by multiple factors, including genetics, environment, and management. Therefore, to better predict pig eye loin area, it's necessary to consider multiple factors, not just the effects of a single molecular marker. Secondly, due to the large number of pig breeds and the significant genetic variation between populations, systematic research across multiple breeds is needed to identify more reliable and effective molecular markers.

[0019] The present application uses genome-wide association analysis (GWAS) to detect genetic marker polymorphisms across the genome of multiple individuals to obtain genotypes, and then conducts statistical analysis of the genotypes and phenotypes at the population level. Based on the significance test, the SNP sites that are most likely to affect the trait are screened to find genes related to pig eye muscle area. The present application found that the single nucleotide polymorphism site (Ensembl database number rs81425149) with A / G occurs at the 3593429 nt position of pig chromosome 10. Among them, the eye muscle area of ​​individuals with the AA genotype at this site is higher than that of other individuals, indicating that the A-type nucleotide at this site can be used as an allele that is beneficial to obtaining the eye muscle area of ​​tall white pigs.

[0020] Based on this, the embodiment discloses a molecular marker associated with the pig eye muscle area, including a nucleotide sequence formed by the A / G single nucleotide polymorphism at position 3593429 of chromosome 10 of the pig international reference genome version 11.1.

[0021] The embodiment discloses a primer pair for detecting the molecular marker, and the nucleotide sequences of the primer pair are shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0022] The embodiment also discloses a kit, comprising the primer pair and reagents required for PCR amplification.

[0023] The embodiment discloses a method for detecting the molecular marker, which includes the following steps: extracting genomic DNA from the pig to be tested; performing PCR amplification on the genomic DNA of the pig to be tested to obtain a PCR amplification product; sequencing the PCR amplification product to obtain a sequencing result; and based on the sequencing result, interpreting the polymorphism of the nucleotide site at position 101 in the sequence shown in pig SEQ ID NO:1.

[0024] The embodiment discloses a method for breeding pig breeds with large eye muscle area, which includes the following steps: detecting the genotype of the 3593429th nucleotide site of chromosome 10 of the pig international reference genome version 11.1, and selecting AA type individuals at the 3593429th nucleotide site as breeding pigs.

[0025] The embodiment discloses a method for breeding pigs with large eye muscle area, comprising: if the nucleotide sequence shown in SEQ ID NO: 3 is detected in the pig genomic DNA, the corresponding homozygous genotype individual is used as a breeding pig.

[0026] The present invention discloses the use of a single nucleotide polymorphism (SNP) site or primer pair associated with intramuscular fat content in Large White pigs for screening pig lines with large loin eye area. The SNP site is located at nucleotide position 3593429 on chromosome 10 of the International Porcine Genome Version 11.1 reference sequence and has an A / G polymorphism. The nucleotide sequences of the primer pair are shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0027] 1. Test sample collection

[0028] The experimental pigs were 744 purebred Large White boars (castrated, weighing approximately 90 kg) from a pig farm in Hubei Province. The pigs had free access to food and water, and the entire feeding method and rearing conditions remained consistent, following conventional methods.

[0029] Before slaughter, ear tissue samples were collected from all experimental pigs and stored in 75% ethanol for use in extracting pig genomic DNA. The specific method was based on the instructions provided by the genomic DNA kit produced by Beijing Tiangen Biochemical Technology Co., Ltd.

[0030] 2. Extraction and Detection of Pig Genomic DNA

[0031] In this experiment, the TIANamp Genomic DNA Kit produced by Beijing Tiangen Biochemical Technology Co., Ltd. was used to extract pig genomic DNA from pig ear tissue. The specific steps are as follows:

[0032] 1) Use alcohol-sterilized ophthalmic surgical scissors to cut the pig ear sample into a paste, add 200 μL of buffer GA (provided with the kit), and shake until thoroughly suspended.

[0033] 2) Add 20 μL of proteinase K solution (provided with the kit), mix well, and digest overnight in a 56°C water bath.

[0034] 3) Add 200 μL of Buffer GB (provided with the kit), mix thoroughly by inversion, and incubate at 70°C for 10 minutes. When the solution becomes clear, centrifuge briefly to remove water droplets on the inner wall of the tube cap.

[0035] 4) Add 200 μL of anhydrous ethanol and vortex thoroughly for 15 seconds. Flocculent precipitation may occur at this time. Centrifuge briefly to remove water droplets on the inner wall of the tube cap.

[0036] 5) Add the solution and flocculent precipitate obtained in the previous step to an adsorption column CB3 (the adsorption column is placed in a collection tube), centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, and return the adsorption column CB3 to the collection tube.

[0037] 6) Add 500 μL of buffer GD (provided with the kit) to the adsorption column CB3, centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, and place the adsorption column CB3 in a collection tube.

[0038] 7) Add 600 μL of rinse solution PW (provided with the kit) to the adsorption column CB3, centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, and place the adsorption column CB3 in a collection tube.

[0039] 8) Repeat step 7.

[0040] 9) Place the adsorption column CB3 back into the collection tube and centrifuge at 12,000 rpm for 2 minutes. Discard the waste liquid and place the adsorption column CB3 at room temperature for several minutes to completely dry the residual rinse solution in the adsorption material.

[0041] 10) Transfer the adsorption column CB3 to a clean centrifuge tube, add 50-200 μL of elution buffer TE dropwise to the middle of the adsorption membrane, let it stand at room temperature for 2-5 minutes, centrifuge at 12000 rpm for 2 minutes, and collect the solution in the centrifuge tube.

[0042] 11) Take 2 μL of the DNA solution obtained in the previous step and mix it with 1 μL of loading buffer. Load it on a 1.2% agarose gel and perform electrophoresis at 120V for about 20 minutes. Observe the electrophoresis results under UV light and take pictures to determine the integrity of the DNA. The extracted DNA is quality tested using a NanoDrop 2000 nucleic acid and protein analyzer (Thermo Fisher Scientific, USA). The A260 / A280 ratio is between 1.7-2.1, and the A260 / A230 ratio is between 1.8-2.2. It is judged to be qualified. The qualified DNA is then concentrated and diluted to 200 ng / μL and stored in a refrigerator at -20°C. Unqualified DNA samples need to be re-extracted.

[0043] 3. SNP chip genotype determination and genotype data quality control

[0044] Genomic DNA samples extracted from 1,066 pig ears were hybridized to the Illumina Porcine SNP60 BeadChip6 whole-genome array. This array contains 61,565 SNP loci. Quality control of the raw genotype data for all individuals was performed using PLINK software. Quality control criteria included a SNP call rate > 90%, a minor allele frequency (MAF) > 0.01, a Hardy-Weinberg equilibrium (HWE) test P value < 10-6, and a sample call rate > 90%.

[0045] 4. Data Collation and Analysis

[0046] 1) Phenotypic data analysis

[0047] R statistical analysis software was used to perform descriptive statistical analysis on pig eye muscle area, including calculating the mean, standard deviation, maximum and minimum values ​​of the trait.

[0048] 2) Genome-wide association analysis

[0049] GWAS analysis was performed using PLINK software. The applicants used the following mixed model to analyze the data.

[0050] The model is: Yij = μ + Gi + εij

[0051] Among them, Yij is the trait value after treatment; μ is the mean of each trait; Gi is the genotype effect; εij is the random effect.

[0052] 3) Test the significance of the association between SNP and traits.

[0053] When a SNP meets P < 10 -4 If the condition is met, it indicates that the SNP has reached genome-wide significance.

[0054] 4) SNP annotation

[0055] Based on the SNP information from the array, the VariantEffectPredictor tool was used to annotate the SNPs in the SusscrofaBuild11.1 database on the Ensembl website (www.ensembl.org). This method determined the chromosome on which the SNP loci were located and their physical location within the chromosome. This allowed the researchers to determine whether these significant SNPs were within or flanking regions of known genes in the Ensembl database. Bioinformatics methods were then used to functionally annotate genes within the target regions based on information on gene structure, gene type, gene function, and pathways provided by Ensembl, NCBI (www.ncbi.nlm.nih.gov), and DAVID (david.abcc.ncifcrf.gov). Finally, the QTLdb website (cn.animalgenome.org / cgi-bin / QTLdb / index) was used to search for reported QTLs associated with meat quality traits, further identifying SNPs associated with pig loin area.

[0056] Table 1 shows the relationship between the genotypes of the SNP sites provided in the Examples and the eye muscle area phenotype. Table 1 shows the eye muscle area of ​​the AA, AG, and GG genotypes, and compares the significance levels of the eye muscle area between these genotypes. A p < 0.05 indicates a significant difference; a p < 0.01 indicates an extremely significant difference.

[0057] As shown in Table 1, among the 744 Large White pigs, 211 had the AA genotype, 288 had the AG genotype, and 245 had the GG genotype. The loin eye area of ​​individuals with the AA genotype was significantly greater than that of individuals with the AG and GG genotypes. There was no significant difference in loin eye area between individuals with the AG genotype and those with the GG genotype. In summary, the A allele is beneficial for loin eye area in Large White pigs.

[0058] Table 1

[0059]

[0060]

[0061] 5. PCR Testing

[0062] PCR amplification was performed on the porcine genomic DNA obtained according to the above steps using the primer pair set forth in SEQ ID NO: 1 and SEQ ID NO: 2. The 12 μL PCR reaction system contained 1 μL porcine genomic DNA, 0.5 μL DNA set forth in SEQ ID NO: 2, 0.5 μL DNA set forth in SEQ ID NO: 3, and 10 μL PCR Mix reagent (2× M5 Taq HiFi PCR Mix, Mei5bio). The PCR reaction steps included 94°C for 3 min; 30 cycles (94°C for 25 s; 60°C for 25 s; 72°C for 10 s); 72°C for 5 min; and 4°C for ∞.

[0063] Figure 2 The following figure shows a 2% agarose gel electrophoresis image of a PCR product obtained by PCR amplification of porcine genomic DNA in the above steps. In the figure, the target fragment amplified by the PCR product is approximately 369 bp in size. This 369 bp was sequenced to obtain the nucleotide sequences shown in SEQ ID NOs: 3 and 4. Figure 3 The results of sequence alignment between the sequence near the SNP site in the nucleotide sequence shown in SEQ ID NO: 3 and the relevant gene fragments of pigs in GenBank, and the results of sequence alignment between the sequence near the SNP site in the nucleotide sequence shown in SEQ ID NO: 4 and the relevant gene fragments of pigs in GenBank are shown.

[0064] The above is only a preferred specific implementation method of the present application, but the scope of protection of the present application is not limited thereto. Any changes or replacements that can be easily thought of by any technician familiar with this technical field within the technical scope disclosed in this application should be covered by the scope of protection of the present application.

Claims

1. A method for detecting molecular markers associated with pig eye muscle area in the selection of pig lines with large eye muscle area, the method comprising: Extracting genomic DNA from the pig to be tested; Performing PCR amplification on the genomic DNA of the pig to be tested to obtain a PCR amplification product; Sequencing the PCR amplification product to obtain a sequencing result; Based on the sequencing results, the polymorphism and genotype of the 3593429nt site on chromosome 10 of the International Porcine Reference Genome 11.1 version were determined, and individuals with the AA genotype were selected as breeding pigs.

2. A method for breeding pigs with large eye loin area, comprising: The genotype of the 3593429th nucleotide position of chromosome 10 of the international porcine reference genome version 11.1 was detected, and individuals with the AA genotype at the 3593429th nucleotide position were selected as breeding pigs.

3. A method for breeding a pig strain with large eye loin area, comprising: If the nucleotide sequence shown in SEQ ID NO: 3 is detected in the pig genomic DNA, an individual having the AA genotype at the 210th nucleotide position of the nucleotide sequence shown in SEQ ID NO: 3 is selected as a breeding pig.

4. The use of a primer pair for detecting a SNP molecular marker associated with pig eye muscle area in screening pig breeds with large eye muscle area. The SNP molecular marker is located at the 3593429th nucleotide site of chromosome 10 of the International Pig Reference Genome Version 11.1 and has an A / G polymorphism. The eye muscle area of ​​individuals with the AA genotype at this site is significantly higher than that of individuals with the AG and GG genotypes. The nucleotide sequences of the primer pair are shown in SEQ ID NO: 1 and SEQ ID NO: 2.

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