A molecular marker associated with traits of pig skin thickness and intramuscular fat content and its application.

Abstract

This application relates to the field of swine technology, specifically to a molecular marker associated with traits of skin thickness and intramuscular fat content in pigs and its application. The molecular marker is a SNP site at nucleotide 7315791 on chromosome 1, NC_010443.5, of the pig reference genome GCF 000003025.6. This molecular marker is closely associated with traits of skin thickness and intramuscular fat content in pigs. This molecular marker can be applied to molecular-assisted selection breeding of high-quality new pig breeds.

Description

Technical Field

[0001] This application relates to the field of pork technology, specifically to molecular markers and their applications associated with traits such as skin thickness and intramuscular fat content in pigs. Background Technology

[0002] The imbalance between supply and demand in the pork market is becoming increasingly apparent, thus, genetic improvement of pork traits is receiving more and more attention from breeders. Meat quality traits such as skin thickness and meat color score are important indicators for evaluating pork traits. Although pork traits are considered moderately heritable, their improvement through conventional breeding is difficult due to the high cost of in vivo testing. Therefore, identifying molecular markers that influence pork traits is crucial for improving them through molecular-assisted breeding.

[0003] Molecular markers used for selection assistance include protein markers, microsatellite markers, and single nucleotide polymorphism (SNP) markers. SNP markers refer to DNA sequence polymorphisms caused by single nucleotide variations in the genome. They are characterized by their large number, high accuracy, and high polymorphism. In breeding practice, SNPs can be used to locate certain superior genes, and combined with phenotypes to determine the association between markers and specific qualities. Molecular markers can also be validated in populations and applied to molecular breeding. Summary of the Invention

[0004] This application has discovered a single nucleotide polymorphism (SNP) of A / C at nucleotide 7315791 on chromosome 1, NC_010443.5, located on chromosome GCF 000003025.6 of the pig reference genome. This molecular marker is closely associated with meat quality traits such as skin thickness and meat color score. This molecular marker can be applied to molecular-assisted selection breeding of new high-quality pig breeds. Therefore, this application discloses at least the following technical solutions:

[0005] In one aspect, the embodiments disclose molecular markers associated with traits of skin thickness and intramuscular fat content in pigs, including nucleotide sequences formed by a single nucleotide A>C mutation at position 7315791 on chromosome 1, NC_010443.5 of the pig reference genome GCF 000003025.6.

[0006] Secondly, the embodiments disclose primer sets. The primer sets comprise DNA molecules as shown in SEQ ID NO:1 and 2. The primer sets are used to amplify nucleotide sequences containing single nucleotide mutation sites in the molecular markers described in the first aspect. In some embodiments, DNA molecules as shown in SEQ ID NO:1 and 2 are used to amplify nucleotide sequences containing single nucleotide A>C mutations at position 7315791 on chromosome 1, NC_010443.5, of the porcine reference genome GCF000003025.6.

[0007] Thirdly, the embodiments disclose a kit. The kit includes the primer set described in the second aspect. The kit is a PCR amplification kit, including the primer set described in the second aspect and other reagents for PCR amplification.

[0008] Fourthly, the embodiments disclose a method for screening individuals with superior pork quality traits using molecular markers. The method includes: extracting genomic DNA from the pig to be tested; performing PCR amplification on the genomic DNA using the primer set described in the second aspect; sequencing the PCR amplification product; determining the genotype of the pig at position 7315791 on chromosome 1 (NC_010443.5) of the reference genome GCF 000003025.6 based on the sequencing base peak diagram results; and identifying the dominant pig individuals in terms of meat quality traits based on the genotype.

[0009] In some embodiments, the meat quality characteristics include at least one of skin thickness and intramuscular fat content.

[0010] In some embodiments, the genotypes at position 7315791 on chromosome 1 (NC_010443.5) of the reference genome GCF 000003025.6 include AA, AC, and CC. Individuals with the CC genotype have significantly less skin thickness than those with the AA and AC genotypes. Individuals with the AA genotype have significantly lower intramuscular fat content than those with the AC and CC genotypes.

[0011] For breeding pigs used to supply pork in common pork dishes, selecting pigs with thin skin is more beneficial for improving the taste and makes them easier to handle and flavor during cooking. Therefore, individuals with the CC genotype are selected as superior pigs.

[0012] For pigs selected for high-quality marbled pork with high intrafat content, AA genotype pigs should be culled.

[0013] Considering the overall meat quality performance, priority should be given to breeding individuals with the CC genotype, which have thin skin and high intramuscular fat content, resulting in better meat quality.

[0014] Fifthly, the embodiments disclose the application of the molecular markers described in the first aspect, the primer sets described in the second aspect, or the kits described in the third aspect in the detection and analysis of pork traits, and in the screening of individuals with superior pork quality traits. Attached Figure Description

[0015] Figure 1 The image provided in this example shows an agarose gel electrophoresis diagram of the PCR amplification product containing the target SNP site.

[0016] Figure 2The genotyping and sequencing peak diagram of porcine SNP chr1:7315791 provided for the example.

[0017] Figure 3 The image shows a visual representation of the porcine SNP chr1:7315791 nucleotide sequence provided in the example (shown as SEQ ID NO:3 or SEQ ID NO:4). The red box indicates the location of the mutation, and the red sequence indicates the primer position. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Reagents not specifically described in detail herein are all conventional reagents and are commercially available; methods not specifically described in detail are all conventional experimental methods and can be learned from the prior art.

[0019] I. Extraction of porcine genomic DNA

[0020] 1. The experimental pig breed used in this application is the Xidu Black Pig, and the samples were obtained from the original breeding pig farm of the Animal Husbandry Institute of Hubei Academy of Agricultural Sciences. Genomic DNA was extracted using a genomic DNA kit produced by Beijing Baitaike Biotechnology Co., Ltd., following the kit's instructions. The concentration and quality of the extracted DNA were tested and then stored at -20℃ for later use. The remaining muscle samples were sealed in bags and stored at 4℃, then sent to the Ministry of Agriculture's Breeding Pig Quality Supervision and Testing Center (Wuhan) of Huazhong Agricultural University within 4 hours for pork trait determination according to the People's Republic of China Agricultural Industry Standard "Technical Specifications for Determination of Pork Traits" (Standard No.: NY / T821-2019).

[0021] II. Preparation of target fragments containing target SNP sites and detection of target SNP sites.

[0022] 1. Prepare the target fragment containing the target SNP site.

[0023] (1) PCR amplification

[0024] Design and synthesize the following primer pairs:

[0025] Forward primer F: 5'-CTCCACCACCAGCACGACCAG-3', SEQ ID NO:1

[0026] Reverse primer R: 5'-CCCAACCTTATTCTCCCCCTA-3', SEQ ID NO:2

[0027] The above primers were used to perform PCR amplification in a pool of mixed genomic DNA from 40 selenium-rich black pigs. The PCR reaction system was 50 μL, and the concentrations of each component in the system were 100 ng template DNA, 4 μL of 10× buffer (containing Mg2+), 0.5 μM of the above upstream and downstream primers, 2.5 μM dNTPs, and 1 U Taq DNA polymerase.

[0028] The PCR procedure was as follows: preheating at 98℃ for 45 seconds; denaturation at 98℃ for 10 seconds, annealing at 62℃ for 30 seconds, extension at 72℃ for 30 seconds, for a total of 34 cycles; extension at 72℃ for 10 minutes; storage at 4℃. PCR products were subjected to 1.5% agarose gel electrophoresis.

[0029] (2) Purification of PCR products

[0030] The PCR products were purified using the Gel Extraction Kit from Shanghai Sangon Biotech Co., Ltd. (following the kit's instructions). The specific steps are as follows: First, cut the gel containing the target fragment from the agarose gel and place it in a 1.5 mL centrifuge tube. Add 400 μL of sol solution and incubate at 50-60°C until the gel is completely melted. While melting the gel, mix every 2 minutes. Cool to room temperature. Place the centrifuge column into a collection tube, transfer the mixture to the column, and incubate at room temperature for 2 minutes. Centrifuge at 12000 rpm for 1 minute. At this point, DNA is adsorbed onto the column. Discard the contents of the collection tube. Collect the waste liquid in the collection tube, place the centrifuge column into the same collection tube, add 700 μL of elution buffer, and centrifuge at 12000 rpm for 1 min; discard the waste liquid in the collection tube, and centrifuge at 12000 rpm for 1 min; place the centrifuge column into a pre-prepared sterile 1.5 mL centrifuge tube, add 40 μL of elution buffer or double-distilled water (pH>7.0), and incubate at room temperature or 37°C for 2-3 min; centrifuge at 12000 rpm for 1 min, and the liquid in the centrifuge tube is the recovered DNA fragment.

[0031] 2. Detection of the target SNP site

[0032] The recovered DNA fragments were sent to Wuhan Aoke Dingsheng Biotechnology Co., Ltd. for sequencing using an ABI 3730XL sequencer, which revealed a single-base mutation site. Figure 2 The mutation, A>C, is located at nucleotide 7315791 on chromosome 1, NC_010443.5, of the pig reference genome GCF 000003025.6. (For example...) Figure 3 As shown, there is a single nucleotide with an A base at 350 bp in SEQ ID NO:3 (i.e., an allelic mutation), and there is a single nucleotide with a C base at 350 bp in SEQ ID NO:4 (i.e., an allelic mutation).

[0033] 3. Genotyping of the target SNP locus

[0034] Using the DNA sample of the individual to be tested as a template, the target fragment containing the target SNP site was amplified according to the method described in step 1 above. The obtained purified PCR product was directly sent to Wuhan Aoke Dingsheng Biotechnology Co., Ltd. for sequencing, and the genotyping results were directly read from the sequencing results. Figure 3 As shown.

[0035] III. Genetic diversity testing and its association with quality

[0036] Using the method provided in the embodiments of this application, genetic diversity and its association with quality were detected in a herd of 277 Xidu Black Pigs (from the original breeding pig farm of the Animal Husbandry Institute of Hubei Academy of Agricultural Sciences). Statistical analysis was performed using SPSS statistical software (Statistical Package for the Social Sciences, Version 26.0) with a general linear model (GLM). The model used is: Y ijklm =μ+G i +A j +X k +S l +e ijklm , where: Y ijklm G represents the phenotypic value of pork traits; μ represents the population mean; G i Indicates genotype effect; A j Indicates the seasonal effect; X k Indicates the gender effect; S l Indicates the paternal effect; e ijklm The values ​​represent random residual effects. Results are expressed as least squares mean ± standard error, and P < 0.05 is considered statistically significant. The association analysis results are shown in Table 1. In Table 1, different lowercase letters in the superscript indicate significant differences between data in the same row (P < 0.05).

[0037] As shown in Table 1, the genotype at position 7315791 on chromosome 1, NC_010443.5, of the reference genome GCF 000003025.6 is... This includes AA, AC, and CC genotypes. Individuals with the CC genotype have significantly less skin thickness than those with the AA and AC genotypes. Individuals with the AA genotype have significantly lower intramuscular fat content than those with the AC and CC genotypes.

[0038] For breeding pigs used to supply pork in common pork dishes, selecting pigs with thin skin is more beneficial for improving the taste and makes them easier to handle and flavor during cooking. Therefore, individuals with the CC genotype are selected as superior pigs.

[0039] For pigs selected for high-quality marbled pork with high intrafat content, individuals with the AA genotype should be culled.

[0040] Considering the overall meat quality, priority is given to breeding individuals with the CC genotype, which have thin skin, high intramuscular fat content, and better meat quality.

[0041] Table 1. Association analysis between mutation at position 7315791 on chromosome 1 in the porcine genome and meat quality traits.

[0042]

[0043] Note: Different letters in the same line indicate significant differences (P<0.05).

[0044] The above description is merely a preferred embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application.

Claims

1. Methods for screening individuals with superior meat quality traits in selenium-rich black pork using molecular markers, including: Genomic DNA was extracted from the selenium-rich black pigs to be tested; The genomic DNA of the selenium-rich black pig was amplified by PCR using a primer set containing DNA molecules with nucleotide sequences as shown in SEQ ID NO:1 and 2. The PCR amplification product was sequenced; Based on the sequencing base peak results, the genotype at position 7315791 on chromosome 1, NC_010443.5 of the reference genome GCF 000003025.6 of Xidu Black Pig was determined; the genotypes include AA, AC, and CC. Based on the genotype, determine the dominant individual pigs of the Xidu Black Pig in terms of meat quality traits, wherein the meat quality traits are at least one of skin thickness and intramuscular fat content, and the specific judgment criteria are as follows: Individuals with the CC genotype had significantly less skin thickness than those with the AA and AC genotypes; individuals with the AA genotype had significantly lower intramuscular fat content than those with the AC and CC genotypes.

2. The application of primer sets for detecting molecular markers associated with traits of pig skin thickness and intramuscular fat content in the detection and analysis of meat quality traits in Xidu Black Pork, and in the screening of individuals with superior meat quality traits in Xidu Black Pork; wherein the molecular marker is a single nucleotide A>C mutation at position 7315791 on chromosome 1, NC_010443.5 of the pig reference genome GCF 000003025.6, and the meat quality trait is at least one of skin thickness and intramuscular fat content.

3. The application according to claim 2, wherein the primer set comprises a DNA molecule with nucleotide sequences as shown in SEQ ID NO:1 and 2.

4. The application of a kit containing primer sets of DNA molecules with nucleotide sequences as shown in SEQ ID NO:1 and 2 in the detection and analysis of meat quality traits of Selenium Capital Black Pork, and in the screening of individuals with superior meat quality traits of Selenium Capital Black Pork, wherein the meat quality traits are at least one of skin thickness and intramuscular fat content.

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