Molecular markers associated with pork color and intramuscular fat content in the porcine SLC22A2 gene and their applications

Abstract

This application relates to the field of swine technology, specifically to a molecular marker in the porcine SLC22A2 gene associated with meat color and intramuscular fat content traits, and its application. The molecular marker is an SNP site at nucleotide 7299982 on chromosome 1, NC_010443.5, GCF 000003025.6 of the porcine reference genome. This molecular marker is closely associated with meat color score and intramuscular fat content traits. This molecular marker can be applied to molecular-assisted selection breeding of new high-quality pig breeds.

Description

Technical Field

[0001] This application relates to the field of swine technology, specifically to molecular markers in the swine SLC22A2 gene that are associated with traits of pork color and intramuscular fat content, and their applications. 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] The porcine SLC22A2 gene (solute carrier family 22member2, Gene ID: 396936, located at 7289278..7323418nt on chromosome 1, NC_010443.5, reference genome GCF 000003025.6) belongs to solute carrier family 22 and encodes an organic cation transporter 2. Although there are few reports directly investigating the relationship between the porcine SLC22A2 gene and growth performance, given its involvement in the transport and metabolic balance of substances in the body, it is speculated that this gene may indirectly affect growth performance indicators such as growth rate and body weight in pigs by influencing nutrient absorption and utilization.

[0005] This application discloses a molecular marker in the porcine SLC22A2 gene associated with meat quality traits. This marker is a single nucleotide polymorphism (SNP) of G / A at nucleotide 7299982 on chromosome 1, NC_010443.5, of the porcine reference genome GCF 000003025.6, specifically located in the third intron of the SLC22A2 gene. This molecular marker is closely associated with meat color score and intramuscular fat content traits. This molecular marker can be applied to molecular-assisted selection breeding for pork quality traits. Therefore, this application discloses at least the following technical solutions:

[0006] In the first aspect, the embodiments disclose molecular markers related to the traits of pork color and intramuscular fat content, including nucleotide sequences formed by a single nucleotide G>A mutation at position 7299982 on chromosome 1, NC_010443.5 of the pig reference genome GCF 000003025.6.

[0007] 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 formed by a single nucleotide G>A mutation at position 7299982 on chromosome 1, NC_010443.5, containing the porcine reference genome GCF000003025.6.

[0008] 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.

[0009] 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 7299982 on chromosome 1 (NC_010443.5) of the reference genome GCF 000003025.6 based on the sequencing base peak results; and identifying the dominant pig individuals in terms of meat quality traits based on the genotype.

[0010] In some embodiments, the meat quality traits include at least one of pork color score 1, meat color score 24, and intramuscular fat content.

[0011] In some embodiments, the genotypes at position 7299982 on chromosome 1 (NC_010443.5) of the reference genome GCF 000003025.6 include GG, GA, and AA. Individuals with the GG genotype have significantly higher flesh color scores 1 and 24 than those with the AA genotype; while individuals with the AA genotype have significantly higher intramuscular fat than those with the GG genotype.

[0012] Taking into account both meat color and intramuscular fat content, and to ensure pork freshness and flavor, the AA genotype should be selected as the dominant pig individual.

[0013] 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

[0014] Figure 1 The image shows an agarose gel electrophoresis diagram of the PCR amplification product containing the target SNP site provided in the example.

[0015] Figure 2 The sequencing peak diagram of the downstream primer for pig SNP chr1:7299982 provided in the example shows that the antisense strand CC genotype corresponds to the sense strand GG genotype, the antisense strand CT genotype corresponds to the sense strand GA genotype, and the antisense strand TT genotype corresponds to the sense strand AA genotype.

[0016] Figure 3 The image shows a visual representation of the porcine SNP chr1:7299982 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

[0017] 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.

[0018] I. Extraction of porcine genomic DNA

[0019] The experimental pig breed used in this application was the Xidu Black Pig, and the samples were obtained from the original breeding pig farm under the Institute of Animal Husbandry and Veterinary Medicine, Hubei Academy of Agricultural Sciences. Genomic DNA was extracted from the pigs using a genomic DNA kit produced by Beijing Baitek 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 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 Specification for Determination of Pork Traits" (Standard No.: NY / T 821-2019).

[0020] II. Preparation of target fragments containing target SNP sites and detection of target SNP sites.

[0021] 1. Prepare the target fragment containing the target SNP site.

[0022] (1) PCR amplification

[0023] Design and synthesize the following primer pairs:

[0024] Forward primer F: 5'-TACACTTCGGTGCCTGTCAT-3', SEQ ID NO:1

[0025] Reverse primer R: 5'-ACGCAAGAATTGGGGTTCCA-3', SEQ ID NO:2

[0026] PCR amplification was performed using the above primers in a pool of mixed genomic DNA from 40 selenium-rich black pigs. The PCR reaction volume was 50 μL, and the concentrations of each component in the volume were 100 ng template DNA and 4 μL 10× buffer (containing Mg). 2+ ), 0.5 μM of the upstream and downstream primers, 2.5 μM of dNTPs, and 1 U Taq DNA polymerase.

[0027] 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.

[0028] (2) Purification of PCR products

[0029] 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.

[0030] 2. Detection of target SNP sites

[0031] 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 is a G>A mutation at nucleotide 7299982 on chromosome 1, NC_010443.5, of the pig reference genome GCF 000003025.6. Figure 3 As shown, there is a single nucleotide mutation of a G base at 176 bp in SEQ ID NO:3, and a single nucleotide mutation of an A base at 176 bp in SEQ ID NO:4 (i.e., an allelic mutation).

[0032] 3. Genotyping of the target SNP locus

[0033] 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.

[0034] III. Genetic diversity testing and its association with quality

[0035] Using the method provided in the embodiments of this application, genetic diversity and its association with quality were detected in a herd of 265 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 points in the same row (P < 0.05). Specifically, meat color score 1 represents the meat color score of pork 1 hour after slaughter, and meat color score 24 represents the meat color score of pork refrigerated for 24 hours after slaughter.

[0036] As shown in Table 1, the genotypes at position 7299982 on chromosome 1 (NC_010443.5) of the reference genome GCF 000003025.6 include GG, GA, and AA. Among them, the flesh color score 1 and flesh color score 24 of individuals with the GG genotype were significantly higher than those of individuals with the AA genotype; while the intramuscular fat of individuals with the AA genotype was significantly higher than that of individuals with the GG genotype.

[0037] Pork color has a significant impact on pork consumption. Consumers prefer pork with a bright red color, and pork with an excessively high color score is not favored by the market. Therefore, when considering both meat color and intramuscular fat content traits to ensure pork freshness and flavor, the AA genotype should be selected as the dominant pig individual.

[0038] Table 1. Association analysis between mutation at position 7299982 on chromosome 1 in the porcine genome and meat quality traits.

[0039]

[0040]

[0041] Note: Different letters in the same line indicate significant differences (P<0.05).

[0042] 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 pork quality traits using molecular markers include: Extract genomic DNA from the pigs to be tested; The porcine genomic DNA was amplified by PCR using primers with nucleotide sequences as shown in SEQ ID NO:1 and SEQ ID NO:

2. The PCR amplification products were sequenced; Based on the sequencing base peak results, the genotype of the pig at position 7299982 on chromosome 1, NC_010443.5 of pig reference genome GCF 000003025.6 was determined; the genotypes included GG, GA and AA. Based on the genotype, the dominant pig individuals in terms of meat quality traits among the candidate pigs are determined, wherein the meat quality traits include meat color score and intramuscular fat, and the specific judgment criteria are as follows: The meat color scores 1 and 24 of individuals with the GG genotype were significantly higher than those of individuals with the AA genotype; the intramuscular fat of individuals with the AA genotype was significantly higher than that of individuals with the GG genotype. Among them, meat color score 1 is the meat color score of pork 1 hour after slaughter, and meat color score 24 is the meat color score of pork refrigerated for 24 hours after slaughter.

2. The application of the method of claim 1 in the detection and analysis of pork traits, and in the screening of individuals with superior pork quality traits.

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