Molecular marker associated with pigskin thickness and intramuscular fat content characters and application

Abstract

The invention relates to the technical field of pigs, in particular to a molecular marker associated with pigskin thickness and intramuscular fat content traits and application. The molecular marker is an SNP (Single Nucleotide Polymorphism) site of nucleotide at a position 7315791 of a chromosome 1 NC010443.5 of a pig reference genome GCF 000003025.6. The molecular marker has the advantages that the molecular marker is an SNP site of nucleotide at the position The molecular marker is closely associated with the characteristics of pigskin thickness and intramuscular fat content. The molecular marker can be applied to molecular-assisted selective breeding of high-quality new pig strains.

Description

Technical Field

[0001] This application relates to the technical field of pork, and specifically relates to molecular markers associated with the traits of pig skin thickness and intramuscular fat content and their applications. Background Art

[0002] The problem of the imbalance between the supply and demand in the pork product market has gradually emerged. Therefore, the genetic improvement of pork traits has increasingly attracted the attention of breeders. Pork quality traits such as pig skin thickness and meat color score are important indicators for evaluating pork traits. Although pork traits belong to medium heritability traits, due to the high cost of in vivo measurement of pork traits, it is difficult to improve them through conventional breeding. Therefore, finding molecular markers that affect pork traits is of great significance for improving pork traits through molecular assisted breeding.

[0003] Molecular markers for assisted selection include protein markers, microsatellite markers, single nucleotide polymorphism (SNP) markers, etc. SNP markers refer to the polymorphisms of DNA sequences caused by single nucleotide variations on the genome. It has the characteristics of a large number, high accuracy, high polymorphism, etc. In breeding practice, SNPs can be used to locate certain excellent genes, and combined with phenotypes to determine the association between markers and specific qualities. Molecular markers can also be verified in populations and applied in molecular breeding. Summary of the Invention

[0004] This application has discovered a single nucleotide polymorphism of A / C at nucleotide position 7315791 on chromosome 1 (NC_010443.5) of the pig reference genome GCF_000003025.6. This molecular marker is closely associated with pork quality traits such as pig skin thickness and meat color score. This molecular marker can be applied to molecular assisted selection breeding of new high-quality pig strains. For this reason, the embodiments of this application at least disclose the following technical solutions:

[0005] In a first aspect, the embodiment discloses a molecular marker associated with the traits of pig skin thickness and intramuscular fat content, including a nucleotide sequence formed by a single nucleotide A>C mutation at nucleotide position 7315791 on chromosome 1 (NC_010443.5) of the pig reference genome GCF_000003025.6.

[0006] In a second aspect, the embodiment discloses a primer set. The primer set includes: DNA molecules as shown in SEQ ID NO: 1 and 2. The primer set is used to amplify a nucleotide sequence containing the single nucleotide mutation site in the molecular marker described in the first aspect. In some embodiments, the DNA molecules as shown in SEQ ID NO: 1 and 2 are used to amplify a nucleotide sequence formed by a single nucleotide A>C mutation at nucleotide position 7315791 on chromosome 1 (NC_010443.5) of the pig reference genome GCF_000003025.6.

[0007] In a third aspect, an embodiment discloses a kit. The kit includes the primer set described in the second aspect. The kit is a PCR amplification kit and includes the primer set described in the second aspect and other reagents for PCR amplification.

[0008] In a fourth aspect, an embodiment discloses a method for screening individuals with excellent pork quality traits using molecular markers. The method includes: extracting genomic DNA of a 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 according to the sequencing base peak map result; and judging the superior pig individuals in terms of pork quality traits based on the genotype.

[0009] In some embodiments, the pork quality traits 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. Among them, the skin thickness of individuals with the CC genotype is significantly less than that of individuals with the AA and AC genotypes. The intramuscular fat content of individuals with the AA genotype is significantly lower than that of individuals with the AC and CC genotypes.

[0011] For breeding pig individuals for pork supply for common pork dishes, screening pig individuals with thin skin is more conducive to improving the taste and is also easier to process and flavor during cooking. Therefore, individuals with the CC genotype are used as the superior pig individuals for screening.

[0012] For screening pig individuals with the quality of snowflake pork with a relatively high intramuscular fat content, pig individuals with the AA genotype should be eliminated.

[0013] For comprehensively considering the meat quality performance level, it is preferred to breed pig individuals with the CC genotype, which have thin skin, high intramuscular fat content, and good meat quality.

[0014] In a fifth aspect, an embodiment discloses the application of the molecular marker described in the first aspect, the primer set described in the second aspect, or the kit described in the third aspect in the detection and analysis of pork traits and the screening of individuals with excellent pork quality traits. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 An agarose gel electrophoresis diagram of the PCR amplification product of the target fragment containing the target SNP site provided for the embodiment.

[0016] Figure 2The typing sequencing peak map of porcine SNP chr1:7315791 provided for the embodiment.

[0017] Figure 3 The nucleotide sequence visualization map of porcine SNP chr1:7315791 provided for the embodiment (shown in SEQ ID NO:3 or SEQ ID NO:4), the red box represents the mutation site, and the red sequence represents the primer position. Specific embodiments

[0018] In order to make the purpose, technical solutions and advantages of the present application clearer, the following further details the present application in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application. The reagents not specifically described in detail in the present application are all conventional reagents and can be obtained from commercial channels; the 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 test pig breed in this application is Xidu Black Pig, and the samples are from the original breeding farm of the Institute of Animal Husbandry, Hubei Academy of Agricultural Sciences. The genomic DNA of pigs was extracted using a genomic DNA kit produced by Beijing Bioteke Corporation and operated according to the instructions of the kit. After detecting the concentration and quality of the extracted DNA, it was stored at -20°C for later use. The remaining muscle samples were sealed in bags and stored at 4°C, and sent to the National Quality Supervision and Inspection Center for Swine (Wuhan), Huazhong Agricultural University within 4 hours, and the pork traits were measured according to the Agricultural Industry Standard of the People's Republic of China "Technical Specification for Measuring Pork Traits" (Standard No.: NY / T 821-2019).

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

[0022] 1. Preparation of target fragments containing the target SNP locus

[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'-CCCAACCTTATTCTCCCCTA-3', SEQ ID NO:2

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

[0028] The running program of PCR was as follows: preheating at 98°C for 45 s; denaturation at 98°C for 10 s, annealing at 62°C for 30 s, extension at 72°C for 30 s, for a total of 34 cycles; extension at 72°C for 10 min; storage at 4°C. The PCR products were electrophoresed on a 1.5% agarose gel.

[0029] (2) Purification of PCR products

[0030] The above PCR products were purified using the Gel Extraction Kit from Sangon Biotech (Shanghai) Co., Ltd. (operated according to the instructions of this kit). The specific steps were 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 solubilization solution and incubate in a water bath at 50 - 60°C until the gel completely melts. Mix every 2 min during heating and cooling to room temperature. Place the centrifugal column in the collection tube, transfer the mixture to the centrifugal column, and let it stand at room temperature for 2 min. Centrifuge at 12,000 r / min for 1 min, and at this time, the DNA is adsorbed onto the column. Pour out the waste liquid in the collection tube, place the centrifugal column in the same collection tube, add 700 μL of elution solution, and centrifuge at 12,000 r / min for 1 min. Pour out the waste liquid in the collection tube and centrifuge at 12,000 r / min for 1 min. Place the centrifugal column in a pre-prepared sterile 1.5 mL centrifuge tube, add 40 μL of elution solution or double-distilled water (pH > 7.0), and let it stand at room temperature or 37°C for 2 - 3 min. Centrifuge at 12,000 r / min for 1 min, and the liquid in the centrifuge tube is the recovered DNA fragment.

[0031] 2. Detection of the target SNP locus

[0032] The DNA fragment recovered above was sent to Wuhan Aoke Dingsheng Biotechnology Co., Ltd. for sequencing using an ABI 3730XL sequencer, and 1 single-base mutation site ( Figure 2 ) was found, which was a nucleotide A > C mutation at position 7315791 on chromosome 1 (NC_010443.5) of the pig reference genome GCF_000003025.6. As Figure 3 shown, there was a single nucleotide of base A (i.e., allelic gene mutation) at 350 bp in SEQ ID NO:3, and there was a single nucleotide of base C (i.e., allelic gene mutation) at 350 bp in SEQ ID NO:4.

[0033] 3. Genotyping of the target SNP locus

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

[0035] III. Genetic diversity detection and correlation analysis with quality

[0036] Using the method provided in the embodiment of the present application, genetic diversity detection and correlation analysis with quality were carried out on 277 Xidu black pigs (from the original breeding farm of the Institute of Animal Science, Hubei Academy of Agricultural Sciences). The general linear model GLM of SPSS statistical software (Statistical Package for the Social Sciences, Version 26.0) was used for statistical analysis. The model used was: Y ijklm = μ + G i + A j + X k + S l + e ijklm , where: Y ijklm represents the phenotypic value of pork traits; μ represents the population mean; G i represents the genotype effect; A j represents the year-quarter effect; X k represents the gender effect; S l represents the paternal effect; e ijklm represents the random residual effect. The results are expressed as least squares mean ± standard error, and P < 0.05 is determined to be significantly different. The correlation analysis results are shown in Table 1. In Table 1, different lowercase letters in the subscripts indicate significant differences between the data in the same row, P < 0.05.

[0037] As shown in Table 1, the genotypes at position 7315791 on chromosome NC_010443.5 of chromosome 1 of the reference genome GCF_000003025.6 include AA, AC, and CC. Among them, the skin thickness of individuals with the CC genotype is significantly less than that of individuals with the AA and AC genotypes. The intramuscular fat content of individuals with the AA genotype is significantly lower than that of individuals with the AC and CC genotypes.

[0038] For breeding pig individuals for pork supply as common pork dishes, screening pig individuals with thin skin is more conducive to improving the taste and is also easier to process and flavor during cooking. Therefore, individuals with the CC genotype are the preferred pig individuals for screening.

[0039] For screening pig individuals with high intramuscular fat content in snowflake pork quality, individuals with the AA genotype should be eliminated.

[0040] For comprehensively considering the meat quality performance level, individuals with the CC genotype should be preferentially selected, with thin skin, high intramuscular fat content, and good meat quality.

[0041] Table 1 Association analysis of the mutation at position 7315791 on chromosome 1 in the pig genome gene with meat quality traits

[0042]

[0043] Note: Different letter superscripts in the same row indicate significant differences, P < 0.05.

[0044] As mentioned above, it is only the preferred specific implementation manner of this application, but the protection scope of this application is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by this application should be covered within the protection scope of this application.

Claims

1. Molecular markers associated with pig skin thickness and intramuscular fat content traits include a nucleotide sequence formed by a single nucleotide A>C mutation at position 7315791 of chromosome 1 NC_010443.5 of the pig reference genome GCF000003025.

6.

2. A primer set for amplifying a nucleotide sequence comprising a single nucleotide mutation site in the molecular marker according to claim 1, wherein the primer set comprises a DNA molecule as shown in SEQ ID NO: 1 and 2.

3. The primer set according to claim 2 is used to amplify the nucleotide sequence formed by the single nucleotide A>C mutation at 7315791 of chromosome 1 NC_010443.5 of the pig reference genome GCF 000003025.6 using the DNA molecule shown in SEQ ID NO: 1 and 2.

4. A kit comprising the primer set according to any one of claims 2 to 3 and other reagents for PCR amplification.

5. Methods for screening individuals with superior pork quality traits using molecular markers, including: Extracting the genomic DNA of the pig to be tested; Performing PCR amplification on the genomic DNA using the primer set described in any one of claims 2 to 3; Sequencing the PCR amplification product; Determine the genotype of the pig at position 7315791 of chromosome 1 NC_010443.5 of the reference genome GCF 000003025.6 according to the sequencing base peak map result; The superior pig individuals in terms of meat quality traits of the selected pigs are determined according to the genotypes.

6. The method according to claim 5, wherein the meat quality trait comprises at least one of skin thickness and intramuscular fat content.

7. The method according to claim 5, wherein the genotype at 7315791 of chromosome 1 NC_010443.5 of the reference genome GCF 000003025.6 includes AA, AC and CC.

8. The method according to claim 7, optionally, the skin thickness of individuals with CC genotype is significantly less than that of individuals with AA and AC genotypes, and optionally, the intramuscular fat content of individuals with AA genotype is significantly lower than that of individuals with AC and CC genotypes.

9. Use of the molecular marker according to claim 1, the primer set according to any one of claims 2 to 3, or the kit according to claim 4 in the detection and analysis of pork traits, and in the screening of individuals with excellent pork quality traits.

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