Molecular marker associated with pork color and intramuscular fat content and application

Abstract

The invention relates to the technical field of pigs, in particular to a molecular marker associated with pork color and intramuscular fat content and application. The molecular marker is an SNP (Single Nucleotide Polymorphism) site of nucleotide at a position 7314350 of a chromosome 1 NC010443.5 of a pig reference genome GCF 000003025.6. The molecular marker is a molecular marker of a pig genome GCF 000003025.6 The molecular marker is closely associated with pork color and intramuscular fat content characters. The molecular marker can be applied to molecular-assisted selective breeding of high-quality pork quality characters.

Description

Technical Field

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

[0002] The problem of imbalance between supply and demand in the pork product market has gradually emerged. Therefore, the genetic improvement of pork traits has attracted increasing attention from breeders. Pork quality traits such as skin thickness and meat color score are important indicators for evaluating pork traits. Although pork traits are 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 affecting 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. They have the characteristics of large quantity, high accuracy, high polymorphism, etc. In breeding practice, SNPs can be used to locate certain excellent genes, determine the association between markers and specific qualities in combination with phenotypes, and also verify molecular markers in populations and apply them in molecular breeding. Summary of the Invention

[0004] This application has discovered a single nucleotide polymorphism of A / G at nucleotide position 7314350 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 low-fat pig lines. For this reason, the embodiments of this application at least disclose the following technical solutions:

[0005] In the first aspect, the embodiment discloses a molecular marker associated with pork color and intramuscular fat content, including a nucleotide sequence formed by a single nucleotide A>G mutation at nucleotide position 7314350 on chromosome 1 (NC_010443.5) of the pig reference genome GCF_000003025.6.

[0006] In the 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>G mutation at nucleotide position 7314350 on chromosome 1 (NC_010443.5) of the pig reference genome GCF_000003025.6.

[0007] In a third aspect, 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] In a fourth aspect, the embodiments disclose a method for screening individuals with excellent pork quality traits using molecular markers. The method includes: extracting genomic DNA of the pigs 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 pigs at position 7314350 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 individuals in terms of pork quality traits among the candidate pigs based on the genotype.

[0009] In some embodiments, the pork quality traits include at least one of muscle color value L1, muscle color value L24, and intramuscular fat content.

[0010] In some embodiments, the genotypes at position 7314350 on chromosome 1 (NC_010443.5) of the reference genome GCF_000003025.6 include AA, AG, and GG. Among them, the muscle color value L1 of GG genotype individuals is significantly higher than that of AA and AG. The muscle color value L24 of GG genotype individuals is significantly higher than that of AA genotype. The intramuscular fat content of AA genotype individuals is significantly lower than that of AG and GG.

[0011] In the breeding of high-quality pigs, GG genotype individuals should be preferentially selected, which have a high intramuscular fat content and good meat color.

[0012] In a fifth aspect, the embodiments disclose 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

[0013] Figure 1 Agarose gel electrophoresis diagram of the PCR amplification product of the target fragment containing the target SNP site provided by the embodiment.

[0014] Figure 2 Genotyping sequencing peak map of pig SNP chr1:7314350 provided by the embodiment.

[0015] Figure 3 Intuitive diagram of the nucleotide sequence of pig SNP chr1:7314350 provided by the embodiment (shown in SEQ ID NO:3 or SEQ ID NO:4), the red frame represents the position of the mutation, and the red sequence represents the primer position. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0016] In order to make the purpose, technical solution and advantages of this application clearer, the following further details this application in combination with embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not used to limit this application. The reagents not specifically described in detail in this 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.

[0017] I. Extraction of Porcine Genomic DNA

[0018] The experimental pigs in this application are Xidu Black Pigs, 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 the extraction was carried out 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 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).

[0019] II. Detection of Target SNP Loci and Target Fragments Containing Target SNP Loci

[0020] 1. Obtaining of Target Fragments Containing Target SNP Loci

[0021] (1) PCR Amplification

[0022] Design and synthesize the following primer pairs:

[0023] Forward primer F: 5’-AGAGGCACACACGTGGCAGT-3’, SEQ ID NO:1

[0024] Reverse primer R: 5’-TCCTCTGGAGGGAAAGGGGT-3’, SEQ ID NO:2

[0025] Use the above primers to perform PCR amplification in a pool of genomic DNA from 40 Xidu Black Pigs. The PCR reaction system is 50 μL, and the concentrations of each component in the system are 100 ng template DNA, 4 μL of 10× buffer (containing Mg2+), 0.5 μM of each of the above upstream and downstream primers, 2.5 μM dNTPs, and 1 U Taq DNA polymerase.

[0026] The running program of PCR is: preheat at 98°C for 45 s; denature at 98°C for 10 s, anneal at 62°C for 30 s, extend at 72°C for 30 s, for a total of 34 cycles; extend at 72°C for 10 min; store at 4°C. The PCR products were electrophoresed on a 1.5% agarose gel.

[0027] (2) Purification of PCR products

[0028] 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 are as follows: First, cut the gel containing the target fragment from the agarose gel, put it into 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. When heating to melt the gel, mix it every 2 minutes and cool to room temperature; place the centrifugal column into the collection tube, transfer the mixture to the centrifugal column, and let it stand at room temperature for 2 minutes; centrifuge at 12,000 r / min for 1 minute, at this time the DNA is adsorbed onto the column; pour out the waste liquid in the collection tube, place the centrifugal column into the same collection tube, add 700 μL of elution solution, and centrifuge at 12,000 r / min for 1 minute; pour out the waste liquid in the collection tube and centrifuge at 12,000 r / min for 1 minute; place the centrifugal column into a pre-prepared sterilized 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 minutes; centrifuge at 12,000 r / min for 1 minute, and the liquid in the centrifuge tube is the recovered DNA fragment.

[0029] 2. Obtaining of SNP sites in the target fragment containing the target SNP site

[0030] The recovered DNA fragment obtained 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 is an A > G mutation at nucleotide position 7314350 on chromosome 1 (NC_010443.5) of the pig reference genome GCF_000003025.6. As Figure 3 shown, there is a single nucleotide of A base (i.e., allelic gene mutation) at 495 bp in SEQ ID NO:3, and there is a single nucleotide of G base (i.e., allelic gene mutation) at 495 bp in SEQ ID NO:4.

[0031] 3. Molecular marker genotyping

[0032] Using the DNA sample of the individual to be detected as a template, amplify the target fragment containing the target SNP site according to the method described in step 1 above, directly send the obtained PCR purified product to Wuhan Aoke Dingsheng Biotechnology Co., Ltd. for sequencing, and directly read the genotyping result from the sequencing result, as Figure 3 shown.

[0033] III. Genetic diversity detection and correlation analysis with quality

[0034] Using the method provided in the embodiments of the present application, genetic diversity detection and association analysis with quality were performed on 272 Xidu black pigs (from the original breeding farm of the Institute of Animal Husbandry, 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-season effect; X k represents the sex effect; S l represents the sire effect; e ijklm represents the random residual effect. The results were expressed as least squares means ± standard errors, and a significant difference was determined when P < 0.05. The results of the association analysis are shown in Table 1. In Table 1, different lowercase letters with superscripts indicate significant differences between the data in the same row, P < 0.05. Among them, the muscle color value L1 of pork refers to the L* value of the muscle color of pork in the Lab color space 1 hour after slaughter. The muscle color value L24 of pork refers to the L* value of the muscle color of pork in the Lab color space 24 hours after slaughter.

[0035] As shown in Table 1, the genotypes at position 7314350 on chromosome 1 (NC_010443.5) of the reference genome GCF_000003025.6 include AA, AG, and GG. Among them, the muscle color value L1 of individuals with the GG genotype was significantly higher than that of AA and AG. The muscle color value L24 of individuals with the GG genotype was significantly higher than that of the AA genotype. The intramuscular fat content of individuals with the AA genotype was significantly lower than that of AG and GG.

[0036] In the breeding of high-quality pigs, individuals with the GG genotype should be preferentially selected, as they have a high intramuscular fat content and good meat color.

[0037] Table 1 Association analysis of the mutation at position 7314350 on chromosome 1 of the pig genome with meat quality traits

[0038]

[0039] Note: Different letters with superscripts in the same row indicate significant differences, P < 0.05.

[0040] As described above, it is only the preferred specific implementation manner of the present application, but the protection scope of the present 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 the present application should be covered within the protection scope of the present application.

Claims

1. A molecular marker associated with pork color and intramuscular fat content, comprising a nucleotide sequence formed by a single nucleotide A>G mutation at position 7314350 of chromosome NC_010443.5 of Sus scrofa chromosome 1 in the porcine reference genome GCF_000003025.

6.

2. A primer set for amplifying a nucleotide sequence containing the single nucleotide mutation site in the molecular marker as described in claim 1, said primer set comprising DNA molecules as shown in SEQ ID NO: 1 and 2.

3. The primer set according to claim 2, using the DNA molecules as shown in SEQ ID NO: 1 and 2 to amplify a nucleotide sequence formed by a single nucleotide A>G mutation at position 7314350 of chromosome NC_010443.5 of Sus scrofa chromosome 1 in the porcine reference genome GCF_000003025.

6.

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

5. A method for screening individuals with excellent pork quality traits using a molecular marker, comprising: Extracting genomic DNA of the pig to be tested; Performing PCR amplification on the genomic DNA using the primer set as described in any one of claims 2 to 3; Sequencing the PCR amplification product; Determining the genotype of the pig at position 7314350 of chromosome NC_010443.5 of Sus scrofa chromosome 1 in the reference genome GCF_000003025.6 according to the sequencing base peak map result; Judging the superior individuals of the pigs to be selected in terms of pork quality traits according to the genotype.

6. The method according to claim 5, wherein the pork quality traits include at least one of muscle color value L1, muscle color value L24, and intramuscular fat content.

7. The method according to claim 5, wherein the genotypes at position 7314350 of chromosome NC_010443.5 of Sus scrofa chromosome 1 in the reference genome GCF_000003025.6 include AA, AG, and GG.

8. The method according to claim 7, optionally, the muscle color value L1 of GG genotype individuals is significantly higher than that of AA and AG, optionally, the muscle color value L24 of GG genotype individuals is significantly higher than that of AA genotype, and optionally, the intramuscular fat content of AA genotype individuals is significantly lower than that of AG and GG.

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

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