Molecular marker related to characters of carcass length and intramuscular fat content of pig and application of molecular marker

Abstract

The invention relates to the technical field of pigs, in particular to a molecular marker related to pig carcass length and intramuscular fat content characters and application. The molecular marker is an SNP (Single Nucleotide Polymorphism) site of nucleotide at a position 7311198 of a chromosome 1 NC010443.5 of a pig reference genome GCF 000003025.6. The molecular marker has the advantages that the molecular marker can be applied to pig breeding; the molecular marker is closely associated with the characteristics of pig carcass length and intramuscular fat content. The molecular marker can be applied to molecular-assisted selective breeding of high-quality pig carcass and meat quality traits.

Description

Technical Field

[0001] This application relates to the technical field of pigs, and specifically relates to a molecular marker related to the traits of pig carcass length and intramuscular fat content and its application. Background Art

[0002] The problem of the imbalance between supply and demand in the pork product market has gradually emerged. Therefore, the genetic improvement of pork traits has attracted more and more attention from 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 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, and high polymorphism. In breeding practice, SNPs can be used to locate certain excellent genes, and combined with phenotypes to determine the correlation 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 / T at nucleotide position 7311198 on chromosome 1 (NC_010443.5) of the pig reference genome GCF_000003025.6. The above-mentioned molecular marker is closely related to pork quality traits such as pig skin thickness, carcass length, and skin rate. The above-mentioned molecular marker can be applied to the 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 related to the traits of pig carcass length and intramuscular fat content, including a nucleotide sequence formed by a single nucleotide A>T mutation at nucleotide position 7311198 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>T mutation at nucleotide position 7311198 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 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 7311198 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 carcass length and intramuscular fat content.

[0010] In some embodiments, the genotypes at position 7311198 on chromosome 1 (NC_010443.5) of the reference genome GCF_000003025.6 include TT, AT, and TT. Among them, the carcass lengths of individuals with TT and AT genotypes are significantly higher than those of AA genotype, and the intramuscular fat contents of individuals with TT and AT genotypes are significantly higher than those of AA genotype.

[0011] In production, it is recommended to preferentially select TT genotype and eliminate AA genotype to facilitate the selection of a population with a long body shape, high meat production, and good meat quality performance.

[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. Description of the Drawings

[0013] Figure 1 Agarose gel electrophoresis map of the PCR amplification product of the target fragment containing the target SNP site provided for the embodiments.

[0014] Figure 2 Sequencing peak map of the downstream primer sequencing typing of pig SNP chr1:7311198 provided for the embodiments. The TT genotype of the antisense strand corresponds to the AA genotype of the sense strand, the TA genotype of the antisense strand corresponds to the AT genotype of the sense strand, and the AA genotype of the antisense strand corresponds to the TT genotype of the sense strand.

[0015] Figure 3Intuitive diagram of the porcine SNP chr1:7311198 nucleotide sequence provided for the example (shown in SEQ ID NO:3 or SEQ ID NO:4), with the red box representing the location of the mutation and the red sequence representing the primer positions. Detailed implementation manners

[0016] In order to make the objectives, technical solutions and advantages of the present application clearer and more understandable, the present application will be further described in detail below in combination 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 described in detail and individually in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail and specifically are all conventional experimental methods and can be known from the prior art.

[0017] I. Extraction of porcine genomic DNA

[0018] The test pig breed in the present 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 were sent to the Quality Supervision, Inspection and Testing Center for Swine Breeds of the Ministry of Agriculture (Wuhan), Huazhong Agricultural University within 4 hours, and the pork traits were determined according to the Agricultural Industry Standard of the People's Republic of China "Technical Specification for Determination of Pork Traits" (Standard No.: NY / T 821-2019).

[0019] II. Detection of the target SNP locus

[0020] 1. Obtaining of the target fragment containing the target SNP locus

[0021] (1) PCR amplification

[0022] The following primer pairs were designed and synthesized:

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

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

[0025] PCR amplification was performed in a pool of 40 Xidu Black Pig mixed genomic DNAs using the above primers. 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 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 as follows: 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 1.5% agarose gel.

[0027] (2) Purification of PCR products

[0028] The above PCR products were purified using the Gel Extraction Kit from Shanghai Sangon Biotech 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 the gel to melt, mix it every 2 min, and cool it to room temperature; place the centrifugal column into the collection tube, transfer the mixed solution to the centrifugal column, and let it stand at room temperature for 2 min; centrifuge at 12000 r / min for 1 min, 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 12000 r / min for 1 min; pour out the waste liquid in the collection tube, and centrifuge at 12000 r / min for 1 min; 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 min; centrifuge at 12000 r / min for 1 min, and the liquid in the centrifuge tube is the recovered DNA fragment.

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

[0030] The above-recovered DNA fragment was sent to Wuhan Aoke Dingsheng Biotechnology Co., Ltd. for sequencing using an ABI3730XL sequencer, and 1 single-base mutation site ( Figure 2 ) was found, which is an A>T mutation at nucleotide 7311198 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 at 251 bp in SEQ ID NO:3 (i.e., allelic gene mutation), and there is a single nucleotide of T base at 251 bp in SEQ ID NO:4 (i.e., allelic gene mutation).

[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 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, such as Figure 3 shown.

[0033] III. Genetic Diversity Detection and Association Analysis with Quality

[0034] Using the method provided in the embodiment of the present application, genetic diversity detection and association analysis with quality were performed on 163 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-quarter effect; X k represents the gender effect; S l represents the sire effect; e ijklm represents the random residual effect. The results are expressed as least squares means ± standard errors, and P < 0.05 is judged as significantly different. The association 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.

[0035] As shown in Table 1, the genotypes at position 7311198 on chromosome 1 (NC_010443.5) of the reference genome GCF 000003025.6 include TT, AT, and AA. Among them, the carcass lengths of individuals with TT and AT genotypes are significantly higher than those of AA genotype individuals, and the intramuscular fat contents of individuals with TT and AT genotypes are significantly higher than those of AA genotype individuals. Therefore, it is recommended to preferentially select TT genotype and eliminate AA genotype in production to facilitate the selection of a population with a long body shape, high meat production, and good meat quality performance.

[0036] Table 1 Association analysis of the mutation at position 7311198 on chromosome 1 of the pig genome with carcass length and intramuscular fat content traits

[0037]

[0038] Note: Different subscripts of letters in the same row indicate significant differences, p < 0.05.

[0039] As described above, the above are only the preferred specific embodiments 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 in the present application should be covered within the protection scope of the present application.

Claims

1. Molecular markers related to pig carcass length and intramuscular fat content traits, including a nucleotide sequence formed by a single nucleotide A>T mutation at position 7311198 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. According to the primer set of claim 2, the DNA molecule shown in SEQ ID NO: 1 and 2 is used to amplify the nucleotide sequence formed by the single nucleotide A>T mutation at 7311198 of chromosome 1 NC_010443.5 of the pig reference genome GCF 000003025.

6.

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 4; Sequencing the PCR amplification product; Determine the genotype of the pig at position 7311198 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 carcass length and intramuscular fat content.

7. The method according to claim 5, wherein the genotype at 7311198 of chromosome 1 NC_010443.5 of the reference genome GCF 000003025.6 includes TT, AT and TT.

8. The method according to claim 7, wherein, optionally, the carcass length of individuals with TT and AT genotypes is significantly higher than that of individuals with AA genotypes. Optionally, the intramuscular fat content of individuals with TT and AT genotypes is significantly higher than that of individuals with AA 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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