Molecular marker associated with pig eye muscle area

Abstract

The invention discloses the technical field of pig eye muscle areas, and particularly relates to a molecular marker associated with the pig eye muscle area. The molecular marker comprises a nucleotide sequence formed by A / G single nucleotide polymorphism at the 3593429nt position of chromosome 10 of the international pig reference genome 11.1 version of pigs. The SNP marker provided by the invention is remarkably related to the eye muscle area of the large white pig muscle, a large white pig strain with the large eye muscle area can be screened by identifying the SNP marker, and the obtained large white pig strain with the large eye muscle area has important economic benefits, edible value and social value.

Description

Technical Field

[0001] The present application relates to the technical field of pig eye muscle area, and in particular to a molecular marker associated with pig eye muscle area. Background Art

[0002] Loin muscle area (LMA) refers to the cross-sectional area of ​​the longissimus dorsi muscle between the last two thoracic vertebrae of livestock, and is of great significance in the study of livestock such as pigs, cattle, and sheep. The loin muscle area of ​​pigs refers to the cross-sectional area of ​​the longissimus dorsi muscle at the last rib of the pig, which is measured by a planimeter. The calculation formula of the loin muscle area is generally loin muscle height (cm) × loin muscle width (cm) × 0.7. Loin muscle area is an important indicator for measuring pig growth performance and carcass meat production capacity. It is directly related to lean meat rate and backfat thickness, which are crucial for the pig industry. Lean pigs occupy a large share of the pork consumption market, and carcass lean meat rate is a very important indicator in evaluating carcass quality. Studies have found that there is a strong correlation between lean meat rate and loin muscle area. Estimating lean meat rate by loin muscle area can significantly reduce costs, so loin muscle area is also a very important indicator in the production and breeding of lean pigs.

[0003] In breeding practice, the determination of eye muscle area plays an important role in selecting pigs with excellent genetic characteristics for breeding, thereby improving the overall meat quality and performance of the pig herd. By measuring the eye muscle area results, farmers can select pigs with larger eye muscle area for breeding, thereby achieving the goal of improving pig production efficiency. Therefore, eye muscle area is not only an important meat quality indicator, but also a key parameter for genetic improvement and breeding in the pig industry. Summary of the invention

[0004] The inventors of the present application used genome-wide association analysis (GWAS) to screen and obtain molecular markers associated with pig eye muscle area, which are located at the single nucleotide polymorphism site A / G at position 3593429 of pig chromosome 10 (Ensembl database number rs81425149), wherein the eye muscle area of ​​individuals with AA genotype at this site is higher than that of other individuals, indicating that the A-type nucleotide at this site can serve as an allele that is beneficial to the eye muscle area of ​​Large White pigs.

[0005] To this end, the embodiments of the present application disclose at least the following technical solutions:

[0006] In a first aspect, the embodiments disclose molecular markers associated with pig eye muscle area, including a nucleotide sequence formed by a single nucleotide polymorphism of A / G at position 3593429 of chromosome 10 of the International Pig Reference Genome 11.1.

[0007] In a second aspect, the embodiment discloses a primer pair for detecting the molecular marker described in the first aspect, and the nucleotide sequence of the primer pair is shown in SEQ ID NO:1 and SEQ ID NO:2.

[0008] In a third aspect, an embodiment discloses a kit comprising the primer pair described in the second aspect and reagents required for PCR amplification.

[0009] In a fourth aspect, an embodiment discloses a method for detecting the molecular markers described in the first aspect, the method comprising the following steps: extracting the genomic DNA of the pig to be tested; performing PCR amplification on the genomic DNA of the pig to be tested to obtain a PCR amplification product; sequencing the PCR amplification product to obtain a sequencing result; based on the sequencing result, interpreting the polymorphism of the 3593429nt site on chromosome 10 of the International Porcine Reference Genome 11.1 version.

[0010] In a fifth aspect, the embodiments disclose molecular markers associated with pig eye muscle area, including nucleotide sequences as shown in SEQ ID NO:3 and SEQ ID NO:4.

[0011] In a sixth aspect, an embodiment discloses a method for breeding a pig breed with a large eye muscle area, the method comprising the following steps: detecting the genotype of the 3593429th nucleotide site of chromosome 10 of the International Pig Reference Genome 11.1 version, and selecting the AA type individuals at the 3593429th nucleotide site as breeding pigs.

[0012] In a seventh aspect, an embodiment discloses a method for breeding a pig strain with a large eye muscle area, comprising: if a nucleotide sequence as shown in SEQ ID NO: 3 is detected in the pig genomic DNA, the corresponding homozygous genotype individual is used as a breeding pig.

[0013] In an eighth aspect, the embodiment discloses the use of a SNP site or primer pair associated with the intramuscular fat content of a Large White pig in screening a pig strain with a large eye muscle area, wherein the SNP site is the 3593429th nucleotide site of chromosome 10 of the International Swine Genome Version 11.1 reference sequence, and has an A / G polymorphism. The nucleotide sequences of the primer pair are shown in SEQ ID NO: 1 and SEQ ID NO: 2. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Manhattan plot of pig eye muscle area trait provided in the embodiment. In the figure, the black circle points to the molecular marker located on chromosome 10 of Large White pig obtained by screening.

[0015] Figure 2The electrophoresis detection diagram of the pig individual genomic DNA amplification product in 2% agarose gel provided in the embodiment. M represents the 1000bp Marker molecular weight standard, and 1 and 2 represent the DNA products amplified from the SNP mutation sites G and A of the Large White pig genomic DNA, respectively. Among them, the 1000bp Marker molecular weight bands are 1000bp, 700bp, 500bp, 400bp, 300bp, 200bp, and 100bp from top to bottom.

[0016] Figure 3 The sequencing peak graph of the pig individual genomic DNA amplification product provided in the example is compared with the sequence of the relevant gene fragments of the pig in GenBank. The upper figure is the sequence comparison result of the 3593429th position of the pig chromosome 10 being G, and the lower figure is the sequence comparison result of the 3593429th position of the pig chromosome 10 being A. DETAILED DESCRIPTION

[0017] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.

[0018] Pig eye muscle area is affected by many factors, including genes, environment, and management. Therefore, in order to better predict pig eye muscle area, it is necessary to consider multiple factors, not just the role of a single molecular marker. Secondly, due to the large number of pig breeds and the large genetic differences between different breed groups, it is necessary to conduct systematic research in multiple different breeds to find more reliable and effective molecular markers.

[0019] The present application uses genome-wide association analysis (GWAS) to detect the polymorphism of genetic markers in multiple individuals across the genome, obtain genotypes, and then perform statistical analysis of genotypes and phenotypes at the population level, screen out the SNP sites that are most likely to affect the trait based on the significance test, and find out the genes related to the eye muscle area of ​​pigs. The present application found that the single nucleotide polymorphism site (Ensembl database number rs81425149) with A / G occurring at the 3593429 nt position of chromosome 10 of pigs, wherein the eye muscle area of ​​individuals with AA genotype at this site is higher than that of other individuals, thereby indicating that the A-type nucleotide at this site can be used as an allele that is conducive to obtaining the eye muscle area of ​​tall large white pigs.

[0020] Based on this, the embodiment discloses molecular markers associated with pig eye muscle area, including a nucleotide sequence formed by a single nucleotide polymorphism A / G at position 3593429 of chromosome 10 of the International Pig Reference Genome 11.1.

[0021] The embodiment discloses a primer pair for detecting the molecular marker, and the nucleotide sequence of the primer pair is shown in SEQ ID NO:1 and SEQ ID NO:2.

[0022] The embodiment also discloses a kit, comprising the primer pair and reagents required for PCR amplification.

[0023] The embodiment discloses a method for detecting the molecular marker, which comprises the following steps: extracting genomic DNA of the pig to be tested; performing PCR amplification on the genomic DNA of the pig to be tested to obtain a PCR amplification product; sequencing the PCR amplification product to obtain a sequencing result; and based on the sequencing result, interpreting the polymorphism of the nucleotide site at position 101 in the sequence shown in pig SEQ ID NO:1.

[0024] The embodiment discloses a method for breeding a pig breed with large eye muscle area, the method comprising the following steps: detecting the genotype of the 3593429th nucleotide site of chromosome 10 of the pig international reference genome version 11.1, and selecting the AA type individuals at the 3593429th nucleotide site as breeding pigs.

[0025] The embodiment discloses a method for breeding pig strains with large eye muscle area, comprising: if a nucleotide sequence as shown in SEQ ID NO: 3 is detected in pig genomic DNA, the corresponding homozygous genotype individual is used as a breeding pig.

[0026] The embodiment discloses the use of a SNP site or primer pair associated with the intramuscular fat content of Large White pigs in screening pig strains with large eye muscle area, wherein the SNP site is the 3593429th nucleotide site of chromosome 10 of the International Swine Genome Version 11.1 reference sequence, and has an A / G polymorphism. The nucleotide sequences of the primer pair are shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0027] 1. Test sample collection

[0028] The experimental pigs were 744 purebred Large White boars (castrated, weighing about 90 kg) from a pig farm in Hubei Province. The pigs were allowed to eat and drink freely, and the entire feeding method and feeding conditions were kept consistent, which was a conventional method.

[0029] Before slaughter, ear tissue samples were collected from all experimental pigs and stored in 75% ethanol for use in extracting pig genomic DNA. The specific method was based on the instructions provided by the genomic DNA kit produced by Beijing Tiangen Biochemical Technology Co., Ltd.

[0030] 2. Extraction and Detection of Pig Genomic DNA

[0031] In this experiment, the genomic DNA kit (TIANamp Genomic DNA Kit) produced by Beijing Tiangen Biochemical Technology Co., Ltd. was used to extract pig genomic DNA from pig ear tissue. The specific operation steps are as follows:

[0032] 1) Use alcohol-disinfected ophthalmic surgical scissors to cut the pig ear sample into a paste, add 200 μL buffer GA (provided in the kit), and shake until thoroughly suspended.

[0033] 2) Add 20 μL of proteinase K solution (provided in the kit), mix well, and digest overnight in a 56°C water bath.

[0034] 3) Add 200 μL of buffer GB (provided with the kit), mix thoroughly by inversion, and place at 70°C for 10 minutes. When the solution becomes clear, centrifuge briefly to remove water droplets on the inner wall of the tube cap.

[0035] 4) Add 200 μL of anhydrous ethanol and vortex thoroughly for 15 seconds. Flocculent precipitation may appear at this time. Centrifuge briefly to remove water droplets on the inner wall of the tube cap.

[0036] 5) Add the solution and flocculent precipitate obtained in the previous step to an adsorption column CB3 (the adsorption column is placed in a collection tube), centrifuge at 12000 rpm for 30 seconds, pour out the waste liquid, and put the adsorption column CB3 back into the collection tube.

[0037] 6) Add 500 μL of buffer GD (provided in the kit) to the adsorption column CB3, centrifuge at 12,000 rpm for 30 s, discard the waste liquid, and place the adsorption column CB3 in a collection tube.

[0038] 7) Add 600 μL of rinsing solution PW (provided in the kit) to the adsorption column CB3, centrifuge at 12000 rpm for 30 s, discard the waste liquid, and place the adsorption column CB3 in a collection tube.

[0039] 8) Repeat step 7.

[0040] 9) Place the adsorption column CB3 back into the collection tube, centrifuge at 12000 rpm for 2 min, discard the waste liquid, and place the adsorption column CB3 at room temperature for several minutes to completely dry the residual rinse liquid in the adsorption material.

[0041] 10) Transfer the adsorption column CB3 into a clean centrifuge tube, add 50-200 μL of elution buffer TE to the middle part of the adsorption membrane, let it stand at room temperature for 2-5 minutes, centrifuge at 12000 rpm for 2 minutes, and collect the solution into the centrifuge tube.

[0042] 11) Take 2 μL of the DNA solution obtained in the previous step and 1 μL of the loading buffer solution, mix them, load them on a 1.2% agarose gel, and perform electrophoresis at 120V for about 20 minutes. Observe the electrophoresis results under ultraviolet light and take pictures to determine the integrity of the DNA. The extracted DNA was quality tested using a NanoDrop 2000 nucleic acid protein analyzer (Thermo Fisher Scientific, USA). The ratio of A260 / A280 was between 1.7-2.1, and the ratio of A260 / A230 was between 1.8-2.2, which was considered qualified. The concentration of qualified DNA was measured, and then the concentration was uniformly diluted to 200 ng / μL and stored in a refrigerator at -20°C. Unqualified DNA samples need to be re-extracted.

[0043] 3. SNP chip genotype determination and genotype data quality control

[0044] The genomic DNA samples extracted from 1066 pig ears were hybridized on the Porcine SNP60 BeadChip6 whole genome chip developed by Illumina. The chip contains 61565 SNP sites. The original genotype data of all individuals were quality controlled using PLINK software, with SNP genotype call rate (SNP call rate)>90%, minor allele frequency (MAF)>0.01, Hardy-Weinberg Equilibrium (HWE) test P value <10-6 and sample call rate (sample call rate)>90% as the standard.

[0045] 4. Data collation and analysis

[0046] 1) Phenotypic data analysis

[0047] R statistical analysis software was used to perform descriptive statistical analysis on pig eye muscle area, including calculating the mean, standard deviation, maximum and minimum values ​​of the trait.

[0048] 2) Genome-wide association analysis

[0049] PLINK software was used to perform GWAS analysis. The applicant used the following mixed model to analyze the data.

[0050] The model is: Yij = μ + Gi + εij

[0051] Among them, Yij is the trait value after treatment; μ is the mean of each trait; Gi is the genotype effect; εij is the random effect.

[0052] 3) Test the significance of the association between SNP and traits.

[0053] When a SNP meets P < 10 -4 If the condition is met, it indicates that the SNP has reached genome-wide significance.

[0054] 4) SNP annotation

[0055] According to the chip SNP information, the Variant Effect Predictor tool was used to annotate the SNP in the SusscrofaBuid11.1 database of the Ensembl website (www.ensembl.org), that is, to determine the chromosome where the SNP site is located and its physical position on the chromosome, and thus determine whether these significant SNPs are inside or flanking regions of known genes in the Ensembl database. Then, using bioinformatics methods, according to the gene structure, gene type, gene function and pathway information provided by Ensembl, NCBI (www.ncbi.nlm.nih.gov), DAVID (david.abcc.ncifcrf.gov), etc., the genes in the target region were functionally annotated. Finally, the QTLdb (cn.animalgenome.org / cgi-bin / QTLdb / index) website was used to search whether the site fell into the QTLs reported to be related to meat quality traits, and further determine the SNPs associated with pig eye muscle area.

[0056] Table 1 shows the relationship between the genotype of the SNP loci provided in the embodiment and the eye muscle area phenotype. In Table 1, the eye muscle area of ​​the AA genotype, the AG genotype and the GG genotype is statistically analyzed, and the significance level of the eye muscle area between these genotypes is compared, p < 0.05 is a significant difference; p < 0.01 is an extremely significant difference.

[0057] As shown in Table 1, among the 744 Large White pigs, there were 211 with AA genotype, 288 with AG genotype, and 245 with GG genotype. The eye muscle area of ​​individuals with AA genotype was significantly higher than that of individuals with AG genotype and GG genotype, and there was no significant difference between the eye muscle area of ​​individuals with AG genotype and GG individuals. In summary, A is an allele that is beneficial to the eye muscle area of ​​Large White pigs.

[0058] Table 1

[0059]

[0060]

[0061] 5. PCR Testing

[0062] The porcine genomic DNA obtained according to the above steps was amplified by PCR using the primer pair shown in SEQ ID NO: 1 and SEQ ID NO: 2. The PCR reaction system contained 1 μL porcine genomic DNA, 0.5 μL DNA shown in SEQ ID NO: 2, 0.5 μL DNA shown in SEQ ID NO: 3, and 10 μL PCR Mix reagent (2×M5TaqHiFiPCRMix, Mei5bio) in 12 μL. The PCR reaction steps included 94°C, 3 min; 30 cycles (94°C, 25 s; 60°C, 25 s; 72°C, 10 s); 72°C, 5 min; 4°C, ∞.

[0063] Figure 2 The electrophoresis detection diagram of the PCR product obtained by PCR amplification of a pig genomic DNA in the above steps in 2% agarose gel is shown. In the figure, the target fragment amplified by the PCR product is about 369 bp in size. The 369 bp is sequenced to obtain the nucleotide sequence shown in SEQ ID NO: 3 and 4. Figure 3 The results of sequence alignment between the sequence near the SNP site in the nucleotide sequence shown in SEQ ID NO:3 and the related gene fragments of pigs in GenBank, and the results of sequence alignment between the sequence near the SNP site in the nucleotide sequence shown in SEQ ID NO:4 and the related gene fragments of pigs in GenBank are shown.

[0064] The above is only a preferred specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed in the present application should be covered within the protection scope of the present application.

Claims

1. Molecular markers associated with pig eye muscle area, including a nucleotide sequence formed by a single nucleotide polymorphism of A / G at position 3593429 of chromosome 10 of the International Pig Reference Genome 11.

1.

2. A primer pair for detecting the molecular marker according to claim 1, wherein the nucleotide sequences of the primer pair are shown in SEQ ID NO: 1 and SEQ ID NO:

2.

3. A kit comprising the primer pair according to claim 2 and reagents required for PCR amplification.

4. A method for detecting the molecular marker according to claim 1, comprising: Extracting genomic DNA from the pig to be tested; Performing PCR amplification on the genomic DNA of the pig to be tested to obtain a PCR amplification product; Sequencing the PCR amplification product to obtain a sequencing result; Based on the sequencing results, the polymorphism of the 3593429nt site on chromosome 10 of the International Pig Reference Genome 11.1 version was determined.

5. Molecular markers associated with pig eye muscle area, including the nucleotide sequences shown in SEQ ID NO:3 and SEQ ID NO:

4.

6. A method for breeding a pig strain with large eye loin area, comprising: The genotype of the 3593429th nucleotide position of chromosome 10 of the International Pig Reference Genome 11.1 version was detected, and the AA type individuals at the 3593429th nucleotide position were selected as breeding pigs.

7. A method for breeding a pig strain with large eye loin area, comprising: If the nucleotide sequence shown in SEQ ID NO: 3 is detected in the pig genomic DNA, the corresponding homozygous genotype individual is used as a breeding pig.

8. Application of a SNP site or primer pair associated with the intramuscular fat content of Large White pigs in screening pig breeds with large eye muscle area, wherein the SNP site is the 3593429th nucleotide site of chromosome 10 of the International Swine Genome Version 11.1 reference sequence, and has an A / G polymorphism, and the nucleotide sequence of the primer pair is shown in SEQ ID NO: 1 and SEQ ID NO: 2.

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