A pair of SNP marker primers associated with backfat thickness in pigs and their applications

Abstract

This invention relates to a primer pair for a SNP marker associated with backfat thickness in pigs and its application. The SNP marker is a molecular marker located at nucleotide 80880668 on chromosome 4 of the pig genome reference sequence (version 11.1), exhibiting A / G polymorphism. This SNP marker is highly significantly correlated with backfat thickness in Suhuai pigs (P < 0.001). A primer pair for detecting the SNP marker is provided, with the upstream primer being SEQ ID NO: 2 and the downstream primer being SEQ ID NO: 3. The SNP marker provided by this invention is highly significantly correlated with backfat thickness in Suhuai pigs. By identifying the genotype of this SNP marker, Suhuai pig breeds with thinner backfat can be screened. The establishment of such breeds can reduce backfat thickness in Suhuai pigs and generate greater social and economic benefits.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology and relates to an SNP marker primer pair related to backfat thickness in pigs and its application. Background Technology

[0002] Backfat thickness is a quantitative trait of significant economic value. Furthermore, backfat thickness in pigs is correlated with numerous traits (such as carcass traits, meat quality traits, and reproductive traits), and shows a strong negative correlation with lean meat percentage. Since measuring lean meat percentage is very complex, using easily measurable backfat thickness as an indicator of lean meat percentage is of great importance in pig production and breeding. Currently, lean-type commercial pig breeds hold a large market share in the pork consumption market, but consumers are increasingly demanding higher quality pork. Current commercial breeds cannot fully meet consumer needs for meat quality. Some high-end consumers prefer to buy premium pork such as local breeds or hybrid pork containing local bloodlines. However, this type of pork often has a thick backfat, making it difficult for most consumers to accept. Therefore, reducing the backfat thickness of local pigs is a key concern for breeders.

[0003] This invention is based on the technology obtained from the research on the carcass traits of the Suhuai pig. The Suhuai pig was developed in 1998 by the Huaiyin Breeding Pig Farm of Huai'an City, Nanjing Agricultural University, Jiangsu Provincial Animal Husbandry Station, and Huai'an Municipal Agricultural Commission, and was approved as a new breed by the National Livestock and Poultry Genetic Resources Committee on March 25, 2011. The Suhuai pig is a new breed developed by introducing Large White pig bloodlines into the Xinhuai pig, through crossbreeding and multi-generational selection, containing 50% Xinhuai pig bloodline and 50% Large White pig bloodline. The Suhuai pig has black hair (very few have white hooves), a medium-sized head, a wide forehead, large ears that tilt slightly forward, a slightly concave face, a straight and long back and loin, a drooping abdomen, well-developed hindquarters, and sturdy limbs. Suhuai pork is delicious and popular with consumers; however, the overall backfat thickness of the pig herd is still relatively high, requiring further selective breeding to reduce it.

[0004] Backfat thickness is a typical quantitative trait with moderate to high heritability (0.12–0.74). Genome-wide association study (GWAS) can locate quantitative trait loci (QTLs) and candidate genes associated with backfat thickness. This method has been used to identify QTLs related to backfat thickness in numerous pig breeds both domestically and internationally. However, most QTL studies focus on Western commercial pig breeds, such as Duroc, Landrace, Large White, and their hybrid populations, with limited research on Chinese breeds like the Suhuai pig. Therefore, using the Suhuai pig as the experimental animal to identify SNPs associated with backfat thickness and to utilize marker-assisted selection technology for genetic improvement of this trait is of great significance. Summary of the Invention

[0005] The purpose of this invention is to address the problems of time-consuming and labor-intensive traditional breeding of pigs with thick backfat and slow breeding results, and to provide a breeding molecular marker developed from SNP markers related to pig backfat thickness.

[0006] Another object of the present invention is to provide primer pairs and detection methods for detecting the above-mentioned SNP markers.

[0007] Another object of the present invention is to provide the use of the above-mentioned SNP markers, molecular markers, and primers.

[0008] The objective of this invention can be achieved through the following technical solutions:

[0009] A molecular marker associated with backfat thickness in pigs, the molecular marker sequence of which is shown in SEQ ID NO: 1, contains a SNP marker site associated with backfat thickness in pigs. This site is the rs80880668 nucleotide site on chromosome 4 of the pig in the international pig genome version 11.1 reference sequence. The SNP marker site described in SEQ ID NO: 1 is located at position 94 and exhibits A / G polymorphism.

[0010] A primer pair for detecting SNP markers associated with backfat thickness in pigs, with the upstream primer being SEQ ID NO: 2 and the downstream primer being SEQ ID NO: 3; the SNP marker is a molecular marker at the rs80880668 nucleotide site on chromosome 4 of the pig genome in version 11.1 of the International Pig Genome Reference Sequence, and exhibits A / G polymorphism; the SNP marker is highly significantly correlated with backfat thickness in Suhuai pigs (P<0.001).

[0011] A method for detecting SNP markers associated with the backfat thickness trait in pigs includes PCR amplification of a sequence at nucleotide site rs80880668 on chromosome 4 of the international pig genome version 11.1 reference sequence, sequencing the amplified product, and interpreting the A / G polymorphism at that site.

[0012] As a preferred embodiment of the present invention, the method includes the following steps:

[0013] (1) Collect ear tissue samples to extract total DNA;

[0014] (2) Using the extracted genomic DNA as a template, PCR amplification was performed using the primer pairs shown in SEQ ID NO: 2 and SEQ ID NO: 3;

[0015] (2) Sequencing of the amplified product, analysis of the sequencing results, and interpretation of the A / G polymorphism at position 94 of SEQ ID NO: 1.

[0016] Application of SNP markers associated with backfat thickness in screening Suhuai pig populations with thin backfat: The SNP markers are molecular markers at the rs80880668 nucleotide site on chromosome 4 of the international pig genome version 11.1 reference sequence, and have A / G polymorphism. The SNP markers are highly significantly associated with backfat thickness in Suhuai pigs (P<0.001).

[0017] The application of the molecular markers described in this invention in screening Suhuai pig populations with thinner backfat.

[0018] The application of the primer pair described in this invention in screening Suhuai pig populations with thinner backfat.

[0019] A method for screening thin-backfat Suhuai pig populations includes detecting the genotype of the rs80880668 nucleotide site on chromosome 4 of the international pig genome version 11.1 reference sequence of Suhuai pigs, and selecting individuals of the GG and / or AG nucleotide types at the rs80880668 nucleotide site as priority for breeding stock.

[0020] Beneficial effects

[0021] This invention develops an SNP marker associated with the backfat thickness of Suhuai pigs and provides primer pairs and methods for detecting the marker. By identifying the genotype of the SNP marker, Suhuai pig breeds with thinner backfat can be screened. The establishment of such breeds can reduce the backfat thickness of Suhuai pigs and generate more social and economic benefits. Attached Figure Description

[0022] Figure 1 Manhattan plot of genome-wide association analysis of backfat thickness at the 6th-7th ribs in Suhuai pigs

[0023] Figure 2 This is a gel image showing the PCR amplification of the rs80880668 site on chromosome 4 of the Suhuai pig.

[0024] Figure 3 The results of the genotyping of the s80880668 site in the staining of Suhuai pig No. 4 are shown in Figure A, which is AG type, Figure B is AA type, and Figure C is GG type. Detailed Implementation

[0025] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0026] Example 1

[0027] 1. Source of experimental animals

[0028] Huaiyin Pig Breeding Farm, Huai'an City, Jiangsu Province

[0029] 2. Extraction of Suhuai pig genomic DNA

[0030] Ear tissue samples were collected from 418 Suhuai pigs for individual DNA extraction.

[0031] Referring to the instructions for the Tissue DNA Extraction Kit from Tiangen Biotech Co., Ltd., the extraction steps are as follows:

[0032] ① First, add 68 mL of buffer GD and 200 mL of anhydrous ethanol to the wash buffer PW, and mix thoroughly.

[0033] ② Collect approximately 100 mg of ear tissue sample and place it in a 2 mL EP tube. After completely cutting it into small pieces, add 200 μL of buffer GA and shake until completely suspended.

[0034] ③ Add 20 μL of proteinase K solution, mix well, and place in a 56°C metal bath for digestion overnight until the tissue sample dissolves. Briefly centrifuge to remove water droplets from the inner wall of the tube cap.

[0035] ④ Add 200 μL of buffer GB, mix thoroughly by inverting, place in a 70℃ metal bath for 10 min, the solution should become clear, and briefly centrifuge to remove water droplets from the inner wall of the tube cap.

[0036] ⑤ Add 200 μL of anhydrous ethanol and shake thoroughly for 15 seconds. At this time, flocculent precipitate may appear. Briefly centrifuge to remove water droplets from the inner wall of the tube cap.

[0037] ⑥ Add the solution and flocculent precipitate obtained in the previous step to an adsorption column CB3, place the adsorption column in the collection tube, then centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, and put the adsorption column CB3 back into the collection tube.

[0038] ⑦ Add 500 μL of buffer GD to the adsorption column CB3, centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, and put the adsorption column CB3 into the collection tube.

[0039] ⑧ Add 600 μL of washing buffer PW to the adsorption column CB3, centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, and place the adsorption column CB3 into the collection tube.

[0040] ⑨ Repeat step ⑧.

[0041] ⑩ Place the adsorption column CB3 back into the collection tube, centrifuge at 12,000 rpm for 2 minutes, and discard the waste liquid. Place the adsorption column CB3 at room temperature for several minutes to thoroughly dry any residual washing liquid in the adsorption material.

[0042] Transfer the adsorption column CB3 into a clean centrifuge tube. Add 100 μL of elution buffer TE to the middle of the adsorption membrane. Incubate at room temperature for 2-5 min, then centrifuge at 12,000 rpm for 2 min. Collect the solution in the centrifuge tube. Add the centrifuged solution back to the adsorption column CB3. Incubate at room temperature for 2 min, then centrifuge at 12,000 rpm for 2 min. Collect the solution in the centrifuge tube.

[0043] The quality and concentration of DNA were determined using a Nanodrop-2000 spectrophotometer. All DNA concentrations were diluted to 50 ng / μL and stored at -20°C for later use.

[0044] 3. Target fragment PCR amplification and sequencing

[0045] PCR amplification was performed using Suhuai pig genomic DNA as a template. The reaction system included 1 μL of DNA template, 1 μL each of the primers shown in SEQ ID NO: 2 and SEQ ID NO: 3, 12.5 μL of PCR mix, and 9.5 μL of ddH2O. The amplification program was as follows:

[0046]

[0047] The amplified product was subjected to agarose gel electrophoresis. The product fragment size was approximately 279 bp. The electrophoresis results are as follows: Figure 1 As shown. The remaining amplification products were sequenced, and the sequencing results were compared and verified for accuracy using DNAman software. The rs80880668 site was genotyped using Chromas software.

[0048] 4. Statistical Analysis

[0049] Association analysis between genotype and phenotype was performed using a general linear model in SAS 9.0 software. The model is as follows:

[0050] y = μ + B + G + e

[0051] Where y is the phenotypic vector of backfat thickness; μ represents the population mean; B represents the fixed effect (sex, batch, and season as fixed effects, carcass weight and age as covariates); G is the fixed effect of individual SNP markers; and e is the random residual effect, following a set order. The normal distribution is given by I, where I is the identity matrix. This represents the residual variance.

[0052] 5 Results

[0053] Table 1 shows the effects of different genotypes at the rs80880668 locus on backfat thickness at the 6th-7th ribs of Suhuai pigs. The results indicate that there were significant differences in backfat thickness among the three genotypes at the rs80880668 locus. Specifically, the backfat thickness of the GG genotype was significantly thinner than that of the AA genotype (P<0.001), the backfat thickness of the AG genotype was significantly thinner than that of the AA genotype (P<0.007), and the backfat thickness of the GG genotype was significantly thinner than that of the AG genotype (P<0.017). Therefore, selective breeding of GG and AG genotype individuals at the rs80880668 locus in Suhuai pigs during successive generations is beneficial for reducing backfat thickness in the Suhuai pig population, thereby improving the economic benefits of Suhuai pigs.

[0054] Table 1. Association analysis between the rs80880668 locus on pig chromosome 4 and backfat thickness in Suhuai pigs.

[0055]

[0056] Note: Different uppercase letters in the same row of numbers indicate extremely significant differences (P<0.001), and lowercase letters indicate significant differences (P<0.05).

Claims

1. The application of primer pairs for detecting SNP markers associated with backfat thickness in screening for thinner backfat-thick Suhuai pig breeds, characterized in that... The SNP marker is located at rs80880668 nucleotides on chromosome 4 of the pig genome in version 11.1 of the International Pig Genome Reference Sequence, and it exhibits A / G polymorphism. The SNP marker is significantly correlated with the backfat thickness of Suhuai pigs. Individuals with the GG type at this site have significantly thinner backfat than individuals with the AA type. The upstream primer of the primer pair is SEQ ID NO: 2, and the downstream primer is SEQ ID NO:

3.

2. A method for screening Suhuai pig breeds with low backfat thickness, characterized in that... This includes using primer pairs with SNP markers as described in claim 1 to PCR detect the genotype of the rs80880668 nucleotide site on chromosome 4 of the Suhuai pig international pig genome version 11.1 reference sequence. Individuals with the GG genotype at this site have significantly thinner backfat than individuals with the AA genotype. Individuals with the GG and / or AG genotypes at the rs80880668 nucleotide site are selected and given priority for breeding.

Images