A snp marker pair related to the pig vulva width trait and application thereof

Abstract

The present application relates to a SNP marker primer pair related to pig vulva width trait and application thereof. The SNP marker site is a molecular marker of nucleotide site rs327735514 of pig chromosome 14 in the international pig genome 11.1 version reference sequence, and has G / T polymorphism. A primer pair for detecting the SNP marker, the upstream primer is SEQ ID NO: 2, and the downstream primer is SEQ ID NO: 3. The SNP marker provided by the present application can be applied to marker assisted selection of pig vulva width trait, and a pig population or strain with wider vulva width is screened by identifying the genotype of the SNP marker. The establishment of the population or strain can improve the reproductive performance of pigs and produce more 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 the pig vulva width trait and its application. Background Technology

[0002] Vulvar traits, as one of the indicators for evaluating sow reproductive performance, are receiving increasing attention. Vulvar traits mainly include vulvar length, width, and vulvar angle score. A larger vulva is more conducive to artificial insemination and normal farrowing in sows, and studies have reported a positive correlation between vulvar size and the total number of piglets born and the number of live piglets. Assessing vulvar size at an appropriate age is an effective method for identifying gilts with maximum reproductive potential. Therefore, selecting sows with large vulvas is crucial for pig production.

[0003] However, vulva width is a quantitative trait, and genetic progress using traditional phenotypic selection is slow. Genome-wide association analysis (GWAS) can be used to identify SNPs significantly associated with vulva width in pigs. Simultaneously, marker-assisted selection (TAG) or genomic selection can be employed for early selection of replacement gilts, accelerating the genetic improvement of vulva width. This invention, using vulva width as a representative example, provides a novel SNP marker influencing vulva width in pigs, which will be of great significance for breeding populations or new strains with wider vulvas and higher litter sizes. Summary of the Invention

[0004] The purpose of this invention is to address the time-consuming and labor-intensive nature of traditional breeding methods for wide vulva in pigs, which results in slow breeding outcomes, by providing a breeding molecular marker developed from SNP markers related to wide vulva in pigs.

[0005] Another object of the present invention is to provide primer pairs and detection methods for detecting the above-mentioned SNP markers. Another object of the present invention is to provide uses for the above-mentioned SNP markers, molecular markers, and primers.

[0006] The objective of this invention can be achieved through the following technical solutions:

[0007] A molecular marker associated with the vulva width trait in pigs is disclosed. The molecular marker sequence is shown in SEQ ID NO: 1, which contains a SNP marker site associated with the vulva width trait in pigs. This site is the rs327735514 nucleotide site on chromosome 14 of the international pig genome version 11.1 reference sequence (this site was identified by the team through genome-wide association analysis). The SNP marker site described in SEQ ID NO: 1 is located at position 501 and exhibits G / T polymorphism. The vulva width of individuals with the GG type is significantly greater than that of individuals with the GT and TT types.

[0008] A primer pair for detecting SNP markers associated with the wide vulva trait in pigs, with upstream primer SEQ ID NO: 2 and downstream primer SEQ ID NO: 3.

[0009] A method for detecting SNP markers associated with the wide vulva trait in pigs includes PCR amplification of a sequence at nucleotide site rs327735514 on chromosome 14 of the international pig genome version 11.1 reference sequence, sequencing of the amplified product, and interpretation of the G / T polymorphism at this site. The method includes the following steps:

[0010] (1) Extract DNA from pig ear tissue samples;

[0011] (2) Using the extracted porcine genomic DNA as a template, PCR amplification was performed using the primer pair described above;

[0012] (3) Sequencing of the amplified product, analysis of the sequencing results, and interpretation of the G / T polymorphism at position 501 of SEQ ID NO: 1.

[0013] The molecular markers described in this invention can be used in screening pig populations or new strains with wider vulvas.

[0014] The application of the primer pairs described in this invention in screening pig populations or new strains with wider vulvas.

[0015] A method for screening PIC Large White pig populations with wider vulvas includes detecting the genotype of the rs327735514 nucleotide site on chromosome 14 of the pig international pig genome version 11.1 reference sequence, and selecting individuals with the GG genotype at the rs327735514 nucleotide site as priority for breeding stock.

[0016] Beneficial effects

[0017] This invention develops a SNP marker associated with vulvar width in pigs and provides primer pairs and methods for detecting this marker. Pig breeds with wider vulvas are screened by identifying the genotype of this SNP marker. Establishing such breeds can improve pig reproductive performance and generate greater social and economic benefits. Attached Figure Description

[0018] Figure 1 This is a gel image showing the PCR amplification of the rs327735514 site on chromosome 14 of PIC Large White pigs.

[0019] Figure 2 Example of a typing diagram for chromosome 14 rs327735514 in PIC Large White pigs.

[0020] Note: A has the genotype GG, B has the genotype GT, and C has the genotype TT. Detailed Implementation Plan

[0021] 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.

[0022] Example 1

[0023] 1. Source of experimental animals

[0024] PIC Core Breeding Farm, Ordos City, Inner Mongolia

[0025] 2. Extraction of pig genomic DNA

[0026] One ear tissue sample was collected from 313 PIC Large White pigs for individual DNA extraction.

[0027] Referring to the instructions for the Tissue DNA Extraction Kit from Tiangen Biotech Co., Ltd., the extraction steps are as follows:

[0028] ① First, add 68 mL of buffer GD and 200 mL of anhydrous ethanol to the wash buffer PW, and mix thoroughly.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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 place the adsorption column CB3 into the collection tube.

[0035] 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.

[0036] ⑨ Repeat step ⑧.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 3. Target fragment PCR amplification and sequencing

[0041] PCR amplification was performed using Large White 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, and 22 μL of PCR mix. The amplification program was as follows:

[0042]

[0043] The amplification products were subjected to agarose gel electrophoresis. The product fragment size was approximately 252 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 rs327735514 site was genotyped using Chromas software.

[0044] 4. Statistical Analysis

[0045] Association analysis between genotype and phenotype was performed using a general linear model in SAS 9.4 software. The model is as follows: Y ijk =μ+B i +G j +W k +e ijk

[0046] Among them, Y ijkB is the phenotypic value of vulvar width; μ is the population mean vulvar width; i The fixation effect of SNP labeling; G j Batch size is used as a covariate; W k Representing age as a covariate; e ijk It is a residual.

[0047] 5 Results

[0048] Table 1 shows the effects of different genotypes at the rs327735514 locus on vulvar width in Large White pigs. The results indicate that individuals with the GG genotype at the rs327735514 locus had a significantly wider vulvar width than those with the GT and TT genotypes (P<0.01). Therefore, selective breeding for individuals with the GG genotype at the rs327735514 locus in Large White pigs is beneficial for increasing vulvar width and thus improving reproductive performance.

[0049] Table 1. Association analysis between the rs327735514 locus on porcine chromosome 14 and vulvar width in Large White pigs.

[0050]

[0051] Note: Different letters in the number subscripts of the same line indicate significant differences (P<0.01).

Claims

1. The application of primer pairs for detecting SNP marker sites associated with vulvar width in screening Large White pig populations with wider vulvas, characterized by... Includes the following steps: (1) Extract DNA from pig ear tissue samples; (2) Using the extracted porcine genomic DNA as a template, PCR amplification was performed using primer pairs; (3) Sequencing of the amplified product, analysis of the sequencing results, and interpretation of the G / T polymorphism at position 501 of SEQ ID NO: 1; The SNP marker site is the rs327735514 nucleotide site on chromosome 14 of the pig genome version 11.1 reference sequence. The vulva width of the GG type individuals is significantly larger than that of the GT and TT type individuals. 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 PIC Large White pig populations or breeds with wider vulvas, characterized in that, This includes detecting the genotype of the rs327735514 nucleotide site on chromosome 14 of the Large White pig international pig genome version 11.1 reference sequence, and selecting GG-type individuals with the rs327735514 nucleotide site as replacement breeding pigs.

Images