Primer pair of snp marker related to piglet vulva width trait on pig chromosome 1 and application thereof

Abstract

The present application relates to a SNP marker primer pair related to pig vulva width trait on pig chromosome 1 and application thereof. The SNP marker site is a molecular marker of nucleotide site rs333681174 on pig chromosome 1 in the international pig genome 11.1 version reference sequence, and has C / A 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 is screened by identifying the genotype of the SNP marker. The establishment of the population or strain can improve the vulva width and 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] In the livestock breeding industry, improving the production and reproductive performance of pigs has always been a crucial concern for farmers. Vulvar width, as an important reproductive trait, significantly impacts the productivity and fertility of sows. The size of the vulvar width not only affects the selection and culling of gilts, the success of mating and farrowing, but is also closely related to postpartum recovery.

[0003] Traditional methods assess vulvar width by manually measuring pig herds, but this approach suffers from inaccuracies, high labor costs, and low efficiency. In recent years, with the development of molecular genetics and genomics, researchers have begun to address this issue by utilizing the association between single nucleotide polymorphism (SNP) markers and pig traits. SNP markers are common genomic genetic markers, possessing the advantages of high polymorphism and widespread distribution throughout the genome, thus becoming important tools for studying pig genetic traits.

[0004] Against this backdrop, this patent proposes a pair of SNP marker primers associated with the vulva width trait in pigs, enabling rapid and accurate assessment of the vulva width trait through genotyping technology. This technology not only improves the accuracy and efficiency of vulva width assessment but also provides important molecular genetic evidence for pig breeding and reproductive performance improvement. Summary of the Invention

[0005] The purpose of this invention is to address the time-consuming and labor-intensive nature of traditional on-site testing for wide vulva in pigs, which results in slow breeding outcomes. This invention provides a breeding molecular marker developed from SNP markers related to wide vulva in pigs.

[0006] 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.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] A molecular marker associated with the wide vulva trait on pig chromosome 1, the molecular marker sequence of which is shown in SEQ ID NO: 1, contains a SNP marker site associated with the wide vulva trait. This site is the rs333681174 nucleotide site on pig chromosome 1 of the international pig genome version 11.1 reference sequence. The SNP marker site described in SEQ ID NO: 1 is located at position 301 and exhibits C / A polymorphism.

[0009] A primer pair for detecting SNP markers on pig chromosome 1 associated with the wide vulva trait, with the upstream primer being SEQ ID NO: 2 and the downstream primer being SEQ ID NO: 3.

[0010] The application of the molecular markers and primer pairs described in this invention in detecting vaginal width traits in pigs and in pig breeding.

[0011] A method for detecting SNP markers on pig chromosome 1 associated with the wide vulva trait includes PCR amplification of a sequence at nucleotide site rs333681174 on pig chromosome 1 of the international pig genome version 11.1 reference sequence, sequencing the amplification product, and interpreting the C / A polymorphism at that site.

[0012] As a preferred embodiment of the present invention, the pig is a Large White pig.

[0013] As a preferred embodiment of the present invention, the primer pair described herein is used to perform PCR amplification of Large White pig genomic DNA.

[0014] As a further preferred embodiment of the present invention, the method includes the following steps:

[0015] (1) Extract DNA from pig ear tissue samples;

[0016] (2) Using the extracted porcine genomic DNA as a template, PCR amplification was performed using the primer pair described in this invention;

[0017] (3) Sequencing of the amplified product, analysis of the sequencing results, and interpretation of the C / A polymorphism at position 301 of SEQ ID NO: 1.

[0018] The application of the molecular markers described in this invention in screening pig populations or new strains with wider vulvas.

[0019] The application of the primer pairs described in this invention in screening pig populations or new strains with wider vulvas.

[0020] A method for screening pig populations with wider vulvas includes detecting the genotype at the rs333681174 nucleotide site on chromosome 1 of the pig international pig genome version 11.1 reference sequence, and selecting individuals with the CC and CA genotypes at the rs333681174 nucleotide site as priority for breeding replacement pigs.

[0021] As a preferred embodiment of the present invention, the pig breed used is the Large White pig.

[0022] As a preferred embodiment of the present invention, the method for detecting the genotype of the rs333681174 nucleotide site on pig chromosome 1 in the International Pig Genome Version 11.1 reference sequence is selected from PCR or gene sequencing.

[0023] Beneficial effects

[0024] This invention develops a SNP marker on pig chromosome 1 that is associated with vulvar width and provides primer pairs and methods for detecting this marker. Pig breeds with wider vulvas can be screened by identifying the genotype of this SNP marker. Establishing such breeds can improve the overall reproductive performance of pigs and generate greater social and economic benefits. Attached Figure Description

[0025] Figure 1 This is a gel image showing the PCR amplification of the rs333681174 site on chromosome 1 of Large White pigs.

[0026] Figure 2 Example of a typing diagram for chromosome 1 of Large White pigs at the rs333681174 locus.

[0027] A is of type CC, B is of type CA, and C is of type AA. Detailed Implementation Plan

[0028] 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.

[0029] Example 1

[0030] 1. Source of experimental animals

[0031] Jiangsu Zhengda Suken Pig Industry Co., Ltd. -- Yixing 5000 Breeding Pig Farm

[0032] 2. Phenotypic Collection Methods

[0033] Vulvar width refers to the distance between the leftmost and rightmost points of the widest part of the vulva. All vulvar width phenotypic data were obtained by the same personnel using a ruler with a minimum scale of 1 mm. 3. Extraction of porcine genomic DNA.

[0034] One ear tissue sample was collected from 439 Canada White pigs for individual DNA extraction.

[0035] Referring to the instructions for the Tissue DNA Extraction Kit from Tiangen Biotech Co., Ltd., the extraction steps are as follows:

[0036] ① First, add 68 mL of buffer GD and 200 mL of anhydrous ethanol to the wash buffer PW, and mix thoroughly.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] ⑨ Repeat step ⑧.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 4. Target fragment PCR amplification and sequencing

[0049] 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:

[0050]

[0051]

[0052] The amplification product was subjected to agarose gel electrophoresis. The product fragment size was approximately 385 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 rs333681174 site was genotyped using Chromas software.

[0053] 5. Statistical Analysis

[0054] Association analysis of 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

[0055] Among them, Y ijk B is the phenotypic value of vulvar width; μ is the population mean vulvar width; i The fixation effect of SNP labeling; G j The sampling batch is represented as a fixed effect; W k Representing age as a covariate; e ijk It is a residual.

[0056] 6 Results

[0057] Table 1 shows the effects of different genotypes at the rs333681174 locus on vulvar width in Large White pigs. The results indicate a highly significant association between the rs333681174 locus genotype and vulvar width phenotype (P<0.01). Specifically, individuals with the CC and CA genotypes had significantly wider vulvas than those with the AA genotype (P<0.01). Therefore, selective breeding for individuals with the CC and CA genotypes at the rs333681174 locus in Large White pigs is beneficial for increasing vulvar width in the population, thereby improving the reproductive performance of Large White pigs.

[0058] Table 1. Association analysis between the rs333681174 locus on chromosome 1 of pigs and the vulva width of Large White pigs.

[0059]

[0060] Note: Different letters in the same row of numbers indicate significant differences (P<0.01).

Claims

1. A molecular marker on pig chromosome 1 associated with the trait of wide vulva, and the application of a primer pair for detecting a molecular marker on pig chromosome 1 associated with the trait of wide vulva in screening Canadian Large White pig populations; the molecular marker sequence is shown in SEQ ID NO: 1, which contains a SNP marker site associated with the trait of wide vulva, which is the rs333681174 nucleotide site on pig chromosome 1 in the international pig genome version 11.1 reference sequence. The SNP marker site described in SEQ ID NO: 1 is located at position 301 and exhibits C / A polymorphism; the primer pair consists of the upstream primer shown in SEQ ID NO: 2 and the downstream primer shown in SEQ ID NO:

3.

2. A method for screening Canadian Large White pig populations with wider vulvas, characterized in that... PCR amplification was performed on a sequence containing a nucleotide locus of rs333681174 on chromosome 1 of the pig genome in version 11.1 of the International Pig Genome Reference Sequence. The amplified product was then sequenced to determine the C / A polymorphism at this locus.

3. The method according to claim 2, characterized in that... The genomic DNA of Canada Large White pigs was amplified by PCR using the primer pair described in claim 1.

4. The method according to claim 3, characterized in that... Includes the following steps: (1) Take tissue samples from Canadian Large White pigs and extract total DNA; (2) Using the extracted porcine genomic DNA as a template, perform PCR amplification using the primer pair described in claim 1; (3) Sequencing of the amplified product, analysis of the sequencing results, and interpretation of the C / A polymorphism at position 301 of SEQ ID NO:

1.

5. A method for screening Canadian Large White pig populations with wider vulvas, characterized in that... This includes detecting the genotype of the rs333681174 nucleotide site on chromosome 1 of the pig in the International Swine Genome Version 11.1 reference sequence, and selecting individuals with the CC and CA nucleotide sites at the rs333681174 nucleotide site as replacement breeding pigs.

6. The method according to claim 5, characterized in that... The method for detecting the genotype at the rs333681174 nucleotide site on chromosome 1 of the pig in the International Pig Genome Version 11.1 reference sequence is PCR amplification and gene sequencing.

Images