Primer pair of snp marker related to rib number trait on pig chromosome 1 and application thereof

Abstract

The present application relates to a SNP marker primer pair related to a rib number trait of a pig on a chromosome 1 of the pig and an application thereof. The SNP marker is located on a nucleotide sequence of a MTHFD1L gene on the chromosome 1 of the pig, the site of the SNP marker is a molecular marker of a nucleotide site of WU_10_2_GL896601_1_56 of the chromosome 1 of the pig in an international pig genome 11.1 version reference sequence, and has a T / G polymorphism. A primer pair for detecting the SNP marker, an upstream primer is SEQ ID NO: 2, and a downstream primer is SEQ ID NO: 3. The SNP marker provided by the present application can be applied to marker-assisted selection of a rib number trait of a pig, and a pig population or strain with a multiple rib number is screened by identifying the genotype of the SNP marker. The establishment of the population or strain can improve the rib number and meat production performance of the pig, 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 trait of rib number in pigs and its application. Background Technology

[0002] Pork accounts for about 60% of meat consumption in China. With continuous population growth and rising living standards, the demand for pork is increasing. Improving pork production is a key goal in breeding, and increasing the production of high-quality pork is an even more important direction. The number of ribs affects carcass length and meat yield, with rib meat being the most popular high-quality cut among consumers. The number of ribs is an important indicator of carcass meat yield. Ribs are connected to the thoracic vertebrae, and the number of thoracic vertebrae determines the number of ribs; one thoracic vertebra corresponds to two ribs. As the number of ribs increases, the body length or carcass length of the pig also increases accordingly. Each additional rib can increase the body length of an adult pig by about 60 mm, thus increasing pork yield. Selecting for the rib number trait has significant economic value. However, the rib number trait is mainly determined post-slaughter, making conventional selection difficult. Compared with traditional breeding, molecular selection using markers related to the trait can effectively accelerate the breeding process. Studying the genetic molecular mechanisms of rib number in pigs and identifying relevant molecular markers that affect rib number for application in breeding has significant economic benefits for selecting populations or new strains with multiple ribs and high meat yield. Summary of the Invention

[0003] The purpose of this invention is to address the problems of difficulty in determining the number of ribs in pigs, time-consuming and labor-intensive breeding, and slow breeding results by providing a breeding molecular marker developed from SNP markers related to the number of ribs in pigs.

[0004] Another object of the present invention is to provide primer pairs and detection methods for detecting the above-mentioned SNP markers.

[0005] Another objective of this invention is to provide the uses of the above-mentioned SNP markers, molecular markers, and primers.

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

[0007] Molecular markers associated with the number of ribs on pig chromosome 1, the molecular marker sequences of which are shown in SEQ ID NO: 1, contain a SNP marker locus associated with the number of ribs in pigs. This locus is the WU_10_2_GL896601_1_56 nucleotide site on pig chromosome 1 in the international pig genome version 11.1 reference sequence. The SNP marker locus described in SEQ ID NO: 1 is located at position 501 and exhibits T / G polymorphism. In Suhuai pigs, individuals with the GG and TG morphisms at this locus have a significantly higher number of ribs than individuals with the TT morphism. In Large White crossbred pigs, individuals with the GG morphism at this locus have a significantly higher number of ribs than individuals with the TT morphism.

[0008] A primer pair for detecting SNP markers on pig chromosome 1 that are associated with the number of pig ribs, with the upstream primer being SEQ ID NO: 2 and the downstream primer being SEQ ID NO: 3.

[0009] The application of the molecular markers and primer pairs described in this invention in detecting the number of ribs in pigs and / or in pig breeding.

[0010] A method for detecting SNP markers on pig chromosome 1 associated with the number of pig ribs includes PCR amplification of a sequence at the WU_10_2_GL896601_1_56 nucleotide site on pig chromosome 1 in the International Pig Genome 11.1 reference sequence, sequencing the amplification product, and interpreting the T / G polymorphism at the site.

[0011] As a preferred embodiment of the present invention, PCR amplification of porcine genomic DNA is performed using the primer pair described herein.

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

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

[0014] (2) Using the extracted porcine genomic DNA as a template, PCR amplification was performed using the primer pair described in this invention;

[0015] (3) Sequencing of the amplified product, analysis of the sequencing results, and interpretation of the T / G polymorphism at position 501 of SEQ ID NO: 1.

[0016] The application of the molecular markers described in this invention in screening pig populations with multiple ribs or new strains.

[0017] The application of the primer pairs described in this invention in screening pig populations with multiple ribs or new strains.

[0018] A method for screening pig populations with multiple ribs includes detecting the genotype of the WU_10_2_GL896601_1_56 nucleotide site on chromosome 1 of the Suhuai pig in the international pig genome version 11.1 reference sequence, and selecting individuals of the GG and TG types at the WU_10_2_GL896601_1_56 nucleotide site as priority for breeding stock.

[0019] As a preferred embodiment of the present invention, the method for detecting the genotype of the WU_10_2_GL896601_1_56 nucleotide site on pig chromosome 1 in the International Pig Genome Version 11.1 reference sequence is selected from PCR or gene sequencing.

[0020] A method for screening pig populations with multiple ribs includes detecting the genotype of the WU_10_2_GL896601_1_56 nucleotide site on chromosome 1 of the pig in the international pig genome version 11.1 reference sequence of the Large White crossbred pig, and selecting individuals with the GG type at the WU_10_2_GL896601_1_56 nucleotide site as replacement breeding pigs.

[0021] As a preferred embodiment of the present invention, the method for detecting the genotype of the WU_10_2_GL896601_1_56 nucleotide site on pig chromosome 1 in the International Pig Genome Version 11.1 reference sequence is selected from PCR amplification or gene sequencing.

[0022] Beneficial effects

[0023] This invention develops a SNP marker on chromosome 1 of pigs that is associated with the number of ribs, and provides primer pairs and methods for detecting this marker. Pig breeds with a higher number of ribs can be screened by identifying the genotype of this SNP marker. The establishment of such breeds can improve the meat production performance of pigs and generate greater social and economic benefits. Attached Figure Description

[0024] Figure 1 This is a gel image showing the PCR amplification of the WU_10_2_GL896601_1_56 site of the MTHFD1L gene on pig chromosome 1.

[0025] Figure 2 Example of a genotyping diagram for the WU_10_2_GL896601_1_56 locus of the MTHFD1L gene on pig chromosome 1.

[0026] A is TT type, B is TG type, and C is GG type. Detailed Implementation Plan

[0027] 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.

[0028] Example 1

[0029] 1. Source of experimental animals

[0030] Suhuai pigs originate from Huaiyin Xinhuai Breeding Pig Farm in Huai'an City, Jiangsu Province, while large crossbred pigs originate from Jiangsu Sushi Meat Products Co., Ltd. in Huai'an City, Jiangsu Province.

[0031] 2. Extraction of pig genomic DNA

[0032] Ear tissue samples from 458 Suhuai pigs and muscle samples from 673 Large White crossbred pigs were collected for DNA extraction.

[0033] Referring to the instructions for the Tissue DNA Extraction Kit from Tiangen Biotech Co., Ltd., the extraction steps are as follows:

[0034] ① First, add 68 mL of buffer GD and 200 mL of anhydrous ethanol to the wash buffer PW, and mix thoroughly.

[0035] ② Cut off about 100mg of ear tissue or muscle sample and place it in a 2mL EP tube. After completely cutting it into small pieces, add 200μL of buffer GA and shake until completely suspended.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] ⑨ Repeat step ⑧.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 3. Target fragment PCR amplification and sequencing

[0047] PCR amplification was performed using porcine 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:

[0048]

[0049] The amplified product was subjected to agarose gel electrophoresis. The product fragment size was approximately 455 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 genotype of the WU_10_2_GL896601_1_56 site was determined using Chromas software.

[0050] 4. Statistical Analysis

[0051] 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 i +B j +G k +e jk

[0052] Where Yijk is the number of ribs in an individual; μ i B represents the mean number of ribs in the group; j The fixed effect representing the slaughter batch; G k For the fixed effect of SNP labeling; e jk It is a residual.

[0053] 5 Results

[0054] Table 1 shows the effects of different genotypes at the WU_10_2_GL896601_1_56 locus on the number of ribs in Su-Huai pigs and Large White crossbred pigs. The results showed that in the Su-Huai pig population, the WU_10_2_GL896601_1_56 locus genotype was significantly associated with the rib number phenotype (P<0.01). Specifically, individuals with the GG and TG genotypes had significantly more ribs than those with the TT genotype (P<0.01), while there was no significant difference in the number of ribs between the GG and TG genotypes (P>0.05). In the Large White crossbred pig population, the WU_10_2_GL896601_1_56 locus genotype was significantly associated with the rib number phenotype (P<0.05). Individuals with the GG type had significantly more ribs than those with the TT type (P<0.05), while there was no significant difference in the number of ribs between individuals with the TG type and those with the GG and TT types (P>0.05).

[0055] Therefore, breeding individuals with the GG and TG morphs at the WU_10_2_GL896601_1_56 locus in the successor generation of Suhuai pigs is beneficial to increasing the number of ribs in the Suhuai pig population, thereby improving the meat production performance of Suhuai pigs. In the Landrace-Large White crossbreeding system, breeding individuals with the GG morph at the WU_10_2_GL896601_1_56 locus in the parent generations of Landrace and Large White pigs is beneficial to increasing the number of ribs in Landrace-Large White crossbred pigs, thereby improving the meat production performance of Landrace-Large White crossbred pigs.

[0056] Table 1. Association analysis between the WU_10_2_GL896601_1_56 locus of the MTHFD1L gene on pig chromosome 1 and the number of ribs in Suhuai pigs.

[0057]

[0058] Note: Different uppercase letters in the same row of numbers indicate extremely significant differences (P<0.01), different lowercase letters indicate extremely significant differences (P<0.05), and n represents the number of individuals under the corresponding genotype.

Claims

1. The application of molecular markers on pig chromosome 1 associated with the number of ribs in screening for Su-Huai pigs or Large White crossbred pigs with multiple ribs, characterized by... The molecular marker sequence is shown in SEQ ID NO: 1, which contains a SNP marker site related to the number of ribs in pigs. This site is the WU_10_2_GL896601_1_56 nucleotide site on chromosome 1 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 501 and exhibits T / G polymorphism. In Suhuai pigs, individuals with the GG and TG morphisms at this site have significantly more ribs than individuals with the TT morphism. In Large White crossbred pigs, individuals with the GG morphism at this site have significantly more ribs than individuals with the TT morphism.

2. The application of a primer pair for detecting SNP marker sites related to the number of ribs in pigs as described in claim 1 in screening multi-rib Su-Huai pigs or Large White crossbred pig populations, characterized in that, The upstream primer is SEQ ID NO: 2, and the downstream primer is SEQ ID NO:

3.

3. A method for screening pig populations with multiple rib counts, characterized in that... This includes detecting the genotype of the WU_10_2_GL896601_1_56 nucleotide site on chromosome 1 of the Suhuai pig in the international pig genome version 11.1 reference sequence, and selecting individuals with the GG and TG nucleotide types at the WU_10_2_GL896601_1_56 nucleotide site as replacement breeding pigs.

4. The method according to claim 3, characterized in that... The method for detecting the genotype at the WU_10_2_GL896601_1_56 nucleotide site on pig chromosome 1 in the International Pig Genome Version 11.1 reference sequence is selected from PCR amplification or gene sequencing.

5. A method for screening pig populations with multiple rib counts, characterized in that... This includes detecting the genotype of the WU_10_2_GL896601_1_56 nucleotide site on chromosome 1 of the pig in the international pig genome version 11.1 reference sequence of the Large White crossbred pig, and selecting individuals with the GG type at the WU_10_2_GL896601_1_56 nucleotide site as replacement breeding pigs.

6. The method according to claim 5, characterized in that... The method for detecting the genotype at the WU_10_2_GL896601_1_56 nucleotide site on pig chromosome 1 in the International Pig Genome Version 11.1 reference sequence is selected from PCR amplification or gene sequencing.

Images