A snp marker primer pair related to the number of pig rib traits and application thereof

Abstract

The present application relates to a SNP marker primer pair related to a pig rib number trait and application thereof. The SNP marker site is a molecular marker of the nucleotide site rs332644388 of pig chromosome 6 in the international pig genome 11.1 version reference sequence, and has A / G 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 rib number trait, and by identifying the genotype of the SNP marker, a pig population or new strain with multiple rib number is screened. The establishment of the population or new strain can improve the rib number and meat production performance of pigs, and produce more social and economic benefits.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of molecular biology, and relates to a SNP marker primer pair related to a pig rib number trait and application thereof. BACKGROUND

[0002] China is a large country in pork production and consumption, and pork is the main source of animal protein in the diet of Chinese people. With the development of economy and the continuous improvement of living quality, people's demand for pork is increasing, and how to improve the pork yield of pig carcass to meet the market demand for pork is the focus of current breeding work.

[0003] The spine of a pig can be divided into cervical vertebrae, thoracic vertebrae, lumbar vertebrae, sacral vertebrae and caudal vertebrae, among which only the number of thoracic vertebrae and lumbar vertebrae varies. The thoracic vertebrae are connected with the ribs, and the number of thoracic vertebrae determines the number of ribs. The number of ribs is significantly correlated with the carcass length of a pig, and each increase of 1 pair of ribs not only increases the amount of spare ribs and tenderloin, but also increases the body length by 10-40 mm, and the pork yield of a pig also increases by about 1%. "Pork spare ribs" is one of the highest-priced carcass parts in the Chinese pork consumption market, and is favored by consumers due to high lean meat rate and good meat quality. Therefore, studying the genetic and molecular mechanism of pig rib number and applying the related molecular markers to breeding work has important economic value for breeding a population or new strain with more rib number and high pork yield. SUMMARY

[0004] The present application aims to provide a breeding molecular marker developed based on a SNP marker related to pig rib number, in view of the time-consuming and labor-consuming traditional pig rib number breeding, the difficulty in phenotype determination, and the slow breeding effect.

[0005] Another object of the present application is to provide a primer pair and a detection method for detecting the above-mentioned SNP marker. Another object of the present application is to provide the use of the above-mentioned SNP marker, molecular marker and primer.

[0006] The object of the present application can be achieved by the following technical solutions:

[0007] A molecular marker related to a pig rib number trait, wherein the sequence of the molecular marker is shown as SEQ ID NO: 1, and the molecular marker contains a SNP marker site related to the pig rib number trait, the site is rs332644388 nucleotide site of pig chromosome 6 in the international pig genome 11.1 version reference sequence, and the SNP marker site in SEQ ID NO: 1 is located at position 301, and there is A / G polymorphism; the rib number of individuals of GG type and AG type is significantly more than that of individuals of AA type, and the rib number of individuals of GG type is significantly more than that of individuals of AG type.

[0008] A primer pair for detecting a SNP marker related to the rib number trait of pigs, wherein the upstream primer is SEQ ID NO: 2, and the downstream primer is SEQ ID NO: 3.

[0009] The molecular marker and the primer pair provided by the present application are used for detecting the rib number trait of pigs and / or pig breeding.

[0010] A method for detecting the SNP marker related to the rib number trait of pigs, comprising the steps of: performing PCR amplification on a sequence of a nucleotide site rs332644388 of a pig chromosome 6 in a pig international pig genome reference sequence version 11.1, and performing sequencing on the amplification product to determine the A / G polymorphism of the site.

[0011] As a preferred embodiment of the present application, the primer pair is used for performing PCR amplification on pig genomic DNA.

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

[0013] (1) extracting total DNA from a pig tissue sample;

[0014] (2) using the extracted pig genomic DNA as a template, and performing PCR amplification using the primer pair provided by the present application;

[0015] (3) performing sequencing on the amplification product, analyzing the sequencing result, and determining the A / G polymorphism at the 301st site of SEQ ID NO: 1.

[0016] The molecular marker provided by the present application is used for screening and establishing a multi-rib pig population or a new strain.

[0017] The primer pair provided by the present application is used for screening and establishing a multi-rib pig population or a new strain.

[0018] A method for screening a multi-rib pig population, comprising the steps of: detecting the genotype of a nucleotide site rs332644388 of a pig chromosome 6 in a pig international pig genome reference sequence version 11.1; and selecting GG type and / or AG type individuals of the nucleotide site rs332644388 as reserve boars for breeding.

[0019] As a preferred embodiment of the present application, the method for detecting the genotype of the nucleotide site rs332644388 of the pig chromosome 6 in the pig international pig genome reference sequence version 11.1 is selected from PCR or gene sequencing.

[0020] Advantages

[0021] The present application develops a SNP marker related to the number of pig ribs, and provides a primer pair and a method for detecting the marker. A new strain of pigs with more ribs is screened by identifying the genotype of the SNP marker. The establishment of the strain can improve the meat performance and the number of ribs of pigs, increase the distribution ratio of high-priced parts in pig carcasses, and generate more social and economic benefits. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Gel map of PCR amplification of the rs332644388 site on pig chromosome 6 of the international pig genome 11.1 version reference sequence.

[0023] Figure 2 Example of genotyping map of the rs332644388 site on pig chromosome 6 of the international pig genome 11.1 version reference sequence.

[0024] Note: the genotype of A is AA type, the genotype of B is AG type, and the genotype of C is GG type. DETAILED DESCRIPTION

[0025] The following examples are used to illustrate the present application, but are not used to limit the scope of the present application. Modifications or replacements of the methods, steps or conditions of the present application, without departing from the spirit and essence of the present application, all belong to the scope of the present application.

[0026] Example 1

[0027] 1 Source of test animals

[0028] Jiangsu Zhengda Suguan Pig Industry Co., Ltd., Huai'an City Su Food Meat Product Co., Ltd.

[0029] 2 Extraction of pig genomic DNA

[0030] One ear tissue sample was collected from each of 413 Canadian Large White pigs and 676 long-term hybrid pigs for individual DNA extraction;

[0031] According to the instructions of the tissue DNA extraction kit of Tian Gen Biological Technology Co., Ltd., the extraction steps are as follows:

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

[0033] ② Collect about 100 mg of ear tissue sample in a 2 mL EP tube, cut it completely, add 200 μL of buffer GA, and shake until it is completely suspended.

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

[0035] (4) Add 200 μL Buffer GB, mix well by inverting the tube, place in a 70°C metal bath for 10 min. The solution should be clear and the tube should be centrifuged briefly to remove water from the inside of the cap.

[0036] (5) Add 200 μL anhydrous ethanol, mix well by inverting the tube, centrifuge briefly to remove water from the inside of the cap.

[0037] (6) Add both the solution and the flocculent precipitate from the previous step to an adsorption column CB3, place the adsorption column in a collection tube, then centrifuge at 12,000 rpm for 30 sec, discard the waste, and place the adsorption column CB3 back into the collection tube.

[0038] (7) Add 500 μL Buffer GD to the adsorption column CB3, centrifuge at 12,000 rpm for 30 sec, discard the waste, and place the adsorption column CB3 in the collection tube.

[0039] (8) Add 600 μL rinse solution PW to the adsorption column CB3, centrifuge at 12,000 rpm for 30 sec, discard the waste, and place the adsorption column CB3 in 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 min, discard the waste, and leave the adsorption column CB3 at room temperature for several minutes to dry the adsorption material completely.

[0042] Transfer the adsorption column CB3 to a clean centrifuge tube, add 100 μL elution buffer TE to the middle of the adsorption membrane, leave at room temperature for 2-5 min, centrifuge at 12,000 rpm for 2 min, collect the solution in the centrifuge tube, and add the solution obtained by centrifugation to the adsorption column CB3, leave at room temperature for 2 min, centrifuge at 12,000 rpm for 2 min, and collect the solution in the centrifuge tube.

[0043] Determine the mass and concentration of the DNA using a Nanodrop-2000 spectrophotometer, dilute the DNA to a concentration of 50 ng / μL, and store at -20°C for later use.

[0044] 3 PCR amplification and sequencing of the target fragment

[0045] 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 primers shown as GGCATTTCTACCTTGGAT (SEQ ID NO: 2) and CACTCAGTTATTGGCTTA (SEQ ID NO: 3), 9.5 μL of ddH2O, and 12.5 μL of PCR mix. The amplification program was as follows:

[0046]

[0047] The amplified product was subjected to agarose gel electrophoresis. The product fragment size was approximately 458 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 rs332644388 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 9.4 software. The model is as follows: Y ijk =μ i +B j +G k +e jk

[0050] 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.

[0051] 5 Results

[0052] Table 1 shows the effect of different genotypes at the rs332644388 locus on the number of ribs in Large White pigs. The results indicate that there were significant differences in the number of ribs among the three genotypes at the rs332644388 locus (P<0.05). Specifically, individuals with the GG and AG genotypes had significantly more ribs than those with the AA genotype (P<0.01), and individuals with the GG genotype had significantly more ribs than those with the AG genotype (P<0.05).

[0053] Table 2 shows the effect of different genotypes at the rs332644388 locus on the number of ribs in Large White crossbred pigs. The results indicate that there were significant differences in the number of ribs among the three genotypes at the rs332644388 locus (P<0.05). Specifically, individuals with the GG and AG genotypes had significantly more ribs than those with the AA genotype (P<0.01), and individuals with the GG genotype had significantly more ribs than those with the AG genotype (P<0.05).

[0054] Therefore, breeding individuals of the GG and AG types at the rs332644388 locus in Large White and Large White crossbred pigs is beneficial to increasing the number of ribs in Large White and Large White crossbred pig populations, thereby improving their meat production performance.

[0055] Table 1. Association analysis between the rs332644388 locus on porcine chromosome 6 and the number of ribs in Large White pigs.

[0056]

[0057] Note: Different letters in the number subscripts of the same line indicate significant differences (P<0.05).

[0058] Table 2. Association analysis between the rs332644388 locus on porcine chromosome 6 and the number of ribs in Large White crossbred pigs.

[0059]

[0060] Note: Different letters in the number subscripts of the same line indicate significant differences (P<0.05).

Claims

1. The application of primer pairs for detecting SNP markers in detecting the number of ribs in pigs, characterized in that, The SNP marker site is the rs332644388 nucleotide site on chromosome 6 of the pig in the international pig genome version 11.1 reference sequence. The number of ribs in GG and AG individuals is significantly higher than that in AA individuals, and the number of ribs in GG individuals is significantly higher than that in AG individuals. The upstream primer of the primer pair is SEQ ID NO: 2, and the downstream primer is SEQ ID NO:

3. The pigs mentioned are Canada Large White pigs or Landrace Large White crossbred pigs.

2. The application according to claim 1, characterized in that, The detection of the number of ribs in pigs includes the following steps: (1) Collect pig tissue samples to 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 A / G polymorphism at position 301 of SEQ ID NO:

1.

3. A method for screening multi-ribbed pig populations, characterized in that, This includes detecting the genotype of the rs332644388 nucleotide site on chromosome 6 of the pig in the international pig genome version 11.1 reference sequence. Individuals with the GG and AG genotypes had significantly more ribs than individuals with the AA genotype, and individuals with the GG genotype had significantly more ribs than individuals with the AG genotype. Individuals with the rs332644388 nucleotide site being of the GG and / or AG genotype were selected as replacement breeding pigs. The pigs mentioned are Canada Large White or Landrace crossbred pigs.

4. The method according to claim 3, characterized in that... The genotype of the rs332644388 nucleotide site on chromosome 6 of the pig in the international pig genome version 11.1 reference sequence is determined by PCR or gene sequencing.

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