Pig snp marker primer pair related to pig chromosome 2 and pig litter size trait and application thereof

Abstract

The application relates to a SNP marker primer pair related to a pig live litter size trait on chromosome 2 of pigs and application thereof. The SNP marker site is a molecular marker of a nucleotide site rs81367175 on chromosome 2 of pigs in an international pig genome 11.1 version reference sequence, and has C / A polymorphism. A primer pair for detecting the SNP marker is as follows: an upstream primer is SEQ ID NO: 2, and a downstream primer is SEQ ID NO: 3. The SNP marker provided by the application can be applied to marker-assisted selection of the pig live litter size trait, and a pig population or strain with more live litter size is screened by identifying the genotype of the SNP marker. The establishment of the population or strain can improve the live litter size of pigs, and more social and economic benefits are generated.

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 live litter size trait and an application thereof. BACKGROUND

[0002] With the global population growth and the improvement of income level, the demand for livestock products is expanding, and higher requirements are put forward for sow farrowing traits. Pig farrowing traits, including total litter size, live litter size, stillbirth number and mummification number, are crucial to the production efficiency and economic benefits of the pig industry. In particular, the live litter size index is the most critical, which determines the key performance indicators of the pig production system, i.e. the number of weaned piglets per sow per year and the number of live pigs sold per sow per year. In the past three decades, developed countries and some pig production enterprises with high production efficiency have increased the number of weaned piglets per sow per year from 20 to 34. This increase is mainly due to the efforts of researchers over the years to improve pig live litter size through genetic means. However, the genetic force of live litter size is low, the accuracy of conventional selection is low, and the selection progress is slow, which requires the combination of molecular selection to speed up the improvement efficiency.

[0003] Based on this background, the present application provides a SNP marker primer pair related to pig live litter size traits, which can quickly and accurately evaluate pig live litter size traits through genotyping technology. This technology not only improves the accuracy and efficiency of live litter size evaluation, but also provides important molecular genetic basis for pig selection and reproductive performance improvement. SUMMARY

[0004] The present application aims to provide a selection molecular marker developed based on a SNP marker related to pig live litter size, which is difficult to genetically select due to the low genetic force of sow farrowing traits, high correlation with gender, and performance in the later life.

[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] The molecular marker associated with the number of live piglets trait on pig chromosome 2, wherein the sequence of the molecular marker is shown in SEQ ID NO: 1, and the SNP marker site associated with the number of live piglets trait is rs81367175 nucleotide site on pig chromosome 2 of the international pig genome version 11.1 reference sequence (the site is identified by the team through whole genome association analysis), and the SNP marker site in SEQ ID NO: 1 is located at position 269, and C / A polymorphism exists.

[0008] A primer pair for detecting the SNP marker associated with the number of live piglets trait on pig chromosome 2, wherein the upstream primer is SEQ ID NO: 2, and the downstream primer is SEQ ID NO: 3.

[0009] The application of the molecular marker and the primer pair of claim 2 in detecting the number of live piglets trait and pig breeding.

[0010] A method for detecting the SNP marker associated with the number of live piglets trait on pig chromosome 2, comprising PCR amplifying a sequence of the rs81367175 nucleotide site on pig chromosome 2 of the international pig genome version 11.1 reference sequence, sequencing the amplification product, and judging the C / A polymorphism of the site.

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

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

[0013] (1) Taking a pig ear tissue sample to extract DNA;

[0014] (2) Using the extracted pig genomic DNA as a template, and using the primer pair of the application for PCR amplification;

[0015] (3) Sequencing the amplification product, analyzing the sequencing results, and judging the C / A polymorphism at position 269 of SEQ ID NO: 1.

[0016] The application of the molecular marker in screening pig populations or new strains with more number of live piglets.

[0017] The application of the primer pair in screening pig populations or new strains with more number of live piglets.

[0018] The application discloses a method for screening a pig population with more live-born piglets, which comprises detecting the genotype of a nucleotide site rs81367175 on a pig chromosome 2 in an international pig genome version 11.1 reference sequence, and breeding individuals with CC and CA types of the nucleotide site rs81367175 as backup boars.

[0019] As a preferred embodiment of the application, the method for detecting the genotype of the nucleotide site rs81367175 on the pig chromosome 2 in the international pig genome version 11.1 reference sequence is selected from PCR or gene sequencing. Advantages

[0020] The application develops a SNP marker on a pig chromosome 2 related to the number of live-born piglets, and provides a primer pair and a method for detecting the marker. The pig strain with more live-born piglets is screened by identifying the genotype of the SNP marker. The establishment of the pig strain can improve the comprehensive reproductive performance of pigs, and generate more social and economic benefits. BRIEF DESCRIPTION OF DRAWINGS

[0021] Fig. 1 is a gel map of PCR amplification of the nucleotide site rs81367175 on a pig chromosome 2 in an international pig genome version 11.1 reference sequence.

[0022] Fig. 2 is an example of a genotyping diagram of the nucleotide site rs81367175 on a pig chromosome 2 in an international pig genome version 11.1 reference sequence.

[0023] Note: the genotype of A is AA type, the genotype of B is CA type, and the genotype of C is CC type. DETAILED DESCRIPTION

[0024] The following examples are used to illustrate the application, but are not used to limit the scope of the application. Modifications or replacements of the method, steps or conditions of the application, without departing from the spirit and essence of the application, all belong to the scope of the application. EXAMPLE

[0025] 1. Source of test animals

[0026] Jiangsu Lihua Animal Husbandry Co., Ltd. - Changzhou Lijia Sow Farm collected 1107 Topigs Large White sows with 4271 pieces of delivery data of multiple parity.

[0027] 2. Phenotype recording method

[0028] According to the national industry standard "Pig Registration Technical Specification" (standard number: NY / T820-2004), the number of live piglets is the number of all surviving piglets born by each sow in one parity, and the number of live piglets less than 5 is regarded as an extreme value and is excluded.

[0029] 3 Extraction of pig genomic DNA

[0030] Pig ear tissue samples were collected for individual DNA extraction;

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

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

[0033] ②Collect 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 completely suspended.

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

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

[0036] ⑤Add 200 μL of absolute ethanol, mix well by shaking for 15 sec. At this time, a flocculent precipitate may appear. Briefly centrifuge to remove water droplets on the inner wall of the tube cap.

[0037] ⑥Add the solution and flocculent precipitate obtained in 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 liquid, and place the adsorption column CB3 back into the collection tube.

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

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

[0040] ⑨Repeat step ⑧.

[0041] d. Put the adsorption column CB3 back into the collection tube, centrifuge at 12,000 rpm for 2 min, and discard the waste liquid. Put the adsorption column CB3 at room temperature for several minutes to completely dry the residual rinse solution in the adsorption material.

[0042] e. Put the adsorption column CB3 into a clean centrifuge tube, and add 100 μL elution buffer TE to the middle of the adsorption membrane by dripping, and place it at room temperature for 2-5 min, centrifuge at 12,000 rpm for 2 min, collect the solution into the centrifuge tube, and add the centrifuged solution to the adsorption column CB3 again, place it at room temperature for 2 min, centrifuge at 12,000 rpm for 2 min, and collect the solution into the centrifuge tube.

[0043] The quality and concentration of the DNA were detected using a Nanodrop-2000 spectrophotometer, and the DNA concentration was diluted to 50 ng / μL and stored at -20 ℃ for standby use.

[0044] 4 PCR amplification and sequencing of the target fragment

[0045] The genomic DNA of Large White pigs was used as the template for PCR amplification, and the reaction system included 1 μL of DNA template, 1 μL of primers shown in SEQ ID NO: 2 and SEQ ID NO: 3, and 22 μL of PCR mix; the amplification program was as follows:

[0046]

[0047] The amplification product was subjected to agarose gel electrophoresis, and the product fragment size was about 269 bp, and the electrophoresis result is shown in Figure 1. The remaining amplification product was sequenced, the sequencing result was verified for sequence accuracy using DNAman software, and the rs81367175 site was judged using SnapGene 6.0.2 software.

[0048] 5 Statistical analysis

[0049] The association analysis of genotypes and phenotypes was performed using the general linear model of SAS 9.4 software, and the model was as follows: Y ijk = μ + B i + G j + W k + e ijk

[0050] wherein Y ijk is the phenotype value of the number of live births; μ is the average number of live births of the population; B i is the fixed effect of the SNP marker; G jRepresenting the parity as a fixed effect; W k Representing the year-season of farrowing as a fixed effect; e ijk is the residual.

[0051] 6 Results

[0052] Table 1 shows the results of the effect of different genotypes of the rs81367175 locus on the number of live piglets of pigs. The results show that the genotype of the rs81367175 locus is significantly associated with the phenotype of the number of live piglets (P < 0.05). Among them, the number of live piglets of CC type and CA type individuals is significantly greater than that of AA type individuals (P < 0.05). Therefore, breeding CC type and CA type individuals of the rs81367175 locus is beneficial to increase the number of live piglets of the pig population.

[0053] Table 1 Association analysis of the rs81367175 locus on chromosome 2 of pigs with the number of live piglets of pigs

[0054]

[0055] Note: n is the number of observations of multiple parities, and different letters in the same column indicate significant differences (P < 0.05).

Claims

1. Use of a primer pair for detecting a SNP marker associated with the litter size trait in Topigs Large White sows in detecting the litter size trait of Topigs Large White sows, wherein the SNP marker is the nucleotide site rs81367175 on pig chromosome 2 of the International Pig Genome 11.1 version reference sequence, and the primer pair consists of an upstream primer as set forth in SEQ ID NO: 2 and a downstream primer as set forth in SEQ ID NO:

3.

2. A method of selecting a Topigs Duroc sow population that produces more live offspring, characterized in that PCR amplification of a sequence containing the nucleotide site rs81367175 on pig chromosome 2 of the International Pig Genome 11.1 version reference sequence, sequencing of the amplification product, and determining the C / A polymorphism at the site.

3. The method of claim 2, wherein PCR amplification of pig genomic DNA using the primer pair of claim 1.

4. A method of selecting a Topigs Duro mother pig population that produces more live offspring, characterized in that Selecting individuals with CC and CA genotypes at the nucleotide site rs81367175 on pig chromosome 2 of the International Pig Genome 11.1 version reference sequence for breeding as reserve boars.

5. The method of claim 4, wherein The method for detecting the genotype of the nucleotide site rs81367175 on pig chromosome 2 of the International Pig Genome 11.1 version reference sequence is PCR amplification and gene sequencing.

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