Primer pair of snp marker related to number of teats trait on pig chromosome 7 and application thereof

Abstract

The present application relates to a SNP marker primer pair related to the number of teats of pig chromosome 7 and its application. The SNP marker is located at the rs3470144376 nucleotide site of pig chromosome 7, the site of the SNP marker is the molecular marker of the rs3470144376 nucleotide site of the international pig genome 11.1 version reference sequence pig chromosome 7, and has G / A polymorphism, the SNP marker is extremely significantly related to the number of teats of large white pigs (P<0.01). 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 is extremely significantly related to the number of teats of large white pigs, and the genotype of the SNP marker is identified to screen large white pig strains with multiple teats. The establishment of the strain can improve the reproductive efficiency of large white 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 teat number trait and application thereof. BACKGROUND

[0002] As one of reproductive traits, pig teat number is an important indicator for measuring the lactation and nursing ability of sows. In recent years, the litter size trait of pigs has been significantly improved through breeding, but the genetic improvement of the teat number trait has not kept pace. The number of effective teats less than the number of live births can cause great trouble for piglet feeding and affect the survival rate of piglets during the lactation period. Sows with more effective teats can feed more piglets. Therefore, breeding to improve the teat number trait of sows is of great significance to improving the reproductive efficiency of sows, especially high-yield sows. The teat number trait is controlled by multiple genes, and the genetic mechanism is complex. The genetic progress of breeding based on the phenotype of the teat number trait is slow, and the combination of molecular marker breeding can effectively accelerate the process of trait improvement.

[0003] Based on this background, the present application provides a SNP marker primer pair related to the teat number trait of pigs, which can quickly and accurately evaluate the teat number trait of pigs through genotyping technology. The present application provides an important molecular genetic basis for the breeding and reproductive performance improvement of pigs. SUMMARY

[0004] The present application aims to provide a breeding molecular marker related to the SNP marker of the teat number of pigs, which can save time and effort in traditional pig teat number breeding, and improve the 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.

[0006] Another object of the present application is to provide the use of the above-mentioned SNP marker, molecular marker and primer.

[0007] The object of the present application can be achieved by the following technical solutions:

[0008] A molecular marker related to the teat number trait of pigs, wherein the sequence of the molecular marker is shown in SEQ ID NO: 1, and a SNP marker site significantly related to the teat number trait of pigs exists at position 301, which corresponds to the international pig genome 11.1 version reference sequence pig chromosome 7 rs3470144376 nucleotide site, and G / A polymorphism exists, the total teat number of GG type individuals is significantly more than that of GA type and AA type individuals, and the total teat number of GA type individuals is significantly more than that of AA type individuals.

[0009] A primer pair for detecting a SNP marker related to the teat number trait of pigs, wherein the upstream primer is:

[0010] SEQ ID NO: 2, the downstream primer is SEQ ID NO: 3

[0011] A method for detecting the SNP marker related to the number of pig teats, comprising PCR amplifying a sequence of the nucleotide site rs3470144376 of the pig chromosome 7 of the international pig genome 11.1 version reference sequence, sequencing the amplification product, and judging the G / A polymorphism of the site.

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

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

[0014] (2) using the extracted pig genomic DNA as a template, and using the primer pair to perform PCR

[0015] amplification;

[0016] (3) sequencing the amplification product, analyzing the sequencing results, and judging the G / A polymorphism at position 301

[0017] of SEQ ID NO: 1.

[0018] The application of the SNP marker related to the number of pig teats in screening a multi-teat pig population, wherein the site of the SNP marker is the molecular marker of the nucleotide site rs3470144376 of the pig chromosome 7 of the international pig genome 11.1 version reference sequence, and has G / A polymorphism, and the SNP marker is extremely significantly related to the number of pig teats (P<0.01).

[0019] The application of the molecular marker in screening a multi-teat pig population.

[0020] The application of the primer pair in screening a multi-teat pig population.

[0021] A method for screening a multi-teat pig population, comprising detecting the genotype of the nucleotide site rs3470144376 of the pig chromosome 7 of the international pig genome 11.1 version reference sequence, and breeding GA type and GG type individuals of the nucleotide site rs3470144376 as reserve boars.

[0022] Advantages:

[0023] The present application develops a SNP marker significantly related to the number of pig teats, and provides a primer pair and a method for detecting the marker. By identifying the genotype of the SNP marker, a pig population with more teats is screened. The establishment of the population can improve the comprehensive reproductive efficiency of pigs, and produce more social and economic benefits. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 PCR amplification gel map of rs3470144376 locus on chromosome 7 of Large White.

[0025] Figure 2 Typing map example of rs3470144376 locus on chromosome 7 of Large White.

[0026] A is AA type, B is GA type, and C is GG type. DETAILED DESCRIPTION

[0027] The following examples are intended to illustrate the present application but not to limit the scope of the present application. Modifications or substitutions of the method, steps or conditions of the present application, which do not depart from the spirit and essence of the present application, all belong to the scope of the present application.

[0028] Example 1

[0029] 1. Source of test animals

[0030] Danish origin Large White from Guizhou Zhiyuan Pig Breeding Co., Ltd.

[0031] 2. Extraction of pig genomic DNA

[0032] Collect 1047 pig ear tissue samples for individual DNA extraction;

[0033] According to the instructions of the tissue DNA extraction kit of Tiangeng Biotechnology Co., Ltd., the extraction steps are as follows:

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

[0035] 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 completely suspended.

[0036] ③ Add 20 μL of proteinase K solution, mix well, and place in a 56°C 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.

[0037] ④ Add 200 μL of buffer GB, mix thoroughly by inverting, and place in a 70°C 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.

[0038] ⑤ Add 200 μL of anhydrous ethanol, mix thoroughly 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.

[0039] (6) The solution and the flocculent precipitate from the previous step were added to an adsorption column CB3, which was placed in a collection tube. The adsorption column CB3 was centrifuged at 12,000 rpm for 30 sec, and the waste liquid was discarded. The adsorption column CB3 was placed back into the collection tube.

[0040] (7) 500 μL of buffer GD was added to the adsorption column CB3, which was centrifuged at 12,000 rpm for 30 sec, and the waste liquid was discarded. The adsorption column CB3 was placed in a collection tube.

[0041] (8) 600 μL of rinse liquid PW was added to the adsorption column CB3, which was centrifuged at 12,000 rpm for 30 sec, and the waste liquid was discarded. The adsorption column CB3 was placed in a collection tube.

[0042] (9) The operation step (8) was repeated.

[0043] (10) The adsorption column CB3 was placed back into the collection tube, which was centrifuged at 12,000 rpm for 2 min, and the waste liquid was discarded. The adsorption column CB3 was placed at room temperature for several minutes to completely dry the residual rinse liquid in the adsorption material.

[0044] The adsorption column CB3 was transferred into a clean centrifuge tube, and 100 μL of elution buffer TE was added to the middle part of the adsorption membrane. The solution was collected into the centrifuge tube after the centrifugation at 12,000 rpm for 2 min. The solution obtained by the centrifugation was added to the adsorption column CB3, which was placed at room temperature for 2 min and centrifuged at 12,000 rpm for 2 min to collect the solution into the centrifuge tube.

[0045] The mass and concentration of the DNA were detected by using a Nanodrop-2000 spectrophotometer. The DNA was diluted to 50 ng / μL and stored at -20 °C for later use.

[0046] 3 PCR amplification and sequencing of the target fragment

[0047] The pig genomic DNA was used as a template for PCR amplification. The reaction system included 1 μL of DNA template, 1 μL of 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 amplification product was subjected to agarose gel electrophoresis. The product fragment was about 325 bp in size, and the electrophoresis result is shown in Figure 1 The remaining amplification product was sequenced. The sequencing result was compared and verified for sequence accuracy by using DNAman software. The rs3470144376 site was judged by using Chromas software.

[0050] 4 Statistical analysis

[0051] Genotype and phenotype association analysis was performed using the general linear model of SAS 9.0 software, and the model was as follows:

[0052] Y i j kl = μ i + S j + YS k + G l + e j kl

[0053] Wherein, Y ijkl is the number of teats of the individual; μ i represents the mean of the number of teats of the population; S j represents the fixed effect of gender; YS k represents the fixed effect of birth year and season; G l is the fixed effect of SNP marker; e jkl is the residual error.

[0054] 5Results

[0055] Table 1 shows the results of the effect of different genotypes of rs3470144376 site on the number of teats of pigs. The results show that there is a very significant difference (P < 0.01) in the number of teats of individuals with three genotypes of rs3470144376 site. The total number of teats of individuals with GG genotype is significantly more than that of individuals with GA and AA genotypes (P < 0.01). The total number of teats of individuals with GA genotype is significantly more than that of individuals with AA genotype (P < 0.01). Therefore, in pigs, breeding individuals with GG and GA genotypes of rs3470144376 site is beneficial to increase the number of teats of the population, and thus improve the reproductive efficiency.

[0056] Table 1 Association analysis of rs3470144376 site on chromosome 7 of pigs with the number of teats of pigs

[0057]

[0058] Note: The same row number with different letters indicates a significant difference (P < 0.01).

Claims

1. Use of reagents for detecting SNP marker loci significantly associated with the number of teats in pigs in the selection of a multi-teat Danish-origin Large White pig population, characterised in that, The SNP marker site corresponds to the nucleotide site rs3470144376 on chromosome 7 of the international pig genome 11.1 version reference sequence, and there is G / A polymorphism, and the total teat number of the GG type individual is extremely significantly more than that of the GA type and AA type individual, and the total teat number of the GA type individual is extremely significantly more than that of the AA type individual.

2. Use of a primer pair for detecting the SNP marker significantly associated with the number of teats trait in pigs as claimed in claim 1 in screening a multi-teat Danish origin Large White pig population, characterized in that, The upstream primer is SEQ ID NO: 2, and the downstream primer is SEQ ID NO:

3.

3. A method of screening a population of Danish Landrace pigs of multiple teat origin, characterized in that, The method comprises detecting the genotype of the nucleotide site rs3470144376 on chromosome 7 of the international pig genome 11.1 version reference sequence, and breeding the GG type and GA type individuals of the nucleotide site rs3470144376 as the reserve breeding pigs.

Images