Pig snp marker primer pair of chromosome 2 associated with small intestine length and application thereof

Abstract

The present application relates to a SNP marker primer pair related to small intestine length of pig chromosome 2 and application thereof. The SNP marker is located on the rs80947993 nucleotide sequence of pig chromosome 2, the site of the SNP marker is the molecular marker of the rs80947993 nucleotide site of pig chromosome 2 in the international pig genome 11.1 version reference sequence, and has G / T 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 small intestine length, and a pig population or strain with long small intestine is screened by identifying the genotype of the SNP marker. The establishment of the population or strain can increase the small intestine length of the pig, improve the time and efficiency of feed digestion and absorption, 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 related to pig small intestine length, a detection method and use thereof. BACKGROUND

[0002] In recent years, the demand for feed grains has been increasing, especially the high dependence on imports of raw materials such as corn and soybean meal. How to improve efficiency and save grains and improve feed utilization is an important problem to be solved. The pig small intestine is the main digestive and absorptive organ of the body, and almost all fat and most of the carbohydrates and proteins are digested and absorbed in the small intestine. The small intestine length is significantly positively correlated with important economic traits such as fat deposition, feed reward, and growth traits such as daily gain and carcass weight. Selecting a pig breed with a longer small intestine length has great economic value.

[0003] However, the small intestine length is mainly determined after slaughter, and the conventional breeding is difficult. Compared with traditional breeding, molecular selection using markers related to traits can effectively accelerate the breeding process. Studying the genetic mechanism of pig small intestine length variation and identifying relevant molecular markers affecting pig small intestine length for breeding has important economic benefits for breeding grain-saving populations or new lines. SUMMARY

[0004] The purpose of the present application is to provide a breeding molecular marker developed from a SNP marker related to pig small intestine length in order to solve the problems of large difficulty in traditional pig small intestine length determination, time-consuming and laborious breeding, and slow breeding effect.

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

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

[0007] The molecular marker of pig chromosome 2 related to small intestine length has a sequence as shown in SEQ ID NO: 1, wherein the molecular marker contains a SNP marker site related to pig small intestine length, the site is rs80947993 nucleotide site of pig chromosome 2 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 G / T polymorphism exists.

[0008] A primer pair for detecting the SNP marker of pig chromosome 2 related to small intestine length, 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 of claim 2 are used for detecting the length of small intestine of pigs and / or pig breeding.

[0010] A method for detecting the SNP marker of the pig chromosome 2 associated with the length of small intestine, comprising PCR amplifying a sequence of the nucleotide site rs80947993 of the pig chromosome 2 in the international pig genome reference sequence version 11.1, sequencing the amplification product, and judging the G / T polymorphism of the site, wherein the length of small intestine of the individuals of TT type and GT type is significantly longer than that of the individuals of GG type.

[0011] As a preferred embodiment of the present application, the pig is a Canadian Landrace.

[0012] As a preferred embodiment of the present application, the pig genomic DNA is subjected to PCR amplification by using the primer pair.

[0013] As a further preferred embodiment of the present application, the method comprises the following steps:

[0014] (1) extracting total DNA from a pig tissue sample;

[0015] (2) using the extracted pig genomic DNA as a template, and performing PCR amplification by using the primer pair of the present application;

[0016] (3) sequencing the amplification product, analyzing the sequencing result, and judging the G / T polymorphism at the 301st site of SEQ ID NO: 1.

[0017] The molecular marker of the present application is used for screening a long small intestine pig population or a new strain.

[0018] The primer pair of the present application is used for screening a long small intestine pig population or a new strain.

[0019] A method for screening a long small intestine pig population, comprising detecting the genotype of the nucleotide site rs80947993 of the pig chromosome 2 in the international pig genome reference sequence version 11.1, and breeding the individuals of TT type and GT type of the nucleotide site rs80947993 as the reserve boars.

[0020] As a preferred embodiment of the present application, the pig population is a Canadian Landrace.

[0021] As a preferred embodiment of the present application, the method for detecting the genotype of the nucleotide site rs80947993 of the pig chromosome 2 in the international pig genome reference sequence version 11.1 is selected from PCR or gene sequencing.

[0022] Beneficial effects

[0023] The application develops a SNP marker site of pig chromosome 2 related to small intestine length, and provides primer pairs and methods for detecting the marker. The genotype of rs80947993 site is significantly associated with small intestine length phenotype (P<0.05). Among them, the small intestine length of individuals of TT and GT types is significantly longer than that of individuals of GG type (P<0.05), and the small intestine length of individuals of GT type is significantly different from that of individuals of GG type (P<0.05). Therefore, in pigs, breeding individuals of TT and GT types of rs80947993 site is beneficial to increase the small intestine length of pig population, and further improve the feed conversion rate of pigs. By identifying the genotype of the SNP marker, a pig strain with long small intestine is screened. The establishment of the strain can improve the feed utilization rate of pigs and produce more social and economic benefits. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 A gel map of PCR amplification of the rs80947993 nucleotide site of pig chromosome 2.

[0025] Figure 2 A gel map of PCR amplification of the rs80947993 nucleotide site of pig chromosome 2.

[0026] A is GG type, B is GT type, and C is TT type. DETAILED DESCRIPTION

[0027] The following examples are used to illustrate the application, but not to limit the scope of the application. Modifications or replacements of the methods, steps or conditions of the application without departing from the spirit and essence of the application shall fall within the scope of the application.

[0028] Example 1

[0029] 1. Source of test animals

[0030] 399 Canadian Large White pigs with an average carcass weight of 99.8±0.6 kg from Jiangsu Zhengda Suguan Pig Industry Co., Ltd.

[0031] 2. Extraction of pig genomic DNA

[0032] 1 portion of pig ear tissue sample was collected for individual DNA extraction;

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

[0034] ① First, add 68 mL and 200 mL of anhydrous ethanol to the 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 Proteinase K solution, mix well and incubate in a 56°C metal bath overnight until the tissue sample is dissolved. Briefly centrifuge to remove water droplets from the inside of the tube cap.

[0037] 4. Add 200 μL Buffer GB, mix well by inverting the tube, and incubate in a 70°C metal bath for 10 min until the solution is clear. Briefly centrifuge to remove water droplets from the inside of the tube cap.

[0038] 5. Add 200 μL absolute ethanol, mix well by inverting the tube, and incubate for 15 sec. A flocculent precipitate may form. Briefly centrifuge to remove water droplets from the inside of the tube cap.

[0039] 6. Add the solution and flocculent precipitate from the previous step to a spin column CB3, which is placed in a collection tube. Centrifuge at 12,000 rpm for 30 sec, discard the waste, and place the spin column CB3 back in the collection tube.

[0040] 7. Add 500 μL Buffer GD to the spin column CB3, centrifuge at 12,000 rpm for 30 sec, discard the waste, and place the spin column CB3 in the collection tube.

[0041] 8. Add 600 μL rinse solution PW to the spin column CB3, centrifuge at 12,000 rpm for 30 sec, discard the waste, and place the spin column CB3 in the collection tube.

[0042] 9. Repeat step 8.

[0043] 10. Place the spin column CB3 back in the collection tube, centrifuge at 12,000 rpm for 2 min, discard the waste, and let the spin column CB3 stand at room temperature for several minutes to dry the spin column material completely.

[0044] Place the spin column CB3 in a clean centrifuge tube, and add 100 μL elution buffer TE to the center of the membrane. Let stand at room temperature for 2-5 min, centrifuge at 12,000 rpm for 2 min, and collect the solution in the centrifuge tube. Add the solution obtained by centrifugation to the spin column CB3, let stand at room temperature for 2 min, centrifuge at 12,000 rpm for 2 min, and collect the solution in the centrifuge tube.

[0045] 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 until use.

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

[0047] The pig genomic DNA was used as a 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, 9.5 μL of dd H2O, and 12.5 μL of PCR mix. The amplification program was as follows:

[0048]

[0049] The amplification product was subjected to agarose gel electrophoresis, and the product fragment size was about 436 bp, and the electrophoresis result is shown in Figure 1 The remaining amplification product was sequenced, the sequencing result was compared and verified the accuracy of the sequence by DNAman software, and the rs80947993 site was judged by Chromas software.

[0050] 4Statistical analysis

[0051] The general linear model of SAS 9.4 software was used for genotype and phenotype correlation analysis, and the model was as follows:

[0052] Y ijk = μ i + B j + G k + e jk

[0053] Wherein, Yijk is the small intestine length of the individual; μ i represents the average small intestine length of the population; B j represents the fixed effect of slaughter batch; G k is the fixed effect of SNP marker; e jk is the residual.

[0054] 5Results

[0055] Table 1 shows the results of the effect of different genotypes of rs80947993 site on the small intestine length of pigs. The results show that the genotype of rs80947993 site is significantly associated with the small intestine length phenotype (P<0.05). Among them, the small intestine length of TT and GT type individuals is significantly longer than that of GG type individuals (P<0.05), and the small intestine length of GT type individuals is significantly different from that of GG type (P<0.05). Therefore, in pigs, breeding TT and GT type individuals of rs80947993 site is beneficial to increase the small intestine length of pig population, and thus improve the feed conversion rate of pigs.

[0056] Table 1 Correlation analysis of rs80947993 site on chromosome 2 of pigs and small intestine length of pigs

[0057]

[0058] Note: Means in the same column followed by different letters are significantly different (P < 0.05).

Claims

1. Use of a detection reagent for a SNP marker in the screening of a Long White Canada pig population, characterized in that, The SNP marker site is the rs80947993 nucleotide site of the international pig genome 11.1 version reference sequence pig chromosome 2, which has G / T polymorphism, the SNP marker is significantly related to the small intestine length trait of Canadian Large White, and the small intestine length of TT type and GT type individuals is significantly longer than that of GG type individuals.

2. Use according to claim 1, characterized in that, The detection reagent of the SNP marker is a primer pair for detecting the SNP marker, and the sequence of the primer pair is shown as SEQ ID NO: 2 and SEQ ID NO:

3.

3. Use of a SNP marker detection reagent in the breeding of Large White swine in Canada, characterized in that, The breeding trait is small intestine length, and the SNP marker site is the rs80947993 nucleotide site of the international pig genome 11.1 version reference sequence pig chromosome 2, which has G / T polymorphism, the SNP marker is significantly related to the small intestine length trait of Canadian Large White, and the small intestine length of TT type and GT type individuals is significantly longer than that of GG type individuals.

4. Use according to claim 3, characterized in that, The detection reagent of the SNP marker is a primer pair for detecting the SNP marker, and the sequence of the primer pair is shown as SEQ ID NO: 2 and SEQ ID NO:

3.

5. A method of screening a population of Large White Canadian Landrace pigs for long small intestine, characterized in that The method for detecting the genotype of the rs80947993 nucleotide site of the international pig genome 11.1 version reference sequence pig chromosome 2 is selected from PCR amplification and gene sequencing.

6. The method of claim 5, wherein The method for detecting the genotype of the rs80947993 nucleotide site of the international pig genome 11.1 version reference sequence pig chromosome 2 is selected from PCR amplification and gene sequencing.

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