Primer pair of snp marker related to rib number trait on pig chromosome 6 and application thereof

Abstract

The present application relates to a SNP marker primer pair related to a pig rib number character and application thereof. The SNP marker site is a molecular marker of the nucleotide site rs341010833 of the international pig genome 11.1 version reference sequence pig chromosome 6, and has T / C 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 character, and the genotype of the SNP marker is identified to screen a pig population or new strain with multiple rib number. 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 rib number trait of a pig and an application thereof. BACKGROUND

[0002] China is the largest pork producer and consumer in the world, and pork is an important source of animal protein in the diet of Chinese people. With the sustained economic growth and the improvement of living standards, the demand for pork is increasing, so improving the pork yield of pig carcass has become the core goal of current pig breeding research.

[0003] The spine of a pig is composed of cervical vertebrae, thoracic vertebrae, lumbar vertebrae, sacral vertebrae and coccyx, and the number of thoracic vertebrae and lumbar vertebrae varies. The thoracic vertebrae are connected with the ribs, so the number of thoracic vertebrae directly determines the number of ribs. The number of ribs is significantly correlated with the carcass length of a pig, and each additional pair of ribs can not only increase the number of spare ribs and tenderloin, but also increase the body length by 10-40 mm, thereby increasing the pork yield by about 1%. Pig spare ribs are one of the most expensive carcass parts on the Chinese market and are deeply loved by consumers. Therefore, studying the genetic and molecular mechanisms of rib number in pigs and applying the relevant molecular markers to breeding practice have important economic significance for breeding pigs or new strains with more ribs and high meat yield. SUMMARY

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

[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 SNP marker related to the rib number trait of a pig, the site being the rs341010833 nucleotide site of pig chromosome 6 in the international pig genome 11.1 version reference sequence, and there being T / C polymorphism; the rib number of individuals of CC type and TC type is significantly more than that of individuals of TT type.

[0008] A molecular marker containing the SNP site, the sequence of the molecular marker being shown as SEQ ID NO: 1, wherein the SNP marker of claim 1 is located at position 301 of SEQ ID NO: 1, and there is T / C polymorphism; the rib number of individuals of CC type and TC type is significantly more than that of individuals of TT type.

[0009] A primer pair for detecting the SNP marker related to the rib number trait of pigs, characterized in that the upstream primer is SEQ ID NO: 2 and the downstream primer is SEQ ID NO: 3.

[0010] The SNP marker, the molecular marker and the primer pair can be used for detecting the rib number trait of pigs and / or pig breeding.

[0011] The primer pair can be used for preparing a reagent for detecting the rib number trait of pigs and / or pig breeding.

[0012] A method for detecting the SNP marker related to the rib number trait of pigs, which comprises PCR amplification of a sequence containing the nucleotide site rs341010833 of pig chromosome 6 in the international pig genome reference sequence 11.1 version of pigs, sequencing of the amplification product, and judgment of the T / C polymorphism at the site.

[0013] As a preferred embodiment of the present application, the primer pair is used for PCR amplification of pig genomic DNA.

[0014] As a further preferred embodiment of the present application, the application of the SNP marker related to the rib number trait of pigs in screening a multi-rib pig population, characterized in that the method comprises the following steps:

[0015] (1) extracting total DNA from a pig tissue sample;

[0016] (2) using the extracted pig genomic DNA as a template, and using the primer pair for PCR amplification;

[0017] (3) sequencing the amplification product, analyzing the sequencing result, and judging the T / C polymorphism at the 301st site of SEQ ID NO: 1.

[0018] A method for screening a multi-rib pig population, which comprises detecting the genotype of the nucleotide site rs341010833 of pig chromosome 6 in the international pig genome reference sequence 11.1 version of pigs, and selecting individuals with CC and / or TC types of the nucleotide site rs341010833 as reserve breeding pigs.

[0019] As a preferred embodiment of the present application, the method for detecting the genotype of the nucleotide site rs341010833 of pig chromosome 6 in the international pig genome reference sequence 11.1 version of pigs 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 the pig carcass, and produce more social and economic benefits. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Gel map of PCR amplification of the rs341010833 locus of pig chromosome 6 for the pig international pig genome 11.1 version reference sequence.

[0023] Figure 2 Example of genotyping map of the rs341010833 locus of pig chromosome 6 for the pig international pig genome 11.1 version reference sequence.

[0024] Note: The genotype of A is TT type, the genotype of B is TC type, and the genotype of C is CC 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] The test pigs were from Huaiyin New Huai Pig Farm in Huai'an City, Jiangsu Province, and the sum of the rib numbers of the left and right carcasses was counted by artificial counting after slaughter.

[0029] 2 Extraction of pig genomic DNA

[0030] One portion of 655 ear tissue samples of Su-Huai pigs was collected for individual DNA extraction;

[0031] According to the instructions of the tissue DNA extraction kit of Tiangen Biosciences 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 overnight digestion until the tissue sample is dissolved. Briefly centrifuge to remove the 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 column in a collection tube, then centrifuge at 12,000 rpm for 30 sec, discard the waste, and place the column CB3 back in the collection tube.

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

[0039] (8) Add 600 μL rinse solution PW to the column CB3, centrifuge at 12,000 rpm for 30 sec, discard the waste, and place the column CB3 in the collection tube.

[0040] (9) Repeat step (8).

[0041] (10) Place the column CB3 back in the collection tube, centrifuge at 12,000 rpm for 2 min, discard the waste, and leave the column CB3 at room temperature for several minutes to dry the adsorption material completely.

[0042] Transfer the 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 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 Suhuai pig genomic DNA as a template. The reaction system included 1 μL of DNA template, 1 μL each of the primers shown in TATCACCAGTAGTAGGTC (SEQ ID NO: 2) and AGGAGAAAAGGATTGTAT (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 amplification product was subjected to agarose gel electrophoresis. The product fragment size was approximately 496 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 rs341010833 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 rs341010833 locus on the number of ribs in Suhuai pigs. The results indicate that there are significant differences in the number of ribs among the three genotypes at the rs341010833 locus. Specifically, the number of ribs in CC and TC genotype individuals was significantly higher than that in TT genotype individuals (P<0.01), while there was no significant difference in the number of ribs between CC and TC genotype individuals.

[0053] Therefore, breeding CC and TC individuals at the rs341010833 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.

[0054] Table 1. Association analysis between the rs341010833 locus on porcine chromosome 6 and the number of ribs in Suhuai pigs.

[0055]

[0056] Note: Means in the same column followed by different letters are significantly different (P < 0.01).

Claims

1. The application of a primer pair for detecting a SNP marker associated with the rib number trait in Suhuai pigs, wherein the SNP marker is the T / C polymorphism at the nucleotide site rs341010833 on chromosome 6 of the international pig genome reference sequence version 11.1, and the rib number of individuals of CC and TC types is significantly more than that of individuals of TT type, and the primer pair consists of an upstream primer shown in SEQ ID NO: 2 and a downstream primer shown in SEQ ID NO:

3.

2. The application of the primer pair in claim 1 in the preparation of a reagent for detecting the rib number trait in Suhuai pigs.

3. A method for detecting the number of ribs trait in Suzhong pig, characterized in that, PCR amplification of a sequence containing the nucleotide site rs341010833 on chromosome 6 of the international pig genome reference sequence version 11.1, sequencing of the amplification product, and determination of the T / C polymorphism at the site.

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

5. The method of claim 4, wherein, The application of a SNP marker associated with the rib number trait in screening a population of Suhuai pigs with more ribs, characterized in that it comprises the following steps: (1) extracting total DNA from pig tissue samples; (2) using the extracted pig genomic DNA as a template and performing PCR amplification using the primer pair in claim 1; (3) sequencing the amplification product, analyzing the sequencing results, and determining the T / C polymorphism at position 301 of SEQ ID NO:

1.

6. A method of screening a population of multi-ribbed pigs, characterized in that The application comprises detecting the genotype of the nucleotide site rs341010833 on chromosome 6 of the international pig genome reference sequence version 11.1, and the rib number of individuals of CC and TC types is significantly more than that of individuals of TT type, and breeding individuals of CC and / or TC types at the nucleotide site rs341010833 as reserve boars.

7. The method of claim 6, wherein The method for detecting the genotype of the nucleotide site rs341010833 on chromosome 6 of the international pig genome reference sequence version 11.1 is PCR or gene sequencing.

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