SNP (Single Nucleotide Polymorphism) marker primer pair related to pig rib number character on pig chromosome 6 and application of SNP marker primer pair

Abstract

The invention relates to an SNP (Single Nucleotide Polymorphism) marker primer pair related to a pig rib number character and application of the SNP marker primer pair. The locus of the SNP marker is a molecular marker of a nucleotide locus of a pig chromosome 6 rs341010833 of a reference sequence of an international pig genome version 11.1, and the SNP marker has T / C polymorphism. According to a primer pair for detecting the SNP marker, an upstream primer is SEQ ID NO: 2, and a downstream primer is SEQ ID NO: 3. The SNP marker provided by the invention can be applied to marker-assisted selection of the pig rib number character, and pig populations or new strains with multiple rib numbers are screened by identifying the genotype of the SNP marker. By establishing the group or the new strain, the rib number and the meat production performance of pigs can be improved, and more social and economic benefits can be generated.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology and relates to a SNP marker primer pair related to the number of pig ribs and its application. Background Art

[0002] China is the world's largest producer and consumer of pork, and pork is an important source of animal protein in the Chinese diet. With the continuous growth of the economy and the improvement of living standards, the demand for pork is increasing continuously. Therefore, improving the meat production rate of pig carcasses has become the core goal of current pig breeding research.

[0003] The spine of a pig consists of cervical vertebrae, thoracic vertebrae, lumbar vertebrae, sacral vertebrae and caudal vertebrae, among which the number of thoracic vertebrae and lumbar vertebrae varies. The thoracic vertebrae are connected to the ribs, so the number of thoracic vertebrae directly determines the number of ribs. The number of ribs is significantly correlated with the length of the pig carcass. For every additional pair of ribs, not only can the number of pork ribs and tenderloin be increased, but also the body length can be increased by 10 - 40 mm, thereby increasing the meat production by about 1%. Pork ribs are one of the highest-priced carcass parts in the Chinese market and are deeply loved by consumers. Therefore, studying the genetic molecular mechanism of the number of pig ribs and applying related molecular markers to breeding practice have important economic significance for breeding pig populations or new strains with more ribs and higher meat production rates. Summary of the Invention

[0004] The purpose of the present invention is to provide a breeding molecular marker developed from SNP markers related to the number of pig ribs, aiming at the time-consuming, laborious, difficult phenotypic determination and slow breeding effect of traditional pig rib number selection.

[0005] Another purpose of the present invention is to provide a primer pair and a detection method for detecting the above SNP markers. Another purpose of the present invention is to provide the uses of the above SNP markers, molecular markers and primers.

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

[0007] A SNP marker related to the number of pig ribs, this locus is the nucleotide locus rs341010833 on chromosome 6 of pigs in the reference sequence of the international pig genome version 11.1, with T / C polymorphism; the number of ribs in CC-type and TC-type individuals is extremely significantly more than that in TT-type individuals.

[0008] A molecular marker containing the above SNP locus, the sequence of the molecular marker is as shown in SEQ ID NO: 1, wherein the SNP marker described in claim 1 is located at position 301 of SEQ ID NO: 1, with T / C polymorphism; the number of ribs in CC-type and TC-type individuals is extremely significantly more than that in TT-type individuals.

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

[0010] Application of the SNP marker, the molecular marker, and the primer pair of the present invention in detecting the number of pig ribs and / or pig breeding.

[0011] Application of the primer pair of the present invention in preparing a reagent for detecting the number of pig ribs and / or pig breeding.

[0012] A method for detecting the SNP marker related to the number of pig ribs, PCR amplifying a sequence containing the nucleotide site rs341010833 on chromosome 6 of the pig in the 11.1 version of the international pig genome reference sequence, sequencing the amplification product, and interpreting the T / C polymorphism at this site.

[0013] As a preference of the present invention, the pig genomic DNA is PCR amplified using the primer pair.

[0014] As a further preference of the present invention, application of detecting the SNP marker related to the number of pig ribs in screening a multi-rib pig population, characterized by including the following steps:

[0015] (1) Taking a pig tissue sample to extract total DNA;

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

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

[0018] A method for screening a multi-rib pig population, including detecting the genotype of the nucleotide site rs341010833 on chromosome 6 of the pig in the 11.1 version of the international pig genome reference sequence. The number of ribs in individuals with the CC type and the TC type is extremely significantly more than that in individuals with the TT type. Selecting individuals with the CC type and / or the TC type at the rs341010833 nucleotide site as reserve breeding pigs for breeding.

[0019] As a preference of the present invention, the method for detecting the genotype of the nucleotide site rs341010833 on chromosome 6 of the pig in the 11.1 version of the international pig genome reference sequence is selected from PCR or gene sequencing.

[0020] Beneficial effects

[0021] The present invention has developed SNP markers related to the number of pig ribs, and provided primer pairs and methods for detecting such markers. By identifying the genotypes of the SNP markers, new pig strains with multiple ribs are screened. The establishment of such strains can improve the meat production performance and the number of ribs of pigs, increase the distribution ratio of high-value parts in pig carcasses, and generate more social and economic benefits. Description of the Drawings

[0022] Figure 1 It is the PCR amplification gel diagram of the rs341010833 locus on chromosome 6 of pigs in the 11.1 version of the International Pig Genome Reference Sequence.

[0023] Figure 2 It is an example of the genotyping diagram of the rs341010833 locus on chromosome 6 of pigs in the 11.1 version of the International Pig Genome 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. Specific Embodiment

[0025] The following examples are used to illustrate the present invention, but not to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement of the methods, steps or conditions of the present invention shall fall within the scope of the present invention.

[0026] Example 1

[0027] 1 Source of Test Animals

[0028] The test pigs are from Huaiyin Xinhuai Breeding Farm in Huai'an City, Jiangsu Province. After slaughter, the sum of the number of ribs on both sides of the carcass is counted manually.

[0029] 2 Extraction of Pig Genomic DNA

[0030] Collect 1 ear tissue sample from each of 655 Suhuai pigs for individual DNA extraction;

[0031] Referring to the instruction manual of the tissue DNA extraction kit of Tiangen Biotech Company, the extraction steps are as follows:

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

[0033] ② Collect about 100 mg of ear tissue sample and place it in a 2 mL EP tube. After completely cutting it into pieces, 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 it in a 56 °C metal bath for digestion overnight 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 of buffer GB, invert thoroughly to mix, place in a 70 °C metal bath for 10 min. The solution should become clear. Centrifuge briefly to remove the water droplets on the inner wall of the tube cap.

[0036] ⑤ Add 200 μL of absolute ethanol, shake thoroughly for 15 sec. Flocculent precipitates may appear at this time. Centrifuge briefly to remove the water droplets on the inner wall of the tube cap.

[0037] ⑥ Add both the solution and the flocculent precipitates 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, pour out the waste liquid, and put the adsorption column CB3 back into the collection tube.

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

[0039] ⑧ Add 600 μL of wash buffer PW to the adsorption column CB3, centrifuge at 12,000 rpm for 30 sec, pour out the waste liquid, and place the adsorption column CB3 in the collection tube.

[0040] ⑨ Repeat operation step ⑧.

[0041] ⑩ Place the adsorption column CB3 back into the collection tube, centrifuge at 12,000 rpm for 2 min, pour out the waste liquid. Place the adsorption column CB3 at room temperature for several minutes to thoroughly dry the residual wash buffer in the adsorption material.

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

[0043] Use a Nanodrop - 2000 spectrophotometer to detect the quality and concentration of DNA. Dilute the DNA concentration to 50 ng / μL and store at - 20 °C for later use.

[0044] 3 PCR Amplification and Sequencing of the Target Fragment

[0045] Perform PCR amplification using the genomic DNA of Suhuai pigs as a template. The reaction system includes 1 μL of DNA template, 1 μL each of the primers shown in TATCACCAGTAGTAGGTC (SEQ ID NO: 2) and AGGAGAAAAGGATTGTAT (SEQ ID NO: 3), ddH 29.5 μL of O, 12.5 μL of PCR mix; the amplification program is as follows:

[0046]

[0047] The amplified products were subjected to agarose gel electrophoresis, and the size of the product fragments was approximately 496 bp. The electrophoresis results are as Figure 1 shown. The remaining amplified products were sequenced, and the sequencing results were compared and verified for sequence accuracy using DNAman software. Chromas software was used to genotype the rs341010833 locus.

[0048] 4 Statistical analysis

[0049] The general linear model of SAS 9.4 software was used for the association analysis of genotypes and phenotypes. The model is as follows: Y ijk = μ i + B j + G k + e jk

[0050] Among them, Yijk is the number of ribs of an individual; μ i represents the mean number of ribs in the population; B j represents the fixed effect of the slaughter batch; G k is the fixed effect of the SNP marker; e jk is the residual.

[0051] 5 Results

[0052] Table 1 shows the results of the effect of different genotypes at the rs341010833 locus on the number of ribs of Suhuai pigs. The results show that there are significant differences in the number of ribs among individuals with the three genotypes at the rs341010833 locus. Among them, the number of ribs of individuals with the CC and TC genotypes is extremely significantly more than that of individuals with the TT genotype (P < 0.01), and there is no significant difference in the number of ribs between individuals with the CC genotype and those with the TC genotype.

[0053] Therefore, continuous selection of individuals with the CC and TC genotypes at the rs341010833 locus in Suhuai pigs is beneficial to increasing the number of ribs in the Suhuai pig population, and thus improving the meat production performance of Suhuai pigs.

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

[0055]

[0056] Note: Different superscript letters for the same row of numbers indicate extremely significant differences (P < 0.01).

Claims

1. A SNP marker associated with the number of pig ribs, characterized in that: This site is the rs341010833 nucleotide site on chromosome 6 of the international pig genome version 11.1 reference sequence, and there is a T / C polymorphism; the number of ribs in CC and TC type individuals is significantly more than that in TT type individuals.

2. A molecular marker comprising the SNP site according to claim 1, characterized in that: The molecular marker sequence is shown in SEQ ID NO: 1, wherein the SNP marker of claim 1 is located at position 301 of SEQ ID NO: 1, and there is a T / C polymorphism; the number of ribs of CC type and TC type individuals is significantly more than that of TT type individuals.

3. A primer pair for detecting the SNP marker associated with the pig rib number trait according to claim 1, characterized in that The upstream primer is: SEQ ID NO: 2, and the downstream primer is: SEQ ID NO:

3.

4. Use of the SNP marker according to claim 1, the molecular marker according to claim 2, and the primer pair according to claim 3 in detecting pig rib number traits and / or pig breeding.

5. Use of the primer pair described in claim 3 in preparing a reagent for detecting pig rib number traits and / or pig breeding.

6. A method for detecting the SNP marker associated with the pig rib number trait according to claim 1, characterized in that: A sequence of the rs341010833 nucleotide site of chromosome 6 of the International Porcine Genome Version 11.1 reference sequence was amplified by PCR, and the amplified product was sequenced to determine the T / C polymorphism of the site.

7. The method according to claim 6, characterized in that The primer pair according to claim 3 is used to perform PCR amplification on pig genomic DNA.

8. The method according to claim 7, wherein the use of detecting SNP markers associated with the number of pig ribs in screening a population of pigs with many ribs is characterized in that The following steps are involved: (1) Extract total DNA from pig tissue samples; (2) using the extracted porcine genomic DNA as a template and performing PCR amplification using the primer pair described in claim 3; (3) The amplified product was sequenced, the sequencing results were analyzed, and the T / C polymorphism at position 301 of SEQ ID NO: 1 was determined.

9. A method for screening a group of pigs with many ribs, characterized in that This includes detecting the genotype of the rs341010833 nucleotide site on chromosome 6 of the International Swine Genome 11.1 version reference sequence. The number of ribs in CC type and TC type individuals is significantly higher than that in TT type individuals. Individuals with CC type and / or TC type at the rs341010833 nucleotide site are selected as reserve breeding pigs.

10. The method according to claim 9, characterized in that The method for detecting the genotype of the rs341010833 nucleotide site of chromosome 6 of the pig international pig genome version 11.1 reference sequence is selected from PCR or gene sequencing.

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