A SNP marker primer pair related to pig litter size character and application thereof

Abstract

The application relates to a SNP marker primer pair related to a pig litter size character and application thereof. The SNP marker is located on a nucleotide sequence on a pig chromosome 3, the site of the SNP marker is a molecular marker containing a rs335892787 nucleotide site on an international pig genome 11.1 version reference sequence pig chromosome 3, and has A / G polymorphism, the SNP marker is significantly related to a pig litter size character. 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 application is significantly related to a pig litter size character, high-litter-size pigs can be screened by identifying the SNP marker, and the obtained high-litter-size pigs have important economic benefits and social values.

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 litter size trait and application thereof. BACKGROUND

[0002] In commercial breeding of pigs, reproductive traits such as litter size, teat number, estrus interval, etc. become the focus of selection, especially in the selection of female pigs. Litter size, as a crucial reproductive trait, is one of the most important production traits of pigs, directly reflecting the production performance of sows and related to the economic benefits of the pig industry.

[0003] As a complex quantitative trait, litter size has a low heritability and is influenced by many factors. From pre-ovulation to postpartum, factors such as breed, age, parity of sows, semen quality of mating boars, and nutritional status of sows during pregnancy all have varying degrees of impact on litter size. Litter size variation is controlled by major genes and micro-effect polygenes, and is influenced not only by genetics but also by the environment. Traditional selection methods are based on phenotypic selection without accurate genetic information, resulting in slow performance improvement during selection. With the development of molecular biology techniques and the discovery of various genetic markers, more powerful companies have used molecular marker-assisted breeding technology to assist in litter size selection, greatly improving the accuracy of selection and the speed of performance improvement.

[0004] Pig litter size is controlled by multiple genes, making it difficult to identify true major genes and mutation sites. Currently, only the estrogen receptor gene (Estrogen receptor, ESR) and the follicle stimulating hormone beta gene (Follicle stimulating hormone beta, FSHβ) are recognized as major genes affecting pig litter size and are widely used in pig breeding. Other genes are micro-effect genes that have been verified to play their respective roles in pig fertilization, ovulation, pregnancy, or embryonic development and ultimately affect pig litter size. SUMMARY

[0005] The present application aims to address the time-consuming and labor-intensive traditional pig litter size selection, which has poor selection results, by providing a SNP marker related to pig litter size and developing it as a molecular marker.

[0006] Another object of the present application is to provide a primer pair and detection method for detecting the above-mentioned SNP marker.

[0007] Another object of the present application is to provide the use of the above-mentioned SNP marker.

[0008] A SNP marker related to litter size trait of Erhualian sows, wherein the SNP marker is located at nucleotide site rs335892787 of pig chromosome 3 in the international pig genome reference sequence version 11.1, and has A / G polymorphism, and the total litter size of an individual with genotype GG at the site is significantly higher than that of an individual with genotype AA.

[0009] A method for developing a molecular marker based on the SNP marker, wherein a primer pair is designed based on the nucleotide sequence of the nucleotide site rs335892787 of pig chromosome 3 in the international pig genome reference sequence version 11.1, and pig genomic DNA is used as a template for PCR amplification, so that the nucleotide site rs335892787 of pig chromosome 3 in the international pig genome reference sequence version 11.1 is converted into a molecular marker.

[0010] As a preferred embodiment of the present application, the primer pair sequence is shown in SEQ ID NO: 2 and SEQ ID NO: 3.

[0011] The molecular marker obtained by the method of the present application.

[0012] As a preferred embodiment of the present application, the molecular marker sequence is shown in SEQ ID NO: 1, and the nucleotide site rs335892787 of pig chromosome 3 in the international pig genome reference sequence version 11.1 is located at position 181 in SEQ ID NO: 1, and has A / G polymorphism.

[0013] A primer pair for detecting the SNP marker, wherein the upstream primer is SEQ ID NO: 2, and the downstream primer is SEQ ID NO: 3.

[0014] A method for detecting the SNP marker related to litter size trait of Erhualian sows, comprising the following steps: PCR amplifying a sequence containing the SNP marker related to litter size trait of Erhualian sows in the genome of an Erhualian sow, sequencing the amplification product, and determining the A / G polymorphism of the SNP marker.

[0015] As a preferred embodiment of the present application, the method comprises the following steps:

[0016] (1) extracting total DNA from a tissue sample of an Erhualian sow;

[0017] (2) using the extracted pig genomic DNA as a template, and using the primer pair for PCR amplification;

[0018] (3) sequencing the amplification product, analyzing the sequencing result, and determining the A / G polymorphism at position 181 in SEQ ID NO: 1.

[0019] The molecular marker and the primer pair provided by the application are used for screening a high-litter-size Erhualian sow population.

[0020] A method for screening a high-litter-size Erhualian sow population, comprising detecting the genotype of a pig international pig genome 11.1 version reference sequence pig chromosome 3 rs335892787 nucleotide site in an Erhualian sow gene, and breeding an individual with a GG type of the rs335892787 nucleotide site as a breeding pig.

[0021] Beneficial effects:

[0022] The SNP marker provided by the application is related to a pig litter size trait, and a molecular marker and a primer developed based on the SNP can be used for SNP detection. Therefore, a high-litter-size pig can be screened by identifying the SNP marker, and the obtained high-litter-size pig has important economic benefits and social value. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Pig 3 chromosome rs335892787 site amplification gel map

[0024] Figure 2 Pig 3 chromosome rs335892787 site genotyping map example DETAILED DESCRIPTION

[0025] 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.

[0026] Example 1:

[0027] 1. Source of test animals

[0028] Changshu Muxiushang Company Pig Farm and Jiaoxi Erhualian Pig Professional Cooperative.

[0029] 2. Extraction of pig genomic DNA

[0030] One tissue sample was collected from each of 373 Erhualian pigs, and individual DNA extraction was performed;

[0031] According to the instructions of the tissue DNA extraction kit of Tiangeng Biotechnology Co., Ltd., extraction was performed in the following order:

[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 tissue sample in a 2 mL EP tube, cut it completely, add 200 μL of buffer GA, and shake until completely suspended.

[0034] 3. Add 20 μL Proteinase K solution, mix well and place in a 56°C metal bath for overnight digestion until the ear sample tissue is dissolved. Briefly centrifuge to remove water droplets from the inside of the tube cap.

[0035] 4. Add 200 μL Buffer GB, mix well by inverting, place 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.

[0036] 5. Add 200 μL absolute ethanol, mix well by inverting for 15 sec. A flocculent precipitate may appear at this point. Briefly centrifuge to remove water droplets from the inside of the tube cap.

[0037] 6. Add both the solution and the flocculent precipitate from the previous step to a column CB3, which is placed in a collection tube. Centrifuge at 12,000 rpm for 30 sec, discard the flow-through, 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 flow-through, and place the column CB3 in the collection tube.

[0039] 8. Add 600 μL Rinse Buffer PW to the column CB3, centrifuge at 12,000 rpm for 30 sec, discard the flow-through, 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 flow-through. Allow the column CB3 to sit at room temperature for several minutes to dry the residual rinse buffer from the adsorption material.

[0042] Place the column CB3 in a clean centrifuge tube, and add 100 μL Elution Buffer TE to the center of the adsorption membrane, allow it to sit at room temperature for 2-5 min, centrifuge at 12,000 rpm for 2 min, collect the solution in a centrifuge tube, and add the solution obtained from the centrifugation to the column CB3, allow it to sit at room temperature for 2 min, centrifuge at 12,000 rpm for 2 min, collect the solution in a centrifuge tube.

[0043] After detecting the mass and concentration using a Nanodrop-100 spectrophotometer, dilute the solution to 50 ng / μL, and store it at -20°C for later use.

[0044] 3. PCR amplification and sequencing of the target fragment

[0045] PCR amplification was performed with the extracted DNA as template according to the designed primers: 1 μL of DNA template, 0.4 μL of each of the primers shown in SEQ ID NO: 2 and SEQ ID NO: 3, 10 μL of PCR Mix reagent, and 7.2 μL of double distilled water; and the PCR amplification system was set as follows:

[0046]

[0047] The PCR product was detected by electrophoresis in a 1.2% agarose gel, and the size of the amplified target fragment was about 343 bp, and the electrophoretogram is shown in Figure 1 The remaining amplified product was sequenced, and the sequencing results were compared and analyzed with the sequence of the related gene fragment of pigs in GenBank by using DNAman software to determine the genotype of the rs335892787 site, and statistical analysis

[0048] The SAS software general linear model was used to analyze the effect of genotype on phenotype. The analysis model was

[0049] Y ijnk = u i + G j + S n + D k + e jnk

[0050] wherein Y ijnk is the total number of piglets trait; G j represents the fixed effect of the jth SNP genotype; S n represents the fixed effect of gender; D k represents the fixed effect of the birth year season; and e jnk is the residual.

[0051] 5Results

[0052] Table 1 shows the effect of the rs335892787 variation site A / G on the total number of piglets trait in the Erhualian pig population. As shown in Table 1, there is a significant difference (P<0.05) in the total number of piglets trait among the three genotypes of the rs335892787 site, and the average total number of piglets of the dominant genotype GG type is 0.96 more than that of the inferior genotype AA type. Therefore, in the Erhualian pig population, the GG type individuals of the rs335892787 site can gradually improve the total number of piglets trait of the Erhualian pig population through successive selection.

[0053] Table 1 shows the effect of the rs335892787 variation site A / G on the total number of piglets trait in the Erhualian pig population. As shown in Table 1, there is a significant difference (P<0.05) in the total number of piglets trait among the three genotypes of the rs335892787 site, and the average total number of piglets of the dominant genotype GG type is 0.96 more than that of the inferior genotype AA type. Therefore, in the Erhualian pig population, the GG type individuals of the rs335892787 site can gradually improve the total number of piglets trait of the Erhualian pig population through successive selection.

[0054]

[0055] Note: Means in the same column followed by different letters are significantly different (P < 0.05).

Claims

1. The application of a SNP marker detection reagent in screening high-producing sow populations, characterized in that, The SNP marker is located at rs335892787 nucleotide site on chromosome 3 of the pig in the international pig genome version 11.1 reference sequence. It has A / G polymorphism. Individuals with the GG genotype at this site have a significantly larger total litter size than individuals with the AA genotype.

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

3.

3. A method for screening high-producing piglet groups among two-flowered sows, characterized in that, This includes detecting the genotype of the rs335892787 nucleotide site on chromosome 3 of the pig in the reference sequence of the 11.1 version of the International Pig Genome, and selecting GG individuals with the rs335892787 nucleotide site as breeding pigs.

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