SNP (Single Nucleotide Polymorphism) marker primer pair related to litter size character of pig and application of SNP marker primer pair

Abstract

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

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology and relates to a SNP marker primer pair related to pig litter size trait and application thereof. Background Art

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

[0003] As a complex quantitative trait, litter size has low heritability and is affected by many factors. From before ovulation to after delivery, factors such as the breed, age, parity, semen quality of the mated boar, and nutritional status of the sow during pregnancy will affect the litter size of the sow to varying degrees. The variation in litter size is controlled by major genes and minor polygenes, and is not only affected by genetics but also by the environment. Traditional breeding methods are based on phenotypic selection and lack accurate genetic information, resulting in slow performance improvement during the breeding process. With the development of molecular biology technology and the discovery of various gene markers, more and more powerful large companies have used molecular marker-assisted breeding technology to assist in litter size selection, greatly improving the accuracy of selection and the speed of improving production performance.

[0004] Pig litter size is controlled by multiple genes, and it is very difficult to identify the true major genes and mutation sites. At present, only the estrogen receptor gene (ESR) and the follicle-stimulating hormone beta gene (FSHβ) are recognized as the major genes that affect pig litter size and are widely used in pig breeding. Other genes are minor genes that have been proven to play their respective roles in pig fertilization, ovulation, pregnancy or embryonic development and ultimately affect pig litter size. Summary of the invention

[0005] The purpose of the present invention is to provide SNP markers related to pig litter size traits and develop them into molecular markers in view of the fact that traditional pig litter size selection is time-consuming and labor-intensive, and the selection effect is not good.

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

[0007] Another object of the present invention is to provide uses of the above-mentioned SNP markers.

[0008] A SNP marker associated with the litter size trait of Erhualian sows, wherein the SNP marker is located at the rs335892787 nucleotide site of chromosome 3 of the international porcine genome version 11.1 reference sequence, has an A / G polymorphism, and the total litter size of individuals with a GG genotype at this site is significantly better than that of individuals with an AA genotype.

[0009] A method for developing molecular markers based on the SNP marker is to design a primer pair using a nucleotide sequence containing the nucleotide site rs335892787 of the international swine genome version 11.1 reference sequence on chromosome 3 as a basic sequence, and to perform PCR amplification using swine genomic DNA as a template to convert the nucleotide site rs335892787 of the international swine genome version 11.1 reference sequence on chromosome 3 into a molecular marker.

[0010] As a preferred embodiment of the present invention, the primer pair sequences are shown as SEQ ID NO: 2 and SEQ ID NO: 3.

[0011] Molecular markers obtained according to the method of the present invention.

[0012] As a preferred embodiment of the present invention, the molecular marker sequence is shown in SEQ ID NO: 1, and the nucleotide site rs335892787 of the international porcine genome version 11.1 reference sequence pig chromosome 3 is located at position 181 in SEQ ID NO: 1, and there is an 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 associated with the litter size trait of the Erhualian sow comprises PCR amplifying a sequence in the genome of the Erhualian sow containing the SNP marker associated with the litter size trait of the Erhualian sow, sequencing the amplified product, and interpreting the A / G polymorphism of the SNP marker.

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

[0016] (1) Extracting total DNA from Erhualian sow tissue samples;

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

[0018] (3) The amplified product was sequenced, the sequencing results were analyzed, and the A / G polymorphism at position 181 of SEQ ID NO: 1 was interpreted.

[0019] The molecular markers and primer pairs of the present invention are used in screening a group of Erhualian sows with high litter size.

[0020] A method for screening a high-litter-number pig population among Erhualian sows, comprising detecting the genotype of the rs335892787 nucleotide site of chromosome 3 of the reference sequence of the International Swine Genome 11.1 version in the Erhualian sow gene, and selecting GG-type individuals at the rs335892787 nucleotide site as breeding pigs.

[0021] Beneficial effects:

[0022] The SNP marker provided by the present invention is related to the pig litter size trait, and the molecular marker and primer developed based on the SNP can be used for SNP detection. Therefore, 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 value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Amplification gel image of the rs335892787 site on pig chromosome 3

[0024] Figure 2 This is an example of the genotyping map of the rs335892787 locus on chromosome 3 of pigs DETAILED DESCRIPTION

[0025] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention all fall within the scope of the present invention.

[0026] Embodiment 1:

[0027] 1. Source of experimental animals

[0028] Changshu Animal Husbandry and Industry Company Breeding Pig Farm and Jiaoxi Erhua Pig Professional Cooperative.

[0029] 2 Extraction of pig genomic DNA

[0030] Tissue samples from 373 Erhua-faced pigs were collected for individual DNA extraction.

[0031] Refer to the instructions of the tissue DNA extraction kit of Tiangen Biotechnology Company, and the extraction is carried out in the following order:

[0032] ① First, add 68 mL and 200 mL of anhydrous ethanol to buffer GD and rinse solution PW respectively, and mix thoroughly.

[0033] ② Collect about 100 mg of tissue sample and place it in a 2 mL EP tube. After completely chopping it, 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 digest overnight in a 56°C metal bath until the ear sample tissue is dissolved. Centrifuge briefly to remove water droplets on the inner wall of the tube cap.

[0035] ④ Add 200 μL of buffer GB, mix thoroughly by inversion, and place in a 70°C metal bath for 10 min. The solution should become clear and centrifuge briefly to remove water droplets on the inner wall of the tube cap.

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

[0037] ⑥ Add the solution and flocculent precipitate 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 seconds, 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 seconds, discard the waste liquid, and place the adsorption column CB3 in the collection tube

[0039] ⑧ Add 600 μL of rinse solution PW to the adsorption column CB3, centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, and place the adsorption column CB3 in a collection tube.

[0040] ⑨Repeat step ⑧.

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

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

[0043] After the mass and concentration were detected by Nanodrop-100 spectrophotometer, the concentration was diluted to 50 ng / μL and stored at -20°C for future use.

[0044] 3. PCR amplification and sequencing of target fragments

[0045] Use the extracted DNA as a template and the designed primers to perform PCR amplification: take 1 μL of DNA template, 0.4 μL of primers shown in SEQ ID NO: 2 and SEQ ID NO: 3, 10 μL of PCRMix reagent, and 7.2 μL of double distilled water; set up the PCR amplification system:

[0046]

[0047] The PCR product was detected by electrophoresis in 1.2% agarose gel. The amplified target fragment was about 343 bp in size. Figure 1 The remaining amplified products were sequenced, and the sequencing results were compared and analyzed with the relevant gene fragment sequences of pigs in GenBank using DNAman software to determine the genotype of the rs335892787 site. 4 Statistical analysis

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

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

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

[0051] 5 Results

[0052] Table 1 shows the effect of the rs335892787 variant site A / G on the total litter size trait in the Erhua pig population. As shown in Table 1, there is a significant difference in the total litter size trait between the three genotypes of the rs335892787 locus (P<0.05), and the average total litter size of individuals with the dominant genotype GG is 0.96 more than that of individuals with the inferior genotype AA. It can be seen that in the Erhua pig population, the successive breeding of GG individuals at the rs335892787 locus can gradually improve the total litter size trait of the Erhua pig population.

[0053] Table 1 Association analysis between rs335892787 locus on chromosome 3 and total litter size of Erhualian pigs

[0054]

[0055] Note: Numbers with different letters in the same column indicate significant differences (P<0.05).

Claims

1. A SNP marker associated with litter size in Erhualian sows, characterized in that: The SNP marker is located at the rs335892787 nucleotide site of chromosome 3 of the international pig genome version 11.1 reference sequence, and has an A / G polymorphism. The total litter size of individuals with a GG genotype at this site is significantly better than that of individuals with an AA genotype.

2. A method for developing molecular markers based on the SNP markers according to claim 1, characterized in that Based on the nucleotide sequence of the rs335892787 nucleotide site on chromosome 3 of the international porcine genome version 11.1 reference sequence, a primer pair was designed, and PCR amplification was performed using porcine genomic DNA as a template to convert the rs335892787 nucleotide site on chromosome 3 of the international porcine genome version 11.1 reference sequence into a molecular marker.

3. The method according to claim 2, characterized in that The primer pair sequences are shown in SEQ ID NO: 2 and SEQ ID NO:

3.

4. Molecular markers obtained according to the method of claim 2 or 3.

5. The molecular marker according to claim 4, characterized in that The molecular marker sequence is shown in SEQ ID NO:

1. The nucleotide site of rs335892787 of pig chromosome 3 in the international pig genome version 11.1 reference sequence is located at the 181st position in SEQ ID NO: 1, and there is an A / G polymorphism.

6. A primer pair for detecting the SNP marker according to claim 1, characterized in that The upstream primer is: SEQ ID NO: 2, and the downstream primer is: SEQ ID NO:

3.

7. A method for detecting the SNP marker associated with the litter size trait of Erhualian sows as claimed in claim 1, characterized in that The method comprises PCR amplifying a sequence in the genome of the Erhualian sow containing the SNP marker related to the litter size trait of the Erhualian sow as described in claim 1, sequencing the amplified product, and interpreting the A / G polymorphism of the SNP marker.

8. The method according to claim 7, characterized in that The following steps are involved: (1) Extracting total DNA from Erhualian sow tissue samples; (2) using the extracted porcine genomic DNA as a template and performing PCR amplification using the primer pair described in claim 6; (3) The amplified product was sequenced, the sequencing results were analyzed, and the A / G polymorphism at position 181 of SEQ ID NO: 1 was interpreted.

9. Use of the molecular markers described in claim 4 or 5 and the primer pairs described in claim 6 in screening a group of Erhualian sows with a high litter size.

10. A method for screening a high litter size pig population among Erhua face sows, characterized in that This includes detecting the genotype of the rs335892787 nucleotide site on chromosome 3 of the International Swine Genome 11.1 version reference sequence in the genes of the Erhualian sow, and selecting GG type individuals at the rs335892787 nucleotide site as breeding pigs.

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