Primer pair of snp marker related to pig chromosome 5 and pig vulva angle score trait and application thereof

Abstract

The present application relates to a SNP marker primer pair related to the pig chromosome 5 and pig vulva angle score trait and its application. The SNP marker site is a molecular marker of the nucleotide site rs322368669 of pig chromosome 5 in the international pig genome 11.1 version reference sequence, 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 vulva angle score traits, and the genotype of the SNP marker is identified to screen pig populations or strains with greater vulva angle score. The establishment of the population or strain can improve the vulva angle score and reproductive performance of pigs, and produce more social and economic benefits.

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 chromosome 5 and a pig vulva angle scoring trait and an application thereof. Background Art

[0002] Improving pig reproductive performance has always been a key concern for livestock farmers. The pig vulva angle score is closely linked to sexual maturity and has a significant impact on sow productivity and fertility. Literature shows that the size of the vulva at day 115 is significantly correlated with the age of first estrus (r = -0.28, P = 0.01). Sows with small vulvas reach first estrus later than those with large vulvas, suggesting that vulva size may be a potential predictor of the timing of first estrus. Furthermore, because secretions from the sow's reproductive tract are discharged through the vagina, sows with higher vulva angle scores are less likely to be culled due to vulvar inflammation and pus discharge. Lower vulva angle scores increase the likelihood of secretions accumulating, leading to infection and inflammation. Therefore, sows with higher vulva angle scores should be selected for breeding.

[0003] Traditionally, pigs have been manually scored for vulvar angle, but this method suffers from inaccurate measurements, high labor costs, and low efficiency. In recent years, with the advancement of molecular genetics and genomics, researchers have begun to leverage the association between single nucleotide polymorphism (SNP) markers and pig traits to better select for vulvar angle through molecular breeding.

[0004] Against this backdrop, this patent proposes a SNP marker primer pair associated with the pig vulva angle score trait, enabling rapid and accurate assessment of the pig vulva angle score trait through genotyping technology. This technology not only improves the accuracy and efficiency of vulva angle score assessments but also provides an important molecular genetic basis for improving pig reproductive performance. Summary of the Invention

[0005] The purpose of the present invention is to provide a breeding molecular marker developed from SNP markers related to pig vulva angle scoring, as the on-site measurement of traditional pig vulva angle scoring is time-consuming and labor-intensive, and the breeding effect is slow.

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

[0007] The purpose of the present invention can be achieved through the following technical solutions:

[0008] A molecular marker related to the pig vulva angle score trait, wherein the sequence of the molecular marker is shown as SEQ ID NO:1, and the molecular marker comprises a SNP marker site related to the pig vulva angle score trait, and the SNP marker site is rs322368669 on chromosome 5 of the international pig genome 11.1 version reference sequence, wherein the SNP marker site is located at position 301 in SEQ ID NO:1, and a T / C polymorphism exists, and the vulva angle score of an individual with a CC type is significantly greater than that of individuals with a TT type and a TC type.

[0009] A primer pair for detecting the SNP marker site related to the pig vulva angle score trait, wherein the upstream primer is SEQ ID NO:2, and the downstream primer is SEQ ID NO:3.

[0010] The molecular marker and the primer pair provided by the application are used for detecting the pig vulva angle score trait and pig breeding.

[0011] A method for detecting the molecular marker related to the pig vulva angle score trait, comprising the following steps: performing PCR amplification on a sequence of the rs322368669 nucleotide site on chromosome 5 of the international pig genome 11.1 version reference sequence of the pig, and performing sequencing on the amplification product, and determining the T / C polymorphism of the site.

[0012] As a preferred embodiment of the application, the pig is a Topigs Landrace.

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

[0014] As a further preferred embodiment of the application, the method comprises the following steps:

[0015] (1) extracting DNA from pig ear tissue samples;

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

[0017] (3) performing sequencing on the amplification product, analyzing the sequencing result, and determining the T / C polymorphism at position 301 in SEQ ID NO:1.

[0018] The molecular marker provided by the application is used for screening a pig population or a new strain with a higher vulva angle score.

[0019] The primer pair provided by the application is used for screening a pig population or a new strain with a higher vulva angle score.

[0020] A method for screening a pig population with higher vulva angle score, comprising detecting the genotype of the pig at the nucleotide site rs322368669 on chromosome 5 of the International Pig Genome 11.1 version reference sequence, and breeding individuals with CC genotype at the nucleotide site rs322368669 as priority for breeding.

[0021] As a preferred embodiment of the present application, the pig breed used is Topigs Large White.

[0022] As a preferred embodiment of the present application, the method for detecting the genotype of the nucleotide site rs322368669 on chromosome 5 of the International Pig Genome 11.1 version reference sequence is selected from PCR or gene sequencing.

[0023] Beneficial effects

[0024] The present application develops a SNP marker on chromosome 5 of pigs associated with the score of vulva angle, and provides a primer pair and a method for detecting the marker. By identifying the genotype of the SNP marker, a pig strain with higher vulva angle score is screened. The establishment of the strain can improve the comprehensive reproductive performance of pigs and generate more social and economic benefits. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Schematic diagram of vulva angle determination

[0026] Figure 2 Gel map of PCR amplification of the site rs322368669 on chromosome 5 of Large White.

[0027] Figure 3 Example of genotyping map of the site rs322368669 on chromosome 5 of Large White.

[0028] A is TT type, B is TC type, and C is CC type. DETAILED DESCRIPTION

[0029] 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.

[0030] Example 1

[0031] 1 Source of test animals

[0032] Jiangsu Lihua Animal Husbandry Co., Ltd. Changzhou Lijia Sow Farm

[0033] 2 Phenotype collection method

[0034] Vulva angle Figure 1) is the angle between the straight line formed by the bottom of the vulva and the bottom of the vulva cleft and the vulva cleft, the angle score is measured with a protractor, 0-60 degrees is 1 point, 61-90 degrees is 2 points, 91-120 degrees is 3 points, 121-150 degrees is 4 points, 151-180 degrees is 5 points. Considering the estrus period, the vulva is red and swollen, and the determination of the vulva needs to be carried out in the state of non-estrus of sows.

[0035] 3Extract pig genomic DNA

[0036] 833 Topigs Large White pigs were collected for individual DNA extraction, and 1 ear tissue sample was collected from each pig;

[0037] According to the instructions of the tissue DNA extraction kit of Tian Gen Biological Technology Co., Ltd., the extraction steps are as follows:

[0038] ①First, add 68 mL and 200 mL of anhydrous ethanol to buffer GD and rinse PW respectively, and mix thoroughly.

[0039] ②Collect 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.

[0040] ③Add 20 μL of proteinase K solution, mix well, and place in a 56°C metal bath for overnight digestion until the tissue sample is dissolved. Briefly centrifuge to remove water droplets on the inner wall of the tube cap.

[0041] ④Add 200 μL of buffer GB, mix well by inverting, and place in a 70°C metal bath for 10 min. The solution should be clear, and briefly centrifuge to remove water droplets on the inner wall of the tube cap.

[0042] ⑤Add 200 μL of anhydrous ethanol, mix well by shaking for 15 seconds. At this time, a flocculent precipitate may appear. Briefly centrifuge to remove water droplets on the inner wall of the tube cap.

[0043] ⑥Add 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. Discard the waste liquid, and place the adsorption column CB3 back into the collection tube.

[0044] ⑦Add 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 into the collection tube

[0045] ⑧Add 600 μL of rinse PW to the adsorption column CB3, centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, and place the adsorption column CB3 into the collection tube.

[0046] ⑨Repeat step ⑧.

[0047] 10. Put the adsorption column CB3 back into the collection tube, centrifuge at 12,000 rpm for 2 min, and discard the waste liquid. Put the adsorption column CB3 at room temperature for several minutes to completely dry the residual rinse solution in the adsorption material.

[0048] Put the adsorption column CB3 into a clean centrifuge tube, and add 100 μL of elution buffer TE to the middle of the adsorption membrane. Place it at room temperature for 2-5 min, centrifuge at 12,000 rpm for 2 min, collect the solution into the centrifuge tube, and add the centrifuged solution to the adsorption column CB3 again. Place it at room temperature for 2 min, centrifuge at 12,000 rpm for 2 min, and collect the solution into the centrifuge tube.

[0049] Use the Nanodrop-2000 spectrophotometer to detect the mass and concentration of the DNA. Dilute the DNA concentration to 50 ng / μL, and store it at -20℃ for later use.

[0050] 4. PCR amplification and sequencing of the target fragment

[0051] Use the Large White pig genomic DNA as the template for PCR amplification. The reaction system includes 1 μL of DNA template, 1 μL of each primer shown in SEQ ID NO: 2 and SEQ ID NO: 3, and 22 μL of PCR mix. The amplification program is as follows:

[0052]

[0053] Perform agarose gel electrophoresis on the amplified product. The product fragment size is about 376 bp, and the electrophoresis result is shown in Figure 2 . Sequence the remaining amplified product, use DNAman software to verify the accuracy of the sequence, and use Chromas software to determine the genotype of rs322368669. An example of the Large White pig chromosome 5 rs322368669 genotyping chart is shown in Figure 3 .

[0054] 5. Statistical analysis

[0055] Use the general linear model of SAS 9.4 software to perform genotype and phenotype correlation analysis. The model is as follows: Y ijk = μ + B i + G j + W k + e ijk

[0056] Where Y ijk is the phenotype value of the vulva angle score; μ is the average value of the vulva angle score of the population; B i is the fixed effect of the SNP marker; G j represents the sampling batch as a fixed effect; Wk Representative age as a covariate; e ijk is the residual.

[0057] 6Results

[0058] Table 1 shows the results of the influence of different genotypes of the rs322368669 site on the vulva angle score of Large White pigs. The results show that the genotype of the rs322368669 site is significantly associated with the vulva angle score phenotype (P<0.05). Among them, the vulva angle score of the CC type individual is significantly higher than that of the TT type and TC type individuals (P<0.05), and there is no significant difference in the vulva angle score between the TT type and TC type individuals. Therefore, in Large White pigs, breeding the CC type of the rs322368669 site in the next generation is beneficial to increase the vulva angle score of the Large White pig population, and thus improve the reproductive performance of the Large White pig.

[0059] Table 1 Association analysis of the rs322368669 site on chromosome 5 of pigs with the vulva angle score of Large White pigs

[0060]

[0061] Note: The same row with different letters indicates a significant difference (P<0.05)

Claims

1. Use of a primer pair for detecting a molecular marker associated with a pig vulva angle score trait in detecting the pig vulva angle score trait and / or in pig breeding, wherein the pig is a Topigs Large White pig; the molecular marker sequence is shown in SEQ ID NO: 1, wherein position 301 is a SNP marker site associated with the pig vulva angle score trait, which is the rs322368669 nucleotide site on chromosome 5 of the International Swine Genome Version 11.1 reference sequence, and has a T / C polymorphism. The vulva angle score of CC type individuals is significantly greater than that of TT and TC type individuals. The pig breeding goal is to screen for pig populations or new breeds with higher vulva angle scores.

2. The use according to claim 1, characterized in that The upstream primer of the primer pair is: SEQ ID NO: 2, and the downstream primer is: SEQ ID NO:

3.

3. A method for selecting a pig population with a higher vulva angle score, characterized in that: The method includes detecting the genotype of the rs322368669 nucleotide site of pig chromosome 5 in the international pig genome version 11.1 reference sequence, and selecting individuals with CC type nucleotide site of rs322368669 as reserve breeding pigs; the pigs are Topigs Large White pigs.

4. The method according to claim 3, wherein The method for detecting the genotype of the rs322368669 nucleotide site of pig chromosome 5 in the international pig genome version 11.1 reference sequence is selected from PCR amplification and gene sequencing.

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