SNP (Single Nucleotide Polymorphism) marker primer pair related to pig chromosome 5 and pig vagina angle scoring character and application of SNP marker primer pair

Abstract

The invention relates to an SNP (Single Nucleotide Polymorphism) marker primer pair related to pig chromosome 5 and pig vagina angle scoring character and application of the SNP marker primer pair. The locus of the SNP marker is a molecular marker of a nucleotide locus rs322368669 of a porcine chromosome 5 in a reference sequence of an international porcine 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 pig pudendum angle scoring characters, and pig groups or strains with higher pudendum angle scores are screened by identifying the genotype of the SNP marker. Establishment of the group or strain can improve the score and reproductive performance of pigs in the aspect of pudendum, and more social and economic benefits can be generated.

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 pig vulva angle scoring traits and an application thereof. Background Art

[0002] In the field of animal husbandry, improving the reproductive performance of pigs has always been an important issue of concern to breeders. There is a close connection between the pig vulva angle score and sexual maturity, which has an important impact on the productivity and fertility of sows. Literature shows that the size of the sow's vulva on the 115th day is significantly correlated with the age of first estrus (r = -0.28, P = 0.01), and the first estrus of sows with small vulvas is later than that of sows with large vulvas, suggesting that the size of the vulva can be used as a potential indicator for predicting the time of the first estrus of sows. In addition, since the secretions from the reproductive tract of the sow will be discharged through the vagina, sows with higher vulva angle scores are less likely to be eliminated due to vulvar inflammation and pus. The lower the vulva angle score, the easier it is for the secretions to be retained, causing infection and inflammation. Therefore, in production, it is necessary to select sows with high vulva angle scores for breeding.

[0003] The traditional method is to manually score the vulva angle of pigs, but this method has problems such as inaccurate measurement, high labor costs and low efficiency. In recent years, with the development of molecular genetics and genomics, researchers have begun to use the association between single nucleotide polymorphism (SNP) markers and pig traits to better carry out the selection of vulva angle through molecular breeding.

[0004] Based on this background, this patent proposes a SNP marker primer pair related to the pig vulva angle scoring trait, which can quickly and accurately evaluate the pig vulva angle scoring trait through genotyping technology. This technology can not only improve the accuracy and efficiency of evaluating the vulva angle score, but also provide an important molecular genetic basis for improving the reproductive performance of pigs. Summary of the invention

[0005] The purpose of the present invention is to provide a breeding molecular marker developed from SNP markers related to the pig vulva angle score, in view of the fact that the on-site measurement of traditional pig vulva angle scoring and breeding 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 associated with a pig vulva angle score trait, wherein the molecular marker sequence is shown in SEQ ID NO: 1, wherein the molecular marker contains a SNP marker site associated with a pig vulva angle score trait, the site is the rs322368669 nucleotide site of chromosome 5 of the international pig genome version 11.1 reference sequence, the SNP marker site in SEQ ID NO: 1 is located at position 301, there is a T / C polymorphism, and the vulva angle score of CC type individuals is significantly greater than that of TT type and TC type individuals.

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

[0010] The molecular markers and primer pairs of the present invention are used in detecting pig vulva angle scoring traits and pig breeding.

[0011] A method for detecting the molecular markers related to the pig vulva angle score trait comprises PCR amplifying a sequence of the rs322368669 nucleotide site of the pig chromosome 5 of the pig international pig genome version 11.1 reference sequence, sequencing the amplified product, and interpreting the T / C polymorphism of the site.

[0012] As a preferred embodiment of the present invention, the pig is a Topigs Large White pig.

[0013] As a preferred embodiment of the present invention, the primer pair of the present invention is used to perform PCR amplification on Large White pig genomic DNA.

[0014] As a further preference of the present invention, the method comprises the following steps:

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

[0016] (2) using the extracted porcine genomic DNA as a template and performing PCR amplification using the primer pair of the present invention;

[0017] (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.

[0018] The molecular markers of the present invention are used in screening pig groups or new strains with higher vulva angle scores.

[0019] The primer pair of the present invention is used in screening pig groups or new strains with higher vulva angle scores.

[0020] A method for screening a pig population with a higher vulva angle score, comprising detecting the genotype of the rs322368669 nucleotide site of chromosome 5 of the pig international porcine genome version 11.1 reference sequence, and selecting CC-type individuals at the rs322368669 nucleotide site as reserve breeding pigs.

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

[0022] As a preferred embodiment of the present invention, the method for detecting the genotype of the nucleotide site rs322368669 of pig chromosome 5 in the international pig genome version 11.1 reference sequence is selected from PCR or gene sequencing.

[0023] Beneficial Effects

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

[0025] Figure 1 Diagram of vaginal angle measurement

[0026] Figure 2 This is the gel image of PCR amplification of the rs322368669 site on chromosome 5 of Large White pig.

[0027] Figure 3 This is an example of the genotyping map of the rs322368669 locus on chromosome 5 of Large White pig.

[0028] A is TT type, B is TC type, and C is CC type. Specific implementation plan

[0029] 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.

[0030] Example 1

[0031] 1. Source of experimental animals

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

[0033] 2 Phenotype collection method

[0034] Vagina angle( Figure 1) is the angle between the straight line formed by the bottom of the vulva and the bottom of the vulvar fissure and the vulvar fissure. The angle score is measured with a protractor, with 0-60 degrees as 1 point, 61-90 degrees as 2 points, 91-120 degrees as 3 points, 121-150 degrees as 4 points, and 151-180 degrees as 5 points. Considering that the vulva becomes red, swollen and enlarged during estrus, vulva measurement needs to be carried out when the sow is not in estrus.

[0035] 3 Extraction of pig genomic DNA

[0036] One ear tissue sample was collected from 833 Topigs Large White pigs for individual DNA extraction;

[0037] Refer to the instructions of the tissue DNA extraction kit of Tiangen Biotechnology Company. The extraction steps are as follows:

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

[0039] ② Collect about 100 mg of ear tissue sample and place it in a 2 mL EP tube. After completely mincing, add 200 μL of buffer GA and shake until completely suspended.

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

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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

[0045] 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.

[0046] ⑨Repeat step ⑧.

[0047] 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.

[0048] 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.

[0049] The quality and concentration of DNA were detected using Nanodrop-2000 spectrophotometer. The DNA concentration was diluted to 50 ng / μL and stored at -20°C for future use.

[0050] 4. PCR amplification and sequencing of target fragments

[0051] PCR amplification was performed using Large White pig genomic DNA as a template. The reaction system included 1 μL of DNA template, 1 μL of primers shown in SEQ ID NO: 2 and SEQ ID NO: 3, and 22 μL of PCR mix. The amplification procedure was as follows:

[0052]

[0053] The amplified product was subjected to agarose gel electrophoresis, and the product fragment size was about 376 bp. The electrophoresis results were as follows Figure 2 The remaining amplified products were sequenced, and the sequencing results were compared with DNAman software to verify the accuracy of the sequence. Chromas software was used to genotype the rs322368669 locus. An example of the genotyping map of the rs322368669 locus on chromosome 5 of Large White pig is shown in Figure 3 .

[0054] 5 Statistical analysis

[0055] The association analysis between genotype and phenotype was performed using the general linear model of SAS 9.4 software. The model was as follows: ijk =μ+B i +G j +W k +e ijk

[0056] Among them, Y ijk is the phenotypic value of vulva angle score; μ is the mean vulva angle score of the group; B i is the fixed effect of SNP marker; G j represents sampling batch as a fixed effect; Wk represents age as a covariate; e ijk is the residual.

[0057] 6 Results

[0058] Table 1 shows the effects of different genotypes of rs322368669 on the vulva angle score of Large White pigs. The results showed that the genotype of rs322368669 was significantly associated with the vulva angle score phenotype (P<0.05). Among them, the vulva angle score of CC type individuals was significantly greater than that of TT type and TC type individuals (P<0.05), and the vulva angle score between TT type and TC type individuals was not significant. Therefore, the selection of CC type at rs322368669 in Large White pigs is conducive to increasing the vulva angle score of the Large White pig population, thereby improving the reproductive performance of Large White pigs.

[0059] Table 1 Association analysis between rs322368669 locus on chromosome 5 and vulva angle score of Large White pigs

[0060]

[0061] Note: Numbers with different letters in the same column indicate significant differences (P<0.05).

Claims

1. A molecular marker associated with the pig vulva angle score trait, characterized in that The molecular marker sequence is shown in SEQ ID NO: 1, and its 301st position is a SNP marker site related to the pig vulva angle score trait. The site is the rs322368669 nucleotide site of pig chromosome 5 in the international pig genome version 11.1 reference sequence. There is a T / C polymorphism, and the vulva angle score of CC type individuals is significantly greater than that of TT type and TC type individuals.

2. A primer pair for detecting the molecular marker associated with the pig vulva angle score 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.

3. Use of the molecular marker described in claim 1 and the primer pair described in claim 2 in detecting the pig vulva angle scoring trait and / or pig breeding, wherein the pig is a Topigs Large White pig.

4. The use according to claim 3, characterized in that Use of the molecular marker described in claim 1 and the primer pair described in claim 2 in screening pig populations or new strains with higher vulva angle scores.

5. A method for detecting the SNP marker associated with the pig vulva angle score trait as claimed in claim 1, characterized in that A sequence containing the rs322368669 nucleotide site of pig chromosome 5 in the international pig genome version 11.1 reference sequence was amplified by PCR, and the amplified product was sequenced to determine the T / C polymorphism of the rs322368669 site.

6. The method according to claim 5, characterized in that The primer pair described in claim 2 is used to perform PCR amplification on the Topigs Large White pig genomic DNA.

7. The method of claim 5, characterized in that The following steps are involved: (1) Take tissue samples of Topigs Large White pigs and extract total DNA; (2) using the extracted porcine genomic DNA as a template and performing PCR amplification using the primer pair described in claim 2; (3) The amplified product was sequenced, the sequencing results were analyzed, and the T / C polymorphism at position 376 of SEQ ID NO: 1 was determined.

8. A method for selecting a pig population with a higher vulva angle score, characterized in that: It includes detecting the genotype of the rs322368669 nucleotide site on pig chromosome 5 in the international pig genome version 11.1 reference sequence, and selecting individuals with CC type at the rs322368669 nucleotide site as reserve breeding pigs.

9. The method according to claim 8, characterized in that The pig is a Topigs Large White pig.

10. The method according to claim 8, characterized in that 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.

Images