SNP (Single Nucleotide Polymorphism) marker primer pair related to porcine chromosome 7 and nipple number character and application of SNP marker primer pair

Abstract

The invention relates to an SNP (Single Nucleotide Polymorphism) marker primer pair related to a pig chromosome 7 and a nipple number character and application of the SNP marker primer pair. The SNP marker is located at an rs3470144376 nucleotide site of a pig chromosome 7, the site of the SNP marker is a molecular marker of the rs3470144376 nucleotide site of the pig chromosome 7 in a reference sequence of an international pig genome version 11.1 and has G / A polymorphism, and the SNP marker is remarkably related to the nipple number of a large white pig (Plt; 0.01) of the substrate. 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 nipple number of the large white pig, and the large white pig strain with multiple nipples is screened by identifying the genotype of the SNP marker. By establishing the strain, the breeding efficiency of the large white pigs can be improved, 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 the pig nipple number trait and an application thereof. Background Art

[0002] As one of the reproductive traits, the number of pig nipples is an important indicator to measure the lactation and feeding ability of sows. In recent years, the pig litter size trait has made relatively good breeding progress, but the genetic improvement of the nipple number trait has not been able to keep up. The number of effective nipples is less than the number of live piglets, which will cause great trouble to piglet feeding and affect the survival rate of piglets during lactation. Sows with more effective nipples can feed more piglets. Therefore, breeding to improve the number of nipples in sows is of great significance to improving the reproductive efficiency of sows, especially high-yielding sows. The nipple number trait is controlled by multiple genes and has a complex genetic mechanism. The genetic progress of breeding the nipple number trait based only on phenotype is slow. Combining molecular marker breeding can effectively accelerate the process of trait improvement.

[0003] Based on this background, this patent proposes a SNP marker primer pair related to the pig nipple number trait, which can quickly and accurately evaluate the pig nipple number trait through genotyping technology, providing an important molecular genetic basis for pig breeding and reproductive performance improvement. Summary of the invention

[0004] The purpose of the present invention is to provide a breeding molecular marker developed from a SNP marker related to the number of pig nipples, in view of the fact that traditional pig nipple breeding is time-consuming and labor-intensive, and the breeding effect is slow.

[0005] Another object of the present invention is to provide a primer pair and a detection method for detecting the above-mentioned SNP marker.

[0006] Another object of the present invention is to provide uses of the above-mentioned SNP markers, molecular markers, and primers.

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

[0008] A molecular marker associated with a pig nipple number trait, wherein the molecular marker sequence is shown in SEQ ID NO: 1, wherein there is a SNP marker site significantly associated with the pig nipple number trait at position 301, the site corresponds to the rs3470144376 nucleotide site of chromosome 7 of the international pig genome version 11.1 reference sequence, there is a G / A polymorphism, the total nipple number of GG type individuals is extremely significantly more than that of GA type and AA type individuals, and the total nipple number of GA type individuals is extremely significantly more than that of AA type individuals.

[0009] A primer pair for detecting a SNP marker associated with the pig nipple number trait, wherein the upstream primer is:

[0010] SEQ ID NO: 2, downstream primer: SEQ ID NO: 3

[0011] A method for detecting the SNP marker associated with the pig nipple number trait comprises PCR amplifying a sequence of the rs3470144376 nucleotide site of the pig chromosome 7 in the international pig genome version 11.1 reference sequence, sequencing the amplified product, and interpreting the G / A polymorphism of the site.

[0012] As a preferred embodiment of the present invention, the method comprises the following steps:

[0013] (1) Extract total DNA from pig tissue samples;

[0014] (2) Using the extracted porcine genomic DNA as a template, PCR was performed using the primers described above.

[0015] Amplification;

[0016] (3) The amplified product was sequenced and the sequencing results were analyzed. The 301st

[0017] G / A polymorphism at the position.

[0018] The application of SNP markers related to the number of pig nipples in screening multi-nippled pig populations, the site of the SNP marker is a molecular marker at the rs3470144376 nucleotide site of chromosome 7 of the international pig genome version 11.1 reference sequence, and has a G / A polymorphism. The SNP marker is extremely significantly correlated with the number of pig nipples (P<0.01).

[0019] The invention discloses an application of the molecular markers in screening a multi-papillary pig population.

[0020] The primer pair of the present invention is used in screening a multi-papillary pig population.

[0021] A method for screening a multi-papillary pig population comprises detecting the genotype of the rs3470144376 nucleotide site of chromosome 7 of the international porcine genome version 11.1 reference sequence, and selecting GA type and GG type individuals at the rs3470144376 nucleotide site as reserve breeding pigs.

[0022] Beneficial effects:

[0023] The present invention develops a SNP marker significantly associated with the number of pig nipples, and provides a primer pair and a method for detecting the marker. A pig population with multiple nipples is screened by identifying the genotype of the SNP marker. The establishment of the population can improve the comprehensive reproductive efficiency of pigs and generate more social and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the gel image of PCR amplification of the rs3470144376 site on chromosome 7 of Large White pig.

[0025] Figure 2 This is an example of the genotyping map of the rs3470144376 locus on chromosome 7 of Large White pig.

[0026] A is AA type, B is GA type, and C is GG type. DETAILED DESCRIPTION

[0027] 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.

[0028] Example 1

[0029] 1. Source of experimental animals

[0030] Danish Large White Pig from Guizhou Zhiyuan Pig Breeding Co., Ltd.

[0031] 2 Extraction of pig genomic DNA

[0032] One sample of ear tissue from 1047 pigs was collected for individual DNA extraction;

[0033] Refer to the instructions of the tissue DNA extraction kit of Tiangen Biotechnology Company. The extraction steps are as follows:

[0034] ① First, add 68 mL and 200 mL of anhydrous ethanol to buffer GD and rinse solution PW respectively, and mix thoroughly.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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

[0041] 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.

[0042] ⑨Repeat step ⑧.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 3. PCR amplification and sequencing of target fragments

[0047] PCR amplification was performed using porcine 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:

[0048]

[0049] The amplified product was subjected to agarose gel electrophoresis, and the product fragment size was about 325 bp. The electrophoresis results were as follows Figure 1 The remaining amplified products were sequenced, and the sequencing results were compared with DNAman software to verify the accuracy of the sequence, and Chromas software was used to type the rs3470144376 site.

[0050] 4 Statistical analysis

[0051] The general linear model of SAS 9.0 software was used for the association analysis between genotype and phenotype. The model was as follows:

[0052] Y i j kl =μ i +S j +YS k +G l +e j kl

[0053] Among them, Y ijkl is the number of papillae of an individual; μ i represents the mean number of nipples in the group; S j represents the fixed effect of gender; YS k represents the fixed effects of birth year and season; G l is the fixed effect of SNP marker; e jkl is the residual.

[0054] 5 Results

[0055] Table 1 shows the effects of different genotypes of rs3470144376 on the number of nipples in pigs. The results showed that there were extremely significant differences in the number of nipples among the three genotypes of rs3470144376 (P<0.01). The total number of nipples in GG-type individuals was significantly higher than that in GA-type and AA-type individuals (P<0.01). The total number of nipples in GA-type individuals was significantly higher than that in AA-type individuals (P<0.01). Therefore, the selection of GG and GA types at rs3470144376 in pigs is beneficial to increase the number of nipples in the group, thereby improving its reproductive efficiency.

[0056] Table 1 Association analysis between rs3470144376 locus on chromosome 7 and nipple number in pigs

[0057]

[0058] Note: Numbers with different letters in the same column indicate significant differences (P<0.01).

Claims

1. A molecular marker associated with the number of pig nipples, characterized in that: The molecular marker sequence is shown in SEQ ID NO: 1, wherein there is a SNP marker site at position 301 that is significantly correlated with the pig nipple number trait, and the site corresponds to the nucleotide site rs3470144376 of chromosome 7 of the international pig genome version 11.1 reference sequence, and there is a G / A polymorphism. The total number of nipples of GG type individuals is extremely significantly more than that of GA type and AA type individuals, and the total number of nipples of GA type individuals is extremely significantly more than that of AA type individuals.

2. A primer pair for detecting the SNP marker significantly associated with the pig nipple number trait as described in claim 1, characterized in that The upstream primer is: SEQ ID NO: 2, and the downstream primer is: SEQ ID NO:

3.

3. A method for detecting the SNP marker associated with the pig nipple number trait as claimed in claim 1, characterized in that It comprises a PCR amplification of a section of the nucleotide site rs3470144376 of the pig chromosome 7 of the international pig genome version 11.1 reference sequence, sequencing the amplified product, and interpreting the G / A polymorphism of the site.

4. The use according to claim 3, characterized in that The pig is a large white pig from Denmark.

5. The method according to claim 3, characterized in that The primer pair described in claim 2 is used to perform PCR amplification on pig genomic DNA.

6. The method according to claim 5, 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 2; (3) The amplified product was sequenced, the sequencing results were analyzed, and the G / A polymorphism at position 301 of SEQ ID NO: 1 was interpreted.

7. Use of the molecular marker described in claim 1 in screening a multi-papillary pig population.

8. Use of a reagent for detecting the SNP marker significantly associated with the pig nipple number trait as claimed in claim 1 in screening a multi-nipple pig population.

9. Use of the primer pair described in claim 2 in screening a multi-papillary pig population.

10. A method for screening a multi-nipple pig population, characterized in that The method comprises detecting the genotype of the rs3470144376 nucleotide site of chromosome 7 of the international pig genome version 11.1 reference sequence described in claim 1, and selecting GG type and GA type individuals at the rs3470144376 nucleotide site as reserve breeding pigs.

Images