Primer pair of snp marker related to piglet number of pig chromosome 1 and application thereof

Abstract

The present application relates to a SNP marker primer pair related to pig teat number trait and application thereof. The SNP marker is a molecular marker containing rs325188308 nucleotide site on pig chromosome 1 of international pig genome 11.1 version reference sequence, and has C / T polymorphism, and the SNP marker is significantly related to pig teat number trait. 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 is significantly related to pig teat number trait, and the multiple teat pig individual can be screened by identifying the SNP marker, and the obtained multiple teat pig population is beneficial to improve the survival rate of nursing piglets of high-yield sows, and has important economic benefits and social value.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology breeding technology, and relates to an SNP marker primer pair related to the trait of pig nipple number and its application. Background Technology

[0002] The number of teats, as an important indicator of a sow's lactation capacity, is one of the most important reproductive traits in pig production. Sows secrete milk through their teats to nurse piglets, providing nutrients for their growth and development, and simultaneously transmitting antibodies to protect them from infection and ensure their healthy growth. Sows with a higher number of effective teats can nurse more piglets and provide more weaned piglets; conversely, a lower number of effective teats than the number of piglets born severely impacts piglet survival rates. Furthermore, there is a correlation between the number of teats and the number of piglets born; the effective combination of a high number of teats and a high number of piglets born is a crucial foundation for achieving high sow productivity. Therefore, selecting a sufficient number of teats to ensure that sows can nurse all their piglets is increasingly valued in pig production. In addition, the number of teats in pigs is strongly genetically correlated with growth and developmental traits such as body length and birth weight, making selective breeding to improve teat count economically valuable. As a quantitative trait, the number of teats is difficult to significantly improve genetically in the short term using traditional breeding techniques, while molecular breeding techniques can improve breeding efficiency. Compared with traditional breeding, molecular selection using trait-related markers can effectively accelerate the breeding process. Studying the genetic molecular mechanisms of pig nipple number and identifying relevant molecular markers affecting nipple number for application in breeding has significant economic benefits for selecting multi-nipple populations or new breeds. Furthermore, if this invention can be widely applied, it can greatly improve the annual productivity of sows in my country, especially the number of weaned piglets (PSY) and the number of market-ready pigs and meat yield (MSY) per sow per year. This means that the number of breeding sows can be reduced, achieving the goal of stable production and supply. Therefore, the social benefits of this invention are considerable. Summary of the Invention

[0003] The purpose of this invention is to address the problems of time-consuming and labor-intensive traditional breeding of pig nipple count, and slow breeding progress, by providing SNP markers related to the trait of pig nipple count and developing them into breeding molecular markers.

[0004] Another object of the present invention is to provide primer pairs and detection methods for detecting the above-mentioned SNP markers.

[0005] Another object of the present invention is to provide the use of the above-mentioned SNP markers, molecular markers, and primers.

[0006] The objective of this invention can be achieved through the following technical solutions:

[0007] Molecular markers associated with the number of pig nipples on chromosome 1 were identified. A C / T polymorphism was found at nucleotide site rs325188308 on chromosome 1 of the international pig genome version 11.1 reference sequence. Individuals with the TT genotype at this site had significantly more nipples than those with the CC genotype. Primers were designed based on the nucleotide sequence containing nucleotide site rs325188308 on chromosome 1 of the international pig genome version 11.1 reference sequence. PCR amplification was performed using pig genomic DNA as a template to convert nucleotide site rs325188308 on chromosome 1 of the international pig genome version 11.1 reference sequence into a molecular marker.

[0008] As a preferred embodiment of the present invention, the molecular marker is as follows:

[0009] ctgacagctc tagatgctag tatacaaggt atctcatgta aattaaactg cttgttggca60atggtacact gaagtagata cattcttcta aaaatctatc ctcccagaat aagctaaaag120aaagcacaag atggtttggg cggtattgct gtaacagtga gaaagtaaaa actacaaaaa180tgtccaacaa tgaaaaacaa yaggctgtca tatagctatt aaaagtttta aataaattaa240tgttgagtta gaaaagtttg taacatgagg aaaaagttta aaattaaata aaaagcataa300aacataatgg tatactgtat gatcgcaata agcaaaaaca aggatttgaa gaaagtctag360aagaaaagat aaaatgctaa tgaaagttgt ttggaggcaa g 401 where the y in the 201st bp represents c or t.

[0010] A primer pair for detecting SNP markers on pig chromosome 1 that are associated with the number of pig nipples, with the upstream primer being SEQ ID NO: 2 and the downstream primer being SEQ ID NO: 3.

[0011] The application of the primer pair in detecting the number of pig nipples and in pig breeding.

[0012] A method for detecting SNP markers on pig chromosome 1 associated with the number of pig nipples includes PCR amplification of a sequence at nucleotide site rs325188308 on pig chromosome 1 in the International Pig Genome 11.1 reference sequence, sequencing the amplified product, and interpreting the C / T polymorphism at that site.

[0013] As a further preferred embodiment of the present invention, the method includes the following steps:

[0014] (1) Collect pig tissue samples to extract total DNA;

[0015] (2) Using the extracted porcine genomic DNA as a template, perform PCR amplification using the primer pair described in claim 5;

[0016] (3) Sequencing of the amplified product, analysis of the sequencing results, and interpretation of the C / T polymorphism at position 201 of SEQ ID NO: 1.

[0017] The application of the molecular markers described in this invention in screening multi-pawed pig populations or new strains.

[0018] The application of the primer pairs described in this invention in screening multi-nipple pig populations or new strains.

[0019] A method for screening multi-nipple pig populations includes detecting the genotype at the rs325188308 nucleotide site on pig chromosome 1 in the International Pig Genome Version 11.1 reference sequence. Individuals with the TT genotype at this site have significantly more nipples than individuals with the CC genotype. Individuals with the TT genotype at the rs325188308 nucleotide site are selected as priority breeding stock.

[0020] As a preferred embodiment of the present invention, the method for detecting the genotype at the rs325188308 nucleotide site on pig chromosome 1 in the International Pig Genome Version 11.1 reference sequence is selected from PCR or gene sequencing.

[0021] Beneficial effects

[0022] The present invention provides a SNP marker on chromosome 1 of pigs that is associated with the number of nipples. Molecular markers and primers developed based on this SNP can be used for SNP detection. Therefore, multi-nippled pig individuals can be screened by identifying this SNP marker, and the resulting multi-nippled pig populations or strains have significant economic and social value. Attached Figure Description

[0023] Figure 1 A gel image of the rs325188308 locus on pig chromosome 1.

[0024] Figure 2 Example of a genotyping diagram for the rs325188308 locus on pig chromosome 1.

[0025] Among them, A: TT type, B: CC type, C: CT type. Detailed Implementation

[0026] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0027] Example 1:

[0028] 1. Source of experimental animals

[0029] Meishan Pig Breeding Co., Ltd., Kunshan City, Jiangsu Province

[0030] 2. Extraction of Meishan pig genomic DNA

[0031] One tissue sample was collected from each of 878 Meishan pigs for individual DNA extraction.

[0032] Referring to the instructions for the Tissue DNA Extraction Kit from Tiangen Biotech Co., Ltd., the extraction was performed in the following order:

[0033] ① First, add 68 mL of buffer GD and 200 mL of anhydrous ethanol to the wash buffer PW, and mix thoroughly.

[0034] ② Collect approximately 100 mg of tissue sample and place it in a 2 mL EP tube. After completely cutting it into small pieces, add 200 μL of buffer GA and shake until completely suspended.

[0035] ③ Add 20 μL of proteinase K solution, mix well, and place in a 56°C metal bath for overnight digestion until the ear-like tissue dissolves. Briefly centrifuge to remove water droplets from the inner wall of the tube cap.

[0036] ④ Add 200 μL of buffer GB, mix thoroughly by inverting, place in a 70℃ metal bath for 10 min, the solution should become clear, and briefly centrifuge to remove water droplets from the inner wall of the tube cap.

[0037] ⑤ Add 200 μL of anhydrous ethanol and shake thoroughly for 15 seconds. At this time, flocculent precipitate may appear. Briefly centrifuge to remove water droplets from the inner wall of the tube cap.

[0038] ⑥ Add the solution and flocculent precipitate obtained in the previous step to an adsorption column CB3, place the adsorption column in the collection tube, then centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, and put the adsorption column CB3 back into the collection tube.

[0039] 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 into the collection tube.

[0040] ⑧ Add 600 μL of washing buffer 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.

[0041] ⑨ Repeat step ⑧.

[0042] ⑩ Place the adsorption column CB3 back into the collection tube, centrifuge at 12,000 rpm for 2 minutes, and discard the waste liquid. Place the adsorption column CB3 at room temperature for several minutes to thoroughly dry any residual washing liquid in the adsorption material.

[0043] Transfer the adsorption column CB3 into a clean centrifuge tube. Add 100 μL of elution buffer TE to the middle of the adsorption membrane. Incubate at room temperature for 2-5 min, then centrifuge at 12,000 rpm for 2 min. Collect the solution in the centrifuge tube. Add the centrifuged solution back to the adsorption column CB3. Incubate at room temperature for 2 min, then centrifuge at 12,000 rpm for 2 min. Collect the solution in the centrifuge tube.

[0044] After measuring the mass and concentration using a Nanodrop-100 spectrophotometer, the concentration was diluted to 50 ng / μL and stored at -20℃ for later use.

[0045] 3. PCR amplification and sequencing of the target fragment

[0046] Using the extracted DNA as a template, PCR amplification was performed according to the designed primers: 1 μL of DNA template, 0.4 μL each of the primers shown in CCTTGACCAAGCCGG AGTAA (SEQ ID NO: 2) and GAATT GCCTG TGGCC CTTAG (SEQ ID NO: 3), 10 μL of PCR Mix reagent, and 7.2 μL of double-distilled water were used to set up the PCR amplification system.

[0047]

[0048] The PCR products were detected by electrophoresis on a 1.2% agarose gel. The amplified target fragment was approximately 600 bp in size. See the electrophoresis image below. Figure 1 The remaining amplification products were sequenced, and the sequencing results were compared and analyzed with relevant pig gene fragment sequences in GenBank using DNAman software to determine the genotype at the rs325188308 locus. Statistical analysis was then performed.

[0049] The effect of genotype on phenotype was analyzed using a general linear model in SAS software. The analysis model was as follows:

[0050] Y ijnk =u i +Gj +S n +D k +e jnk

[0051] Where: Y ijnk The total number of teats in pigs; G j S represents the genotype fixation effect of the j-th SNP; n The fixed effect of gender; D k The fixed effect representing the birth season; e jnk This is the residual.

[0052] 5 Results

[0053] Table 1 shows the effect of the C / T variant at the rs325188308 locus on the total teat number trait in Meishan pigs. As shown in Table 1, there were highly significant differences in the total teat number trait among the three genotypes at the rs325188308 locus (P<0.01). Individuals with the dominant TT genotype had an average of 0.5 more total teats than individuals with the inferior CC genotype. Therefore, in the Meishan pig population, successive generations of breeding for individuals with the TT genotype at the rs325188308 locus can gradually improve the total teat number trait in the Meishan pig population.

[0054] Table 1. Association analysis between the rs325188308 locus on chromosome 1 of pigs and the total number of teats in Meishan pigs.

[0055]

[0056] Note: Different letters in the same row's number subscript indicate significant differences (P<0.05).

[0057] Example 2:

[0058] 1. Source of experimental animals

[0059] A breeding company in Taizhou City, Jiangsu Province

[0060] 2. Extraction of genomic DNA from Sujiang pigs

[0061] Genomic DNA was extracted from 365 Sujiang pigs using the method described in Example 1.

[0062] 3. Target fragment PCR amplification and sequencing

[0063] The target fragment was amplified, sequenced, and identified according to the method in Example 1, and the genotype of the 201st site of SEQ ID NO: 1 (i.e., the rs325188308 variant site) was determined.

[0064] 4. Statistical Analysis

[0065] The effect of genotype on phenotype was analyzed using a general linear model in SAS software. The analysis model was as follows:

[0066] Y ijnk =u i +G j +S n +D k +e jnk

[0067] Where: Y ijnk The total number of teats in pigs; G j S represents the genotype fixation effect of the j-th SNP; n The fixed effect of gender; D k The fixed effect representing the birth season; e jnk This is the residual.

[0068] 5 Results

[0069] Table 2 shows the effect of the C / T variant at the rs325188308 locus on the total teat number trait in the Sujiang pig population. As shown in Table 2, there were significant differences in the total teat number trait among the three genotypes at the rs325188308 locus (P<0.05). Individuals with the dominant TT genotype had an average of 0.3 more total teats than individuals with the inferior CC genotype. Therefore, in the Sujiang pig population, successive generations of breeding for individuals with the TT genotype at the rs325188308 locus can gradually improve the total teat number trait in the Sujiang pig population.

[0070] Table 2. Association analysis between the rs325188308 locus on chromosome 1 of pigs and the total nipple trait in Sujiang pigs.

[0071]

[0072] Note: Different letters in the same row of numbers indicate significant differences (P<0.05).

Claims

1. Application of primer pairs for detecting SNP marker related to pig teat number trait in screening Meishan pig or Sujiang pig population with high teat number, the SNP marker site is the nucleotide site rs325188308 on pig chromosome 1 of international pig genome 11.1 version reference sequence, which has C / T polymorphism, and the teat number of TT type individual is significantly more than that of CC type individual, the primer pair sequence is upstream primer: SEQ ID NO: 2, downstream primer: SEQ ID NO:

3.

2. A method of selecting a multi-toothed pig individual, establishing a multi-toothed pig population or line, characterized in that It comprises detecting the genotype of pig rs325188308 nucleotide site on pig chromosome 1 of international pig genome 11.1 version reference sequence, which has C / T polymorphism, and the teat number of TT type individual is significantly more than that of CC type individual, and breeding TT type individual of rs325188308 nucleotide site as breeding pig, and the pig is Meishan pig or Sujiang pig.

3. The method according to claim 2, wherein the method for detecting the genotype of rs325188308 nucleotide site on pig chromosome 1 of international pig genome 11.1 version reference sequence is PCR or gene sequencing.

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