KR20200004108A - Composition for prediction of swine fecundity using methylation status of ZPBP gene and method for predictioin of swine fecundity using the same - Google Patents

Abstract

The present invention relates to a composition for predicting the litter size of swine using the methylation of ZPBP gene and to a prediction method using the same. More specifically, the present invention is capable of early selecting prolific swine and predicting the litter size of swine by using the methylation of the ZPBP gene.

Description

Composition for prediction of swine fecundity using methylation status of ZPBP gene and method for predictioin of swine fecundity using the same}

The present invention relates to a composition for predicting the litter size of pigs using methylation of ZPBP gene and a prediction method using the same. In more detail, the present invention can select the fertility pigs early and accurately predict the number of litter, DNA methylation marker of the litter associated ZPBP gene, composition for predicting the litter size of the pig using the same and method for predicting litter size using the same It is about.

Maintaining optimum sow productivity and conception efficiency is a very important factor in the economics of the hog industry. However, the number of litters in pigs is influenced by several factors, such as ovulation rate, fertility, embryo viability and fetal viability due to complex physiological characteristics. Due to this complex factor, heredity to live births in pigs is low, around 10%.

Therefore, epigenetics approaches are needed to study the effects of environmental factors and genetic factors on pig live numbers. In particular, DNA methylation is a chemically very stable epigenetic modification that is inherited over generations, which is useful for the study.

Although methylation of DNA is known to occur in certain mammalian developmental processes such as early embryonic or germline development, DNA methylation patterns on somatic cells remain stable.

 Abnormal DNA methylation is also known to be linked to cancer and other diseases. This DNA methylation can be used as a major marker for the selection of genetically fertile sows, and can increase litter count by selecting superior individuals in a short period of time compared to long-term and expensive breeding techniques through conventional breeding. There is a need for technology.

Over the years, hormonal treatments have developed a variety of techniques to increase ovulation rate and number of embryos. However, as the number of embryos increased, the survival and live weight of piglets appeared to decrease. This is because the number of piglets that a pig's uterus can sustain during pregnancy is limited, and in order to increase the number of litters, it is important to select individuals having excellent capacity of the uterus. For example, the native Chinese species, the Meishan species, are already well known for their high uterine production capacity.

As a background art of the present invention, Republic of Korea Patent No. 10-185666 (2018.05.01) describes a SNP marker for predicting the total number of pigs and a prediction method using the same.

Disclosure of the Invention An object of the present invention is to provide epigeneome markers of ZPBP genes related to litter number and to predict the litter size of pigs using the same, which enables early selection of fertility pigs and accurately predicts litter size. will be.

Another object of the present invention to provide a kit for predicting the number of live pigs comprising the composition.

Still another object of the present invention is to provide a method for predicting live number of pigs capable of early selecting the fertility pigs using the predictive composition.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to one aspect, there is provided a composition for predicting the litter size of a pig, including an agent for measuring the expression level and the methylation level of the ZPBP gene.

According to one embodiment, the composition may further comprise an agent capable of amplifying the methylation site.

According to one embodiment, the agent may comprise a primer pair represented by the nucleotide sequence of SEQ ID NO: 1 and 2.

According to one embodiment, the composition can measure the degree of methylation of the CpG site.

According to one embodiment, the composition may comprise HpaII and MspI, which are methylation specific restriction enzyme pairs.

According to one embodiment, the pig may be a black pig.

According to one embodiment, the black pig may be Berkshire.

According to another aspect, there is provided a kit for predicting the litter size of a pig comprising the composition.

According to one embodiment, the kit may be an RT-PCR kit, a microarray chip kit or a protein chip kit.

According to another aspect, extracting DNA from the subject; Measuring the expression level and methylation level of ZPBP gene from the DNA; And predicting pigs having a lower number of methylation than pigs having an average methylation level as pigs having a higher number of pigs than pigs that are not.

According to one embodiment, the subject may be blood, hair root or endometrial tissue.

According to one embodiment, the step of measuring the expression level and methylation degree of the ZPBP gene may further comprise amplifying the gene with a primer pair represented by the nucleotide sequence of SEQ ID NO: 1 and 2.

According to one embodiment, the step of measuring the degree of methylation is methylation specific PCR (realization methylation specific PCR), real time methylation specific PCR (pyro sequencing, PCR using methylated DNA specific binding protein or Quantitative PCR and DNA chips, methylation sensitivity restriction endonucleases, bisulfite sequencing or PMP assay (Porcine methylation-specific restriction enzyme PCR assay) can be used.

According to one embodiment, by using the predictive composition of the present application, it is possible to easily obtain a subject through the blood or hair root of the pig, to quickly and accurately predict the number of living pigs.

According to one embodiment, using the composition and prediction method for the prediction of the number of pigs of the present application, it is possible to early select, breed, and improve the system of the excellent pig breeding, through this, pig industry Can greatly increase the competitiveness and added value of

1 is a view showing a workflow for selecting a DMR differs depending on the number of live pigs.
FIG. 2 is a graph showing the distribution of methylation levels in the whole CpG region and the CHG region in percentage (a) and inferior group (b).
FIG. 3 is a graph showing the distribution of methylation levels of CpG regions in the group (a) and the inferior group (b) of pigs at the genome level.
4 is a diagram showing the results of classifying genes in regions showing differences in methylation according to function.
5 is a diagram showing the methylation level of ZPBP gene by methylation-specific polymerase chain reaction (PMP assay) from blood samples of the good and inferior group of pigs. U refers to DNA that has not been treated with restriction enzymes (Uncutted DNA) as a control.
FIG. 6 is a graph showing the results of correlation between methylation markers of ZPBP gene and total number of litters (TNB) and number of litters (NBA) through pig linear regression analysis.

As the invention allows for various changes and numerous embodiments, particular embodiments will be illustrated in the drawings and described in detail in the written description.

However, this is not intended to limit the present invention to specific embodiments, it should be understood to include all transformations, equivalents, and substitutes included in the spirit and scope of the present invention.

The terminology used herein is for the purpose of describing particular example embodiments only and is not intended to be limiting of the present invention. In this application, the terms "comprise" or "have" are intended to indicate that there is a feature, number, step, operation, component, part, or combination thereof described in the specification, and one or more other features. It is to be understood that the present invention does not exclude the possibility of the presence or the addition of numbers, steps, operations, components, components, or a combination thereof.

In the following description of the present invention, if it is determined that the detailed description of the related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

As used herein, the term "methylation" refers to a methyl group is requested to the base constituting the DNA. In the present invention, whether or not methylation means whether methylation occurs in the cytosine of a specific CpG region of a specific gene. When methylation occurs, this disrupts the binding of transcription factors, thereby inhibiting the expression of certain genes, and conversely, when demethylation (without methylation) or hypomethylation occurs, the expression of certain genes increases.

As used herein, the term 'hypermethylation' means that the amount of 5-methylcytosine in one or more CpG dinucleotides in the DNA sequence of the DNA sample being tested is found in the corresponding CpG dinucleotide in the normal control DNA sample. By increased methylation status compared to the amount of 5-methylcytosine.

As used herein, the term 'hypomethylation' refers to the amount of 5-methylcytosine in one or more CpG dinucleotides in the DNA sequence of the DNA sample being tested, found in the corresponding CpG dinucleotide in the normal control DNA sample. Means a reduced methylation state compared to the amount of 5-methylcytosine.

As used herein, the term “probe” is an oligonucleotide that is capable of binding to a target nucleic acid in a base specific manner as a nucleic acid strand that is partially or completely complementary to the target nucleic acid. Although not limited thereto, oligonucleotides capable of binding to the target nucleic acid in a base specific manner may be suitable. The probe includes not only nucleic acids, but also any known nucleic acid derivative capable of complementary binding, including peptide nucleic acids.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings.

The present invention finds methylation markers of pig's litter-related genes by using genome wide bisulfite sequencing method in black pig species, and predicts the litter size of pigs. I was able to select.

In the present invention, the black pig species, the genomic DNA (gDNA) was isolated from the uterine tissues of pigs having excellent live numbers and inferior pigs, respectively. The isolated gDNA was genome wide bisulfite sequencing (Genome wide bisulfite sequencing) to establish the correlation between the number of pigs and the methylation of the ZPBP gene. The blood samples were then separated from the gDNA to analyze the methylation level through a PMP assay for the degree of methylation of the litter-associated genes and correlated with the two properties related to litter size (average total litter and average litter). By analyzing the relationship statistically, the present invention was completed by confirming that the degree of methylation of ZPBP, which is a related gene of piglet number, is related.

According to one aspect, a composition for predicting the litter size of a pig may be provided comprising an agent for measuring the expression level and methylation level of the ZPBP (NC — 010451.3) gene.

The zona pellucida binding protein (ZPBP) gene is a binding protein that binds to the egg's zona pellucida protein. It shows hypomethylation in pigs with excellent litter size and hypermethylation in pigs with poor litter size. ).

The 'measurement of the expression level of the gene' may be to measure the mRNA level or protein level. The 'measuring level of mRNA' is not limited thereto, but may be analyzed by RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay, Northern blotting, DNA microarray or any method known in the art. Can be. This may be achieved by hybridizing mRNA isolated from the biological sample or cDNA derived therefrom onto a microarray in which a probe specific for one or more genes selected from the gene is immobilized, and measuring the resulting degree of hybridization. The degree of hybridization can be measured by any measurement method known in the art, such as fluorescence measurement and electrical measurement. In this case, the probe or target nucleic acid may be labeled with a suitable detectable label. The cDNA may be directly amplified by RT-PCR using a sense and antisense primer pair as a primer targeting one or more marker genes selected from the group consisting of the genes.

The 'measurement of the level of protein' may be any protein measurement or detection method known in the art. For example, an assay using an antibody that specifically binds to a protein expressed from one or more marker genes selected from the group consisting of the above genes can be used. Protein analysis methods using antibodies include, but are not limited to, Western blotting, ELISA, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunity staining, immunoprecipitation assay, complement Fixed assays and FACS and the like. The ELISA may include a direct ELISA, an indirect ELISA, a direct sandwich ELISA, and an indirect sandwich ELISA.

Western blotting is to separate the whole protein, electrophoresis, the protein is separated according to the size, and then transferred to the nitrocellulose membrane to react with the antibody, the amount of the generated antigen-antibody complex using a labeled antibody How to check. In addition, as a method for measuring protein level, a method using an enzyme, a substrate, a coenzyme, a ligand, or the like that specifically binds to a target protein may be used.

In addition, the expression level of the gene may be determined by measuring the amount of amplification products obtained by nucleic acid amplification performed by reverse transcriptase polymerase chain reaction (RT-PCR), based on RNA isolated from the sample. .

In the present invention, the "agent for measuring the degree of methylation" is not particularly limited as long as the methylation level of the gene can be detected or amplified, and known means can be used. Although not limited thereto, the composition may be used to predict methylated markers by amplification and PMP assay. It may mean a primer capable of specifically amplifying a polynucleotide including the methylation marker and a methylation specific restriction enzyme used in a PMP assay method.

According to one embodiment of the invention, the composition may comprise HpaII and MspI, which are methylation specific restriction enzyme pairs.

In the present invention, the methylation level of the gene is amplification obtained by nucleic acid amplification performed by polymerase chain reaction (PCR) in which gDNA isolated from a subject is a DNA cleaved by methylation specific restriction enzyme as a template. It may be determined by measuring the amount of product.

The composition may further include a reagent required for hybridization with the marker gene or a nucleic acid expression product expressed therefrom in a sample. In addition, the composition may further include a buffer, a solvent, and the like, which stabilize the probe and serve as a reaction medium.

Binding (generally hybridization) of the probe to the target nucleic acid is sequence dependent and can be performed under a variety of conditions. In general, the hybridization reaction occurs at a temperature of about 5 ° C. below the Tm for a particular sequence at a particular ionic strength and pH. The Tm means a state in which 50% of the probes complementary to the target sequence bind to the target sequence. Examples of hybridization conditions may be 0.01 to 1.0 M Na ⁺ ion concentration at pH 7.0 to 8.3. In addition, in order to increase the specificity of the target nucleic acid and the probe may be carried out in the presence of conditions that destabilize the binding of the target nucleic acid and the probe, for example, high temperature, high concentration of destabilizing agent (for example formamide). .

Measurement of the methylation degree of the present invention can utilize the following method.

1.Methylation specific PCR

When bisulfite is treated on genomic DNA, cytosine at the 5′-CpG-3 ′ site remains cytosine as it is methylated and becomes uracil when unmethylated. Therefore, PCR primers corresponding to methylation of the 5'-CpG-3 'nucleotide sequence after the bisulfite treatment and two primers corresponding to the non-methylation When the genomic DNA is converted to bisulfite, and PCR is performed using the two kinds of primers, the PCR product is made by using the primer corresponding to the methylated sequence when methylated. In the case of using a primer corresponding to the unmethylated PCR product is made, so can be qualitatively confirmed by agarose gel electrophoresis method.

2. Real time methylation specific PCR

Real-time methylation-specific PCR converts the methylation-specific PCR method into a real-time measurement method. After treating bisulfite on genomic DNA, a PCR primer corresponding to methylation is designed and real-time PCR is performed using these primers. It is. At this time, the detection method using the TanMan probe complementary to the amplified base sequence and the detection using LCgreen. Real-time methylation specific PCR can selectively quantitate only methylated DNA.

3. Pyro Sequencing

The pyro sequencing method is a method of converting the bisulfite sequencing method into quantitative real-time sequencing. As with bisulfite sequencing, genomic DNA is converted by bisulfite treatment, and then PCR primers corresponding to sites without the 5'-CpG-3 'sequencing are prepared and the genomic DNA is treated with bisulfite. After amplification with the PCR primers, real-time sequencing using the sequencing primers can quantitatively analyze the amount of cytosine and thymine at the 5'-CpG-3 'site to indicate the degree of methylation as an index.

4. PCR or Quantitative PCR and DNA Chips Using Methylated DNA Specific Binding Proteins

When a protein that specifically binds to methylated DNA is mixed with DNA, only the methylated DNA can be selectively separated because the protein specifically binds to methylated DNA. After genomic DNA is mixed with methylated DNA-specific binding protein, only methylated DNA can be selectively isolated and amplified using PCR primers, and then methylated by agarose electrophoresis.

In addition, methylation can also be determined by quantitative PCR, and methylated DNA isolated from methylated DNA-specific binding proteins can be labeled with a fluorescent dye and hybridized to a DNA chip in which a complementary probe is integrated to determine methylation. Can be.

5. Methylation Sensitivity Restriction Endonucleases

The determination of differential methylation can be performed by contacting the nucleic acid sample with a methylation sensitive restriction endonuclease that cleaves only unmethylated CpG sites, thereby cleaving the non-methylated nucleic acid.

The "methylation sensitivity restriction endonuclease" is a restriction enzyme that contains CG at the recognition site and has activity when C is methylated compared to when C is not methylated. For example, SmaI may be suitable. Non-limiting examples of such methylation sensitive restriction endonucleases may be used alone or in combination with MspI, HpaII, BssHII, BstUI and NotI enzymes. Other methylation sensitivity limiting endonucleotides include, but are not limited to, SacII and EagI.

Isochizomers of methylation sensitive restriction endonucleases are restriction endonucleases that have the same recognition site as methylation sensitive restriction endonucleases and cleave both methylated and non-methylated CGs. For example, there is MspI.

6. Bisulfite Sequencing

Other methods of detecting nucleic acids containing methylated CpG include contacting a sample containing nucleic acid with an agent that modifies non-methylated cytosine and amplifying the CpG-containing nucleic acid of the sample using CpG-specific oligonucleotide primers. It includes. Here, the oligonucleotide primer may be characterized by detecting the methylated nucleic acid by distinguishing the modified methylated and non-methylated nucleic acid. Although not limited thereto, the amplifying step may be further included. The method relies on a PCR reaction that distinguishes between modified (eg, chemically modified) methylated and non-methylated DNA. U.S. Patent 5,786,146 describes bisulfite sequencing for the detection of methylated nucleic acids.

According to one embodiment, the composition may further comprise an agent that can amplify the degree of methylation. The agent may comprise a pair of primers. The primer may be a pair of primers that can be specifically amplified cytosine that is methylated and not modified by bisulfate and a pair of primers that can specifically amplify cytosine that is not methylated and modified by bisulfite. The primer is not limited thereto, but may be a primer pair represented by the nucleotide sequences of SEQ ID NOs: 1 and 2 (see Table 6).

According to one embodiment, the composition can measure the degree of methylation of the CpG site. The CpG site of the gene means a CpG site present on the DNA of the gene. The DNA of the gene is a concept that includes all of a series of structural units required for the gene to express and are operably linked to each other, but is not limited thereto, and includes a promoter region and an open reading frame (ORF). And a terminator region. In the present invention, the degree of methylation occurring at a specific CpG position of the ZPBP gene was measured, and it was confirmed that when the degree of methylation is above the average degree of methylation, it can be predicted as a fertility pig. To modify the unmethylated cytosine base of the gene, bisulfite or a salt thereof may be used, but is not limited thereto. The bisulfite or salt thereof can be used to detect unmethylated cytosine residues to detect methylation of the CpG site.

According to one embodiment, the pig may be a black pig. Although not limited to this, the black pork may be more suitable Berkshire.

According to another aspect, there may be provided a kit for predicting the litter size of a pig comprising the composition. The kit may further comprise a reagent required for methylation detection. The composition and kit may further include a polymerase, a buffer solution required for agarose electrophoresis, etc. in addition to the formulation.

According to one embodiment, the kit is not limited thereto, but may be an RT-PCR kit, a microarray chip kit or a protein chip kit, but may be a kit well known in the art.

According to another aspect, extracting DNA from the subject; Measuring the expression level and methylation level of ZPBP gene from the DNA; And predicting swine having a lower degree of methylation than the average methylation level as pigs having a higher number of litters than pigs that are not.

According to one embodiment, the subject may be blood, hair root or endometrial tissue.

According to one embodiment, the step of measuring the expression level and methylation degree of the ZPBP gene may further comprise amplifying the gene with a primer pair represented by the nucleotide sequence of SEQ ID NO: 1 and 2.

According to one embodiment, the step of measuring the degree of methylation is methylation specific PCR (realization methylation specific PCR), real time methylation specific PCR (pyro sequencing, PCR using methylated DNA specific binding protein or Quantitative PCR and DNA chips, methylation sensitivity restriction endonucleases, bisulfite sequencing or PMP assay (Porcine methylation-specific restriction enzyme PCR assay) assays can be used.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these Examples are only for illustrating the present invention, and the scope of the present invention will not be construed as being limited by these Examples.

Example

I. DNA Methylation Profiling

1. Endometrial Tissue Extraction and Genomic DNA Extraction

For DNA methylation profiling, Berkshire sows were selected as three heads, each divided into two groups, which had an excellent litter size (more than 11 heads) and an inferior group (less than 7 heads, as shown in Table 1). Endometrial tissue was excised from the uterine angle immediately after slaughter and washed with phosphate buffer (PBS) and frozen in liquid nitrogen.

 Genomic DNA (gDNA) was isolated from endometrial tissue collected using a DNeasy Tissue Kit (Qiagen, Valencia, CA, USA), and NanoDrop ™ ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) Through quantitative and qualitative analysis of genomic DNA was prepared.

2. Genome wide bisulfite sequencing (GWBS) analysis

Sodium bisulfite was treated with 6 μg of genomic DNA samples using the EZ DNA Methylation-Gold ™ Kit (Zymo Research, Orange, Calif., USA). Converted DNA products by Bisulfite were quantified using Quant-iT ™ dsDNA High Sensitivity Assay Kit (Life Technologies, Rockville, MD, USA) and illuminated on the HiSeq 2500 platform (Illumina, San Diego, CA, USA) Analyzed.

3. DNA Methylation Profiling and DMR Screening Associated with Pig Live Number

As for the whole genome DNA methylation profiling, as a result of counting the sites where methylation occurred, as shown in FIG. 2, methylation was superior at the CG site in both the group having the highest number and the inferior number. Relatively CHG sites showed a very low methylation tendency. The sites where methylation occurred were identified as 1,249 million and 1,217 million, respectively, with an excellent number of inferior and inferior groups in raw reads, of which 53.08% and 50.48% were mapped to the pig genome, respectively.

As shown in FIG. 3, as a result of measuring the average CG methylation of each structure at the genome level, the methylation level was lowered around the transcription start site (TSS), and the average was 75 at the transcribed site (gene body) part. It was observed to appear at the% level.

In addition, a slightly lower methylation frequency was observed at the transcription termination site (TTS) and downstream 1 kb than the transcribed reaction (gene body). In conclusion, the methylation frequency distribution patterns in the transcribed gene bodies according to the litter number groups were generally similar.

According to the workflow shown in FIG. 1, differentially methylated regions (DMRs), which differ according to the number of live pigs, were analyzed. As a result, as shown in Table 2, 1,566 hypermethylated regions and 1,703 hypomethylated regions were identified.

In particular, the distribution of DMR at the CG site in the genome structure is 92 at the promoter (up1kb) and 1,313 at the transcription sequence (CDS), intron (see Table 3).

As shown in FIG. 4, gene ontology (GO) analysis was performed to analyze genes by function using DMR. The hypermethylated region is composed of genes corresponding to cytoskeleton organization, phosphorylation, and cell adhesion. The hypomethylated region is typically the gene corresponding to the plasma membrane part. And embryonic morphogenesis and negative reulation of gene expression.

In order to select methylation markers associated with pig litter number, genes corresponding to reproduction were selected according to methylation difference of 0.2 or more, P value <0.01 and FDR ( Q value) <0.01, As a result, ZPBP gene was selected (see Table 4).

II. Validation and Reproducibility of DNA Methylation Markers of ZPBP Gene

1. Blood collection and animal information collection

To verify the reproducibility of DNA methylation of selected genes, blood was drawn from 137 sows of Berkshire species, and the average of total number of born (Avg. Avg. Of NBA (Average of number of born alive) was identified (see Table 5).

2. Porcine methylation-specific restriction enzyme PCR assay (PMP assay)

Methylation specific polymerase chain reaction (PMP assay) is a simple way to directly assess the degree of methylation of a specific CpG site using methylation specific restriction enzymes.

For reproducibility verification, genomic DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA). 10 μg / μL of genomic DNA was cleaved using Hpa II (methylated specific restriction enzyme) and Msp I (methylated nonspecific restriction enzyme), which are methylation-sensitive isoschizomers, followed by ZPBP in Table 6. Amplification was performed using gene specific oligonucleotide primers. The amplified PCR product was electrophoresed to determine the intensity of the band and analyzed for the degree of methylation (see Table 6).

3. Statistical Analysis

The correlation between the methylation of the ZPBP gene and the number of live birth traits (total and live) in pigs was determined using the SPSS (IBM Statistics SPSS Statistics, Inc., Somers, NY, USA) program (version 20.0). Simple linear regression analysis was used.

4. Validation of ZPBP gene as methylation marker in blood samples

ZPBP genes showing methylation differences according to the number of litters in pig uterine tissues were verified in pig blood samples. This is an essential process for the early selection of pigs with excellent live numbers using blood samples. We confirmed that the methylation pattern of ZPBP gene in the genomic DNA of blood is consistent with the results in uterine tissue.

As shown in FIG. 5, the degree of methylation of the ZPBP gene by methylation specific polymerase chain reaction (PMP assay) in blood samples was the same as that obtained by bisulfite sequcing in uterine tissues. The hypomethylation was confirmed. These results show that methylation of the ZPBP gene may be useful as a marker for early selection of pigs with high litter count using blood.

5. Validation of DNA methylation marker of ZPBP gene by PMP assay

To verify DNA methylation markers of ZPBP genes, blood samples of 137 sows from Berkshire species were subjected to a PMP assay to determine the degree of methylation of ZPBP genes and the number of live births (TNB) and live counts (NBA). The correlation of was analyzed.

As shown in Table 7 below, the degree of methylation and average number of TNB of the ZPBP gene were determined by the unstandardized coefficients (y) = -6.6545 * ZPBP (x) + 11.096 and The same regression was derived and the regression coefficient for the degree of methylation of the ZPBP gene was negative and statistically significant.

Therefore, the degree of methylation of ZPBP gene affects the average total number of sows in black pig sows, and in particular, since the regression coefficient has a negative value, it is confirmed that there is a negative correlation with the increase in total number as the methylation of ZPBP gene decreases. It became. In this case, the t value is a value obtained by dividing the nonstandardized coefficient by the standard error.

In addition, as shown in Table 8, the methylation marker of the ZPBP gene and the average number of NBAs were determined by unstandardized coefficients (y) = -5.1436 * ZPBP (x) + A regression equation such as 9.5116 was derived, and the regression coefficient for the methylation marker of the ZPBP gene was negative and statistically significant.

Therefore, the methylation marker of the ZPBP gene affects the average number of live pups in black pig sows, and in particular, since the regression coefficient has a negative value, it was confirmed that the number of live digits increases with decreasing methylation of the ZPBP gene. .

Although the present invention has been described in detail through specific examples, it is intended to describe the present invention in detail, and the present invention is not limited thereto, and should be understood by those skilled in the art within the technical spirit of the present invention. It is clear that the deformation and improvement can be made. Simple modifications and variations of the present invention are all within the scope of the present invention, and the specific scope of protection of the present invention will be apparent from the appended claims.

Claims (13)

A composition for predicting the litter size of a pig, comprising an agent for measuring the expression level and the methylation level of the ZPBP gene. The method of claim 1,
The composition is a composition for predicting the litter size of pigs comprising an agent capable of amplifying a methylation site. The method of claim 2,
The formulation comprises a primer pair represented by the nucleotide sequence of SEQ ID NO: 1 and 2, composition for predicting the litter size of the pig. The method of claim 1,
The composition is a composition for predicting the litter size of pigs, measuring the degree of methylation of the CpG site. The method of claim 1,
Comprising a methylation specific restriction enzyme pair HpaII and MspI, pig composition predictive composition. The method of claim 1,
The pig is a black pig, composition for predicting the number of pigs. The method of claim 6,
The black pig is Berkshire, composition for predicting the number of pigs. A kit for predicting the litter size of a pig, comprising the composition for predicting litter size of the pig according to any one of claims 1 to 7. The method of claim 8,
The kit is RT-PCR kit, microarray chip kit or protein chip kit for predicting the number of pigs. Extracting DNA from a pig subject;
Measuring the expression level and methylation level of ZPBP gene from the DNA; And
Predicting pigs having a lower degree of methylation than the average methylation level as pigs having a higher number of litters than pigs that are not. The method of claim 10,
Wherein the subject is blood, hair root or endometrial tissue, piglet predicting method. The method of claim 10,
Measuring the expression level and the degree of methylation of the ZPBP gene further comprises amplifying the gene with a primer pair represented by the nucleotide sequence of SEQ ID NO: 1, 2, pig number prediction method. The method of claim 10,
Measuring the degree of methylation may include methylation specific PCR, real time methylation specific PCR, pyro sequencing, PCR or quantitative PCR using methylated DNA-specific binding proteins, DNA chips, A method for predicting litter count in swine using methylation sensitivity restriction endonucleases, bisulfite sequencing or PMP assay.

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