KR102114697B1 - 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 birth number of pigs using methylation of the ZPBP gene and a prediction method using the same. More specifically, the present invention can predict the early selection and number of births of a fertile pig using methylation of the ZPBP gene.

Description

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

The present invention relates to a composition for predicting the birth number of pigs using methylation of the ZPBP gene and a prediction method using the same. In more detail, the present invention is a DNA methylation marker of a ZPBP gene associated with an abundance that can select a fertile pig at an early stage and accurately predict the number of litters, a composition for predicting the number of pigs using the same, and a method of predicting the number of pigs using the same It is about.

Maintaining optimal productivity and sowing efficiency of sows is a very important factor in the economics of the pig industry. However, due to the complex physiological properties of pigs, the number of piglets is affected by several factors such as ovulation rate, fertility, embryo viability and fetal viability. Because of these complex factors, the heritability of pigs to livestock is around 10%, low.

Therefore, an epigenetics approach is needed to study the effects of environmental factors and genetic factors on swine production. In particular, DNA methylation (Mehtylation) is a very chemically stable epigenetic modification that is inherited over generations and is useful in this study.

It is known that methylation of DNA is generated in a specific developmental process in mammals, such as early embryonic development or germ cell development, but the DNA methylation pattern on somatic cells remains stable.

 In addition, it is known that abnormal DNA methylation is also associated with cancer and various diseases. This DNA methylation can be used as a major marker to select genetically fertile sows, and can increase the number of livestock by selecting superior individuals in a short period of time compared to long-term and expensive breeding techniques through conventional mating. There is a need for a skill.

Over the years, various techniques have been developed to increase ovulation rates and the number of embryos through hormone treatment. However, as the number of embryos increased, the survival rate and live weight of piglets decreased. 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 births, it is important to select individuals with excellent uterine capacity. For example, the Chinese native species, Meishan, is already well-known for its high uterine production capacity and large number of litters.

As a background technology for the present invention, Korean Patent Registration No. 10-185666 (2018.05.01) describes an SNP marker for predicting the total number of pigs in pigs and a prediction method using the same.

It is an object of the present invention to provide a composition for predicting the number of pigs using the epigenome marker of the ZPBP gene associated with the number of litters and pigs using the same, which can select fertile pigs early and accurately predict the number of litters. will be.

Another object of the present invention is to provide a kit for predicting litter size of pigs comprising the composition.

Another object of the present invention is to provide a method for predicting the number of pigs in a pig that can select a fertile pig early using the composition for prediction.

Other objects and advantages of the present invention will be further clarified by the following detailed description of the invention, claims and drawings.

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

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

According to one embodiment, the preparation may include 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 include a methylation specific restriction enzyme pair HpaII and MspI.

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 birth numbers of pigs 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 the DNA from the subject; Measuring the expression level and methylation level of the ZPBP gene from the DNA; And predicting a pig having a lower methylation level than an average methylation level as a pig having a higher number of digits than a pig having a lower level of methylation.

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 the degree of methylation of the ZPBP gene may further include amplifying the gene with a primer pair represented by the base sequences of SEQ ID NOs: 1 and 2.

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

According to one embodiment, using the composition for prediction herein, it is possible to quickly and accurately predict the number of pigs in the pig by easily obtaining a subject through the blood or hair root of the pig.

According to one embodiment, using the composition and prediction method for predicting the number of pigs in the present application, it is possible to early select and breed a multiplicity of individuals, and improve the excellent pig lineage, through which the pig industry Can greatly enhance its competitiveness and added value.

1 is a view showing a workflow for selecting a DMR that differs according to the number of live pigs.
FIG. 2 is a diagram showing the distribution of methylation levels in the whole CpG region and CHG region as a percentage in the group (a) and the inferior group (b) having excellent pig numbers.
Figure 3 is a diagram showing the distribution of methylation levels of the CpG region in the group (a) and inferior group (b) having a good number of pigs at the genome level.
4 is a diagram showing results of classifying genes in regions showing methylation differences according to function.
Figure 5 is a diagram showing the methylation level of the ZPBP gene by methylation specific polymerase chain reaction (PMP assay) from blood samples of groups with poor pig numbers and inferior groups. U refers to DNA (Uncutted DNA) without restriction enzymes as a control.
FIG. 6 is a diagram showing the results of analyzing the correlation between the methylation marker of the ZPBP gene and the total number of characters (TNB) and the number of actual numbers (NBA), which are traits of pigs, through simple linear regression analysis.

The present invention can be applied to various transformations and may have various embodiments, and specific embodiments will be illustrated in the drawings and described in detail in the detailed description.

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

The terms used in the present application are only used to describe specific embodiments, and are not intended to limit the present invention. In this application, terms such as “include” or “have” are intended to indicate that a feature, number, step, operation, component, part, or combination thereof described in the specification exists, one or more other features. It should be understood that the existence or addition possibilities of fields or numbers, steps, operations, components, parts or combinations thereof are not excluded in advance.

In the description of the present invention, if it is determined that a detailed description of known technologies related to the present invention may obscure the subject matter of the present invention, the detailed description will be omitted.

The term 'methylation' in the present specification means that a methyl group is requested to the base constituting the DNA. In the present invention, methylation means whether methylation occurs in the cytosine of a specific CpG subunit of a specific gene. When methylation occurs, the binding of transcription factors is interrupted thereby, and the expression of a specific gene is suppressed. Conversely, when unmethylation (without methylation) or hypomethylation occurs, expression of a specific gene increases.

The term 'hypermethylation' herein 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. It means an increased methylation state compared to the amount of 5-methylcytosine.

The term 'hypomethylation' as used herein 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. Refers to a reduced methylation state compared to the amount of 5-methylcytosine.

The term “probe” herein is a nucleic acid strand that is partially or completely complementary to a target nucleic acid, and is an oligonucleotide capable of binding the target nucleic acid in a base-specific manner. Although not limited thereto, an oligonucleotide capable of binding 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 derivatives capable of complementary binding, including peptide nucleic acids.

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

The present invention uses a genome wide bisulfite sequencing method in a black pig species to discover methylation markers of a pig's litter number-related gene to predict the pig litter number, thereby predicting the number of pig litters early. It was made possible to select.

In the present invention, gDNA (genomic DNA) was isolated from the uterine tissues of pigs and inferior pigs, each of which had an excellent number of pigs in a black pig species. The isolated gDNA was genome wide bisulfite sequencing, and the correlation between the number of pigs and the methylation of the ZPBP gene was established. Then, from the blood sample, gDNA was separated, and the methylation level of the gene related to the acid number was analyzed through a PMP assay and correlated with two properties related to the number of the acid (average total number of operators and average number of operators). The present invention was completed by statistically analyzing the relationship and finally confirming that the degree of methylation of ZPBP, a gene related to piglet number, is related.

According to one aspect, a composition for predicting the number of pigs in pigs including an agent for measuring the expression level and methylation level of the ZPBP (NC_010451.3) gene may be provided.

The zona pellucida binding protein (ZPBP) gene is a binding protein that binds to the oocyte's transparent protein, showing hypomethylation in pig groups with excellent fertility, and hypermethylation in pig groups with inferior fertility. ).

The 'measurement of the expression level of a gene' may be to measure mRNA level or protein level. The 'measurement level of mRNA' is not limited to this, but can 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. It may be achieved by hybridizing mRNA or cDNA derived therefrom from the biological sample on a microarray in which a probe specific for one or more genes selected from the gene is immobilized, and measuring the degree of hybridization obtained as a result. 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 gene.

The 'measurement of protein level' may be any known protein measurement or detection method. For example, an analysis method using an antibody specifically binding to a protein expressed from one or more marker genes selected from the group consisting of the gene may be used. The protein analysis method using the antibody is not limited to, but is not limited to, Western blotting, ELISA, radioimmunoassay, radioimmunoassay, Oukteroni immunodiffusion method, rocket immunoelectrophoresis, tissue immunohistostaining, immunoprecipitation assay, complement Fixed assays and FACS. The ELISA may include direct ELISA, indirect ELISA, direct sandwich ELISA, and indirect sandwich ELISA.

With the Western blotting, the whole protein is separated, electrophoresed, the protein is separated according to size, and then transferred to a nitrocellulose membrane to react with the antibody, and the amount of the generated antigen-antibody complex is used as a labeled antibody. It is a method to confirm. In addition, as a method for measuring the protein level, a method using an enzyme, a substrate, a coenzyme, a ligand, etc. 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 chain reaction (RT-PCR) using RNA isolated from the sample as a template. .

In the present invention, "an 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. Without being limited thereto, a methylation marker may be read through an amplification and PMP assay method to mean a composition capable of predicting the number of pigs. It may mean a primer capable of specifically amplifying the polynucleotide containing the methylation marker and a methylation specific restriction enzyme used in the PMP assay method.

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

In the present invention, the level of methylation 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 a 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 reaction with the marker gene or a nucleic acid expression product expressed therefrom in the sample. In addition, the composition may further include a buffer, a solvent, etc., which stabilize the probe and serve as a medium for reaction.

The binding of the probe to the target nucleic acid (generally, hybridization) is sequence-dependent and can be performed under various conditions. In general, the hybridization reaction takes place at a temperature of about 5 ° C. below Tm for a particular sequence at a specific ionic strength and pH. The Tm means a state in which 50% of the probe complementary to the target sequence is bound to the target sequence. Examples of hybridization reaction 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, conditions that destabilize the binding of the target nucleic acid and the probe, for example, may be performed in the presence of a high temperature and a high concentration of a destabilizing agent (for example, formamide). .

The following method can be used to measure the methylation degree of the present invention.

1. Methylation specific PCR

When bisulfite is treated with genomic DNA, the cytosine at the 5'-CpG-3 'site remains cytosine when it is methylated, and when it is non-methylated, it changes to uracil. Therefore, for the base sequence converted after bisulfite treatment, PCR primers corresponding to the methylated region and 5 kinds of primers corresponding to the non-methylated region of the 5'-CpG-3 'base sequence are used. Producing, converting genomic DNA into bisulfite, and then PCR using the above two types of primers, in the case of methylation, PCR products are made from the primers corresponding to the methylated base sequence. In this case, since PCR products are made from primers corresponding to non-methylation, it can be qualitatively confirmed by agarose gel electrophoresis.

2. Real time methylation specific PCR

Real-time methylation-specific PCR is a conversion of a methylation-specific PCR method to a real-time measurement method. After bisulfite is processed on genomic DNA, PCR primers corresponding to methylated cases are designed and real-time PCR is performed using these primers. Is to do. At this time, it can be detected by using a method and LCgreen to detect using a tanMan probe complementary to the amplified base sequence. Real-time methylation specific PCR can selectively quantitatively analyze methylated DNA.

3. Pyrosequencing

The pyrosequencing method is a method of converting the bisulfite sequencing method into quantitative real-time sequencing. Similar to bisulfite sequencing, genomic DNA is converted by treatment with bisulfite, and then PCR primers corresponding to regions without a 5'-CpG-3 'base sequence are prepared and genomic DNAs are treated with bisulfite. After amplification with the PCR primers, real-time sequencing using 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 chip using methylated DNA specific binding protein

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

In addition, methylation can be measured by quantitative PCR method, and methylated DNA separated with methylated DNA-specific binding protein is labeled with a fluorescent dye and hybridized to DNA chips with complementary probes to measure methylation. Can be.

5. Restriction of methylation sensitivity endonuclease

Measurement of differential methylation can be accomplished by cleaving a non-methylated nucleic acid by contacting a nucleic acid sample with a methylation sensitive restriction endonuclease that cleaves only the unmethylated CpG site.

The "methylation sensitive 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. As a non-limiting example of the methylation sensitivity limiting endonuclease, MspI, HpaII, BssHII, BstUI and NotI enzymes may be used alone or in combination. Other methylation sensitivity limiting endonucleotides include, but are not limited to, SacII and EagI.

The isochizomer of the methylation sensitive restriction endonuclease is a restriction endonuclease having the same recognition site as the methylation sensitive restriction endonuclease, and cleaves both methylated and non-methylated CGs, for example For example, there is MspI.

6. Bisulfite sequencing

Other methods of detecting a nucleic acid 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 methylated nucleic acids by distinguishing modified methylated and non-methylated nucleic acids. Although not limited thereto, the amplification step may be further included. The method relies on PCR reactions to differentiate modified (eg, chemically modified) methylated and non-methylated DNA. No. 5,786,146 describes bisulfite sequencing for the detection of methylated nucleic acids.

According to one embodiment, the composition may further include an agent capable of amplifying the degree of methylation. The formulation may include a primer pair. The primers may be a pair of primers capable of specifically amplifying cytosine modified by bisulfate and methylated, and a pair of primers capable of specifically amplifying cytosine modified by bisulfite not methylated. The primer is not limited thereto, and may be a primer pair represented by 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 required to express the gene, and is a concept including all of a series of structural units operably linked to each other, but is not limited thereto, a promoter region, a protein coding region (open reading frame, ORF) And a terminator region. In the present invention, by measuring the degree of methylation occurring at a specific CpG position of the ZPBP gene, it was confirmed that when the degree of methylation is higher than the average degree of methylation, it can be predicted as a polyacid pig. In order to modify the non-methylated cytosine base of the gene, but not limited to, bisulfite or a salt thereof can be used. When the bisulfite or a salt thereof is used, the methylation of the CpG site can be detected by modifying a non-methylated cytosine residue.

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

According to another aspect, a kit for predicting litter size of pigs comprising the composition may be provided. The kit may further include reagents required for methylation detection. The composition and kit may further include a polymerase, a buffer solution required for agarose electrophoresis, and the like in addition to the agent.

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

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

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 the degree of methylation of the ZPBP gene may further include amplifying the gene with a primer pair represented by the base sequences of SEQ ID NOs: 1 and 2.

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

Hereinafter, the present invention will be described in more detail through examples. However, these examples are only for illustrating the present invention, it will be said that the scope of the present invention is not interpreted to be limited by these examples.

Example

I. DNA methylation profiling

1. Endometrial tissue collection and genomic DNA extraction

For DNA methylation profiling, black pigs (Berkshire) sows were divided into three groups, each of which was divided into a group with an excellent number of births (average number of births of 11 or more) and an inferior group (average number of births of 7 or less), as shown in Table 1. The endometrial tissue was cut off from the angle of the uterus immediately after slaughter, washed with phosphate buffer solution (PBS) and frozen with liquid nitrogen.

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

2. Genome wide bisulfite sequencing (GWBS) analysis

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

3. DNA methylation profiling and DMR screening related to swine production

As a result of counting the site where methylation occurred for the whole genome DNA methylation profiling, as shown in FIG. 2, methylation occurred predominantly at the CG site in both the group with excellent acid number and the group with inferiority. The CHG site showed a relatively low tendency to methylation. In the methylation site, 1,249 million and 1,217 million were identified in the raw read group and inferior group, respectively, in raw read, and 53.08% and 50.48%, respectively, were mapped to the pig genome.

As shown in FIG. 3, as a result of measuring the average CG methylation for each structure at the genome level, the methylation level is lowered around the transcription start site (TSS), and the average is 75 in the transcribed site (gene body) region. % Levels.

In addition, a slightly lower frequency of methylation was observed at the transcription termination site (TTS) and downstream 1 kb than the transcribed resion (gene body). In conclusion, the pattern of frequency distribution of methylation in the gene body according to the number group is generally found to be similar.

According to the workflow shown in FIG. 1, different methylation sites (DMRs) with different numbers of 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 in the CG region on the genome structure is 92 at the promoter (up1kb), 1,313 at the transcribed position (cording sequence (CDS), intron) (see Table 3).

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

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

II. Validation and reproducibility of DNA methylation marker of ZPBP gene

1. Blood collection and animal information collection

To verify the reproducibility of DNA methylation of the selected gene, blood was collected from 137 sows of Berkshire species and the average total number of born (Avg. Of TNB) and average missing Number (Avg. Of NBA; Average of number of born alive) was confirmed (see Table 5).

2. Methylation specific polymerase chain reaction (PMP assay; Porcine methylation-specific restriction enzyme PCR assay)

The methylation specific polymerase chain reaction (PMP assay) is a simple method that can directly evaluate the degree of methylation of a specific CpG site using a methylation specific restriction enzyme.

For reproducibility verification, genomic DNA was extracted using DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA). The 10 μg / μL of genomic DNA was cleaved using methylation-sensitive isoschizomer ( HPA II), a methylation-sensitive isoschizomer pair, and Msp I (non-specific methylation restriction enzyme), followed by ZPBP in Table 6. It was amplified using gene-specific oligonucleotide primers. The amplified PCR product was subjected to electrophoresis to confirm the strength of the band, and the degree of methylation was analyzed (see Table 6).

3. Statistical Analysis

The correlation between the methylation level of the ZPBP gene and the number of piglet traits (total number of livestock, number of livestock) was determined using the SPSS (IBM Statistics SPSS Statistics, Inc., Somers, NY, USA) program (version 20.0). It was analyzed through simple linear regression analysis.

4. Confirmation of the usefulness of the ZPBP gene as a methylation marker in blood samples

ZPBP gene showing methylation difference according to the number of ions in the uterine tissue of pig was verified as a methylation marker in pig blood samples. This is an essential process for early selection of pigs with high birth numbers using blood samples from pigs, and it was confirmed that the methylation pattern of the ZPBP gene in the genomic DNA of blood is consistent with the results in uterine tissue.

As shown in FIG. 5, the methylation degree of the ZPBP gene by methylation-specific polymerase chain reaction (PMP assay) in blood samples is the same as the result of bisulfite sequcing in the uterine tissue. It was confirmed that the hypomethylation (Hypermethylation) pattern. These results show that the methylation of the ZPBP gene can be useful as a marker for early selection of pigs with high birth numbers using blood.

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

To verify the DNA methylation marker of the ZPBP gene, the methylation level of the ZPBP gene and the total number of characters (TNB) and the number of actual numbers (NBA) of the pig's arithmetic through PMP assay in blood samples of 137 sows of Berkshire species The correlation was analyzed.

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

Therefore, the degree of methylation of the ZPBP gene affects the average total number of piglets sow, and in particular, since the regression coefficient has a negative value, it is confirmed that as the methylation of the ZPBP gene decreases, the number of total numbers increases. Became. At this time, the value of t is the value obtained by dividing the non-standardization coefficient by the standard error.

In addition, as shown in Table 8 below, the methylation marker and average number of NBAs of the ZPBP gene are averaged number of y (y) = -5.1436 * ZPBP (x) + by unstandardized coefficients. A regression equation such as 9.5116 was derived, and the regression coefficient for the methylation marker of the ZPBP gene had a negative value and was found to be statistically significant.

Therefore, it was confirmed that the methylation marker of the ZPBP gene affects the average number of miscarriage of sow pigs, and in particular, as the regression coefficient has a negative value, it has been confirmed that the methylation of the ZPBP gene has a negative correlation with the increase in the number of miscarriers. .

Although the present invention has been described in detail through specific examples, the present invention is specifically for describing the present invention, and the present invention is not limited to this, and by those skilled in the art within the technical spirit of the present invention. It is clear that it can be modified or improved. All simple modifications and variations of the present invention belong to the scope of the present invention, and the specific protection scope of the present invention will be clarified by the appended claims.

<110> GYEONG NAM NATIONAL UNIVERSITY OF SCIENCE AND TECHNOLGY <120> Composition for prediction of swine fecundity using methylation          status of ZPBP gene and method for predictioin of swine fecundity          using the same <130> NPF32194 <160> 2 <170> PatentIn version 3.2 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 atcaggtgag gcgtcggcat 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 cgtcatcaat gtccagtcct 20

Claims (13)

Contains an agent that measures the degree of methylation of the ZPBP gene,
The formulation comprises a primer pair represented by the nucleotide sequence of SEQ ID NO: 1 and 2, the composition for predicting the number of pigs in pigs. According to claim 1,
The composition is capable of amplifying the methylation site, a composition for predicting the number of pigs in the mountains. delete According to claim 1,
The composition is to measure the degree of methylation of the CpG site, the composition for predicting the number of pigs living. According to claim 1,
A composition for predicting the number of pigs in a pig, comprising a pair of methylation-specific restriction enzymes HpaII and MspI. According to claim 1,
The pig is a black pig, a composition for predicting the number of pigs. The method of claim 6,
The black pig is Berkshire, a composition for predicting the number of pigs. A kit for predicting the birth number of a pig, comprising the composition for predicting the birth number of a pig according to any one of claims 1, 2, and 4 to 7. The method of claim 8,
The kit is an RT-PCR kit, a microarray chip kit, or a protein chip kit. Extracting DNA from a pig subject;
Measuring the degree of methylation of the ZPBP gene from the DNA; And
And predicting a pig having a lower methylation level than the average methylation level as a pig having a higher number of digits than a pig not having the same,
The step of measuring the degree of methylation of the ZPBP gene further comprises amplifying the gene with a primer pair represented by the base sequences of SEQ ID NOs: 1 and 2, the method for predicting the number of pigs in the pig. The method of claim 10,
The subject is blood, hair root or endometrial tissue, method for predicting the number of pigs in the pig. delete The method of claim 10,
The step of measuring the degree of methylation includes methylation specific PCR, real time methylation specific PCR, pyrosequencing, PCR using a methylated DNA specific binding protein or quantitative PCR and DNA chip, A method for predicting the number of pigs in pigs, using a methylation sensitivity limiting endonuclease, bisulfite sequencing, or PMP assay analysis.

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