KR102001528B1 - Gene marker for discrimination of Korean Native pig and use thereof - Google Patents

Abstract

The present invention relates to a genetic marker and kit for identifying Korean native pigs, and a method for identifying Korean native pigs using the same. According to the present invention, even if the hair color is similar, by using the unique nucleotide sequence of Korean native pigs, it is possible to quickly, accurately and economically identify the Korean native pigs.

Description

Gene marker for discrimination of Korean Native pig and use approximately}

The present invention relates to a gene marker for identifying a Korean native pig and its use. More specifically, the present invention relates to a composition, kit for identifying Korean native pigs, and a method for identifying Korean native pigs using the same, which can quickly and accurately and economically identify Korean native pigs using SNP markers unique to Korean native pigs. will be.

Korean native pigs (KNP) have been reported to have higher consumer preference because they have hard fat tissue, white fat color, rich and light gravy, chewy and soft texture, red meat, and marbling, compared to general breeds. .

In particular, the black pigs on Jeju Island, among Korean traditional pigs, are very oily and tender in quality, so they are highly preferred and their selling prices are relatively high.

As such, black Berkshire is often sold as Jeju black pork.

On the other hand, the groping is known as a qualitative trait, but there is a difficulty in identifying the breed at the breed or individual level. Therefore, there has been a need for a method that can identify Korean native pigs at the genetic level quickly, accurately and economically.

Background art of the present invention is disclosed in Korean Patent Laid-Open No. 10-2016-006348 for a single base polymorphic marker useful for discriminating black pork and non-black pork and its use.

Throughout this invention, many patent documents are referred and the citation is shown. The disclosure of the cited patent document is incorporated by reference in its entirety as a whole, and the level of the technical field to which the present invention belongs and the content of the present invention are more clearly explained.

An object of the present invention is to provide a genetic marker for identifying Korean native pigs.

Another object of the present invention is to provide a composition for identification of Korean native pigs comprising an agent capable of detecting or amplifying the genetic markers.

Still another object of the present invention is to provide a kit for identifying a Korean native pig comprising the composition.

Still another object of the present invention is to provide a method for identifying Korean native pigs by checking the genotype by the genetic marker.

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

According to an aspect of the present invention, in the polynucleotide represented by SEQ ID NO: 1, the 386th base is A or G, and the base sequence of SEQ ID NO: 1 includes 5 to 600 consecutive bases including the 386th base. Provided is a composition for identifying Korean native pigs, comprising an agent capable of detecting or amplifying a SNP marker comprising a polynucleotide or a complementary polynucleotide thereof.

According to an embodiment of the present invention, the preparation may include a primer set represented by SEQ ID NOs: 2 and 3.

According to one embodiment of the present invention, it is possible to identify the land race, Duroc, Great Yorkshire, and Berkshire varieties and Korean native pig varieties.

According to one embodiment of the present invention, it is possible to identify the Berkshire varieties and Jeju native pig varieties.

According to another aspect of the present invention, there is provided a kit for identifying Korean native pigs, comprising the composition.

According to another aspect of the present invention, there is provided a microarray for identifying a Korean native pig, comprising the composition.

According to another aspect of the invention, a) extracting a nucleic acid from a pig sample; b) amplifying a polymorphic site of said SNP marker from said extracted nucleic acid; And c) determining the base of the amplified polymorphic site.

According to one embodiment of the present invention, amplification in step b) may use a primer set represented by SEQ ID NOs: 2 and 3.

According to one embodiment of the present invention, if the allele of the SNP corresponding to the 386th base of the polynucleotide of SEQ ID NO: 1 in step c) is A / A type, it may be determined as a Korean native pig breed.

According to one embodiment of the present invention, if the base type of the SNP corresponding to the 386th base of the polynucleotide of SEQ ID NO: 1 in step c) can be determined as a Korean native pig breed.

According to an embodiment of the present invention, when the base type of the SNP corresponding to the 386th base of the polynucleotide of SEQ ID NO: 1 is G, it may be determined as a Landrace, Duroc, Great Yorkshire, or Berkshire variety.

According to an embodiment of the present invention, the Korean native pigs can be quickly, accurately and economically identified by using a gene sequence unique to Korean native pigs.

According to one embodiment of the present invention, it is possible to prepare a basis for a genetic diagnosis system for identifying Korean native pigs.

According to one embodiment of the present invention, even if the groping is similar, Korean native pig breeds can be accurately identified with foreign pig breeds, which can help establish a transparent distribution system of pork.

Figure 1 is a photograph showing the classification of the offspring coming from crossing the white race land race and the black pig Jeju native pig.
Figure 2 is a graph showing the results of the GWAS analysis for whole body black color in Jeju traditional pig and land race breeding herds.
Figure 3 is a sequencing result showing a c.586 G> A novel mutation of the exon2 region of the KIT gene according to an embodiment of the present invention.
Figure 4 is a diagram showing the results of the network analysis between varieties using the base mutation of the pig KIT gene according to an embodiment of the present invention.
5 is a view showing the position of the SNP markers in the swine KIT exon 2 nucleotide sequence (SEQ ID NO: 1) according to an embodiment of the present invention.
FIG. 6 is a diagram showing the results of confirming that allelic types are shown as G / G and A / A types as the mutation of the 386th base in the base sequence of SEQ ID NO: 1 is represented by A or G according to one embodiment of the present invention. to be.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Certain terms are defined herein for convenience of understanding the invention. Unless defined otherwise herein, scientific and technical terms used herein have the meanings that are commonly understood by one of ordinary skill in the art. Also, unless specifically indicated in the context, the singular forms "a", "an", and "the" are intended to include their plural forms as well.

According to one aspect of the present invention, there is provided a Single Nucleotide Polymorphism (SNP) marker that can be distinguished from Korean pigs.

The SNP marker is located at exon 2 of the KIT gene and can accurately identify Korean native pigs and foreign pigs, and thus can be usefully used as a gene marker for identifying Korean native pigs. The SNP marker may be all or part of a polynucleotide including the SNP region of the KIT gene.

The term "KIT" gene of the present invention, which plays an important role in melanocyte migration and survival and encodes the mast / stem cell growth factor receptor, pig KIT gene is chromosome 8 Encoded in, it consists of 21 exons. The SNP markers of the present invention derived from the KIT gene, which can accurately identify Korean native pig varieties and foreign varieties, are not known and were first discovered by the present inventors.

The term "Single Nucleotide Polymorphism" (SNP) in the present invention refers to single nucleotide polymorphism, which occurs when a single nucleotide (A, T, C, or G) in the genome differs between members of a species or between paired chromosomes of an individual. Refers to the diversity of DNA sequences.

As used herein, the term "polymorphism" refers to a case in which two or more alleles exist in one locus, and among polymorphic sites, only a single base differs according to an individual. It is called polymorphism.

As used herein, the term “allele” refers to several types of genes that exist at the same locus of homologous chromosomes. Alleles are also used to indicate polymorphism, for example SNPs have two kinds of alleles.

Land races, which are white varieties, and Jeju native pigs of black color are produced by producing more than 1200 F2 offspring. There are various types of offspring, which can be classified into six types (Figure 1).

Analysis of genome wide association study (GWAS) using genotypes of cross-breed reference stocks of white races, Landrace and Jeju black pigs, showed that the specific genes of Korean native pigs were on chromosome 8. It could be confirmed (Fig. 2).

Sequence analysis of the KIT gene confirmed that the c.586 G> A mutation was present in the exon2 region of the KIT gene (FIG. 3).

In addition, as a result of analyzing the network between varieties using the base mutation of the pig KIT gene according to an embodiment of the present invention it was confirmed that the Jeju native pigs are classified into a genetically independent group compared to the foreign pig breed (Fig. 4).

According to an embodiment of the present invention, the genotype by the SNP marker of KIT exon 2 is present in the G / G genotype and A / A genotype, in the case of A / A type can be determined as a Korean native pig breed (Fig. 5, 6 and Table 1). As shown in Table 1, Jeju native pigs and inland native pigs are A / A genotypes, while foreign pig varieties all have G / G genotypes.

Therefore, by amplifying the SNP marker region of the present invention and confirming the genotype of the SNP marker by Pyro-sequencing method, it is possible to accurately identify whether or not Korean native pig.

According to one embodiment of the present invention, it is possible to identify the land race, Duroc, Great Yorkshire, and Berkshire varieties and Korean native pig varieties.

According to one embodiment of the present invention, it is possible to identify the Berkshire breed and Jeju native pig breed having a similar black color.

In the present invention, "pyrosequencing" does not require a labeled primer and does not require gel electrophoresis, and by sequencing the base sequence of the DNA only about 30-40 base pairs (bp) This is a quick and accurate way to analyze. Pyro sequencing method is a sequencing method performed in real time, without electrophoresis, pyrophosphate is released by the addition and merging of sequential nucleotides one at a time in primer-directed PCR and released as the nucleotides merge It is paired with sulfurylase and luciferase enzymes to emit light, which is done by detecting this light. The emitted light exhibits signal peaks in the order of reaction of each nucleotide sequentially entered, and the light is shown as a peak having a relative height in proportion to the number of merged nucleotides, which can be determined in real time.

Patterns of peaks differ between homo and hetero, wild-type, and mutant samples, so you can compare the nucleotide sequence of the wild-type with the analyzed sequencing to identify and determine the type and exact location of the mutation through sequence differences. The advantage is that a large amount of sample can be processed in a simple and economic manner.

Although the conventional direct DNA sequencing method is currently performed by an automated sequencing apparatus, a DNA sample such as a PCR product to be sequenced is obtained, purified, and then subjected to a sequencing reaction, and then DNA generated by the sequencing reaction. While the time required for sequencing is much shorter because it is precipitated and loaded into an automated DNA sequencing apparatus, pyro sequencing has a sequencing primer if only PCR products to be sequenced are prepared. After the reaction, the sequence can be analyzed in approximately 10 minutes by a pyro sequencing device.

In addition, since nucleotide dispensation orders can be designed, sequence variations that vary by a single nucleotide are generated as distinct pyograms that appear in different patterns at different peaks. Moreover, the use of specific nucleotide distribution strategies can reduce the number of pyro sequencing cycles, thereby increasing the quality of the sequence and the read length.

Brief description of the processes that can be used as one embodiment of the present invention is as follows. Conjugation of a biotin to the 5 'end of the forward or reverse primer for pyro-PCR primer set (single-stage purification of one strand for isolation and purification of one strand) of the gene containing the sequence to be detected by pyro sequencing (Mutation or Variation) Amplified by RT-PCR and PCR, and the amplified PCR product is reacted with the conjugated beads of avidin, and then only one strand of the DNA amplified by PCR using affinity between avidin and biotin is separated into the beads. Fix it. After annealed with the pyro sequencing primer using a single strand of DNA template thus separated and purified, the resulting nucleotides were reacted with the substrate / enzyme with the DNA template according to the sequence of nucleotide distribution in the pyro-sequencer. Pyrogram is used to analyze the degree of variation in the nucleotide sequence.

According to another aspect of the present invention, in the polynucleotide represented by SEQ ID NO: 1, the 386th base is A or G, and the base sequence of SEQ ID NO: 1 includes 5 to 600 consecutive bases including the 386th base. Provided is a composition for identifying Korean native pigs, comprising an agent capable of detecting or amplifying a SNP marker comprising a polynucleotide or a complementary polynucleotide thereof.

In the present invention, the agent capable of amplifying or detecting the SNP marker refers to a composition capable of identifying Korean native pigs by detecting such a genetic mutation site, that is, the SNP marker through amplification and pyro sequencing methods. , A primer capable of specifically amplifying a polynucleotide including the SNP marker, a pyro sequencing primer, and an amplification composition used in a PCR method.

According to an embodiment of the present invention, the preparation may include a primer set represented by SEQ ID NOs: 2 and 3.

As used herein, the term “primer” refers to a short sequence that functions as a starting point for copying a template strand with a base sequence having a short free 3 ′ hydroxyl group. Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (ie, DNA polymerase or reverse transcriptase) at appropriate buffers and temperatures. The length of PCR conditions, sense and antisense primers can be modified based on what is known in the art.

Figure 5 shows the position for the production of SNP markers and analytical primers in swine KIT exon 2 nucleotide sequence (SEQ ID NO: 1) according to an embodiment of the present invention.

According to another aspect of the present invention, there is provided a kit for identifying Korean native pigs, comprising the composition.

In the present invention, the kit may be, but is not limited to, an RT-PCR kit or a microarray chip kit including an agent capable of detecting or amplifying SNP markers.

The kit of the present invention can identify whether or not Korean native pig by amplifying a gene that can identify Korean native pig and then cutting the band with restriction enzymes. The PCR kit includes a test tube or other suitable container, reaction buffer (variable pH and magnesium concentrations), deoxynucleotides (dNTPs) in addition to each primer pair specific for a polynucleotide comprising a SNP marker or its complementary polynucleotide. ), Enzymes such as Taq-polymerase, DNase, RNAse inhibitors, DEPC-water, sterile water, and the like. Specifically, the PCR kit may be a kit for performing PCR-RFLP (PCR-restriction fragment length polymorphism).

In the present invention, the PCR-RFLP method is a technique for analyzing the restriction fragment length polymorphism of the gene fragments using PCR technology among various DNA analysis techniques for improving the genetic characteristics or ability of the livestock, a specific point mutation (point mutation) When there is a restriction enzyme recognition site at the site of mutation, it is a method to determine the desired genotype of each individual more quickly and accurately by using various fragment lengths generated by restriction enzymes depending on the genotype difference of each individual. .

According to another aspect of the present invention, there is provided a microarray for identifying a Korean native pig, comprising the composition.

In the present invention, "microarray" refers to a microarray in which a group of polynucleotides is immobilized at a high density on a substrate, and each of the polynucleotide groups is immobilized in a predetermined region. Such microarrays are well known in the art. Microarrays are disclosed, for example, in US Pat. Nos. 5,445,934 and 5,744,305, the contents of which are incorporated herein by reference.

According to another aspect of the invention, a) extracting a nucleic acid from a pig sample; b) amplifying a polymorphic site of said SNP marker from said extracted nucleic acid; And c) determining the base of the amplified polymorphic site.

In the present invention, the method of extracting the nucleic acid from the swine sample in step a) and b) and amplifying the polymorphism site of the SNP marker may be made through a method known in the art. For example, it can be done by directly purifying DNA from tissue or cells or by specifically amplifying and isolating specific regions using amplification methods such as PCR. In the present invention, a nucleic acid is meant to include not only DNA but also cDNA and RNA molecules synthesized from mRNA. Extracting the nucleic acid from the sample of interest may include, for example, PCR amplification, ligase chain reaction (LCR) (Wu and Wallace, Genomics 4, 560 (1989), Landegren et al., Science 241, 1077 (1988)), transcription Transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA 86, 1173 (1989)) and self-sustaining sequence replication (Guatelli et al., Proc. Natl. Acad. Sci. USA 87, 1874 (1990)) And nucleic acid based sequence amplification (NASBA) and the like.

In the present invention, the sample may be a hair, urine, blood, various body fluids, separated tissues, samples such as isolated cells or saliva, but is not particularly limited thereto. It is not limited thereto, but may be skin tissue.

In the present invention, the amplification in step b) may use a primer set represented by SEQ ID NO: 2 and 3.

In the present invention, the method of determining the base of the amplified polymorphic site in step c) comprises sequencing, hybridization by microarray, allele specific PCR, dynamic allele hybridization technique (dynamic allele-specifichybridization (DASH), PCR prolongation analysis, PCR-SSCP, PCR-RFLP analysis or TaqMan technique, SNPlex platform (Applied Biosystems), mass spectrometry (e.g. Sequenom's MassARRAY system), mini-sequencing using sequencing methods, Bio-Plex systems (BioRad), CEQ and SNPstream systems (Beckman), Molecular Inversion Probe array technologies (e.g., AffymetrixGeneChip), and BeadArray Technologies (e.g., Illumina GoldenGate and Infinium assays) It may be carried out by, but is not particularly limited thereto. One or more alleles can be identified in the SNP markers by the methods or other methods available to those skilled in the art.

Hybridization refers to a process in which two complementary strands of nucleic acids combine to form a double stranded molecule (hybrid). In the process of the invention said hybridization is a process carried out under high stringency hybridization conditions.

To detect the degree of hybridization, the target sequence can be labeled with a detectable label. In one embodiment, the labeling material may be, but is not limited to, a material that emits fluorescence, phosphorescence, or radioactivity. Preferably, the labeling substance is Cy-5 or Cy-3. When amplifying the target sequence, PCR is performed by labeling Cy-5 or Cy-3 at the 5'-end of the primer to allow the target sequence to be labeled with a detectable fluorescent labeling substance. In addition, the label using the radioactive material may add radioactive isotopes such as P32 or S35 to the PCR reaction solution during the PCR, while the amplification product is synthesized and the radioactive material is incorporated into the amplification product, thereby amplifying the radiolabeled product.

The TaqMan method comprises the steps of: (1) designing and constructing a primer and a TaqMan probe to amplify a desired DNA fragment; (2) labeling probes of different alleles with FAM dye and VIC dye (Applied Biosystems); (3) using the DNA as a template and performing PCR using the primers and probes described above; (4) after the PCR reaction is completed, analyzing and confirming the TaqMan assay plate with a nucleic acid analyzer; And (5) determining the genotype of the polynucleotides of step (1) from the analysis result.

The sequencing may use conventional methods for sequencing and may be performed using an automated genetic analyzer. In addition, allele-specific PCR refers to a PCR method for amplifying a DNA fragment in which the mutation is located with a primer set including a primer designed with the base at which the mutation site is located as the 3 'end. The principle of the method is, for example, when a specific base is substituted from A to G, PCR by designing a primer containing A as the 3 'terminal base and a reverse primer capable of amplifying a DNA fragment of a suitable size. When the reaction is performed, when the base of the mutation position is A, the amplification reaction is normally performed, and a band of the desired position is observed. When the base is substituted with G, the primer may complementarily bind to template DNA. The amplification reaction is not performed properly because the 3 'end is not complementary.

DASH may be performed by a conventional method, preferably by a method by Prince et al.

PCR extension analysis, on the other hand, first inactivates a DNA fragment containing a base on which a single nucleotide polymorphism is located by amplifying with a primer set, and then deactivating all nucleotides added to the reaction by inactivating the specific extension primer, dNTP. By adding a mixture, didioxynucleotide, reaction buffer and DNA polymerase to effect primer extension. At this time, the extension primer is a 3 'end of the base immediately adjacent to the 5' direction of the base where the mutation site is located, the dNTP mixture excludes a nucleic acid having the same base as the didioxynucleotide, the didioxynucleotide is a mutation It is selected from one of the base types shown. For example, if there is a substitution from A to G, when a mixture of dGTP, dCTP and TTP and ddATP are added to the reaction, the primer is extended by DNA polymerase at the base where the substitution takes place and after several bases A The primer extension is terminated by ddATP at the position where the base first appears. If the substitution has not occurred, since the extension reaction is terminated at the position, it is possible to determine the base type showing the mutation by comparing the length of the extended primer.

In this case, as the detection method, when the fluorescent primer is labeled with an extended primer or didioxynucleotide, the mutation can be detected by detecting fluorescence using a gene analyzer (for example, Model 3700, manufactured by ABI, etc.) used for general sequencing. In the case of using unlabeled extension primers and didioxynucleotides, the variation can be detected by measuring molecular weight using a matrix assisted laser desorption ionization-time of flight (MALDI-TOF) technique.

6, the alleles were analyzed to appear as G / G and A / A types as the mutation of the 386th base in the base sequence of SEQ ID NO: 1 is represented by A or G.

According to one embodiment of the present invention, if the allele of the SNP corresponding to the 386th base of the polynucleotide of SEQ ID NO: 1 in step c) is A / A type, it may be determined as a Korean native pig breed.

According to one embodiment of the present invention, if the base type of the SNP corresponding to the 386th base of the polynucleotide of SEQ ID NO: 1 in step c) can be determined as a Korean native pig breed.

According to an embodiment of the present invention, when the base type of the SNP corresponding to the 386th base of the polynucleotide of SEQ ID NO: 1 is G, it may be determined as a Landrace, Duroc, Great Yorkshire, or Berkshire variety.

Therefore, by detecting the genotype of the SNP of the present invention or by using a primer capable of detecting the genotype, it is possible to selectively and accurately identify only Korean native pig varieties.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example  1: Korean traditional pig GWAS (genome-wide association study) analysis

GWAS analysis was performed using a SNP chip (Porcine 60K Beadchip, Illumina) for black color in the cross reference reference group of white breeds of Landrace pigs and black color Jeju native black pigs (FIG. 2). Figure 2 is a graph showing the results of the GWAS analysis for whole body black color in Jeju traditional pig and land race breeding herds. As shown in FIG. 2, GWAS analysis showed the highest significance in chromosome 8, and the region had a KIT gene related to the groping of pigs and was selected as a candidate gene.

The porcine KIT gene is known to be an important color gene used to identify whites and colors. Specifically, white-grown varieties include land races and large Yorkshire varieties, and these varieties have CNV (copy number variation) with at least two KIT genes. Therefore, in order to select the fastest method for searching for a variant because the gene structure is complex and its length is large, pig skin was collected, the mRNA sequence was amplified by RT-PCR method, and the base sequence was decoded by the sequencing method. As a result, non-synonymous SNPs (c.586G> A) with a protein sequence change in exon2 were identified (FIG. 3).

Example  2: base mutation of the pig KIT gene Breed  Network analysis

In the preliminary experiments, variants were identified in the 5'-UTR, exon, and 3'-UTR regions of the KIT gene for six varieties (land race, large Yorkshire, Dourok, Jeju native pig, Berkshire, and Hampshire) in the preliminary experimental phase. The genetic distance was calculated using the sequence homology of the variants, and network analysis was performed.

Figure 4 is a diagram showing the results of the network analysis between varieties using the base mutation of the pig KIT gene according to an embodiment of the present invention. As shown in FIG. 4, it is confirmed that Jeju native pigs are divided into genetically independent populations compared to foreign pig varieties, and thus, Jeju native pigs, in particular, sequences are more likely to exist.

Example  3: SNP genotyping

Genotyping using a Pyro-sequencer was performed to prepare primers capable of detecting the SNP (c.586G> A) mutation as follows.

KIT_new_ex2F: biotin-GGC-CAT-TCA-AAC-TCC-TGA-TT (SEQ ID NO: 2)

KIT_ new_ex2R: CAG-AAG-CAA-GCC-AGC-AAG-AG (SEQ ID NO: 3)

KIT_ new_ex2_MINI: GAT-ACT-CGC-GCT-TCA-CGT-TT (SEQ ID NO: 4)

Input [C / T] TGATGGTGATGCC (SEQ ID NO: 5)

PCR product size: 344bp

One pair of primers were prepared as described above for population analysis. KIT_new_ex2F was prepared by combining biotin-dye.

After PCR amplification, genotyping was performed according to the manual using a Pyro-Sequencer to read the genotype. At this time, gene amplification was performed at 95 ° C for 5 minutes, followed by denaturation at 95 ° C for 1 minute, at 60 ° C for 1 minute, and at 72 ° C for 1 minute for a total of 35 cycles.

As a result of the pyro-sequencing analysis, as shown in FIG. 6, allelic types are shown as G / G type and A / A type as the 386th mutation of the 386th base is represented as A or G. Could know.

Example  4: KIT by pig breed Exxon 2  Genotype Population Analysis

KIT exon 2 genotypes of each of the typical pig breeds, such as Landrace, Dourok, Great Yorkshire, and dark brown Berkshire breeds and conventional pork breeds, are analyzed and shown in Table 1.

As shown in Table 1, a total of 49 dogs, including 44 Jeju native pigs and 5 inland native pigs, are fixed to the A / A genotype, while the remaining four breeds have the G / G genotype. have.

Therefore, by detecting the genotype of the SNP of the present invention or by using a primer capable of detecting the genotype, it is possible to selectively and accurately identify only Korean native pig varieties.

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Claims (11)

In the polynucleotide represented by SEQ ID NO: 1, the 386th base is A or G, and the polynucleotide consisting of 5 to 600 consecutive bases including the 386th base as the base sequence of SEQ ID NO. A composition for identifying Korean native pigs, comprising an agent capable of detecting or amplifying SNP markers comprising polynucleotides, said composition capable of identifying landrace, Duroc, Daeyokshire, and Berkshire varieties and Korean native pig varieties. That is, the composition.
The method of claim 1,
The formulation comprises a primer set represented by SEQ ID NO: 2 and 3, Korean traditional pig identification composition.
delete The method of claim 1,
Korean native pig identification composition, which can identify Berkshire varieties and Jeju native pig varieties.
Korean traditional pig identification kit containing the composition of any one of Claims 1, 2, and 4.
A microarray for identifying Korean native pigs, comprising the composition according to any one of claims 1, 2 and 4.
a) extracting nucleic acid from a pig sample;
b) amplifying the polymorphic site of the SNP marker of claim 1 from the extracted nucleic acid; And
and c) determining the base of the amplified polymorphic site, the method of identifying Korean native pigs from Landrace, Duroc, Great Yorkshire, and Berkshire varieties.
The method of claim 7, wherein
Amplification in step b) using a primer set represented by SEQ ID NO: 2 and 3, the method of identifying Korean native pigs.
The method of claim 7, wherein
If the allele of the SNP corresponding to the 386th base of the polynucleotide of SEQ ID NO: 1 in step c) is A / A type, it is determined that the Korean native pig breed, Korean native pig.
The method of claim 7, wherein
If the base type of the SNP corresponding to the 386th base of the polynucleotide of SEQ ID NO: 1 in step c) is determined to be a Korean native pig breed, a method for identifying Korean native pigs.
The method of claim 7, wherein
If the base type of the SNP corresponding to the 386th base of the polynucleotide of SEQ ID NO: 1 in step c) is G, judging by the land race, Dourok, Dae Yorkshire or Berkshire varieties, Korean native pigs.

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