KR20170055818A - Novel SNP marker for discriminating reduction of backfat thickness and use thereof - Google Patents

Abstract

The present invention relates to a SNP marker for judging reduction of the backfat thickness of a pig, and a use thereof. More specifically, the present invention relates to a SNP marker capable of judging reduction in backfat thickness of pigs, a SNP marker capable of detecting or amplifying the SNP marker A composition for judging reduction of the backfat thickness of a pig including a preparation, a kit for judging reduction of backfat thickness of a pig containing the composition, a method for judging reduction of backfat thickness of a pig, A method for reducing the thickness of the back layer of a pig, and a microarray for determining the back layer thickness reduction of a pig. The SNP marker of the present invention is a SNP marker that can be used to judge the reduction of backing thickness of pigs and is used as a means for objectively evaluating the reduction of the back thickness not discriminated by the naked eye. You can do it.

Description

SNP Markers for Reducing the Backing Layer Thickness of Pigs and Their Use [

The present invention relates to a SNP marker for judging reduction of the backfat thickness of a pig, and a use thereof. More specifically, the present invention relates to a SNP marker capable of judging reduction in backfat thickness of pigs, a SNP marker capable of detecting or amplifying the SNP marker A composition for judging reduction of the backfat thickness of a pig including a preparation, a kit for judging reduction of backfat thickness of a pig containing the composition, a method for judging reduction of backfat thickness of a pig, A method for reducing the thickness of the back layer of a pig, and a microarray for determining the back layer thickness reduction of a pig.

Since breeding in the eastern part of India about 9,000 years ago, pigs have been raised as the most basic animal for the worldwide consumption of protein that people need, according to their age, situation and people's preferences. European varieties and Asian varieties are derived from Susscrofa on each continent. There are currently 200 varieties of varieties presently available. According to a recent FAO (Finance and Accounts Office) report, Asian cultivars account for 30% , And European varieties were reported to be around 33%. Differences in phenotype among these varieties are sought, size, and body shape.

Recently, in order to improve the quality of the pork meat, the pork is raised in a certain standard and is scientifically managed, and the pork meat obtained from the pork meat is branded, and various brands of pork meat are already commercially sold. However, since the branded pork meat and unbranded pork meat are difficult to identify with the naked eye, they can be used to sell the unbranded pork meat into branded pork meat, Disturbing events are occurring frequently. Indeed, since it is very difficult to distinguish between branded pigs and unbranded pork meat at the level of experts, studies are actively being conducted to establish objective criteria to judge the quality of genuine pork meat.

As a part of this research, a method for judging pig meat of a genuine brand was developed by analyzing the gene of pork meat. Randomly amplified polymorphic DNA (RAPD), single strand conformation polymorphisms, and other DNA analysis techniques to develop and develop a method for discriminating pork meat varieties. For example, Japanese Patent Application Laid-Open No. 2004-0039059 discloses a gene assay method capable of selecting pigs having excellent traits of pigs by using specific DNA markers related to the daily gain of body weight, backfat thickness, and meat quality traits of pigs. Patent Publication No. 2007-0113336 discloses a DNA marker for confirming the increase of porcine myocyte count using a mutation (SNP) caused by a single base sequence difference in the promoter region of the 5 'end of Myogenin gene known to be involved in the myocyte differentiation of pigs Japanese Patent Application Laid-Open No. 2011-0011443 discloses a technique for detecting an accurate Korean rice variety and other breeder pigs by detecting a Korean native pig-specific DNA marker for the KIT gene, -0050261 discloses a method of discriminating the varieties of black-bred pigs using haplotypes estimated from SNPs in the KIT gene region of black-bred pigs Patent Publication No. 2011-0139011 discloses a method for evaluating meat quality using a single-trait polymorphic biomarker for diagnosing fat content in pigs. In Patent Publication No. 2012-0046968, And a method of screening pigs of a trait of good quality using a gene of a pig is disclosed in Patent Publication No. 2012-0049624, (SNP) site of the PPARGC1A gene, which is involved in the meat quality of the pig, has been disclosed. In the patent publication No. 2012-0072871, there is disclosed a single gene for confirming the content of unsaturated fatty acids in pigs A method for identifying high quality pork meat using a base polymorphism (SNP) marker is disclosed.

However, in addition to low economic efficiency such as slow growth and low number of habitats, traditional pigs are too thick to be industrialized because they are too thick in the carcass. In the backfat thickness, the pigs are about 20mm in the finishing pigs, but the native pigs are at least 40mm in length and some individuals are found in 80mm. So far, the characteristics of traditional pork have been preserved, and it has been preserved as a species maintenance rather than the selection until now. However, as a genetic resource of native species, it has become an important resource for industrialization with increasing value. The development of regulatory methods at the genetic level is required for the purpose.

Under these circumstances, the present inventors have made intensive researches to develop a method for reducing the backfat thickness of pigs. As a result, when the SNPs present on chromosome 5 are used, reduction in the back layer thickness of genetically determined pigs And confirmed that the quality of pigs can be judged. Thus, the present invention has been completed.

It is an object of the present invention to provide a SNP marker for determining the backing thickness reduction of a pig.

It is another object of the present invention to provide a composition for determining the backfat thickness reduction of a pig, which comprises a preparation capable of detecting or amplifying the SNP marker.

It is still another object of the present invention to provide a kit for determining the backfat thickness reduction of pigs comprising the composition.

It is yet another object of the present invention to provide a method for determining backfill thickness reduction.

It is another object of the present invention to provide a method for producing pigs with reduced backfat thickness.

It is yet another object of the present invention to provide a method for reducing the thickness of the back side of a pig.

Yet another object of the present invention is to provide a microarray for determining the backfill thickness reduction.

The present inventors searched for a quantitative trait loci (QTL) related to the back thickness of pigs in order to develop a method for reducing the backfat thickness of pigs. As a result, It was confirmed that the SNP (ALGA0031489) present at 35 MB was associated with the reduction of the backfat thickness, and it was found that the SNP can be used as a genetic marker capable of determining the backfat thickness reduction.

In one embodiment, the present invention provides a polynucleotide comprising a nucleotide sequence of SEQ ID NO: 1, wherein the 501st base is A or G, 5 to 1000 consecutive bases containing the 501st base , Or a complementary polynucleotide thereof, for the determination of the backing thickness reduction of pigs.

The term "polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1" of the present invention is a polymorphic sequence comprising a polymorphic site of a gene involved in reducing the backfat thickness of a pig. The polymorphic sequence is a polynucleotide sequence Quot; means a sequence comprising a polymorphic site comprising a SNP. The polynucleotide sequence may be DNA or RNA.

The term "polymorphism " of the present invention means a case where two or more alleles exist in one locus, and among polymorphic sites, only one single base is different from the single It is called single nucleotide polymorphism (SNP). Specific polymorphic markers have two or more alleles that exhibit an incidence of 1% or more, more preferably 5% or 10% or more in the selected population.

The term "allele " of the present invention refers to various types of a gene existing on the same locus of a homologous chromosome. Alleles are also used to represent polymorphisms, for example, SNPs have two kinds of bialles.

The term "backfat thickness" of the present invention refers to the thickness of the back side fat, and pigs generally have a lot of subcutaneous fat, but they are thickly adhered particularly to the shoulder and the back, The thickness of the back room is measured between the 11th to 12th ribs, the last frame and the first lumbar spine, and the average value is evaluated.

In the present invention, the backfat thickness is affected by the genotype of the SNP contained in the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, and when the allele of the 501st nucleotide of the SNP is all A, Of pigs were lower than those of normal pigs.

Specifically, pigs to be subjected to the reduction of the backfat thickness of the pigs are not particularly limited. Specifically, pigs may be crossbreeds of Jeju native pigs, land races, or Jeju native pigs and land races.

According to one embodiment of the present invention, the phenotype of the backfat thickness was analyzed using a Jeju native porcine conductor. As a result, the backfat thickness was more than 40 mm and more than 80 mm when 90 kg was reached 1). In addition, there was almost no edible part because of high fat content between loin and deltoid.

According to another embodiment of the present invention, it was found that the SNPs present in the 30 th to 35 th Mb of chromosome 5 are related to the reduction of the backfat thickness (FIG. 2, FIG. 3, and Table 1). As a result of analyzing the SNP, it was confirmed that the SNP (501 (A / G)) present in the polynucleotide of SEQ ID NO: 1 was associated with the reduction of back layer thickness ). As a result of analyzing the relationship between SNP and reduction of backfat thickness in actual pigs, it was found that the backfat thickness was reduced when the polynucleotide of SEQ ID NO: 1 (A / G) at 501 was all A (Table 2).

Therefore, it has been found that SNPs of SEQ ID NO: 1 can be used as a genetic marker capable of determining the backfat thickness reduction as well as determining the backfat thickness. The SNP markers can be used by the present inventors It was first identified.

As another embodiment for achieving the above object, the present invention provides a composition for determining the backfat thickness reduction of a pig, which comprises a preparation capable of detecting or amplifying the SNP marker for determining the backfat thickness reduction.

The term "agent capable of detecting or amplifying" as used herein means a preparation capable of specifically recognizing binding to a polymorphic site containing a SNP in a polynucleotide or amplifying the polymorphic site, Specifically, it may be a probe capable of specifically binding to a polymorphic site containing the SNP, a polynucleotide comprising the SNP marker, or a primer capable of specifically amplifying a complementary polynucleotide thereof.

More specifically, it may include, but is not limited to, a primer pair consisting of SEQ ID NO: 3 and SEQ ID NO: 4 capable of amplifying a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1.

In the present invention, a probe used to recognize and bind to a SNP marker includes a sequence complementary to a polynucleotide sequence including a SNP, and may be a DNA, RNA, or DNA-RNA hybrid form . Further, fluorescent markers, radiation markers, and the like can be additionally attached to the 5 'or 3' ends of the probe so as to be visually recognizable.

The term " primer " of the present invention means a base sequence having a short free 3 'hydroxyl group and can form base pairs with a complementary template, It means a short sequence functioning as a point. The primers used in the present invention for the amplification of SNP markers can be amplified by PCR using appropriate conditions in suitable buffers (for example, 4 different nucleoside triphosphates and polymerase such as DNA, RNA polymerase or reverse transcriptase) Stranded oligonucleotide that can serve as a starting point for the directed DNA synthesis. The appropriate length of the primer may vary depending on the purpose of use, but it may be generally used in a size of 15 to 30 nucleotides. The primer sequence is not necessarily completely complementary to the polynucleotide comprising the SNP marker or its complementary polynucleotide, and can be used if it is sufficiently complementary to hybridize.

The primers can also be modified, for example, by methylation, capping, substitution of nucleotides or modifications between nucleotides such as uncharged linkers (e.g., methylphosphonate, phosphotriester, phosphoramidate, Carbamates, etc.) or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).

According to another aspect of the present invention, there is provided a kit for determining the backfat thickness reduction of a pig comprising the composition. Specifically, the kit may be a PCR (Polymerase Chain Reaction) kit or a DNA analysis kit (for example, a DNA chip).

The kit of the present invention can be used to determine the genotype of SNP markers provided by the present invention by amplification or to determine the expression level of mRNA using the above composition to determine the back thickness reduction of pigs. The kit provided in the present invention may be a kit containing essential elements necessary for performing RT-PCR.

For example, in addition to the respective primer pairs specific for the haplotype gene, the RT-PCR kit can also include a test tube or other appropriate container, reaction buffer (pH and magnesium concentrations vary), deoxynucleotides (dNTPs), enzymes such as Taq polymerase and reverse transcriptase, DNase, RNAse inhibitors, DEPC-water, sterile water, and the like. It may also contain a primer pair specific for the gene used as a quantitative control.

As another example, the kit of the present invention may be a DNA chip kit for judging reduction of the backing thickness of a pig, which includes elements necessary for carrying out a DNA chip.

The term "DNA chip" of the present invention means one of DNA microarrays capable of confirming each base of hundreds of thousands of DNAs at a time.

The DNA chip kit is formed by attaching nucleic acid species to a glass surface, which is generally not larger than a flat solid support plate, typically a slide for a microscope, in a gridded array. The nucleic acid is uniformly arranged on the chip surface, It is a tool that enables multiple parallel hybridization reactions between the nucleic acid on the chip and the complementary nucleic acid contained in the treated solution on the chip surface.

According to another aspect of the present invention, there is provided a method of detecting a polynucleotide comprising: (a) amplifying a polymorphic site of the SNP marker from DNA of a sample isolated from an individual; And (b) determining the base of the amplified polymorphic site of step (a). At this time, DNA of the separated sample can be obtained from a sample isolated from an individual.

In the present invention, the term "individual" refers to a pig to which reduction of backfat thickness is to be confirmed. By analyzing the genotype of the polymorphic site of the SNP using the sample obtained from the pig, can do. Examples of the specimen include, but are not limited to, hair, urine, blood, various body fluids, separated tissues, separated cells or saliva, and the like.

The step of amplifying the polymorphic site of the SNP contained in the marker from the DNA of step (a) can be used in any method known to a person skilled in the art. For example, the target nucleic acid can be obtained by PCR amplification and purification thereof. Other ligase chain reaction (LCR) (Wu and Wallace, Genomics 4, 560 (1989), Landegren et al., Science 241, 1077 (1988)), transcription amplification (Kwoh et al., Proc. Natl. Acad. Sequence amplification based on nucleic acids (NASBA) can be used as well as self-sustaining sequence replication (Guatelli et al., Proc. Natl. Acad. Sci. USA 87, 1874 (1990)).

Determination of the base of the amplified polymorphic site in step (b) of the above method can be carried out by sequencing, hybridization by microarray, allele specific PCR, dynamic allelespecifichybridization, DASH), PCR extension analysis, PCR-SSCP, PCR-RFLP analysis or TaqMan technique, SNPlex platform (Applied Biosystems), mass spectrometry (e.g. MassenRAY system of Sequenom), mini- (E.g., BioRad), CEQ and SNPstream system (Beckman), Molecular Inversion Probe array technology (e.g. Affymetrix GeneChip), and BeadArray Technologies (e.g. Illumina GoldenGate and Infinium assay) But is not particularly limited thereto. One or more alleles in a SNP marker comprising the mutation site can be identified by the methods described above or other methods available to those skilled in the art to which the invention pertains. Determination of the base at such a mutation site can be carried out specifically through a DNA chip.

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

The sequencing analysis can be performed using a conventional method for nucleotide sequencing, and can be performed using an automated gene analyzer. In addition, allele-specific PCR means a PCR method in which a DNA fragment in which the mutation is located is amplified with a primer set including a primer designed with the base at the 3 'end at which the mutation site is located. The principle of the above method is that, for example, when a specific base is substituted by A to G, an opposite primer capable of amplifying a primer containing the A as a 3 'terminal base and a DNA fragment of an appropriate size is designed, When the base at the mutation position is A, the amplification reaction is normally performed and a band at a desired position is observed. When the base is substituted with G, the primer can be complementarily bound to the template DNA, And the amplification reaction is not performed properly due to the inability of complementary binding at the terminal. DASH can be carried out by a conventional method, specifically, by the method by Prince et al.

On the other hand, in the PCR extension analysis, first, a DNA fragment containing a base in which a single nucleotide polymorphism is located is amplified with a pair of primers, and all the nucleotides added to the reaction are deactivated by dephosphorylation, and a specific extension primer, dNTP And then performing a primer extension reaction by adding a mixture, a digoxinucleotide, a reaction buffer and a DNA polymerase. At this time, the extension primer has the 3 'end immediately adjacent to the 5' direction of the base where the mutation site is located, and the nucleic acid having the same base as the didyoxynucleotide is excluded in the dNTP mixture, and the didyoxynucleotide has a mutation ≪ / RTI > For example, when dGTP, dCTP and TTP mixture and ddATP are added to the reaction in the presence of substitution from A to G, the primer is extended by the DNA polymerase in the base in which the substitution has occurred, The primer extension reaction is terminated by ddATP at the position where the base first appears. If the substitution has not occurred, the extension reaction is terminated at the position, so that it is possible to discriminate the kind of base showing the mutation by comparing the length of the extended primer.

At this time, as a detection method, when the extension primer or the didyxin nucleotide is fluorescently labeled, the mutation is detected by detecting fluorescence using a gene analyzer (for example, Model 3700 manufactured by ABI Co., Ltd.) used for general nucleotide sequence determination And when the unlabeled extension primer and the didyxin nucleotide are used, the genetic variation of the SNP can be detected by measuring the molecular weight using MALDITOF (matrix assisted laser desorption ionization-time of flight) technique.

Specifically, if the allele of the 501st nucleotide sequence of SEQ ID NO: 1 contained in the marker of step (b) is all A, the pig containing the allele of the 501th base of SEQ ID NO: Can be judged to be reduced.

According to another aspect of the present invention, there is provided a method for fixing a SNP trait involved in reducing the back layer thickness of a pig, comprising the steps of: contacting a polynucleotide comprising a nucleotide sequence of SEQ ID NO: And a step of immobilizing all of the genes with A, wherein the thickness of the back layer of the pig is reduced.

According to another aspect of the present invention, there is provided a method of fixing SNP traits of a gene involved in reducing the back layer thickness of a pig, comprising the steps of: (1) isolating a polynucleotide comprising a nucleotide sequence of SEQ ID NO: Wherein all of the alleles of the pigs are fixed to A.

Specifically, the step of immobilizing the SNP trait may be carried out by crossing an individual having the SNP trait and selecting an individual having the desired trait. More specifically, an individual having a 501st base involved in reducing the backing thickness of a pig is crossed with an individual having A / A, and the 501st nucleotide of the polynucleotide consisting of SEQ ID NO: 1 among the progeny obtained therefrom is fixed with A / A By selecting a pig, pigs with reduced backfat thickness can be obtained. The pig may be a hybrid of Jeju native pig, land lace or Jeju native pig and land lace.

According to another aspect of the present invention, there is provided a microarray for determining the backfat thickness reduction of a pig comprising a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 of the pig.

The microarray may comprise DNA or RNA polynucleotides. The microarray comprises a conventional microarray except that the polynucleotide of the present invention is contained in the probe polynucleotide.

Methods for producing microarrays by immobilizing probe polynucleotides on a substrate are well known in the art. The probe polynucleotide means a polynucleotide capable of hybridizing, and means an oligonucleotide capable of binding to the complementary strand of the nucleic acid in a sequence-specific manner. The probe of the present invention is an allele-specific probe in which a polymorphic site exists in a nucleic acid fragment derived from two members of the same species and hybridizes to a DNA fragment derived from one member but does not hybridize to a fragment derived from another member . In this case, the hybridization conditions show a significant difference in the intensity of hybridization between alleles, and should be sufficiently stringent to hybridize to only one of the alleles. This can lead to good hybridization differences between different allelic forms. The probe of the present invention can be used for a method of determining the backing thickness reduction of pigs by detecting an allele. The determination method includes detection methods based on hybridization of nucleic acids such as Southern blot, and may be provided in a form pre-bonded to a substrate of a DNA chip in a method using a DNA chip. The hybridization can usually be performed under stringent conditions, for example, a salt concentration of 1 M or less and a temperature of 25 ° C or higher. For example, conditions of 5x SSPE (750 mM NaCl, 50 mM Na Phosphate, 5 mM EDTA, pH 7.4) and 25-30 < 0 > C may be suitable for allele-specific probe hybridization.

The process of immobilizing the probe polynucleotide on the substrate related to the determination of the backfat thickness reduction of the pig of the present invention can also be easily carried out using this conventional technique. In addition, hybridization of nucleic acids on a microarray and detection of hybridization results are well known in the art. The detection can be accomplished, for example, by labeling the nucleic acid sample with a labeling substance capable of generating a detectable signal comprising a fluorescent material, such as Cy3 and Cy5, and then hybridizing on the microarray and generating The hybridization result can be detected.

SNP markers capable of judging reduction of the backfat thickness of pigs according to the present invention can be used as a means for objectively evaluating the reduction of backfat thickness which is not distinguished by the naked eye and thus can contribute to establishment of the distribution order of pork meat .

Fig. 1 is a photograph showing the phenotype of the back side thickness of a conductor of Jeju native pig.
FIG. 2 is a graph showing the result of analyzing a genome wide association study (GWAS) using a DNA chip as a chromosome containing a gene related to the back thickness of a pig.
FIG. 3 is a schematic diagram showing various gene groups in the locus of the backswing thickness-related gene of chromosome 5.
4 is a nucleotide sequence of 1001 nucleotides centered on the SNP of chromosome 5.
FIG. 5 is a graph showing the results of confirming the genotype of the 501 (A / G) SNP contained in the polynucleotide of SEQ ID NO: 1 using pyrosequencing.

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  One. Jeju's traditional pig Backfill thickness  Phenotypic analysis

Using the Jeju pork carcass, the phenotype of backfat thickness was analyzed (Fig. 1). Fig. 1 is a view showing the phenotype of the backfill thickness of a conductor of Jeju native pig. Fig. As shown in Fig. 1, the backfill thickness was more than 40 mm when 90 kg was reached, and was much more varied up to 80 mm. In addition, there was almost no edible part because of high fat content between loin and deltoid.

Therefore, it was found that backfat reduction is necessary to utilize the traditional pig.

Example  2. Backfill thickness  relation Gene locus  Search

For all the subjects who were slaughtered more than about 1000 in 2nd generation descendants for the reference group of Jeju traditional pig and land race, three regions, ie, 4-5 rib region, 11-12 rib region, last thoracic region and lumbar region And the quantitative trait loci (QTL) were searched by analyzing GWAS with a large-capacity DNA chip (FIG. 2).

As a result, the highest QTL was confirmed between the last rib and the lumbar vertebra, and as shown in FIG. 2, the QTL was found to exist on chromosome 5.

Table 1 below shows a list of the top 10 SNPs having a high phenotypic association significance with respect to the back ground thickness in the chromosome 5. Of these, the SNPs located at 31,444,249 BP showed the highest significance with respect to the backbone thickness, and 1001 nucleotides (SEQ ID NO: 1) were obtained centering on the SNP (FIG. 4). As a result of confirming the obtained nucleotide sequence, it was confirmed that the SNP present at the base No. 501 of the ensured nucleotide sequence is related to the back thickness.

chromosome SNP BP P-VALUE LOG 5 ALGA0031489 31,444,249 5.39E-15 14.26849182 5 MARC0065416 31,339,549 5.63E-15 14.24926025 5 ASGA0025386 36,568,996 5.85E-15 14.2327699 5 ASGA0025185 29,697,723 2.46E-14 13.6099485 5 ASGA0025253 33,437,040 3.68E-14 13.43438828 5 ASGA0025355 34,915,630 3.87E-14 13.41217683 5 DRGA0005608 32,804,318 8.29E-14 13.08170749 5 ALGA0031991 58,351,846 1.04E-13 12.98505965 5 ASGA0025277 34,074,022 1.07E-13 12.97183558 5 ASGA0025237 32,661,210 1.12E-13 12.94923369

FIG. 3 is a diagram showing various gene groups in the locus of the backswing thickness-related gene of chromosome 5.

As shown in FIG. 3, various genes (MON2, ssc-let-7i, ENSSSCG00000021558, ENSSSCG00000029004, ENSSSCG00000025211, ENSSSCG00000021070, ENSSSCG00000024879, HSBP1, POLR2L, U6, TMEM5, SRGAP1, C12orf56, c12orf66, XPOT, ENSSSCG00000023715, TBC1D30, ENSSSCG00000030998, ENSSSCG00000000468, LEMD3, MSRB3, ENSSSCG00000024846, HMGA2, ENSSSCG00000018696, ENSSSCG00000000474, 5S_rRNA, LLPH, ENSSSCG00000000473, IRAK3, ENSSSCG00000000476, HELB, GRIP1, U1, ENSSSCG00000023676, ENSSSCG00000028869) 30 Mb to 35 Mb.

Example  3. Pyro-sequencing ( Phyro -sequencing Backfill thickness  Extraction and analysis of base sequences containing related SNPs.

The SNP having the highest significance in relation to the backfat thickness was identified as ALGA0031489 in the reference region of the Jeju native pig and the land lace mating group. Therefore, 1001 nucleotides (SEQ ID NO: 1) centered on the SNP were amplified and SNP To analyze the sequence.

First, in order to amplify the polynucleotide of SEQ ID NO: 1, PCR was performed using the DNA obtained from a number of pigs as a template and the following primers to obtain a polynucleotide fragment of SEQ ID NO: 1 (SEQ ID NO: 2, 299 bp) .

BF_F: 5'-GGC-ATT-CAT-GAG-TTC-AGT-ACA-CC-3 '(SEQ ID NO: 3)

BF_R: 5'-biotin-AAG-CTG-AAT-GGA-AAC-AAA-ACA-3 '(SEQ ID NO: 4)

The nucleotide sequence of the polynucleotide fragment of SEQ ID NO: 1 obtained using the above primer was determined and the genotype of the SNP contained therein was confirmed.

In order to confirm the 501 (A / G) SNP, the nucleotide sequence of the polynucleotide fragment was determined using the following primer (Mini-seq), and the primer of the input was used. / G) SNP was confirmed (Fig. 5).

5-GCT-CTT-AGC-TGG-CAA-ATT-CT-3 '(SEQ ID NO: 5)

Input: 5 '- [A / G] AACTCTTAT-3' (SEQ ID NO: 6)

FIG. 5 is a graph showing the results of confirming the genotype of the 501 (A / G) SNP contained in the polynucleotide of SEQ ID NO: 1 using pyrosequencing. As shown in FIG. 4, it was confirmed that the amplified polynucleotides of SEQ ID NO: 1 contained the AA, AG, and GG genotypes, respectively.

Example  4. Genotype of chromosome 5 SNP Backfat  Association analysis

The changes in isthmus thickness according to the genotype of confirmed SNPs in Example 3 were compared (Table 2).

Analysis of association between SNP (ALGA0031489) genotype and backfat thickness genotype Analysis Backing Thickness (mm) AA 326 24.8282 + - 7.1571 AG 525 26.7524 + - 7.3770 GG 194 26.7474 + - 7.4760

As shown in Table 2, when the genotype of the SNP (ALGA0031489) was AA, the backfat thickness was about 24.8282 mm, and when the genotype was AG or GG, it was about 26.7524 and 26.7474 mm, And it was confirmed that it is the lowest one of the genotypes.

In addition, since there is no difference in backfat thickness according to genotype of AG or GG, it can be deduced that genetic aspect is dominant effect rather than additive effect.

Therefore, when mating with consideration of the genotype, pigs with reduced backfat thickness can be selected and cultivated.

In summary, among the polynucleotides of SEQ ID NO: 1, SNP No. 501 is directly related to the reduction of the back layer thickness of pigs. Therefore, when all or part of the SNPs are detected, .

It is to be understood that the invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Novel SNP marker for discriminating reduction of backfat          thickness and use thereof <130> KPA151110-KR <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 1001 <212> DNA <213> Artificial Sequence <220> <223> SNP nucleotide <400> 1 gatcctattt ttagaatttc cagatattag tgttataaga agttagtaag cttgctactc 60 attacagtga gtgaaagctt ttatagataa tattaatggt tacgctatat ttaagtcata 120 tactggacca tggatcaata cagatctcaa atttttcgtt ggtaaacaaa gacaattcag 180 ttactttttc ataatgccct ttctcccact ccagccaagc tagtatgagg gtgggaacat 240 tccatcataa acactgaact gggaagatgt agagtcatgg cagttatgct ggccacagaa 300 atggatggat cgcagcagct gtttaaaagt aagaggcatt catgagttca gtacacctat 360 tttataaaac tgattttttc taaaactatt tattaaccaa aaatgatata gaattcagta 420 actgggagcc atgtggactg ttcagctttt gcgcttactc tttatcaagg cattggccag 480 gctcttagct ggcaaattct raactcttat aactcaatat aacaatagtg tgtctgtaca 540 tagcatttat tcagtagcat gtgttgaata tattaaaaaa taaacaataa ttatattgga 600 ttctaaaaaa tatgttttgt ttccattcag cttagaactc aatatttgaa ttatttctct 660 gggaatcatt ttttaagtat tgaaagtact tcatgggcag gaagttttta actgagccaa 720 gcagaaaaag aataaatact aaaaattaga aactctagtg tatgctaaga aatcttgaag 780 agcaagtcac tgttttaaaa ttctatatat gagctcctta aatatgttca ttcttcaatt 840 aatggatatt tgtgattaat gattgaataa taactttagg atcctaaaac acatctttca 900 tcatgcatga atatactata atatctcata taagccctac tttatataat gtgtagctag 960 aaagttacaa ttaaattgaa tttttgtaac ttgattcata t 1001 <210> 2 <211> 299 <212> DNA <213> Artificial Sequence <220> <223> SNP nucleotide fragment by PCR <400> 2 ggcattcatg agttcagtac acctatttta taaaactgat tttttctaaa actatttatt 60 ggatgatgat ttactcttta tcaaggcatt ggccaggctc ttagctggca aattctraac tcttataact 180 caatataaca atagtgtgtc tgtacatagc atttattcag tagcatgtgt tgaatatatt 240 aaaaaataaa caataattat attggattct aaaaaatatg ttttgtttcc attcagctt 299 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> BF_F primer <400> 3 ggcattcatg agttcagtac acc 23 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> BF_R primer <400> 4 aagctgaatg gaaacaaaac a 21 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mini-seq primer <400> 5 gctcttagct ggcaaattct 20 <210> 6 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 raactcttat 10

Claims (13)

A polynucleotide comprising a nucleotide sequence of SEQ ID NO: 1, wherein the nucleotide at position 501 is A or G, the polynucleotide consisting of 5 to 1000 consecutive bases comprising the nucleotide at position 501, or a complementary polynucleotide thereof. SNP markers for judgment of reducing the back room thickness of pigs.
The method according to claim 1,
The pigs are SNP markers for judging reduction of the back layer thickness of pigs, which are Jeju native pigs, land races or Jeju native pigs and land race hybrids
A composition for judging reduction in backfat thickness of pigs comprising the agent according to claim 1 or 2 capable of detecting or amplifying SNP markers for determining the backfat thickness reduction of pigs.
The method of claim 3,
Wherein the preparation comprises a primer pair consisting of SEQ ID NOS: 3 and 4 capable of amplifying a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1.
A kit for determining the reduction of the backfat thickness of a pig, comprising the composition of claim 3.
6. The method of claim 5,
Wherein the kit is an RT-PCR kit or a DNA chip kit.
(a) amplifying the polymorphic site of the SNP marker of claim 1 from DNA of a sample isolated from the individual; And
(b) determining the base of the amplified polymorphic site of step (a).
8. The method of claim 7,
The polynucleotide of SEQ ID NO: 1 judges that the backbone thickness of the pig is reduced compared to when the allele of the 501 th base is all A in the polynucleotide of SEQ ID NO: 1, A method for determining the thickness reduction.
A method for fixing an SNP trait involved in reducing the backing thickness of a pig, comprising the step of fixing all the alleles of the 501st nucleotide of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 to A, By weight or less.
10. The method of claim 9,
Wherein the step of immobilizing the SNP trait is carried out by crossing a subject having the SNP trait and selecting an individual having a trait of interest, wherein the thickness of the back trabecula of the pig is reduced.
A method for fixing an SNP trait of a gene involved in reducing the backing thickness of a pig, comprising the step of fixing all the alleles of the 501st nucleotide of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 to A, A method for reducing the thickness of a room.
12. The method of claim 11,
Wherein the step of immobilizing the SNP trait is carried out by crossing an individual having the SNP trait to select an individual having the desired trait.
A microarray for reducing the backfat thickness of pigs comprising the polynucleotide of claim 1.

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