KR20160054147A - SNP for regulating cortisol secretion level of Pig and use thereof - Google Patents

Abstract

The present invention relates to a composition for determining a level of meat quality traits of a pig containing an agent capable of detecting a single nucleotide polymorphism (SNP) of an intron region of inositol-tetrakisphosphate 1-kinase (ITPK1) gene for regulating a cortisol secretion level of a pig; a kit for determining a level of meat quality traits of a pig, containing the composition; a method of determining a level of meat quality traits of a pig; a method of producing a pig in which a level of meat quality traits of a pig is increased; a method of reducing a cortisol secretion level of a pig; a haplotype marker for determining a level of meat quality traits of a pig; and a microarray for determining a level of meat quality traits of a pig containing the haplotype marker. According to the present invention, the haplotype marker is a specific gene marker determining a level of meat quality traits through an amount of secretion of cortisol, stress hormone secreted by a pig. In addition, the haplotype marker is used as a means for objectively evaluating a level of meat quality traits of a pig not distinguished with the naked eye, thereby contributing to establishment of a distribution order of a pig meat.

Description

SNPs that regulate cortisol secretion levels in pigs and their uses {SNP for regulating cortisol secretion levels of Pig and use thereof}

The present invention relates to a SNP regulating the level of cortisol secretion in pigs and a use thereof. More particularly, the present invention relates to a SNP of the intron region of an inositol-tetrakisphosphate 1-kinase (ITPK1) gene controlling the cortisol secretion level of pigs nucleotide polymorphism), a kit for determining a meat quality trait level of a pig, a method for determining a meat quality trait level of a pig, a meat quality trait of a pig, The present invention relates to a method for producing pigs with increased levels, a method for lowering the cortisol secretion level of pigs, a haplotype marker for judging the level of meat quality of pigs, and a microarray for determining the meat quality trait level of pigs including the haplotype marker.

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, methods for accurately determining the meat quality traits of pigs have not yet been developed.

Under these circumstances, the inventors of the present invention have conducted intensive research to develop a method for judging the quality of pork meat based on the meat quality of pigs. As a result, it has been found that a gene regulating the secretion amount of cortisol, The present inventors confirmed that the quality of pigs can be judged by judging the level of meat quality traits of genetically determined pigs when they are used, and completed the present invention.

It is an object of the present invention to provide a composition for judging the quality of meat quality of pigs.

Another object of the present invention is to provide a kit for judging a meat quality trait level of a pig, which comprises the above composition.

It is a further object of the present invention to provide a method for determining the meat quality trait level of a pig.

It is another object of the present invention to provide a method for producing pigs with increased meat quality traits of pigs.

It is another object of the present invention to provide a method for lowering the cortisol secretion level of a pig.

It is another object of the present invention to provide a haplotype marker for judging the quality of meat quality of pigs.

Yet another object of the present invention is to provide a microarray for judging the quality of a meat quality of a pig, which comprises an haplotype marker for judging a meat quality trait level of the pig.

The present inventors paid attention to cortisol, a stress-resistance hormone, while carrying out various studies to develop a method for judging the quality of pork meat based on the meat quality of pigs. Generally, pigs are weak to stress because they do not have sweat glands. Sudden deaths occur when they are exposed to excessive stress. In addition, the secretion of cortisol, a stress-resistant hormone in the endocrine system, increases. It is known that when cortisol is excessively secreted due to excessive stress, the meat is pale and the juice is discharged in a large quantity, which adversely affects meat quality and meat taste. Therefore, if the gene capable of controlling the secretion amount of cortisol can be identified, it is expected that the quality of pork meat can be judged more effectively. As a result of searching for a gene related to the cortisol secretion amount, it was found that three SNPs present in the intron region (SEQ ID NO: 2) of ITPK1 (inositol-tetrakisphosphate 1-kinase) gene located at 119 Mb to 121 Mb of chromosome 7 correspond to cortisol And that the SNP can be used as a gene marker capable of predicting the secretion amount of cortisol.

In one embodiment of the present invention, the polynucleotide having the nucleotide sequence of SEQ ID NO: 2 is A or G at position 457, T or C at position 470, and C Or A, and is capable of detecting or amplifying a polynucleotide consisting of 5 to 1,000 consecutive bases comprising a single nucleotide polymorphism (SNP) located at the 457, 470 or 501 base, or a complementary polynucleotide thereof A composition for judging the level of meat quality of pigs.

The polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2 is a polymorphic sequence comprising a polymorphic site of the gene involved in the regulation of the cortisol secretion level of the pig. The polymorphic sequence includes the SNP in the polynucleotide sequence Polymorphic < / RTI > site. 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). Preferred polymorphic markers have two or more alleles with a frequency of occurrence 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 "agent capable of detecting or amplifying a polynucleotide" of the present invention refers to an agent capable of specifically recognizing binding to a polymorphic site containing a SNP in a polynucleotide and amplifying the polymorphic site As a preparation, specifically, a probe capable of specifically binding to a polymorphic site containing a SNP, a polynucleotide comprising the SNP marker, or a primer capable of specifically amplifying a complementary polynucleotide of the polynucleotide may be used.

More specifically, it may include, but is not limited to, primer pairs consisting of SEQ ID NOS: 3 and 4 capable of amplifying the polynucleotide of SEQ ID NO: 2.

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.).

The term "meat quality trait " of the present invention means various expression traits indicating the state of slaughtered products obtained from swine. The expression traits include, but are not limited to, muscle fat content, meat color, water holding capacity, shearing force and the like.

In the present invention, the meat quality trait is affected by the genotype of the three SNPs contained in the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2, wherein the 457th nucleotide of the SNP is G, the 470th nucleotide is C , And when the 501st base is A, it can be judged that the pig containing the same has a lower level of cortisol secretion than the general pig. It is known that cortisol is a stress hormone, and when its secretion amount is increased, the color of pork is reduced, poultry is pale, and the juice contained in meat is rapidly released, resulting in deterioration of meat quality and meat taste. Therefore, when the 457th nucleotide of the SNP is G, the 470th nucleotide is C, and the 501st nucleotide is A, the pig containing the same has a lower level of cortisol secretion than the common pig, It can be judged.

In particular, pigs to be used for judging the level of meat quality traits through the cortisol secretion level of the pigs are not particularly limited, but may be preferably hybrids of Jeju native pig, duroc or jeju native pig and duroc.

According to one embodiment of the present invention, when a pig gene related to the secretion amount of cortisol was searched, it was confirmed that SNPs present in 119 Mb to 121 Mb of chromosome 7 were related to the secretion amount of cortisol (FIGS. 1 and 2) (A / G), 470 (T / C) and 501 (T / C) residues in the intron region (SEQ ID NO: 2) of the inositol-tetrakisphosphate 1-kinase gene of ITPK1 (Fig. 3, 4a, 4b and 4c). As a result of analyzing the relationship between the amount of cortisol secreted and the SNP in actual pigs, it was found that the ratio of cortisol to cortisol was 457 The cortisol secretion amount is minimized when the A / G SNP is A, the 470 T / C SNP is T and the genetic trait of the 501 (C / A) SNP is C, / G) SNP of G, 470 (T / C) SNP of C and 501 (C / A) SNP of genotype A showed that cortisol secretion was maximized (Table 1).

Therefore, the SNP containing the SNP of SEQ ID NO: 2 can not only predict the secretion amount of cortisol but also can be used as a genetic marker capable of evaluating the meat quality of pigs directly affected by the cortisol secretion amount And the gene markers were first identified by the present inventors.

As another embodiment for achieving the above object, the present invention provides a kit for determining a meat quality trait level of a pig comprising the composition. Specifically, the kit may be, but not limited to, a PCR (Polymerase Chain Reaction) kit or a DNA analysis kit (for example, a DNA chip).

The kit of the present invention can determine the level of meat quality of a pig by confirming the genotype of the three SNP markers provided by the present invention using the above composition by amplification or by checking the expression level of mRNA. As a specific example, 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 the quality of a pig meat quality trait including essential 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.

(A) a polymorphic site of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2 or its complementary polynucleotide from the DNA of the sample isolated from the individual, ; And (b) determining the base of the amplified polymorphic site. At this time, DNA of the separated sample can be obtained from a sample isolated from an individual.

The term "individual" of the present invention refers to a pig to which a meat quality trait level is to be confirmed. Using the sample obtained from the pig, the genotype of the polymorphic site of the SNP is analyzed to determine the meat quality trait level of the pig . 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 haplotype marker from the DNA of step (a) may be any method known to those 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 allele- PCR-SSCP, PCR-RFLP analysis or TaqMan technique, SNPlex platform (Applied Biosystems), mass spectrometry (e.g., Sequenom's MassARRAY system), mini-sequencing method , The Bio-Plex system (BioRad), the CEQ and SNPstream system (Beckman), the Molecular Inversion probe array technology (e.g. Affymetrix GeneChip), and BeadArray Technologies (e.g. Illumina GoldenGate and Infinium analysis) But is not limited thereto. One or more alleles in a haplotype that includes 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. The base at such a mutation site can be determined preferably 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 polynucleotides of step (1) from the analysis results.

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 performed by a conventional method, preferably by a method such as 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) 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 a MALDI-TOF (matrix assisted laser desorption ionization-time of flight) technique .

Preferably, in the base sequence determined in step (b), when the 457th base of SEQ ID NO: 2 contained in the haplotype marker is A, the 470th base is T, and the 501st base is C, It can be concluded that pigs have a lower level of cortisol secretion than common pigs and have excellent meat quality traits.

In another aspect of the present invention, the present invention provides a method of isolating a SNP trait of a gene involved in the cortisol secretion level of a porcine, comprising the steps of: contacting the 457th nucleotide of the polynucleotide comprising the nucleotide sequence of SEQ ID NO: The secretion level of the cortisol secreted from the pig is reduced, which comprises fixing the gene to A, fixing the allele of the 470th base to T, or fixing allele of the 501th base to C, And provides a method for producing an excellent pig.

In another aspect of the present invention, the present invention provides a method of isolating a SNP trait of a gene involved in the cortisol secretion level of a porcine, comprising the steps of: contacting the 457th nucleotide of the polynucleotide comprising the nucleotide sequence of SEQ ID NO: A step of fixing the gene to A, fixing the allele of the 470th base to T, or fixing the allele of the 501st base to C, in order to reduce the secretion level of the cortisol secreted in the pig .

In particular, the step of immobilizing the SNP trait may be carried out by crossing an individual having all of the SNP traits, and selecting an individual having the desired trait. More specifically, individuals having A / A, T / T, and C / C bases at 457, 470, and 501 bases, which are genes involved in the cortisol secretion level of pigs, were crossed, and in the progeny obtained therefrom, Pigs with a reduced level of cortisol secretion in pigs can be obtained by selecting pigs in which polynucleotides 457, 470 and 501 of the polynucleotide are fixed at A / A, T / T and C / C, respectively. The pig may be a hybrid of Jeju native pig, Duroc or Jeju native pig and duroc.

As another embodiment for achieving the above object, the present invention provides a method for determining a meat quality trait level of a pig including SNP (single nucleotide polymorphism), which is a single nucleotide polymorphism of the intron region of the ITPK1 (inositol-tetrakisphosphate 1-kinase) Provides a haplotype marker.

Specifically, the haplotype marker for determining the meat quality trait level of the pig is A or G at the 457th base of the intron region (SEQ ID NO: 2) of the inositol-tetrakisphosphate 1-kinase (ITPK1) gene and the 470th base is T or C , And the polynucleotide of which the 501st base is C or A or a complementary polynucleotide thereof.

The term "ITPK1 (inositol-tetrakisphosphate 1-kinase) gene" of the present invention means a gene encoding ITPK1 (inositol-tetrakisphosphate 1-kinase) involved in the inositol signaling pathway regulating the chlorine channel activated by calcium do. The nucleotide sequence of the ITPK1 gene can be obtained from a known database such as NCBI's GenBank. NM_001142593, NM_172584, and the like, preferably, a polynucleotide of SEQ ID NO: 1.

The term "haplotype marker" of the present invention refers to a marker comprising a combination of alleles of each locus on the same chromosome when polymorphic loci of the polymorphism exist on one chromosome it means. By using the haplotype, the gene region can be identified, and the alleles forming the haplotype can have a chain disequilibrium relationship.

In the present invention, the haplotype may be all or some of the polynucleotides including the SNP region of the intron region of the ITPK1 gene, more preferably the polynucleotide of SEQ ID NO: 2 The 457th nucleotide at the SNP position is A or G, the 470th nucleotide is T or C, and the nucleotide at position 457 is A or G. The polynucleotide of SEQ ID NO: Can be a haplotype marker that can determine the meat quality trait level of a pig, including a polynucleotide whose 501th base is C or A, or a complementary polynucleotide thereof.

In the haplotype marker of the present invention, when the allele of the 457th base is A, the allele of the 470th base is T or the allele of the 501th base is C, the allele of the 457th base is G, the 470th base , The secretion level of cortisol secreted from the pig is lower than that of the allele of C or the allele of the 501 < th > base is A, and the meat quality of the pig is excellent.

In another aspect of the present invention, there is provided a microarray for determining a meat quality trait level of a pig including an haplotype marker for determining the meat quality trait level 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 determining the level of meat quality 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.

Immobilization of the probe polynucleotide on the substrate associated with the determination of the meat quality trait level 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.

The haplotype marker of the present invention is a specific gene marker that determines the level of meat quality traits through the secretion amount of cortisol, which is a stress hormone secreted from pigs, and is used as a means of objectively evaluating the level of meat quality traits Therefore, it will contribute to establishing the order of distribution of pork meat.

FIG. 1 is a graph showing the results of genome wide association study (GWAS) analysis of a chromosome containing a gene related to cortisol secretion from pigs.
Fig. 2 is a chart showing the results of searching for genes related to cortisol secretion in pigs. Fig.
3 is an electrophoresis picture showing the polynucleotide fragment of SEQ ID NO: 2 amplified from a number of pigs.
FIG. 4A is a graph showing the result of confirming the genotype of the 457th (A / G) SNP contained in the polynucleotide of SEQ ID NO: 2.
4B is a graph showing the result of confirming the genotype of 470 (T / C) SNP contained in the polynucleotide of SEQ ID NO: 2.
4C is a graph showing the result of identifying the genotype of the 501 (C / A) SNP contained in the polynucleotide of SEQ ID NO: 2.

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: Cortisol  Search for genes related to secretion

The content of cortisol in the serum of the second-generation offspring of pigs with reference to the Jeju native pig and the duroc cross was measured and the locus that determines the cortisol secretion amount through GWAS (genome wide association study) analysis was searched (Fig. 1) .

FIG. 1 is a graph showing the results of genome wide association study (GWAS) analysis of a chromosome containing a gene related to cortisol secretion from pigs. As shown in FIG. 1, it was confirmed that the gene related to the cortisol secretion of pigs was present at the end of chromosome 7.

In order to identify the gene related to the secretion amount of cortisol among the genes located at the end of the determined chromosome 7, the content of cortisol in the serum of the second generation progeny pig was measured using a large-capacity DNA chip, and the correlation between the genotype and phenotype (Fig. 2).

Fig. 2 is a chart showing the results of searching for genes related to cortisol secretion in pigs. Fig. As shown in FIG. 2, it was confirmed that about 65 SNPs in the chromosome 7 were associated with cortisol secretion in pigs. In particular, it was confirmed that 10 genes showing a high degree of significance exist in 119 Mb to 121 Mb of chromosome 7 .

Example  2: Cortisol  Verification of candidate gene related to secretion amount

Example  2-1: Candidate gene securing

As a result of screening for the gene related to the secretion of cortisol among the genes existing in the 119Mb to 121Mb of the chromosome 7 determined in Example 1-1, it was found that the inositol-tetrakisphosphate 1-kinase (ITPK1) The gene (SEQ ID NO: 1) was predicted to be directly related to stress and was selected as a candidate gene. As a result of analyzing the nucleotide sequence of the gene and the amino acid sequence of the protein expressed therefrom, it was confirmed that the mutation sequence exists at 141 and 209 but does not affect the amino acid sequence.

Based on the gene analysis data of 8 selected individuals, the SNPs located at 121,017,897b on chromosome 7 showed the highest correlation with the cortisol secretion trait And 1001 nucleotide sequences (SEQ ID NO: 2) containing the SNP as a center were obtained. (A / G), 470 (T / C) and 501 (C / A) bases of the ensured nucleotide sequence, and the nucleotide sequence of the obtained nucleotide sequence was confirmed to be the sequence of the intron region of the ITPK1 gene. Were associated with cortisol secretory traits.

Example  2-2: PCR  analysis

The above SEQ ID NO: 2 was amplified and the SNP sequence contained therein was analyzed.

First, to amplify the polynucleotide of SEQ ID NO: 2, a polynucleotide fragment of SEQ ID NO: 2 was obtained (FIG. 3) by performing PCR using the following primers, using chromosomal DNA obtained from a number of pigs as a template.

CTC-ACA-A-3 ' (SEQ ID NO: 3)

TGT-TTG-C-3 ' (SEQ ID NO: 4): 5'-biotin-ACC-AGA-TCT-GAG-CTG-

3 is an electrophoresis picture showing the polynucleotide fragment of SEQ ID NO: 2 amplified from a number of pigs.

The nucleotide sequence of the obtained polynucleotide fragment of SEQ ID NO: 2 was determined and the genotype of the SNP contained therein was confirmed.

First, in order to confirm the 457th (A / G) SNP, the nucleotide sequence of the polynucleotide fragment was determined using the following primer (mini_seq1), and the nucleotide sequence of the 457th (A / G) SNP was confirmed (Fig. 4A).

GTG-CCT-GTC-TT-3 ' (SEQ ID NO: 5)

Input 1: 5 '- [A / G] CGTTTACC-3' (SEQ ID NO: 6)

FIG. 4A is a graph showing the result of confirming the genotype of the 457th (A / G) SNP contained in the polynucleotide of SEQ ID NO: 2. As shown in FIG. 4A, it was confirmed that the amplified polynucleotide of SEQ ID NO: 2 contained AA, AG, and GG genotypes, respectively.

Next, in order to confirm the 470th (T / C) SNP, the nucleotide sequence of the polynucleotide fragment was determined using the following primer (mini_seq2), and the primer of input 2 was used to determine 470 / C) SNP genotype (Fig. 4B).

TAT-TAC-CCT-GC-3 ' (SEQ ID NO: 7)

Input 2: 5 '- [C / T] TGGGACTC-3' (SEQ ID NO: 8)

4B is a graph showing the result of confirming the genotype of 470 (T / C) SNP contained in the polynucleotide of SEQ ID NO: 2. As shown in FIG. 4B, it was confirmed that the amplified polynucleotides of SEQ ID NO: 2 contained CC, CT and TT genotypes, respectively.

Finally, in order to confirm the 501 (C / A) SNP, the nucleotide sequence of the polynucleotide fragment was determined using the following primer (mini_seq3), and the primer of the input 3 was used to identify 501 / A) SNP genotype (Fig. 4C).

ATCC-TGG-AT-3 '(SEQ ID NO: 9)

Input 3: 5 '- [A / C] AGAAACAG-3' (SEQ ID NO: 10)

4C is a graph showing the result of identifying the genotype of the 501 (C / A) SNP contained in the polynucleotide of SEQ ID NO: 2. As shown in FIG. 4C, the amplified polynucleotide of SEQ ID NO: 2 contained AA, AC, and CC genotypes, respectively.

As a result of analyzing the genotype of each SNP, it was confirmed that the genotypes of the three SNPs were completely related. That is, the genotypes that can be formed by the combination of three SNPs can theoretically represent 8 genotypes (ATC, ATA, ACC, ACA, GTC, GTA, GCC and GCA by 457-470-501) It was confirmed that the genotypes actually detected were only two genotypes (457-470-501: ATC and GCA), and the genotypes of the three SNPs were found to be completely mutually independent and not mutually independent.

Example  2-3: Cortisol  Secretion analysis

The changes in cortisol secretion according to the genotypes of the three SNPs were compared according to the result that the genotypes of the three SNPs identified in Example 2-2 were completely related (Table 1).

Analysis of association between SNP genotype and cortisol secretion SNP location genotype Analysis Cortisol secretion (unit) 457 AA 73 16.01 + - 10.79 AG 162 23.80 ± 12.81 GG 69 28.75 ± 18.74 470 TT 72 16.00 ± 10.76 TC 161 23.83 + - 12.82 CC 67 28.64 ± 18.71 501 CC 73 16.01 + - 10.79 CA 164 23.84 ± 12.84 AA 69 28.75 ± 18.74

As shown in Table 1, when the genetic trait of the 457th (A / G) SNP is AA, the genetic traits of 470 (T / C) SNP are TT and the genetic traits of 501 (C / When the trait was CC, all showed about 16 units of cortisol secretion; When the genetic trait of the 457th (A / G) SNP is GG and the genetic trait of the 470th (T / C) SNP is CC and the genotype of the 501th (C / A) SNP is AA Showed about 28.70 units of cortisol secretion; When the genetic trait of the 457th (A / G) SNP is AG, when the genotype of the 470th (T / C) SNP is TC and the genotype of the 501st (C / A) SNP is CA It was confirmed that the amount of cortisol secreted was about 23.80 units.

(A / G) SNP is A, 470 (T / C) SNP is T and 501 (C / A) SNP is A, (A / G) SNP is G, 470 (T / C) SNP is C and 501 (C / A) SNP is the genetic trait of SNP 457 In this case, the secretion amount of cortisol was maximized.

(A / G), 470 (T / C) and 501 (C / A) SNPs of the polynucleotides of SEQ ID NO: 2 including the intron region of the ITPK1 gene of pigs, It was found that when the whole or a part of the SNP was detected, the amount of cortisol, which is a stress-resistant hormone, was small and the pigs having improved meat quality could be distinguished.

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> SNP for regulating cortisol secretion level of Pig and use          the <130> KPA140941-KR <160> 10 <170> Kopatentin 2.0 <210> 1 <211> 1351 <212> DNA <213> Artificial Sequence <220> <223> SNP nucleotide <400> 1 atgcagacct ttctgaaagg gaagagagtt ggctactggc tgagcgagaa gaaaatcaag 60 aagctgaatt tccaggcctt cgccgagctg tgcaggaagc gagggatcga agtcgtgcag 120 ctgaacctca gccggccgat ygaggagcag ggccccctgg atgtcatcat ccacaagctg 180 accgatgtca tcctcgaagc cgaccagaay gacagccagt ccctggagct ggtgcacagg 240 ttccaggaat acatcgatgc ccaccccgag accattgtcc tggaccccct tcctgccatc 300 aggaccctgc tcgatcggtc caagtcctac gagctcatcc ggaagatcga agcctacatg 360 caagatgaca ggatctgctc gccgcccttc atggagctca cgagcctgtg cggggacgac 420 accatgcagc tcctggagaa gaacggcctg gccttcccct tcatttgcaa aaccagagtg 480 gctcatggca ccaactccca cgagatggcc atcgtgttca accaggaggg cttgagcgcc 540 atccagccgc cctgcgtggt ccagaatttc atcaaccaca acgccgtcct gtacaaggtg 600 tttgtagtcg gcgagtccta caccgtggtc cagagaccct cgctcaagaa cttctccgcg 660 ggcacgtcag accgtgagtc catcttcttc aacagccaca acgtgtcgaa gccagagtcg 720 tcgtcggtcc tgaccgcgct ggacaagatc gagggcgtgt tcgagcgacc gagcgacgag 780 gtcatccggg cgctgtcccg ggccctgcgg caggcgctgg gcgtgtcgct cttcgggatc 840 gacatcatta tcaacaacca gacggggcag cacgcggtca tcgacatcaa cgccttcccg 900 ggctacgagg gcgtgagcga gttcttcacc gacctcctga accacatcgc ctcggtgctg 960 cagggccaga gcgcggccgc ggcggcctcg ggggacgtgg ccccgctgag gcacagcaag 1020 ctcctggcag agcaggcggg tggcctggcg gctgagcgga cgtgcagcgc cagccccggc 1080 tgctgcagca gcatgatggg ccaggagccg ccctggacgt ccgaggctga tgtgggcggc 1140 gtgggcgcgg gcagcaccgc caagctgccg caccagagac tcggctgcac cgccgccgtg 1200 tcgcccagct tccagcagca ttgcgtggcc tcgctggcca ccaaggcctc ctcccagtag 1260 ccacggagcc cgggacccag aggggtggcg cacggagcac acgcgctggg caagcagctc 1320 ccgacggtgc cgctactact aagaattccc c 1351 <210> 2 <211> 1001 <212> DNA <213> Artificial Sequence <220> <223> SNP nucleotide <400> 2 cattcatccc agttcgttgg ggaaagccca cctccccatt tggtaaccag cctccttctc 60 tctagcagcc cttcctgtcc ccaacccaag gaagaggctg tcacatggga acacggaagt 120 gccgcactag gggtaggacg ggacctcact gccagaggct aaggaaggga gggaagggca 180 gggcagggca gggcagggca gggcagggca gggcagggca gggctgggca gagctgggct 240 caggggagca gcactgctct actttttgac ctggagctgg gtacatggtg tatatagctg 300 gttggtaaaa actgatcaag ctgctcacaa tgtgtagacc ttactctgtg tatattatac 360 tgcaattaaa aaaatagaag gaagagagaa agcaagcact gaaccaaaga gcaaggccgt 420 gggttgtggt cccagctctg cagtgtggcc tgtcttrcgt ttaccctgcy tgggactcca 480 gctttccaca tgcggtggat magaaacaga aaatggggag tttctattgt ggcgcagcag 540 aaacaaatcc aactaggaat catgaggttg caggttcgat ccctggtctt gctcagtggg 600 ttaaggatcc agtgttgccg tgagctatgg tgtaggtttg caaacacagc tcagatctgg 660 tgtggctgtg gcgcaggcca aaagcaacag ctctaattag actcctagcc tgagaacctc 720 cacatgccac gggtgcagcc ctagaaggac aaaagaccaa aaagaaaaaa agaaaaaaga 780 aaagaaatgg aaaacagggc ccagttccac caggcctccc ccacagccct ggcagccacc 840 accctgagca tggtggcggc gggcacttgg gaattacagc aaccgtttgt gctcatctca 900 gcaggacttc agaatcctgg ttgccacttg gttctagaag taggtaagtg tcggcactca 960 agacaaacac cacaggccgt gcacagaaga aatcagagct g 1001 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 aaactgatca agctgctcac aa 22 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 accagatctg agctgtgttt gc 22 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 tctgcagtgt ggcctgtctt 20 <210> 6 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 rcgtttacc 9 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 ctgtcttacg tttaccctgc 20 <210> 8 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 ytgggactc 9 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 gctttccaca tgcggtggat 20 <210> 10 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 magaaacag 9

Claims (14)

The 457th base is A or G in the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2, the 470th base is T or C, the 501st base is C or A, the SNP located at the 457th, 470th or 501th base a composition capable of detecting or amplifying a polynucleotide consisting of 5 to 1,000 contiguous bases comprising a single nucleotide polymorphism or a complementary polynucleotide thereof.
The method according to claim 1,
Wherein said preparation comprises a primer pair consisting of SEQ ID NOS: 3 and 4, capable of amplifying a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2.
The method according to claim 1,
Wherein the pig is a hybrid of Jeju native pig, Duroc or Jeju native pig and duroc.
The method according to claim 1,
Wherein the meat quality of the pig is judged to be excellent as the secretion level of cortisol secreted from the pig is lowered.
A kit for determining a meat quality trait level of a pig, comprising the composition of any one of claims 1 to 4.
(a) amplifying a polymorphic site of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2 or its complementary polynucleotide from the DNA of the sample separated from the individual; And
(b) determining the base of the amplified polymorphic site.
The method according to claim 6,
In the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2, when the allele of the 457th base is A, the allele of the 470th base is T or the allele of the 501th base is C, the cortisol secreted from the pig The secretion level is lowered and the meat quality of the pig is judged to be excellent.
The method according to claim 6,
Wherein said pig is a hybrid of Jeju native pig, Duroc or Jeju native pig and duroc.
A step of fixing the allele of the 457th base of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2 to A or the allele of the 470th nucleotide of the nucleotide sequence of SEQ ID NO: Is fixed to T, or the allele of the 501st base is fixed to C, whereby the secretion level of the cortisol secreted from the pig is lowered and the meat quality trait is excellent.
10. The method of claim 9,
Wherein the step of immobilizing the SNP trait is carried out by crossing an individual having all of the SNP traits and selecting an individual having the desired trait.
A step of fixing the allele of the 457th base of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2 to A or the allele of the 470th nucleotide of the nucleotide sequence of SEQ ID NO: To T, or to fix the allele of the 501 &lt; th &gt; base to C, in order to reduce the secretion level of cortisol secreted from the pig.
12. The method of claim 11,
Wherein the step of immobilizing the SNP trait is carried out by crossing an individual having all of the SNP traits and selecting an individual having the desired trait.
The polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2 is A or G at position 457, T or C at position 470, and C or A at position 501, Haplotype markers for judging the quality of meat quality.
A microarray for judging a meat quality level of a pig, comprising the haplotype marker of claim 13.

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