KR101821542B1 - SNP marker regulating palmitic acid level in the pork and uses thereof - Google Patents

Abstract

The present invention relates to a composition for determining the content of palmitic acid in pork comprising an agent capable of detecting or amplifying the SNP associated with the content of palmitic acid in pork, a kit for determining the content of palmitic acid in pork containing the composition, Methods for determining the content of palmitic acid, methods for producing pigs with reduced palmitate content in pork, methods for decreasing palmitic acid content in pork, markers and microarrays for determining content of palmitic acid in pork . Since the SNP marker capable of determining the content of palmitic acid in the pork of the present invention is used as a means for objectively evaluating the content of palmitic acid in the pork which is not visually distinguishable, It will be possible.

Description

SNP markers for regulating the content of palmitic acid in pork and uses thereof Field of the Invention < RTI ID = 0.0 >

The present invention relates to a SNP marker for controlling the content of palmitic acid in pork and a use thereof. More particularly, the present invention relates to an SNP marker for controlling the content of palmitic acid in pork, A composition for determining an acid content, a kit for determining the content of palmitic acid in a pork containing the composition, a method for determining a content of palmitic acid in a pork, a method for producing a pork having a reduced amount of palmitic acid in pork, A method for decreasing the acid content, a marker for determining the content of palmitic acid in pork, and a microarray.

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 using a base polymorphism (SNP) marker is disclosed.

On the other hand, palmitic acid is one of the typical saturated fatty acids in pork, and it can be judged that the higher the content of palmitic acid is, the lower the quality of pork. Thus, the content of palmitic acid in pork can be judged as a criterion However, a method for evaluating the content of palmitic acid in pork using a gene has not yet been developed.

Under these circumstances, the present inventors have made extensive efforts to develop a method for evaluating the content of palmitic acid in pork using a gene. As a result, when SNPs present on chromosome 8 and chromosome 12 were used, the content of palmitic acid in pork And the present invention has been completed.

One object of the present invention is to provide a composition for determining the content of palmitic acid in pork comprising a preparation capable of detecting or amplifying the SNP associated with the content of palmitic acid in pork.

Another object of the present invention is to provide a kit for determining palmate content in pork comprising the above composition.

It is a further object of the present invention to provide a method for determining the content of palmitic acid in pork using the composition.

It is another object of the present invention to provide a method for producing pigs with reduced palmitate content in pork.

It is another object of the present invention to provide a method for reducing palmate content in pork.

Another object of the present invention is to provide a marker for determining palmitate content in pork.

It is still another object of the present invention to provide a microarray for determining the content of palmitic acid in pork comprising the marker.

The present inventors searched for quantitative trait loci (QTL) related to saturated and unsaturated fatty acid contents of pigs in order to develop a method for reducing the saturated fatty acid content of pigs. As a result, SNPs located on chromosome 8 (MARC0091202) and the SNP located on chromosome 12 (ALGA0067072) are related to the content of palmitic acid, and that the SNP can be used as a genetic marker for determining the content of palmitic acid in pork I could. The technique of using the SNP (MARC0091202) located on chromosome 8 and the SNP (ALGA0067072) located on chromosome 12 to determine the content of palmitic acid in pork has not been known until now and was first developed by the present inventor.

In one embodiment, the present invention provides a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, wherein the base 501 is T or C, and the single nucleotide polymorphism (SNP) located at the base 501 is included A polynucleotide consisting of 5 to 1000 consecutive bases or an agent capable of detecting or amplifying a complementary polynucleotide of the polynucleotide or its complementary polynucleotide.

The term "polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1" of the present invention is a polymorphic sequence including a polymorphic site of a gene involved in the content of palmitic acid in pork, and polymorphic sequence means 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 terms of the invention "palmitate (palmitic acid) (C16: 0 )" is, CH ₃ (CH ₂₎ and the general formula of ₁₄ COOH, had have a molecular weight of between 256Da, melting of 62.65 ℃ having up to 16 carbon atoms , A boiling point of 351.5 DEG C, and a fat-soluble solid fatty acid.

In the present invention, the palmitic acid is used as an object capable of predicting the content of pork meat using the genotype of the SNP (MARC0091202) located on chromosome 8. The pork meat containing palmitic acid is not particularly limited, Can be pork derived from Jeju native pigs, land lace or their hybrid pigs.

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.

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.). As an example, the primer may be a primer base pair consisting of SEQ ID NOS: 4 and 5 capable of amplifying a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, but is not limited thereto.

In the present invention, the content of palmitic acid in the pork is the same as that of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, It can be judged that palmitic acid is contained in a higher content than the derived pork.

The composition for determining the content of palmitic acid in pork according to the present invention is a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2, wherein the base 501 is A or G and the single nucleotide polymorphism (SNP) A polynucleotide consisting of 5 to 1000 consecutive bases or a preparation capable of detecting or amplifying a complementary polynucleotide of the polynucleotide, wherein the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2 is amplified The agent that can be used is not particularly limited, but may include a pair of primers consisting of SEQ ID NOS: 9 and 10.

According to one embodiment of the present invention, in order to investigate the fatty acid content, the fattening of the fattened shrimp was sampled for the offspring of the Korean traditional pig and the land race reference group F2, The content of palmitic acid was the second most abundant in the total fatty acids. The content of the palmitic acid was found to be 25%, which is the major component of fatty acids in pork. In addition, analysis of the locus of pigs related to palmitic acid in pork confirmed that SNP (MARC0091202) located on chromosome 8 was associated with the content of palmitic acid in pork (Fig. 2 and Table 1) As a result of analysis of the content of palmitic acid in the pork, the content of palmitic acid was the highest when the genotype of the SNP was TT, and the content of palmitic acid when the genotype was CC And the content of palmitic acid was found to be a median value when the genotype was TC (FIG. 5 and Table 2).

Therefore, it can be seen that the SNP of SEQ ID NO: 1 can be used as a gene marker for determining the content of palmitic acid in pork.

As another embodiment for achieving the above object, the present invention provides a kit for determining the content of palmitic acid in pork 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 determine the content of palmitic acid in pork by confirming the genotype of the SNP marker provided by the present invention using the above composition by amplification or by checking the expression level of mRNA. 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 SNP for determining the content of palmitic acid in the pork, the RT-PCR kit also includes a test tube or other appropriate container, a reaction buffer (pH and magnesium concentration: Deoxyinucleotides (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 determining the content of palmitic acid in pork containing 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 polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 or a complementary nucleotide thereof, from the DNA of the sample separated from the individual, Amplifying; 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" in the present invention means a porcine which is a target to be assayed for the content of palmitic acid in pork. By analyzing the genotype of the polymorphic site of the SNP using the sample obtained from the pig, The content of palmitic acid in pork can be judged. 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 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 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, the pork pork in which the allele of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 in the base sequence determined in the step (b) is all the C-501 allele is the pork pork of all the T alleles It can be judged that the palmitic acid content is reduced.

After the step (b), a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2 or a polymorphic site of the complementary nucleotide thereof is amplified from the DNA of the sample isolated from the individual, and the base of the amplified polymorphic site And that the pork pork of all G alleles of the identified polymorphic site is less palmitate than the pork of the alleles of alleles A .

According to another aspect of the present invention, there is provided a method of isolating SNP traits involved in the content of palmitic acid in pork, comprising the steps of: isolating a polynucleotide comprising a nucleotide sequence of SEQ ID NO: 1 And fixing all the alleles to C, wherein the content of palmitic acid in the pork is reduced.

According to another aspect of the present invention, there is provided a method for fixing an SNP trait of a gene involved in the content of palmitic acid in pork, comprising the steps of: detecting a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 And fixing all of the alleles of the base to C in the pork meat.

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, a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, which participates in the content of palmitic acid in pork, is crossed with an individual having C / C at the 501st nucleotide, and among the progeny obtained therefrom, A pig with a reduced content of palmitic acid in the pork can be obtained by selecting a pig in which the nucleotide 501 base is fixed with C / C. The pig may be a hybrid of Jeju native pig, land lace or Jeju native pig and land lace.

In another embodiment for achieving the above object, the present invention provides a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, wherein the base 501 is T or C, the base of the palm palmitic acid Provide markers for content determination.

In the present invention, using the marker, the pork pork in which the allele of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 is all C base 501 is palmy than the pork pork in which the allele is all T, It can be judged that the content of acid is reduced.

According to another aspect of the present invention, there is provided a microarray for determining the content of palmitic acid in pork comprising a marker for determining the content of palmitic acid in the pork.

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 content of palmitic acid in pork 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 in connection with the determination of the content of palmitic acid in the pork 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.

Since the SNP marker capable of determining the content of palmitic acid in the pork of the present invention is used as a means for objectively evaluating the content of palmitic acid in the pork which is not distinguished by the naked eye, There will be.

1 is a schematic view showing the composition of fatty acids in pork;
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 content of palmitic acid in pork.
FIG. 3 is a schematic diagram showing various gene groups in the locus of the content of palmitic acid in the pork of chromosome 12.
Fig. 4 is a schematic diagram showing various gene groups in the locus of the content of palmitic acid in pork of chromosome 8; Fig.
5 is a graph showing a result of identifying a genotype of the 501 (T / C) SNP contained in the polynucleotide of SEQ ID NO: 1 of chromosome 8 using pyrosequencing.
FIG. 6 is a graph showing the result of checking the genotype of the 501 (T / C) SNP contained in the polynucleotide of SEQ ID NO: 1 of chromosome 12 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: In pork  Fatty acid composition analysis

In order to analyze the composition of saturated and unsaturated fatty acids in pigs, the content of fatty acid composition was investigated in about 1,000 dogs in the second generation offspring of Jeju traditional pig and landrace mating reference group.

1 is a schematic view showing the composition of fatty acids in pork; As shown in Fig. 1, the content of palmitic acid is 25%, which is the second highest content of total fatty acids.

Example  2: In pork  Palmitate content association Gene locus  Search

In order to analyze the gene region regulating the content of palmitic acid in pork, GWAS was analyzed with a large DNA chip (illumina 60k SNPchip) to search for the saturated fatty acid content-related gene locus (Fig. 2).

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 content of palmitic acid in pork. As shown in FIG. 2, the locus associated with the content of palmitic acid in pork was found to have the highest significance on chromosome 8 and chromosome 12 of pigs. In addition, the effect of genotype on the content of palmitic acid in pork was higher in chromosome 12 than in chromosome 8.

Example  3. In pork  Securing palmitate content-related SNP

From the above example 2, it was confirmed that the content of palmitic acid in the pork was determined by the loci of two chromosomes corresponding to chromosome 8 and 12. Based on this, SNPs having high correlation with the content of palmitic acid in pork were selected Table 1).

SNPs that are highly related to palmitate content in pork chromosome SNP BP LOG 12
12
12
12
12
8
8
8
8
8 ALGA0067072
M1GA0017062
ASGA0055256
ASGA0096092
ASGA0096381
MARC0091202
MARC0109916
ALGA0049249
ALGA0049254
MARC0025408 57,831,831
57,787,203
58,105,269
59,699,634
59,752,770
119,721,311
99,661,930
120,972,820
120,996,107
120,836,105 16.90309
15.592949
15.44916
14.215098
14.102648
9.461552
9.172308
8.601366
8.601366
8.597911

As shown in the above Table 1, the higher SNPs correlated with the palmitate content in pork from chromosome 8 and 12 were ALGA0067072 on chromosome 12 and MARC0091202 on chromosome 8.

Example  4: In pork  Palmitate content association Chromosomes 8 and 12 Gene locus  Identification of Genes in the Region

Based on the SNP list having a high correlation with the palmitic acid content in the pork of Example 3, various gene groups of gene locus regions of chromosome 8 and 12 associated with palmitic acid content in pork were identified (FIGS. 3 and 4) .

FIG. 3 is a schematic diagram showing various gene groups in the locus of the content of palmitic acid in the pork of chromosome 12. As shown in FIG. 3, it was found that the genes related to palmitic acid content in the pork chromosome 12 contained MYH13, MYH1, MYH3, SCO1, TMEM220 and the like.

Fig. 4 is a schematic diagram showing various gene groups in the locus of the content of palmitic acid in pork of chromosome 8; Fig. As shown in FIG. 4, the genes related to palmitic acid content in the pork chromosome 8 contained PITX2C, ELOVL6, EGF, CASP6, LRIT3, PLA2G12A, SEC24B, RRH and CCDC109B.

Example  5: Pyro-sequencing ( Phyro -sequencing In pork  Extraction and analysis of nucleotide sequences containing palmitic acid content-related SNPs

In Example 3, ALGA0067072 on chromosome 12 and MARC0091202 on chromosome 8 were found to have the highest significance in relation to the content of palmitic acid in pork, and it was confirmed that the SNPs on chromosomes 8 and 12 1001 nucleotides (SEQ ID NO: 1 and SEQ ID NO: 2), respectively, were obtained and their nucleotide sequences were analyzed.

Example  5- 1: 8 times  SNP analysis of chromosomes

In order to amplify the polynucleotide of SEQ ID NO: 1 of the chromosome 8, PCR was carried out 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: 3, 385 bp).

Forward: 5'-GGG-TGA-AAA-CTG-TGT-GTG-TCA-3 '(SEQ ID NO: 4)

Reverse: 5'-biotin-TTT-TAT-CAA-AGG-CTC-CGA-AGA-3 '(SEQ ID NO: 5)

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 (T / C) SNP, the nucleotide sequence of the polynucleotide fragment was determined using the following primer (Mini-seq), and the primer of the input was used. / C) SNP genotype (Fig. 5).

ATCC-TGG-CCT-CTC-TCT-AA-3 '(SEQ ID NO: 6)

Input: 5 '- [T / C] GATCTTAACTTT-3' (SEQ ID NO: 7)

5 is a graph showing a result of identifying a genotype of the 501 (T / C) SNP contained in the polynucleotide of SEQ ID NO: 1 of chromosome 8 using pyrosequencing. As shown in FIG. 5, it was confirmed that polynucleotides of amplified SEQ ID NO: 1 contained TT, TC, or CC genotypes, respectively.

Example  5- 2: 12  SNP analysis of chromosomes

In order to amplify the polynucleotide of SEQ ID NO: 2 of the chromosome 12, 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: 2 (SEQ ID NO: 8, 397bp).

Forward: 5'-CAG-CAG-AAG-TAG-AGG-GAT-GC-3 '(SEQ ID NO: 9)

Reverse: 5'-biotin-TTC-GCT-CCC-GTC-TAA-GAG-AC-3 '(SEQ ID NO: 10)

The nucleotide sequence of the polynucleotide fragment of SEQ ID NO: 2 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 genotype (Fig. 6).

AA-ACA-GGG-CC-3 ' (SEQ ID NO: 11), 5'-TTT-GCC-

Input: 5 '- [A / G] GGAGTGTGC-3' (SEQ ID NO: 12)

6 is a graph showing the result of checking the genotype of the 501 (A / G) SNP contained in the polynucleotide of SEQ ID NO: 2 of chromosome 12 using pyrosequencing. As shown in FIG. 6, it was confirmed that the amplified polynucleotide of SEQ ID NO: 1 contained the AA, AG, or GG genotypes, respectively.

Example 6: 8  And the genotype of the SNP of chromosome 12 In pork  Analysis of association with palmitic acid content

The changes in the content of palmitic acid in pork according to the genotypes of chromosome 8 SNPs of 8 and 12 identified in Example 5 were compared (Tables 3 and 4).

Analysis of the relationship between the genotype of pig chromosome 8 SNP and the content of palmitic acid in pork genotype Analysis Palmitic acid content TT
CT
CC 354
510
106 25.3316 ± 1.3049
24.9404 + 1.3036
24.5085 ± 1.3148

As shown in Table 2, when the genotype of chromosome 8 SNP was TT, the content of palmitic acid in the pork was the highest, and the content of palmitic acid in the pork was relatively high when the genotype was CC And the content of palmitic acid in the pork was the intermediate level when the genotype was TC.

Analysis of the relationship between the genotype of chromosome 12 of pork and the content of palmitic acid in pork genotype Analysis Palmitic acid content AA
AG
GG 248
455
267 25.3594 + - 1.3148
25.1256 ± 1.3148
24.5923 ± 1.3148

As shown in Table 3, when the genotype of chromosome 12 was AA, the content of palmitic acid in the pork was the highest, and the content of palmitic acid in the pork was relatively high when the genotype was GG And it was confirmed that the content of palmitic acid in pork was a medium level when the genotype was AG.

Example 7: 8  Effect of combination of chromosome and chromosome 12 SNP

In Example 6, changes in the content of palmitic acid in the pork according to the genotypes of chromosomes 8 and 12 were confirmed, and the change in the content of palmitic acid in the pork according to the combination of the two SNPs was confirmed (Table 4).

Effect of combination of chromosome 8 and 12 SNP in pigs Chromosome 8 SNP Chromosome 12 SNP Analysis Palmitic acid content CC GG
GA
AA 25
50
31 24.3372 ± 1.3148
24.3548 ± 1.5187
24.8945 ± 1.5187 TT GG
GA
AA 31
133
84 24.8945 ± 1.6947
25.2058 ± 1.6947
25.7446 ± 1.6947

As shown in Table 4, regardless of the SNP of chromosome 12, when the SNP of chromosome 8 is C, the palmitate content is decreased compared to that of T, and when the SNP of chromosome 8 is the same, 12 And that the palmitic acid content was decreased when the SNP of the chromosome No. G was G,

Therefore, the palmitate content in pork was determined by the genotype of the SNP of chromosome 8, and when the genotype of SNP of chromosome 8 was the same, the genotype of SNP of chromosome 12 was determined.

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> SNP marker regulating palmitic acid levels in the pork and uses          the <130> KPA151106-KR <160> 12 <170> Kopatentin 2.0 <210> 1 <211> 1001 <212> DNA <213> Artificial Sequence <220> <223> SNP <400> 1 actcaaaatt taaatagatg gcaagtaaat ggtacagaaa tgttccccct ggagcacttg 60 tcctgaaact gatgtcagca cgtaggctgt ctcctcttga tcctgtttta agtggaattg 120 tttggactta tgcacgaggc agacccatgc ttataactca actctccgaa atgtcttcag 180 tcaatacttt ggaggaccag gcatgttttc atgttgtatg atctctgagg gcaatgcaga 240 acatttaaca tatcaacata acaatgcaaa gaaatgactg gcttaagcct taggtaatca 300 tgttgctaca gggtgaaaac tgtgtgtgtc agagttcctt ttacaacaaa tacactcaca 360 acccctcttt ttgattatca aagttgtcac gtatgcgctg aattatacta gcaggagaac 420 tgattagagg ttttctattt cattaatatt aactccctct ttggactttg cagaccctct 480 gt; ctgccctttc tggttttctt gctctcgttg ctgttaccaa ctccaagtga cttgtcttcc 600 cctcacgcat ctgtctgtgg ttctctgccc taaaagtctt catttttttt tttttccttt 660 cttgcttcaa gagctcttcg gagcctttga taaaaagctt taactctttt ccagaaaatt 720 caatcgcatt taaaattttg catgcaacct tagggcatcc aaagatctga cgtcattttt 780 ggtcttcagc cccaccctga ggatatgagt ctctgataaa tcctttaaga cccaacaaaa 840 gtcttattc tccatgaaac taagcttttg caattcatag tgtggactat tttgtgtaac 900 acttagtgga gaaggcctca gggtttaaga gcctgtgttc aagccccctct ttctactaac 960 tctaagatct agcctctctg gaatttcctg ccccataaat t 1001 <210> 2 <211> 1001 <212> DNA <213> Artificial Sequence <220> <223> SNP <400> 2 aagaggagga gagaggcctt cgcacaaacc ggccctgcca gcaccctgat cttgaacatc 60 cagcctccag aaccgcgaaa aaataaatgg ctattatttc agtgcccagc ctgtggaact 120 ttattctggc agtctgagca aacccacaca gcatgtgcgc tgggctcccc aggcaaagcg 180 agtgcggcag ctcgagctgc tgagtgtggg gagtgggggt ggggggagcc tattggaggc 240 ggcctacccc acacttccag ggctgagggc tgggggggcc ctggtcaggt ggtagacccc 300 acccccagca gaagtagagg gatgcccaga gaggtcggcc agcggcccac cgagatgtga 360 tcacaggaga ccctggcagc tccaaggact ccaaagcccc cggcaaaggc tggcgattgg 420 ttgcatctgg gctcctacgg ggagcggagg actctgctgc tgagcccact acactcctca 480 tttgccccta aaacagggcc rggagtgtgc cctgaccaca gctgccaaag ggctcgccaa 540 aaggaaggca cccacccatc cacctcctag ggacctctgc ctcctatgga cagtgctggg 600 tccagggaaa ccctccccaa gggaacacga atgactctaa tcagagcagc acagacctca 660 ggatgcttgc aggggggtgg ggtctcttag acgggagcga attccacgca aagctgtgag 720 ctcagctcta agtcaccaat ctctgcagga cccctgctat cgggtaagag aggtgtggaa 780 aatgactttg taaatcagta tctatggaca ctgagttttt aagaaaaagt atcatcttta 840 agctagaaga aaagtatatg gcagatacaa ttggccagat cagtagctcc tgcctgtctc 900 ctgaccagaa tcctctggaa accaaacccc taatcagaaa caggcgctgg gggaggaagg 960 acagaaagca aatgacagaa gccacctggt cccgaagacc a 1001 <210> 3 <211> 385 <212> DNA <213> Artificial Sequence <220> <223> template <400> 3 gggtgaaaac tgtgtgtgtc agagttcctt ttacaacaaa tacactcaca acccctcttt 60 ttgattatca aagttgtcac gtatgcgctg aattatacta gcaggagaac tgattagagg 120 ttttctattt cattaatatt aactccctct ttggactttg cagaccctct gtaatctggc 180 ctctctctaa ygatcttaac ttttcttcac cccagcactt accacgtgtt ctgccctttc 240 tggttttctt gctctcgttg ctgttaccaa ctccaagtga cttgtcttcc cctcacgcat 300 ctgtctgtgg ttctctgccc taaaagtctt catttttttt tttttccttt cttgcttcaa 360 gagctcttcg gagcctttga taaaa 385 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 gggtgaaaac tgtgtgtgtc a 21 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 ttttatcaaa ggctccgaag a 21 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 gtaatctggc ctctctctaa 20 <210> 7 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 ygatcttaac ttt 13 <210> 8 <211> 397 <212> DNA <213> Artificial Sequence <220> <223> template <400> 8 cagcagaagt agagggatgc ccagagaggt cggccagcgg cccaccgaga tgtgatcaca 60 ggagaccctg gcagctccaa ggactccaaa gcccccggca aaggctggcg attggttgca 120 tctgggctcc tacggggagc ggaggactct gctgctgagc ccactacact cctcatttgc 180 ccctaaaaca gggccrggag tgtgccctga ccacagctgc caaagggctc gccaaaagga 240 aggcacccac ccatccacct cctagggacc tctgcctcct atggacagtg ctgggtccag 300 ggaaaccctc cccaagggaa cacgaatgac tctaatcaga gcagcacaga cctcaggatg 360 cttgcagggg ggtggggtct cttagacggg agcgaat 397 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 cagcagaagt agagggatgc 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 ttcgctcccg tctaagagac 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 tttgccccta aaacagggcc 20 <210> 12 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 rggagtgtgc 10

Claims (16)

A polynucleotide consisting of 5 to 1000 consecutive bases containing a single nucleotide polymorphism (SNP) located at the 501 base, wherein the polynucleotide having the nucleotide sequence at position 501 is T or C in the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1 or a complementary A polynucleotide consisting of 5 to 1000 consecutive bases comprising a SNP at position 501 and a nucleotide at position 501 of SEQ ID NO: 2 or A or G, or a complementary polynucleotide thereof Wherein the composition is capable of detecting or amplifying a palmate content in a pork meat.
The method according to claim 1,
Wherein said preparation comprises a primer pair consisting of SEQ ID NOS: 4 and 5 capable of amplifying a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1.
The method according to claim 1,
Wherein the agent comprises a primer pair consisting of SEQ ID NOS: 9 and 10 capable of amplifying a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2.
The method according to claim 1,
Wherein the formulation is used to determine the palmitic acid content in pork obtained from Jeju native pig, land lace or Jeju native pig and land race hybrids.
delete A kit for determining the content of palmitic acid in pork comprising the composition of any one of claims 1 to 4.
(a) amplifying a polymorphic site of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 or its complementary nucleotide from the DNA of the sample separated from the individual, and determining the base of the amplified polymorphic site; And (b) amplifying the polymorphic site of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2 or its complementary nucleotide from the DNA of the sample isolated from the individual, and determining the base of the amplified polymorphic site. As a method for determining intracellular palmitic acid content,
In the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, the content of palmitic acid in the pork having all of the alleles of the polymorphic site 501 base was found to be lower than the content of palmitic acid in the pork having the allele of T And when the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2 has the same allele, the content of palmitic acid in the pork in which the allele of the polymorphic site 501 base is all G is not less than the palm palmitic acid Content is judged to be lower than the content

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