KR101432164B1 - Novel haplotype marker for discriminating level of meat quality of Pig and use thereof - Google Patents

Abstract

The present invention relates to a composition for determining a meat quality trait level of a pig, comprising a haplotype marker comprising a SNP capable of determining a meat quality trait level of a pig, an agent capable of detecting or amplifying the haplotype marker, A kit or a microarray for determining a meat quality trait level of a pig, and a polymorphic site of a SNP contained in the haplotype marker. The haplotype of the present invention is a specific gene marker for judging the level of meat quality of pigs. Since the haplotype is used as a means of objectively evaluating the level of meat quality of pigs not visually distinguished, it contributes to establishment of distribution order of pork meat It will be possible.

Description

[0001] The present invention relates to a haplotype marker for judging a meat quality of a pig, and a use thereof,

More particularly, the present invention relates to a haplotype marker comprising a SNP capable of determining the level of a meat quality of a pig, a haplotype marker for detecting the haplotype marker, A kit for determining a meat quality trait level of a pig comprising an agent capable of amplifying, a kit or a microarray for determining a meat quality trait level of a pig including the composition, and a step of determining a polymorphic site of a SNP contained in the haplotype marker The present invention relates to a method for determining the level of meat quality traits in pigs.

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 5'promoter region of Myogenin gene known to be involved in the myocyte differentiation of pigs , Patent Publication No. 2011-0011443 discloses a technique for detecting the Korean native pig-specific DNA marker for the KIT gene to discriminate an accurate variety of native Korean pig and other improved pig from the genome, and discloses a technique disclosed in Patent Publication No. 2011-0050261 Discloses a method for identifying a variety of black-bred pigs using haplotypes estimated from SNPs of the KIT gene region of black-bred pigs, Korean Patent Laid-Open 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, Discloses a method for screening pigs having excellent color quality using a gene of a pig, and Patent Publication No. 2012-0049624 discloses a method for screening pigs having an excellent color of meat. In Patent Publication No. 2012-0052796, (SNP) site of the PPARGC1A gene involved in the characteristics of the pig, and Patent Publication No. 2012-0072871 discloses a DNA marker for confirming the increase in the meat quality of a pig, and a single base polymorphism (SNP) markers are used to identify high quality pork meat. However, methods for accurately determining the meat quality traits of pigs have not yet been developed.

Under these circumstances, the present inventors have made extensive efforts to develop a method for judging the quality of pork meat based on the meat quality of pigs. As a result, the present inventors have found that a mash of the MYH13 gene and the MYH4 gene The present inventors confirmed that the quality of pigs can be judged by judging the level of the meat quality traits of the pigs genetically determined when the body shape is used.

One object of the present invention is to provide a haplotype marker comprising a SNP capable of determining the level of meat quality traits of a pig.

Another object of the present invention is to provide a composition for judging the quality of meat quality of a pig, which comprises a preparation capable of detecting or amplifying the haplotype marker.

It is still another object of the present invention to provide a kit or microarray for judging the quality of meat quality of pigs comprising the composition.

Yet another object of the present invention is to provide a method for determining a meat quality trait level of a pig, comprising determining a polymorphic site of SNP contained in the haplotype marker.

In one aspect, the present invention provides a haplotype marker comprising a single nucleotide polymorphism (SNP) that is a single nucleotide polymorphism capable of determining the level of meat quality of a pig.

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. For the purpose of the present invention, the haplotype may be all or some of the polynucleotides including the SNP region of the MYH13 gene and the SNP region of the MYH4 gene, and more preferably the polynucleotide of SEQ ID NO: 1 MYH13 gene) and a polynucleotide comprising all or a part of a polynucleotide (MYH4 gene) consisting of SEQ ID NO: 2, and most preferably a SNP located in the 5'-UTR region of the MYH13 gene A polynucleotide consisting of 5 to 100 consecutive bases including the SNP position and the 201th base of the polynucleotide consisting of SEQ ID NO: 1 corresponding to the 4,999th base is G or A, and It is composed of SEQ ID NO: 2 corresponding to base 3,378 base sequence based on mRNA which is the SNP position of MYH4 gene Wherein the 603rd base of the polynucleotide is T or C and the polynucleotide comprises a polynucleotide consisting of 5 to 100 consecutive bases including the SNP position or a complementary polynucleotide thereof, It can be a haplotype marker that can judge the level.

The term "MYH13 gene" of the present invention means a gene encoding MYH13, which is myosin expressed in external eye muscles. The MYH13 gene is composed of 40 exons and has a length of 5,737 bp based on the mRNA nucleotide sequence And has a length of about 58,600 bp based on the DNA base sequence. The base sequence of the MYH13 gene can be obtained from a known database such as NCBI's GenBank. The gene may be a gene represented by NM_003802.2, preferably a polynucleotide of SEQ ID NO: 1. MYH13 is known as a specialized myosin responsible for the ocular movement function.

The term "MYH4 gene" of the present invention refers to a gene encoding Myosin-4, which is one of the heavy chain proteins contained in myosin constituting the muscle of an animal. The MYH4 gene is composed of 40 exons, Has a length of 5,807 bp based on the sequence and has a length of 1,936 aa based on the amino acid sequence. The base sequence of the MYH4 gene can be obtained from a known database such as NCBI's GenBank. The gene may be a gene represented by NM_017533.2, preferably a polynucleotide of SEQ ID NO: 2. Myosin-4 is one of the 10 heavy chain proteins contained in myosin, and is known to be involved in embryonic development.

The term "polymorphism " of the present invention refers to the case where two or more alleles exist in one locus. Of the polymorphic sites, only a single base is different from a polymorphic site, 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.

SEQ ID NO: 1 "and" SEQ ID NO: 2 "are polymorphic sequences comprising polymorphic sites. A polymorphic sequence means a sequence comprising a polymorphic site comprising a SNP in a polynucleotide sequence. The polynucleotide sequence may be DNA or RNA.

In the haplotype marker of the present invention, it can be judged that when the SNP region of the MYH13 gene is G and the SNP region of the MYH4 gene is T, the pig having the MYH13 gene has a higher level of meat quality than the common pig.

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. For the purpose of the present invention, the meat quality trait is affected by the genotype of the haplotype marker of the present invention. When the mutation site of the MYH13 gene constituting the haplotype marker is G and the mutation site of the MYH4 gene is T, The mutation site of the MYH13 gene is T, the mutation site of the MYH13 gene is G, and the mutation site of the MYH4 gene is G, C, it can be judged that it has an intermediate quality meat quality. If the mutation site of the MYH13 gene is A and the mutation site of the MYH4 gene is C, it can be judged that it has the lowest level of meat quality traits.

According to one embodiment of the present invention, progeny obtained by crossing Korean traditional black pork and land race pig with MS markers or large-capacity SNP chips are used to measure the meat quality (intramuscular fat content, meat color, water holding capacity and shearing force) As a result of QTL analysis, it was confirmed that a gene involved in the meat quality of pigs was present on chromosome 12 (FIGS. 1 and 2). MYH13, MYH1, MYH3, MYH2, SCO1, ENSSSCG00000029441, ENSSSCG00000018006, ADPRM or TMEM220 were identified as the genes involved in the meat quality traits of pigs (FIG. 3) The SNP of the MYH4 gene indicates that the level of the meat quality trait is changed. As a result of analyzing the SNP genotypes of the MYH13 gene and the MYH4 gene using the Pyro-sequencer, it was found that the G / G genotype, the A / G genotype and the A / A genotype, which are SNP mutations of the MYH13 gene, (Fig. 6), there are T / T genotypes, T / C genotypes and C / C genotypes, which are SNP mutations of the MYH4 gene, and that the fat characteristics of pigs having respective genotypes are different from each other (Fig. 7). As the G copy number in the MYH13 gene SNP increased, the level of the meat quality trait was significantly increased (Fig. 8). As the T copy number increased in the SNP of the MYH4 gene, (FIG. 9). It was confirmed that high meat quality pigs having genotype of haplotype marker including GT of MYH13 gene and MYH4 gene were able to provide pig meat of higher quality than common pigs (Table 2).

Therefore, when the genotype of the haplotype marker of the present invention is detected or a primer capable of detecting the genotype of the haplotype marker is used, it is expected that the level of the meat quality trait of the pig can be accurately discriminated, Were first identified by the present inventors.

In another aspect, the present invention provides a composition for determining a meat quality trait level of a pig comprising an agent capable of detecting or amplifying the haplotype marker.

The term "agent capable of detecting or amplifying a haplotype" of the present invention means a composition capable of determining the level of a meat quality of a pig by confirming a mutation site of the gene by amplification, Means a primer capable of specifically amplifying a haplotype polynucleotide. The primers used in the haplotypes amplification can be amplified by PCR using appropriate conditions in suitable buffers (e. G., 4 different nucleoside triphosphates and polymerase such as DNA, RNA polymerase or reverse transcriptase) and template- directed DNA Stranded oligonucleotide which can serve as a starting point of synthesis. The appropriate length of the primer may vary depending on the intended use, but is usually 15 to 30 nucleotides. Short primer molecules generally require a lower temperature to form a stable hybrid with the template. The primer sequence need not be completely complementary to the haplotype, but should be sufficiently complementary to hybridize with the haplotype, preferably a polynucleotide sequence of SEQ ID NOS: 3 to 5 capable of amplifying the MYH13 gene, or a polynucleotide sequence of MYH4 The polynucleotide sequences of SEQ ID NOS: 6 to 8 capable of amplifying the gene, but are not particularly limited thereto.

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 primer can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i.e., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures. At this time, the PCR conditions, the lengths of the sense and antisense primers can be modified based on those known in the art.

The primers of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using many means known in the art. Non-limiting examples of such modifications include, but are not limited to, methylation, "capping ", replacement of natural nucleotides with one or more homologues, and modifications between nucleotides, such as uncharged linkers, such as methylphosphonate, Phosphoamidates, carbamates, etc.) or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).

In another aspect, the present invention provides a kit for determining a meat quality trait level of a pig comprising the composition. The kit may be an RT-PCR kit or a kit for DNA analysis (e.g., a DNA chip).

The kit of the present invention can determine the level of meat quality of pigs by confirming the genotype of the haplotype marker, which is a marker for determining the meat quality trait level of the pig, or by checking the expression level of the haplotype marker in the expression level of the mRNA . As a specific example, in the present invention, the kit for measuring the mRNA expression level of the haplotype marker for determining the meat quality trait level of pigs may be a kit containing essential elements necessary for performing RT-PCR. In addition to the respective primer pairs specific for the haplotype gene, the RT-PCR kit also includes 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. Also preferably, the kit of the present invention may be a kit for judging the quality of a pig meat quality trait including essential elements necessary for carrying out a DNA chip. DNA chip kits are those in which nucleic acid species are attached in a gridded array on a generally flat solid support plate, typically a glass surface not larger than a slide for a microscope, and nucleic acids are uniformly arranged on the chip surface, Hybridization reaction occurs between the nucleic acid on the surface and the complementary nucleic acid contained in the solution treated on the surface of the chip to enable a mass parallel analysis.

In another aspect, the present invention provides a microarray for judging a meat quality trait level of a pig, which comprises a polynucleotide of an haplotype marker for judging a 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.

(A) amplifying a polymorphic site of each SNP contained in the haplotype marker from DNA of a sample isolated from an individual; And (b) determining the base of the amplified polymorphic site of step (a).

The term "individual" of the present invention means a pig to which a level of meat quality traits is to be confirmed. By analyzing a genotype of a polymorphic site of SNP contained in the haplotype marker using the sample obtained from the pig, It is possible to judge the quality level of the meat quality. 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 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 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 Co., Ltd.) used for general nucleotide sequence determination And when the unlabeled extension primer and the dideoxy nucleotide are used, the mutation can be detected by measuring the molecular weight using a MALDI-TOF (matrix assisted laser desorption ionization-time of flight) technique.

Preferably, in the polynucleotide of SEQ ID NO: 1 contained in the haplotype marker among the nucleotide sequences determined in the step (b), the allele of the 201 th base as the polymorphism site is G, and in the polynucleotide of SEQ ID NO: If the allele of the polymorphic site 603 base is T, it can be judged that the pig has a higher level of meat quality than the general pig.

The haplotype marker of the present invention is a specific gene marker for judging the level of meat quality of pigs, and is used as a means of objectively evaluating the level of meat quality of pigs not visually distinguished. Therefore, You can do it.

1 is a graph showing the results of performing QTL analysis on the meat quality of offspring pigs using MS markers.
FIG. 2 is a graph showing the results of QTL analysis of the meat quality of offspring pigs using a large-capacity SNP chip.
Fig. 3 is a schematic diagram showing the results of gene analysis in the QTL region involved in porcine meat quality traits.
Figure 4 is a schematic diagram showing the IBD mapping results for pig meat quality traits.
5 is a schematic diagram showing the results of LD block analysis in a meat quality trait QTL region.
FIG. 6 is a graph showing the results of genotyping of the MYH13 gene using Pyro-sequencer.
FIG. 7 is a graph showing the results of genotyping of the MYH4 gene using Pyro-sequencer.
8 is a graph showing the results of analyzing the relationship between the genotype of the MYH13 gene and the fat content of pigs.
FIG. 9 is a graph showing the results of analyzing the relationship between the genotype of the MYH4 gene and the fat content of pigs.

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

Example  1: pig's Flesh trait QTL  Analysis

QTL analysis of pig meat quality (intramuscular fat content, meat coloring, water holding capacity and shearing force) was carried out using MS (microsatellite) marker or large SNP chip in offspring obtained by crossing Jeju native black pig and land race pig 1 and 2). FIG. 1 is a graph showing the results of QTL analysis of the meat quality of progeny pigs using MS markers, wherein EMA represents meat quality traits, ALLEMA represents the cross sectional area of whole meat, CFAT represents crude fat content in sirloin , SHEAR indicates the shear force, MOIST indicates the crude moisture content, DRIPL indicates the water holding capacity, and MARB indicates the intramuscular fat content.

FIG. 2 is a graph showing the results of QTL analysis of the meat quality of offspring pigs using a large-capacity SNP chip.

As shown in FIGS. 1 and 2, it was confirmed that the gene involved in the meat quality of pigs was present on chromosome 12.

On the other hand, IBD mapping of pig meat quality traits was used to compress the locus of the fleshy trait regulatory gene in the gene region from MYH13 to MYH2 (FIGS. 3 to 5). FIG. 3 is a schematic diagram showing the results of gene analysis in the QTL region related to the pig meat quality trait, FIG. 4 is a schematic diagram showing IBD mapping results of pig meat quality traits, and FIG. Fig.

As shown in FIGS. 3 to 5, MYH13, MYH1, MYH3, MYH2, SCO1, ENSSSCG00000029441, ENSSSCG00000018006, ADPRM or TMEM220 are present as genes involved in porcine meat quality traits. Especially, by mutation of MYH13 gene and MYH4 gene, And the level of education.

Therefore, it was found that the meat quality of pigs can be determined by confirming haplotypes composed of alleles of MYH13 gene and MYH4 gene.

Example  2: Pyro - sequencer  Through analysis MYH13  Gene and MYH4  Genotype analysis of genes

Example  2-1: MYH13  Genotype analysis of genes

A primer capable of detecting the trait of the MYH13 gene by carrying out genotyping analysis using a Pyro-sequencer was constructed as follows:

MYH13_F: 5'-TCTGTTGTTTCTGGAGGGCT-3 '(SEQ ID NO: 3)

MYH13_R: 5'-ACTCAGGTTGTAGGAGGACA-3 '(SEQ ID NO: 4)

MYH13_seq: 5'-ACGTCTCCCAGGGAGTGGCA-3 '(SEQ ID NO: 5)

Of the above primers, MYH13_R was prepared in such a form that biotin was bound to its 5'-terminal.

The amplified MYH13 gene samples were obtained (PCR amplification conditions: 96 ° C → 30s, 60 ° C → 30s, 72 ° C → 30s 30cycles) by using the primers and genomic DNA obtained from pigs as a template. Next, the amplified MYH13 gene samples were subjected to genotype analysis using Pyro-sequencer (FIG. 6). FIG. 6 is a graph showing the results of genotyping of the MYH13 gene using Pyro-sequencer. As shown in FIG. 6, the G / G genotype, the A / G genotype, and the A / A genotype exist on the basis of the -4999 base on the basis of the ATG start site, which is the SNP position in the 5'-UTR region of the MYH13 gene , And the meat quality of pigs with each genotype were different.

Example  2-2: MYH4  Genotype analysis of genes

A primer capable of detecting the trait of the MYH4 gene by performing a genotyping analysis using a Pyro-sequencer was constructed as follows:

MYH4 F: 5'-TCCATCAGTCTGGCTCAAAG-3 '(SEQ ID NO: 6)

MYH4 R: 5'-AGTGCGCTGTATAAACCGTG-3 '(SEQ ID NO: 7)

MYH4_Seq: 5'-GAGGAGCTGGAGGAAGAGAT-3 '(SEQ ID NO: 8)

Of the above primers, MYH4R was prepared in a form in which biotin was bound to its 5'-terminal.

The amplified MYH4 gene samples were obtained (PCR amplification conditions: 96 ° C → 30s, 66 ° C → 30s, 72 ° C → 30s, 30cycles) by using the above primers and genomic DNA obtained from pigs as a template. Next, the amplified MYH4 gene samples were subjected to genotype analysis using Pyro-sequencer (FIG. 7). FIG. 7 is a graph showing the results of genotyping of the MYH4 gene using Pyro-sequencer. As shown in FIG. 7, there exist T / T genotypes, T / C genotypes and C / C genotypes based on mRNA sequence base 3,378 bases, which are SNP positions of MYH4 gene, Respectively.

Example  3: MYH13  Gene and MYH4  Genotype and pig Flesh trait  Level relevance analysis

Genotype analysis using the primers was performed on second generation offspring obtained by crossing Jeju native black pig and land race pig, and the content of muscle fat in one of the meaty traits of each genotype was compared (FIGS. 8 and 9) .

8 is a graph showing the results of analyzing the relationship between the genotype of the MYH13 gene and the fat content of pigs. As shown in FIG. 8, when the MYH13 gene has a G / G genotype, the content of intramuscular fat is relatively high, whereas the content of A / A genotype has a relatively low level of intramuscular fat content. / A genotypes, intermediate values of intramuscular fat content were shown.

FIG. 9 is a graph showing the results of analyzing the relationship between the genotype of the MYH4 gene and the fat content of pigs.

As shown in FIG. 9, when the MYH4 gene had a T / T genotype, it had a relatively high level of intramuscular fat content, while a C / C genotype had a relatively low level of intramuscular fat content, and T / C genotype, the median value of intramuscular fat content was shown.

Thus, it was expected that the haplotype in which the MYH13 gene has the G genotype and the MYH4 gene has the T genotype will have an additive effect.

Example  4: MYH13  Gene and MYH4  Gene On hips  Following Sirloin  Comparison of fat content

From the results of Example 3, it was expected that the MYH13 gene would have the G genotype and the MYH4 gene would have the additive effect in the haplotype having the T genotype.

Specifically, the second generation offspring obtained by crossing Jeju native black pig and land race pig were classified according to the haplotype of MYH13 gene and MYH4 gene (Table 1).

Classification according to haplotype of MYH13 gene and MYH4 gene MYH13 genotype MYH4 genotype Meat grade G
G
A
A T
C
T
C High meat type
Intermediate type
Intermediate type
Low meat type

Among the meat quality grades shown in Table 1, the high meat type represents the case where the crude fat content in the sirloin is 10% or more, the low meat type represents the case where the crude fat content in the sirloin is 1-2% Indicates a case where the crude fat content in the sirloin is less than 2 to 10%.

As shown in Table 1, it was found that the high meat type of the present invention had a G / G genotype in MYH13 and a T / T genotype in MYH4.

Example  5: High meat type MYH13  Gene and MYH4  Gene Haplotype  Sensory evaluation of porcine sirloin

The 7-point evaluation method (7 points) was used to compare the pork belly areas from the high meat type pigs having the haplotype GT of the MYH13 gene and the MYH4 gene in terms of flavor, year, juiciness, : Very good, 6 points: good, 5 points: slightly good, 4 points: normal, 3 points: slightly unfavorable, 2 points: poor, 1 point: very uncomfortable).

Sensory Evaluation Results of Porcine Sirloin with High Meat Type Haplotypes Evaluation items Plain pig High-fat pig Flavor
year
Juiciness
Purity 4.8 ± 1.1
4.9 ± 1.0
5.1 ± 1.0
5.0 ± 1.1 5.8 ± 0.6
5.8 ± 0.9
5.8 ± 0.7
5.9 ± 0.6

As shown in Table 2 above, it was found that the high meat type pork meat having a haplotype of the MYH13 gene and the MYH4 gene of the present invention exhibited better sensory characteristics than the normal pork meat.

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Novel haplotype marker for discriminating level of meat quality          of Pig and use thereof <130> PA121035 / KR <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 402 <212> DNA <213> 5'-UTR of MYH13 gene <400> 1 gcgggcaggg tggagaggga tccggcgcag acctgccgct ggcgtgatcc atccttgcag 60 agtcccgtgc ccttggcctt ctcctggttt gtgggggagg tgggaggagg acggtctgtt 120 gtttctggag ggctggaccc caagggggaa ggctggaggg aggtggttgg aaagtggctg 180 acgtctccca gggagtggca rggtttcctg ttaaccggct tcctagtcaa tatccctgtg 240 ttccagctga gccagggacc ccaggcttgg ggctggagtc ccacacttcc tagcctcgcg 300 tggccctcca cggtgtcctc ctacaacctg agtcccatgg agccatctcc actggctccc 360 cactgtcccg agagtcgggc tgctccttgc tgggggacag ga 402 <210> 2 <211> 1535 <212> DNA <213> MYH4 gene <400> 2 gaccctggat gacctgcagg cagaagagga caaagtcaac accctgacca aagctaaaac 60 caagctagag cagcaagtgg atgacgtaag tctaggatta tcaaggaaat tattttcttc 120 aaaaggaagc taagcatccc ttttgactca gcattatttc tatttagctt gaagggtcct 180 tggagcaaga aaagaaactt cgcatggact tagagagagc caagaggaaa ctggaaggtg 240 acctcaagtt ggctcaagag tccataatgg acattgagaa tgaaaaacag caacttgatg 300 aaaagcttaa aaagtaagta ggaaagaatt gcttatgaac ttgcatctaa tgttgcaaat 360 ggactcaact gttatctttt tctacttttc taaaaggaaa gagtttgaaa taagcaattt 420 acaaagcaag attgaagatg aacaagcact tgccattcag ctgcaaaaga agatcaaaga 480 gttgcaagta agttcatatt tccatcagtc tggctcaaag aggcacagac gtgacataaa 540 gaagctaatt ttgggcattc ccccactgcc aggcccgcat tgaggagctg gaggaagaga 600 tcygaggcag agcgggcctc cagggccaaa gcagagaagc agcgctccga cctctcccgg 660 gaactggagg agatcagcga gaggctggaa gaagccggcg gggcgacgtc agcccagatt 720 gagatgaaca agaagcgcga ggctgagttc cagaagatgc gccgggacct ggaggaggcc 780 accctgcagc acgaggccac agcggccgcg ttgaggaaga agcacgcgga cagcgtggct 840 gagctggggg agcagatcga caacctgcag agggtcaagc agaagctgga gaaggagaag 900 agtgagatga agatggagat cgacgacctt gctagcaaca tggagaccgt ctccaaagcc 960 aaggtaccac agagtctggg aaattgttat tcacaatgat cctttacgta catatcacgg 1020 tttatacagc gcactttaca cacatcaaat catttgtttt tttgcaataa tccaggcagt 1080 aaagcaagaa cgatattagt acttgtgtta ttttcattta accatatgaa gaaactaaaa 1140 ttcaaagaca catcggcgcc cctcatagca ctaacaaatg ccagcaccag cactgaacat 1200 aggtcatcag tgttcccttg ctctccaact ggagaacata ggaccatatt ctcaattatc 1260 cagtacggaa tgtacataaa ggcagagttc ccatcttggc tcagcggaga tgaatctgac 1320 tagtatccat gagacatggg ttctatccct ggccttgctc agtgggttaa ggatccagcg 1380 ttgccatgag ctgtggtgta agtcacagat gcagctcaga tcctgcgttg ttgtggctgc 1440 ggtgtaggcc ggtggctaca gatctgatta gacccctagc ctgggaacct ccatatgctc 1500 catatgctcc aaggacaaaa agacaaaaaa aaaaa 1535 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MYH13_F primer <400> 3 tctgttgttt ctggagggct 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MYH13_R primer <400> 4 actcaggttg taggaggaca 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MYH13 primer <400> 5 acgtctccca gggagtggca 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MYH4 F primer <400> 6 tccatcagtc tggctcaaag 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MYH4R primer <400> 7 agtgcgctgt ataaaccgtg 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MYH4 primer <400> 8 gaggagctgg aggaagagat 20

Claims (10)

(a) a polynucleotide consisting of 5-100 consecutive bases, wherein the 201st base is G or A in the polynucleotide derived from the MYH13 gene represented by SEQ ID NO: 1, and the 201st base, or a complementary polynucleotide thereof; And
(b) a polynucleotide consisting of 5 to 100 consecutive bases, wherein the 603rd base is T or C in the MYH4 gene-derived polynucleotide shown in SEQ ID NO: 2, or a complementary polynucleotide thereof A haplotype marker that can be used to determine the level of meat quality of the pigs involved.
The method according to claim 1,
Wherein the meat quality of the pig is a trait selected from the group consisting of intramuscular fat content, meat color, water holding capacity and shearing force of pork meat.
A composition for judging the quality of a meat quality of a pig, comprising an agent capable of detecting or amplifying a haplotype marker capable of judging the meat quality trait level of the pig of claim 1.
The method of claim 3,
Wherein the preparation is a primer represented by a polynucleotide selected from the group consisting of SEQ ID NOS: 3 to 5 capable of amplifying the polynucleotide represented by SEQ ID NO: 1.
The method of claim 3,
Wherein the agent is a primer represented by a polynucleotide selected from the group consisting of SEQ ID NOS: 6 to 8 capable of amplifying the polynucleotide represented by SEQ ID NO: 2.
A kit for determining a meat quality trait level of a pig comprising the composition of any one of claims 3 to 5.
The method according to claim 6,
Wherein the kit is an RT-PCR kit or a DNA chip kit.
A microarray for judging a meat quality trait level of a pig comprising the polynucleotide of claim 1.
(a) amplifying the polynucleotide consisting of the polynucleotide consisting of SEQ ID NO: 1 and the polynucleotide consisting of SEQ ID NO: 2 contained in the haplotype marker of claim 1 from the DNA of the sample separated from the individual; And
(b) determining the base of each amplified polymorphic site of step (a).
10. The method of claim 9,
In the polynucleotide of SEQ ID NO: 1, when the allele of the 201st base as the polymorphism site is G and the allele of the polymorphism site 603 base is T in the polynucleotide of SEQ ID NO: 2, &Lt; / RTI &gt;

Images