KR102083672B1 - Composition for identifying a bovine marbling trait comprising an agent capable of detecting or amplifying a haplotype - Google Patents

Abstract

The present invention relates to a composition for discriminating a carcass trait of cows, comprising a preparation capable of detecting or amplifying haploids for discriminating the carcass trait of cows composed of single nucleotide polymorphism markers. According to the present invention, it is possible to accurately determine the carcass trait of cows using the haploid of the present invention.

Description

Composition for identifying a bovine marbling trait comprising an agent capable of detecting or amplifying a haplotype}

The present invention relates to a composition for judging marbling of a cow comprising an agent capable of detecting or amplifying a haploid for judging marbling among bovine carcasses consisting of a single nucleotide polymorphism (SNP) marker. A composition for judging marbling of a cow comprising a formulation capable of detecting or amplifying a haploid for discrimination, a kit for judging marbling of a cow including a formulation capable of detecting or amplifying the bovine marbling for a haploid, and a method for judging marbling of a cow It is about.

Marbling is an important economic trait for farmers, which is the distribution of fats that make meat lighter and more succulent. It is known to be the primary standard for the quality of cattle.

Recent advances in sequencing technology have led to an increasing number of studies using Next-Generation Sequencing decoding and full-field genetic association analysis to select biomarkers for marbling, one of the bovine complex traits. In particular, when biomarkers are selected for bovine marbling using next-generation sequencing and full-length genetic association analysis, it is more precisely associated with bovine carcass traits than commercial biochips that select specific biomarkers for bovine marbling. There is an advantage to finding markers.

However, when a single nucleotide polymorphism marker is selected and used as a marker for predicting marbling, which is a complex trait, a large number of factors that determine the marbling, which is a complex trait, is likely to exist. There was a problem of low accuracy.

Accordingly, the present inventors completed the present invention by selecting haploids, which are combinations of single nucleotide polymorphism markers, which are more significant in predicting marbling, which is a complex trait, than conventional single nucleotide polymorphism markers.

Republic of Korea Patent Publication No. 10-2015-0047672

SUMMARY OF THE INVENTION An object of the present invention is to provide a composition for discriminating bovine carcass trait comprising an agent capable of detecting or amplifying bovine carcass morphology determinants.

Another object of the present invention is to provide a kit for discriminating bovine carcass traits.

Another object of the present invention is to provide a method for discriminating bovine carcass quality.

In order to achieve the above object, there is provided a composition for the determination of bovine carcass trait comprising an agent capable of detecting or amplifying a bovine carcass morphology haploid selected from the group consisting of monobasic polymorphism.

The present invention also provides a kit for discriminating bovine carcass traits.

In addition, the present invention provides a method for discriminating bovine conductor quality.

By using the composition for determining the carcass shape of the cow of the present invention, it is possible to accurately determine the carcass trait of the cow, and through this, high-quality meat can be easily and accurately determined.

Figure 1 shows the results of the full-length genome association analysis to select Hanwoo haploid markers associated with Hanwoo carcass traits.

Hereinafter, the present invention will be described in detail.

The present invention relates to a composition for determination of bovine carcass trait comprising an agent capable of detecting or amplifying a haplotype for carcass trait discrimination selected from the group consisting of the following (a) and (b).

(a) a single nucleotide polymorphism (SNP) in which the 201 base is A or G in the polynucleotide described in SEQ ID NO: 1, or a single A or G in the polynucleotide described in SEQ ID NO: 2 Nucleotide polymorphism; And

(b) a single nucleotide polymorphism in which the 201 base is A or G in the polynucleotide of SEQ ID NO: 3, or a single nucleotide polymorphism in which the 201 nucleotide of SEQ ID NO: 4 is A or G.

SEQ ID Nos. 1 to 2 included in (a) may include two SNP markers (the 117431264th and 117434023 bases of the 3rd Korean beef chromosome) existing between 117,431,264bp and 117,434,023bp of the Hanwoo Hanwoo chromosome 3 , SEQ ID NOs: 3 to 4 included in (b) may include two SNP markers (14509316th and 14511623th bases of Hanwoo chromosome 12) existing between 14,509,316bp and 14,511,623bp of Hanwoo chromosome 12 .

As used herein, the term "nucleotide" is a deoxyribonucleotide or ribonucleotide that exists in single- or double-stranded form, and includes analogs of natural nucleotides unless otherwise specified.

In the present invention, the term "Single Nucleotide polymorphism" (SNP) refers to a single nucleotide polymorphism, SNP refers to one or several dozen nucleotide variations representing the individual deviation of the three billion nucleotide sequence of the chromosome in the cell nucleus, This results in differences in phenotype, responsiveness to drugs and resistance to disease.

As used herein, the term “haplotype” has the same meaning as a haplotype, and refers to a bundle of a series of SNPs located and inherited on one chromosome during meiosis, and such haploid or haplotypes are on the same chromosome. The pattern of a certain SNP is shown as a combination of several SNPs present in.

The term "Linkage Disequilibrium" in the present invention means a non-random association of alleles in two or more loci that are not necessarily on the same chromosome in population genetics. That is, linkage disequilibrium is a measure of binding between alleles at different positions, and if each gene is combined completely independently, the gene set will probably maintain a linkage equilibrium, but some alleles are found together. There are many examples, that is, they are not randomly mixed up, and these alleles are in an imbalanced state. This linkage disequilibrium is interpreted as a result of natural selection when alleles are located close together or when alleles are clustered together.

As used herein, the term "nucleic acid" has the meaning of comprehensively including DNA (gDNA and cDNA) and RNA molecules, and the nucleotides that are the basic structural units in nucleic acid molecules are natural nucleotides, as well as analogs in which sugar or base sites are modified. We include (analogue)

In this document, the term "marbling" refers to the distribution of fat that makes meat light and succulent, an important economic trait for farmers. It is one of the traits that directly affects the price formation of carcasses. It becomes the car standard.

As used herein, the term "agent capable of detecting or amplifying haploids or SNPs" refers to specifically binding to a site containing an SNP in a polynucleotide constituting a haploid so as to recognize or amplify a site containing the SNPs. As an agent which can be used, specifically, a probe capable of specifically binding to a site containing the SNP, a polynucleotide comprising the site containing the SNP, or a complementary polynucleotide thereof can be specifically amplified. It may be a primer.

The primers can be chemically synthesized using phosphoramidite solid support methods, or other well known methods. Such primers can be modified using a number of means known in the art, as long as they exhibit the effect of detecting the site containing the SNP. Examples of such modifications include methylation, capping, substitution of one or more homologs of natural nucleotides, and modifications between nucleotides, eg, uncharged linkages (eg, methyl phosphonates, phosphoesteres, phosphoroamidates). , Carbamate, and the like) or charged linkers (eg, phosphorothioate, phosphorodithioate, etc.). Nucleic acid may be one or more additional covalently linked residues, such as proteins (eg, nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), inserts (eg, acridine , Proralenes, and the like), chelating agents (eg, metals, radioactive metals, iron, oxidizing metals, etc.) and alkylating agents. In addition, the primer may include a label which can be detected directly or indirectly by spectroscopic, photochemical, biochemical, immunochemical or chemical means, if necessary. Examples of labels include enzymes (eg horseradish peroxidase, alkaline phosphatase), radioisotopes (eg ³² P), fluorescent molecules and chemical groups (eg biotin), and the like. There is this.

As used herein, the term "probe" refers to a nucleic acid fragment corresponding to several to several hundred bases capable of specifically binding to DNA or RNA, and includes oligonucleotide probes and single stranded DNA probes. It may be prepared in the form of a double stranded DNA (double stranded DNA) probe or RNA probe.

The probe may be chemically synthesized using phosphoramidite solid support methods, or other well known methods. Such probes can be modified using a number of means known in the art, as long as the probes have the effect of detecting the site containing the SNP. Examples of such modifications include methylation, capping, substitution of one or more homologs of natural nucleotides, and modifications between nucleotides, eg, uncharged linkages (eg, methyl phosphonates, phosphoesteres, phosphoroamidates). , Carbamate, and the like) or charged linkers (eg, phosphorothioate, phosphorodithioate, etc.). Nucleic acid may be one or more additional covalently linked residues, such as proteins (eg, nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), inserts (eg, acridine , Proylene, etc.), chelating agents (eg, metals, radioactive metals, iron, oxidizing metals, etc.) and alkylating agents.

The probe may be further conjugated to a reporter at its 5 'end. The reporter is FAM (6-carboxyfluorescein), Texas red (texas red), fluorescein (fluorescein), fluorescein chlorotriazinyl, HEX (2 ', 4', 5 ', 7'-tetrachloro -6-carboxy-4,7-dichlorofluorescein, rhodamine green, rhodamine red, tetramethylrhodamine, fluorescein isothiocyanate (FITC), oregon green, Alexa Fluorine (alexa fluor), JOE (6-Carboxy-4 ', 5'-Dichloro-2', 7'-Dimethoxyfluorescein), ROX (6-Carboxyl-X-Rhodamine), TET (Tetrachloro-Fluorescein), TRITC ( at least one selected from the group consisting of tertramethylrodamine isothiocyanate, TAMRA (6-carboxytetramethyl-rhodamine), NED (N- (1-Naphthyl) ethylenediamine), cyanine-based dyes, and thiadicarbocyanine However, in addition to the known materials that can be used as a reporter in the art, any one can be used.

The probe may be further conjugated to a quencher at its 3 'end. The quencher is TAMRA, black hole quencher (BHQ) 1, BHQ2, BHQ3, nonfluorescent quencher (NFQ), dabcyl, Eclipse, deep dark quencher (DDQ), Blackberry Quencher, Iowa black It may be any one or more selected from the group consisting of), but any other known material that can be used as a quencher in the art can be used.

The "agent capable of detecting or amplifying haploids" may include reverse transcriptase, DNA polymerase, co-factors such as Mg ²⁺ , dATP, dCTP, dGTP and dTTP. Various DNA polymerases can be used in the amplification step of the present invention to amplify reverse transcribed cDNA, and examples of DNA polymerases include Klenow fragments of E. coli DNA polymerase I, thermostable DNA polymerase or bacteriophage T7 DNA polymerisation. There is an enzyme. Polymerases can be obtained from cells that are isolated from the bacteria themselves or purchased commercially or express high levels of cloning genes encoding the polymerase.

In the present invention, a bovine chromosomal determinant haploid is a combination of SNP marker groups (2) existing between 117,431,264 bp and 117,434,023 bp of Hanwoo chromosome 3 and SNP marker group (2, 14,509,316 bp and 14,511,623 bp of chromosome 12). Dog).

The combination of two groups of SNP markers existing between 117,431,264 bp and 117,434,023 bp of the Hanwoo chromosome 3 is a single nucleotide polymorphism (SNP) in which the 201 base is A or G in the polynucleotide described in SEQ ID NO: 1. Or a polynucleotide according to SEQ ID NO: 2, wherein the 201st base is A or G.

The combination of the two groups of SNP markers existing between 14,509,316 bp and 14,511,623 bp of the Hanwoo chromosome 12 is a single nucleotide polymorphism in which the 201st base is A or G in the polynucleotide of SEQ ID NO: 3, or SEQ ID NO: 4 The 201st base described may consist of monobasic polymorphisms of A or G.

The conductor shape may be marbling, and the cattle may be Korean beef, but is not limited thereto.

The present invention relates to a kit for determining the carcass shape of a cow comprising a composition for judging the carcass quality of a cow comprising an agent capable of detecting or amplifying a haplotype for carcass shape determination.

The composition may have the features as described above. In one example, the composition comprises a single nucleotide polymorphism (SNP) wherein the 201 base is A or G in the polynucleotide described in SEQ ID NO: 1, or the 201 base is A or a polynucleotide described in SEQ ID NO: It may include an agent capable of detecting or amplifying any one or more SNPs selected from the group consisting of a single nucleotide polymorphism, which is G, and the single nucleotide of the 201 base in the polynucleotide of SEQ ID NO: 3 is A or G. Polymorphism or a single base polymorphism in which the 201st base described in SEQ ID NO: 4 is A or G may include an agent capable of detecting or amplifying any one or more SNPs selected from the group.

In addition, the agent capable of detecting or amplifying the polynucleotide is an agent capable of specifically binding to and recognizing a site containing an SNP in the polynucleotide or amplifying a site containing the SNP. May be a probe capable of specifically binding to the SNP-containing site, a primer capable of specifically amplifying a polynucleotide including the SNP-containing site, or a complementary polynucleotide thereof.

The kit may be a PCR kit or a kit for DNA analysis, but is not limited thereto.

The kit of the present invention can be confirmed by amplifying the genotype of the SNP marker provided in the present invention using the composition. The kit provided by the present invention may be a kit including essential elements necessary for performing RT-PCR.

For example, the RT-PCR kit can be used in tubes or other suitable containers, reaction buffers (pH and magnesium concentrations vary) in addition to specific primer pairs for polynucleotides or their complementary polynucleotides comprising the site containing the SNPs. Enzymes such as deoxynucleotides (dNTPs), Taq-polymerases and reverse transcriptases, DNase inhibitors, RNase inhibitors, DEPC-water and sterile water, and the like. On the other hand, the components included in the kit may be prepared in a liquid form, or may be prepared in a dried form in order to improve the stability of the product by lowering the degree of freedom of the included components. In order to produce such a dried form, it is necessary to apply a drying step, in which warming drying, natural drying, reduced pressure drying, freeze drying, or a combination thereof may be used.

In the present invention, when applied to a PCR amplification process, a "kit" may optionally contain reagents necessary for PCR amplification, such as buffers, DNA polymerases (eg, Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis). , Thermisflavus, Thermococcus literalis or thermally stable DNA polymerase obtained from Pyrococcus furiosus (Pfu)), DNA polymerase cofactors and dNTPs. The kit can be made in a number of separate packaging or compartments containing the reagent components described above.

In addition, the present invention is a step of amplifying a region comprising the SNP of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, and the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2 from the DNA of the sample isolated from the bovine ,

The SNP is a base 201 of a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 1, or a base 201 of a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 2; And

2) provides a method for determining the carcase shape, comprising the step of determining the base type of the SNP at the site containing the amplified SNP.

In addition, the present invention 1) any one or more poly selected from the group consisting of a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 3 from the DNA of a sample isolated from cattle, and a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4 Amplifying a site comprising an SNP of a nucleotide or complementary polynucleotide thereof,

The SNP is base 201 of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3, or base 201 of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4; And

2) provides a method for determining the carcase shape, comprising the step of determining the base type of the SNP at the site containing the amplified SNP.

Amplifying the site including the SNP of step 1) can be used by any method known to those skilled in the art. For example, the target nucleic acid can be amplified by PCR and purified. Ligase chain reaction (LCR) (Wu and Wallace, Genomics 4, 560 (1989), Landegren et al., Science 241, 1077 (1988)), transcription amplification (Kwoh et al., ProcNatlAcad Sci USA 86, 1173) (1989)), self-sustaining sequence replication (Guatelli et al., ProcNatl Acad Sci USA 87, 1874 (1990)) and nucleic acid based sequence amplification (NASBA).

Determining the base type of the SNP at the site containing the amplified SNP of step 2) is sequencing, hybridization by microarray, allele specific PCR, dynamic allele hybridization technique (dynamic allelespecifichybridization (DASH), PCR prolongation analysis, PCR-SSCP, PCR-RFLP analysis or TaqMan technique, SNPlex platform (Applied Biosystems), mass spectrometry (e.g. Sequenom's MassARRAY system), mini-sequencing method , Bio-Plex system (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) It may be, but is not limited thereto.

In the method for determining the bovine conductor morphology of the present invention, when the 201st base of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 is G, it can be determined that a lot of marbling is present in the bovine.

In the above method for determining the carcass shape of a cow of the present invention, when the 201st base of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2 is A, it can be determined that a lot of marbling is present in the cow.

In the method for determining the bovine conductor morphology of the present invention, when the 201st base of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3 is A, it can be determined that a lot of marbling is present in the bovine.

In the method for determining the bovine conductor morphology of the present invention, when the 201st base of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4 is A, it can be determined that a lot of marbling is present in the bovine.

In addition, in the present invention, when there is a lot of marbling in the cow, it can be determined that the quality meat is superior.

In one embodiment of the present invention, the present inventors screened 611,782 monobasic polymorphic markers out of 777,962 monobasic polymorphism (SNP) markers present in Illumina 770K SNP Chip data in 898 heads of Hanwoo, and used 975,945 Hanwoo haploids. The information was constructed (see Example 1).

Further, in an embodiment of the present invention, full-length genetic association analysis of the 975,945 haploid information was performed to select Hanwoo haploid markers associated with marbling traits of Hanwoo carcasses, and 117,431,264bp of Hanwoo chromosome 3 of the selected Hanwoo haploid markers. The haploid marker (HH1_GA), a combination of two SNP marker groups present between 117,434,023 bp, had a -log10P-value of 6.277957 and the SNP marker group of two (14) present between 14,509,316 bp and 14,511,623 bp of chromosome 12. Since the haploid marker (HH2_AA), which is a combination, had a -log10P-value of 6.65339, it was found that the selected Hanwoo haploid markers exhibited high significance (see Example 2).

Therefore, when using a composition for discriminating bovine carcass trait comprising an agent capable of detecting or amplifying bovine carcass morphology haploid consisting of a single nucleotide polymorphism marker of the present invention, a small carcass is compared with the case of using a conventional monobasic polymorphism marker. The trait (marbling) can be more accurately discriminated, and through this, high-quality meat can be easily and accurately discriminated.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following Examples are intended to illustrate the present invention, and the content of the present invention is not limited by the Examples.

Example 1 Hanwoo Haploid Information Structure

Among the 777,962 single nucleotide polymorphism (SNP) markers in Illumina 770K SNP Chip data in 898 heads of Hanwoo, 611,782 single nucleotide polymorphisms passing the criteria with MAF value of 0.01 or more, HWE value of 0.000001 or more, and genotyping rate of 0.05 or more ( SNP) markers were used for analysis. Apply 611,782 single nucleotide polymorphism (SNP) marker information that passes the above criteria to the R package GHap software (version 1.2.2, https://cran.r-project.org/web/packages/GHap/index.html) Based on the linkage disequilibrium value of 0.2, 975,945 haploid information, which is a combination of SNPs, was constructed. As a result of analyzing the correlation inequality from 0.2 to 0.7, the haploid information was constructed based on 0.2 as the optimal value for selecting significant haploid markers.

Example 2: Selecting Hanwoo Haploid Markers Associated with Hanwoo Carcass Traits

In order to select Hanwoo haploid markers related to Hanwoo carcass traits, a genome wide association study (GWAS) based on the next generation nucleotide sequence was performed using the analysis model of Equation 1 below.

[Equation 1]

y = Wα + xβ + u + ε

In the above analysis model, y is a phenotype for marbling, W is a covariance matrix for fixed effects, α is a coefficient including intercepts, x is a genotype vector, β is an effective size of the marker, u is a random effect, and ε is an error. The value for.

Specifically, the association of the Hanwoo marbling traits with the 975,945 haploid markers constructed in Example 1 using a mixed linear model based association analysis (MLMA) and a restricted maximum likelihood (REML) analysis method. Significance was confirmed, and the gender and slaughter age were significantly corrected by adding covariates.

GEMMA (version 0.96, http://www.xzlab.org/software.html) statistical software was used to verify the significance of each haploid marker for traits. Carcass weight, fillet cross-sectional area, and marbling information of Hanwoo carcass It was provided by the National Agricultural Cooperative Federation Hanwoo Improvement Office. Significant haploid markers for each trait were selected by markers having a p-value of 0.05 or less through a significance test.

As a result, two haploid markers with the highest significance were selected (combination of SNP marker groups (2, SEQ ID Nos. 1 to 2) existing between 117,431,264 bp and 117,434,023 bp of chromosome 3) and between 14,509,316 bp and 14,511,623 bp of chromosome 12 SNP marker group (combination of two, SEQ ID NO: 3 to 4) present in the (Fig. 1).

SEQ ID NO: 1 to 2 is a nucleotide sequence of about 400 bp comprising two SNP markers existing between 117,431,264 bp and 117,434,023 bp of Hanwoo chromosome 3, wherein SEQ ID NOs 3 to 4 are 14,509,316 bp and 14,511,623 of Hanwoo chromosome 12 A base sequence of about 400 bp comprising two SNP markers present between bp.

The combination of the SNP marker group (two) present between the 117,431,264 bp and 117,434,023 bp of the Hanwoo chromosome 3 is the 117431264th and 117434023th bases of the 10th cow's chromosome in order 'GA' (Table 1),

The combination of two SNP marker groups existing between 14,509,316 bp and 14,511,623 bp of Hanwoo chromosome 12 is 'AA' in the order of the 14509316 and 14511623 bases of Hanwoo chromosome 10 (Table 2).

Haplotype ID SNP Marker ID chromosome location Genotype information HH1_GA BovineHD0300034180 3 117431264 A / G BovineHD0300034181 3 117434023 A / G

Haplotype ID SNP Marker ID chromosome location Genotype information HH2_AA BovineHD1200004311 12 14509316 A / G BovineHD1200004312 12 14511623 A / G

Among the selected Hanwoo haploid markers, the haploid marker (HH1_GA), which is a combination of two groups of SNP markers existing between 117,431,264 bp and 117,434,023 bp of chromosome 3, had a -log10P-value of 6.277957, and 14,509,316 bp of 14,509,316, of chromosome 12 The haploid marker (HH2_AA), which is a combination of 10 groups of SNP markers present between bp, had a -log10P-value of 6.65339 (Table 3).

Haplotype ID chromosome Average location Haplotype Allele frequency P-value -log ₁₀ P-value HH1_GGAGA 10 81997757 0.013 4.70E-07 6.32763047 HH2_GGGGAAACAG 10 82033004 0.13 4.70E-07 6.32763047

In other words, the selected haploid markers were found to have a high degree of significance with the Hanwoo carcass trait (marbling).

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Composition for identifying a bovine marbling trait comprising an          agent capable of detecting or amplifying a haplotype <130> 2018P-10-021 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 402 <212> DNA <213> Artificial Sequence <220> <223> No1 <400> 1 taggtaatga agcttaagtg cctggtatag tctccagcaa acagcagggt cctcaatgtt 60 aactgttatt tctagcaaca ataatagcaa ctagccaaca ttggctccat ccctctttca 120 tgtgcccagg tccagaaagg gatttaacaa caaaacaagc aatcacttcc agaagatttt 180 aaaagtcctg ccaatcacag agctctgaca caccatcagt aatgatttat tttgtgaata 240 attgaatgca tgaataactg actgcctgaa actatccttt tgagaaacat gcgtggtggc 300 acttccctag cggtccaacg gttaagaatc cgcctgccag tgcagggggc ctcaggttca 360 atccctggac caggaagatc ccacatgcct cagggcaacg aa 402 <210> 2 <211> 402 <212> DNA <213> Artificial Sequence <220> <223> No2 <400> 2 agagctactg tgaggctcca ggtccaagca tcctggctgg attccacccc caggatacac 60 ccacggctgc cctcgacatc tcatcccagt gtgtggctgc cgagggtcag gctgtcctct 120 cgtgggctag ccagcatctt tccgtccagc tgtgcctgct cgcctctcac cgtcactgga 180 ctaaccatcg cccattaaat agctcccctc agttaaaccc ctgactctgc cttcttctcc 240 tggttcaacc agcccaaccc ttcaacatgt tctttcaggg attattttcc ttcctgcctt 300 atcttcccca ctcttctttg aacttcatca atttcaatgt cagaagacag taccaggaga 360 aattcaagca aggaaacaaa gacgtttgta atgaaggatg tt 402 <210> 3 <211> 402 <212> DNA <213> Artificial Sequence <220> <223> No3 <400> 3 ggagaattct atggacagag gagcctgggg agctacagtc catggggtgg caaagagtca 60 gacacatctg agtaactaag cgtgcacaca cacacacaca cacacacaca cacacagtaa 120 ggcatctcag gaaaattttt tccaggtgga tctaacccaa agacatacct gctctgattc 180 tttttctgtc ctaatccttt agaggaccct ctctcatctt catcacttca ttattttggt 240 ctctggtcct ttcccatgta cttcaccaat atccagccca cactgatcta cctggaacca 300 tccttccacc tcagacaagt ttccaattgc agctaattct tgttttattt ttcaaaatta 360 tacatacatg tatatatatg catatatata ttagagttgc tc 402 <210> 4 <211> 402 <212> DNA <213> Artificial Sequence <220> <223> No4 <400> 4 tccatggcct cttccatccc aagtaaccct atccttgttc tgtggatgtt gtggagaaaa 60 gggtgagcag ggtacgatgc atacagactc aggtccggcc ttgacaatga gcagagtgga 120 ggtttctcaa gagtgcagtg gtatgtccag ggtatacgca gtttctctct caggggtctc 180 agaaccctca aggaaaactc agaagagact ctggtgtgct tccaatccat gctgccacca 240 ctctctgtgt tagttgctca gccatgtcgg attctttgcg accccatgga ctgtagccca 300 ccaggcttct ctaccctggg attttccagg caagaatact ggagtggatt gccatttcct 360 tctccaggga atcctcccga tgcagggact gaacctgcgt ct 402

Claims (9)

Detecting haplotypes for the determination of marbling of Korean cattle of polynucleotides according to SEQ ID NO: 3 with a single nucleotide polymorphism of which the 201 base is A or G and monobasic polymorphisms with a 201 base of A or G described in SEQ ID NO: 4 Or a composition for determining the marbling of Hanwoo comprising a preparation that can be amplified.
delete delete According to claim 1, wherein the agent capable of detecting or amplifying the haploid is characterized in that the primer or probe, the composition for marbling determination of Hanwoo.
A kit for determining the marbling of a Korean beef comprising the composition of claim 1.
delete delete delete 1) amplifying a region comprising a SNP of a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 3 and a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 4 or a complementary polynucleotide thereof from DNA of a sample isolated from Hanwoo ,
The SNP is base 201 of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3 and base 201 of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4; And
2) comprising the step of determining the base type of the SNP at the site containing the amplified SNP, marbling determination method of Korean cattle.

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