KR102083673B1 - Composition for identifying a bovine eye muscle area trait comprising an agent capable of detecting or amplifying a haplotype - Google Patents

Abstract

The present invention relates to a composition for discriminating carcass traits in cows, the composition comprising an agent capable of detecting or amplifying a haploid for discriminating carcass traits composed of single nucleotide polymorphism markers. The haploid of the present invention can be used to accurately determine the carcass traits of cows.

Description

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

The present invention relates to a composition for discriminating loin sectional area of a bovine, comprising an agent capable of detecting or amplifying the locus of cross-sectional area of a haploid in a bovine carcass constituting a single nucleotide polymorphism (SNP) marker. A composition for discriminating bovine loin cross-sectional area comprising a preparation capable of detecting or amplifying bovine loin cross-section determining haploids, a kit for discriminating bovine loin cross-sectional area comprising a preparation capable of detecting or amplifying bovine loin cross-sectional area determining haploids, And a method for determining the sirloin cross-sectional area.

Sirloin section (longest root area) is an important economic trait for farmers. It is a value measured in cm ² using the area ruler expressed in units. It is one of the traits that directly affect the price formation of carcasses.

Recent developments in sequencing technology have led to an increasing number of studies using Next-Generation Sequencing detoxification and full-length genetic association to select biomarkers for sirloin cross-sections, one of the complex traits of cattle. In particular, when biomarkers are selected for bovine fillet area using next-generation sequencing and full-length genetic association analysis, cattle conductors are more accurately compared to the selection of specific biomarkers for fillet area of bovine using commercial chips. There is an advantage in finding markers associated with traits.

However, when a single nucleotide polymorphism marker is selected and used as a marker for predicting the lumber cross-sectional area of the complex trait, a large number of factors that determine the lumber cross-sectional area of the complex trait are highly likely to exist. There was a problem that the accuracy was low when predicting.

Thus, the present inventors completed the present invention by selecting a haploid, which is a combination of a single nucleotide polymorphism marker that is more significant in predicting the wick sectional area that is a complex trait than a conventional single nucleotide polymorphism marker.

Republic of Korea Patent No. 10-1522579

SUMMARY OF THE INVENTION An object of the present invention is to provide a composition for discriminating loin cross-sectional area in a carcass shape of a bovine including a preparation capable of detecting or amplifying a haploid for determining lobe cross-sectional area in a bovine carcass shape.

Another object of the present invention is to provide a kit for determining the fillet cross-sectional area of a bovine carcass.

Another object of the present invention is to provide a method for discriminating the fillet cross-sectional area of a small conductor type.

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 of SEQ ID NO: 1, a single base in which the 201 base is A or G in the polynucleotide of SEQ ID NO: 2 Polymorphism, monobasic polymorphism wherein the 201 base is A or G in the polynucleotide of SEQ ID NO: 3, monobasic polymorphism wherein the 201 base is A or G in the polynucleotide according to SEQ ID NO: 4, poly Monobasic polymorphism wherein the 201 base is A or C in nucleotides, monobasic polymorphism where the 201 base is A or G in the polynucleotide described in SEQ ID NO: 6, and the 201 base is the polynucleotide shown in SEQ ID NO: 7 Or monobasic polymorphism of C, monobasic polymorphism of which the 201st base is A or G in the polynucleotide of SEQ ID NO: 8, or Monobasic polymorphisms in which the 201st base is A or C in the polynucleotide of SEQ ID NO: 9; And

(b) a monobasic polymorphism of which the 201 base is A or G in the polynucleotide of SEQ ID NO: 10; a monobasic polymorphism of the 201 base of SEQ ID NO: 11 or A; Single nucleotide polymorphism with base A or C, single nucleotide polymorphism with 20 or 20 base in SEQ ID NO: 13, single nucleotide polymorphism with 20 or nucleotide base in SEQ ID NO: 14, in SEQ ID NO: 15 A monobasic polymorphism wherein the 201st base described is A or G.

SEQ ID Nos. 1 to 5 included in (a) are five SNP markers (81990480, 81991722th, 81995899th, 82003284th, and 82005034th) between 81,990,480bp and 82,005,034bp of Hanwoo chromosome 10. And base numbers 10 to 15 included in (b) include -10 SNP markers (82011799th and 82013193th of Hanwoo chromosome 10) which exist between 82,011,799bp and 82,054,208bp of Hanwoo chromosome 10 , 82024014 th, 82027222 th, 82030321 th, 82036157 th, 82043067 th, 82047253 th 82051139 th, and 82054208 th base).

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 means a bundle of a series of SNPs located and inherited on one chromosome during meiosis of germ cells, and the haplotype or haplotype is on the same chromosome. The combination of several SNPs present shows a pattern of constant SNPs.

As used herein, the term "Linkage Disequilibrium" refers to non-random association of alleles in two or more loci that are not necessarily on the same chromosome in population genetics. do. 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. (analogue) is also included.

The term "loan cross-sectional area (longest root area)" of the present invention is an important economic quality for farmers, 1 cm horizontally and vertically on the rating plate. It is a value measured in cm ² using the area ruler expressed in units. It is one of the traits that directly affect the price formation of carcasses.

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 in addition, any material known to 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, the bovine carcass morphology, the haploid for the sectional area of the loin, is a combination of the SNP marker group (five) present between 81,990,480 bp and 82,005,034 bp of Hanwoo chromosome 10 and the SNP present between 82,054,208 bp of 82,011,799 bp of chromosome 10 It means a combination of 10 marker groups.

The combination of the SNP marker groups (five) existing between 81,990,480 bp and 82,005,034 bp of Hanwoo No. 10 is a single nucleotide polymorphism (SNP) in which the 201st base is A or G in the polynucleotide described in SEQ ID NO: 1. , Polynucleotide according to SEQ ID NO: 2, monobasic polymorphism having 201 base is A or G, polynucleotide according to SEQ ID NO: 3, monobasic polymorphism having 201st base A or G, polynucleotide described in SEQ ID NO: 4 In which the 201 base is A or G, or in the polynucleotide described in SEQ ID NO: 5, the 201 base is A or C.

The combination of the SNP marker group (10) present between 82,011,799 bp and 82,054,208 bp of the Hanwoo chromosome 10 is a single nucleotide polymorphism of which the 201 base is A or G in the polynucleotide of SEQ ID NO: 6, Single nucleotide polymorphism, where the 201 base is A or G, single nucleotide polymorphism, where the 201 base is SEQ ID NO: 8, or A, G single nucleotide polymorphism, where the 201 base is SEQ ID NO: 9 is A or G, the sequence Monobasic polymorphism wherein the 201 base of SEQ ID NO: 10 is A or G, monobasic polymorphism wherein the 201 base of SEQ ID NO: 11 is A or G, single which the 201 base of SEQ ID NO: 12 is A or G Nucleotide polymorphism, or a single nucleotide polymorphism in which the 201st base described in SEQ ID NO: 13 is A or G, a single nucleotide polymorphism in which the 201th base described in SEQ ID NO: 14 is A or G, or 201 described in SEQ ID NO: 15 The base may consist of a single nucleotide polymorphism of A or G.

The conductor shape may be a sirloin cross-sectional area, and the cattle may be Hanwoo, 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) in which the 201 base is A or G in the polynucleotide described in SEQ ID NO: 1, and the 201 base in the polynucleotide described in SEQ ID NO: 2 is A or G. Monobasic polymorphism, single nucleotide polymorphism in which the 201 base is A or G in the polynucleotide described in SEQ ID NO: 3, monobasic polymorphism in which the 201 base is A or G in the polynucleotide described in SEQ ID NO: 4, or SEQ ID NO: It may include an agent capable of detecting or amplifying any one or more SNPs selected from the group consisting of monobasic polymorphisms in which the 201st base is A or C in the polynucleotide of 5, and the polynucleotide of SEQ ID NO: 6 Monobasic polymorphism, where the 201st base is nucleotide A or G, and the 201th base of SEQ ID NO: 7 is A or Is a monobasic polymorphism of G, a monobasic polymorphism of which the 201 base of SEQ ID NO: 8 is A or C, a monobasic polymorphism of 82027222 of the base of SEQ ID NO 9 of A or G, the 201 th of SEQ ID NO: 10 Monobasic polymorphism of which the base is A or G, monobasic polymorphism of which the 201 base of SEQ ID NO: 11 is A or G, monobasic polymorphism of which the 201 base of SEQ ID NO: 12 is A or G, or SEQ ID NO: Monobasic polymorphism wherein the 201 base described in 13 is A or G, monobasic polymorphism wherein the 201 base described in SEQ ID NO: 14 is A or G, or a single polymorphic form wherein the 201 base described in SEQ ID NO: 15 is A or G It may include an agent capable of detecting or amplifying any one or more SNPs selected from the group consisting of nucleotide polymorphisms.

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 1) polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 from the DNA of the sample isolated from bovine, polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2, polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3 Site comprising a SNP of any one or more polynucleotides selected from the group consisting of a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 4, and a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 5, or a complementary polynucleotide thereof As a step of amplification,

The SNP is a base 201 of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, base 201 of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2, base 201 of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3 , Base 201 of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4, or base 201 of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 5; 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) polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 6 from the DNA of the sample isolated from bovine, polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 7, polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 8 , Polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 9, polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 10, polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 11, polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 12, sequence At least one polynucleotide selected from the group consisting of a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 13, a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 14, and a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 15, or a complement thereof Amplifying a region containing an SNP of a specific polynucleotide As the step,

The SNP is a base 201 of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 7, base 201 of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 7, base 201 of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 8 , Base 201 of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 9, base 201 of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 10, base 201 of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 11, sequence Base 201 of the polynucleotide consisting of the nucleotide sequence of No. 12, base 201 of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 13, base 201 of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 14, or SEQ ID NO: A base number 201 of the polynucleotide consisting of the nucleotide sequence of 15; 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 above method for discriminating bovine carcass 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 the bovine loin cross-sectional area is wide.

In the method for determining the bovine conductor trait of the present invention, when the 201st base of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2 is G, it can be determined that the bovine loin cross-sectional area is wide.

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 the bovine loin cross-sectional area is wide.

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 G, it can be determined that the bovine loin cross-sectional area is wide.

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: 5 is A, it can be determined that the bovine loin cross-sectional area is wide.

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: 6 is G, it can be determined that the bovine loin cross-sectional area is wide.

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: 7 is G, it can be determined that the bovine loin cross-sectional area is wide.

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: 8 is G, it can be determined that the bovine loin cross-sectional area is wide.

In the above method for discriminating bovine carcass of the present invention, when the 201st base of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 9 is G, it can be determined that the bovine loin cross-sectional area is wide.

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: 10 is A, it can be determined that the bovine loin cross-sectional area is wide.

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: 11 is A, it can be determined that the bovine loin cross-sectional area is wide.

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: 12 is A, it can be determined that the bovine loin cross-sectional area is wide.

In the above method for discriminating bovine carcass of the present invention, when the 201st base of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 13 is C, it can be determined that the bovine loin cross-sectional area is wide.

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: 14 is A, it can be determined that the bovine loin cross-sectional area is wide.

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: 15 is G, it can be determined that the bovine loin cross-sectional area is wide.

In addition, in the present invention, when the loin sectional area of the cow is wide, it can be judged as high-quality meat having a high meat index or grade.

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 related to loin cross-sectional traits of Hanwoo carcasses, and 81,990,480bp of Hanwoo chromosome 10 of the selected Hanwoo haploid markers. Haploid marker (HH1_GGAGA), a combination of five SNP marker groups (82) between 82,005,034 bp, had a -log10P-value of 6.32763, and a group of SNP markers (82,005,034 bp) located between 81,990,480 bp of chromosome 10 Since the haploid marker (HH2_GGGGAAACAG) which is a combination of -log10P-value was 6.32763, 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. It is possible to more accurately determine the trait (loin cross-sectional area), thereby making it easier and accurate to discriminate high-quality meat.

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 the fillet cross-sectional area, W is a covariance matrix for fixed effects, α is a coefficient including the intercept, x is a genotype vector, β is an effective size of the marker, u is a random effect, and ε is It means the value for error.

Specifically, using the mixed linear model based association analysis (MLMA) and restricted maximum likelihood (REML) analysis method of the Hanwoo loin cross-sectional trait and 975,945 haploid markers constructed in Example 1 Association significance was confirmed, and gender and slaughter age were greatly corrected by adding covariates to the Hanwoo loin cross-sectional phenotype.

GEMMA (version 0.96, http://www.xzlab.org/software.html) statistical software was used to verify the significance of each haploid marker for traits, and carcass weight, fillet cross-sectional area, and marbling index information of Hanwoo carcasses were used. 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 (a combination of SNP marker groups (5, SEQ ID Nos. 1 to 5) existing between 81,990,480 bp and 82,005,034 bp of chromosome 10 and between 82,011,799 bp and 82,054,208 bp). SNP marker group (10 combinations, SEQ ID NOs: 6-13)) (FIG. 1).

SEQ ID NO: 1 to 5 is a base sequence of about 400 bp including five SNP markers present between 81,990,480 bp and 82,005,034 bp of Korean beef chromosome 10, and SEQ ID NOs 6 to 15 are 82,054,208 at 82,011,799 bp of Korean beef chromosome 10 A base sequence of about 400 bp comprising 10 SNP markers present between bp.

The combination of the SNP marker groups (five) present between 81,990,480 bp and 82,005,034 bp of the Hanwoo chromosome 10 is the 81990480th, 81991722th, 81995899th, 81995899th, 82003284th, and 82005034th bases of the Hanwoo chromosome 10 in order of 'GGAGA. (SEQ ID NO: 16) (Table 1),

The combination of the SNP marker group (10) existing between 82,011,799 bp and 82,054,208 bp of the Korean beef chromosome 10 was 82011799, 82013193, 82024014, 82027222, 82030321, 82036157, 82043067, 82047253 Bases in the 82051139th, and 82054208th are 'GGGGAAACAG' (SEQ ID NO: 17) in that order (Table 2).

Haplotype ID SNP Marker ID chromosome location Genotype information HH1_GGAGA BovineHD1000023368 10 81990480 A / G BovineHD1000023369 10 81991722 A / G BovineHD1000023370 10 81995899 A / G BovineHD1000023372 10 82003284 A / G BTB-01373360 10 82005034 A / G

Haplotype ID SNP Marker ID chromosome location Genotype information HH2_GGGGAAACAG BovineHD1000023374 10 82011799 A / G BovineHD1000023375 10 82013193 A / G BovineHD1000023378 10 82024014 A / G BovineHD1000023379 10 82027222 A / C BTB-01373370 10 82030321 A / G BovineHD1000023380 10 82036157 A / G BovineHD1000023382 10 82043067 A / G BovineHD1000023383 10 82047253 A / C BovineHD1000023384 10 82051139 A / C BovineHD1000023385 10 82054208 A / G

The haploid marker (HH1_GGAGA), which is a combination of SNP marker groups (5) present between 81,990,480 bp and 82,005,034 bp of chromosome 10 among the selected Hanwoo haploid markers, had a -log10P-value of 6.32763, and 82,054,208 at 82,011,799 bp of chromosome 10 The haploid marker (HH2_GGGGAAACAG), which is a combination of 10 groups of SNP markers present between bp, had a -log10P-value of 6.32763 (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 carcass traits (loin area).

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Composition for identifying a bovine eye muscle area trait          configuring an agent capable of detecting or amplifying a          haplotype <130> 2018P-10-022 <160> 17 <170> KoPatentIn 3.0 <210> 1 <211> 402 <212> DNA <213> Artificial Sequence <220> <223> No1 <400> 1 ctcactgaag atcagctcag atgtcatgat gcagagtgca gccttccagg tgggactgcc 60 tgggcattgg ctgtgctgtc ctgggcaagt aatgcccttt cttgttggca agatataagt 120 gcaaatacca caagctgtac agatagattg aagaccatgc tcaatgtcct tcacaggtgc 180 ctggcatcaa gtcagagaac agtgagaacc agaggcggat gctcgtttca ctcttcccat 240 cttgcttcag gcggtcaagg ttctgtgcct ctctgtgtgc tcaggcgtag tatgtgctgc 300 ttctggagat gctgggatgg gccctggggt tagctgagcc ctgctgcttt gtgggggaag 360 tcacagctca ggttgaccgt ctgctgttgc cctggaccct tg 402 <210> 2 <211> 402 <212> DNA <213> Artificial Sequence <220> <223> No2 <400> 2 tggtcctgga tgctctattt ttctcttgac gatctcatcg tatcccatga ctttaaatct 60 gtgccctggt gactctaaca tttgcatccc tagccctcat tccaacctcc tatgctagct 120 gcctagggca catcttcatt taggtcctgt cttacttgtg ttccctgctg cagacagagc 180 ctgagacagg ggttctggcg agagggttta tcaaagcacg gtctctgggg aaagggaatg 240 agggagacgg ggtagggcag ggaagaggct aggcaaggat gtggtcccac ttgcttctgt 300 gtggaatctg agaaagtgaa actcacagaa acagtagaat ggtgtggggt tgggagcagg 360 tgggggaagt ggggagatac tgatcaaagg gcacacactt cc 402 <210> 3 <211> 402 <212> DNA <213> Artificial Sequence <220> <223> No3 <400> 3 taaatatagc tgtgtgtacc tattgatccc catttttttt caattagggg tgaatcttta 60 aaaatcttgt atcaaatcat atcatttcct tgggtagact ctttcagtgc ctttccatgg 120 tacttaggat gaaatccaaa ctcctcagcc ctgtgtcaag tcctacataa tctgaccttt 180 gtcccctctc cccttcccat aggtctccca cctcctgcct caggtagggt atggtaaagt 240 cggtaaggtg tgacatgtct ctcgtgtttg tgcttctttt ctcctcagct tggctgggag 300 gttcttgaag gaggagccat gattgtccct ggattttgtg tgggcagcac attcttgaca 360 tttaagtctg tgctcaaatg tcatccaccc tccagtcttt ct 402 <210> 4 <211> 402 <212> DNA <213> Artificial Sequence <220> <223> No4 <400> 4 atgcaaaacc ttgccagggc taaaactttg gagccatcct cttgttttgg gcatccccaa 60 ccaaacagtg gcttatgcca tcctgtcccc tgagaccaga gcagttcatc aaccagcagc 120 caggtgtgta gttgttggaa ggtgccagca tctgtcccca ccttccccca actcaacttg 180 gccaccaggg aagtgggagg agggctttgg ctttccagag atgtgtggaa ccacgggttt 240 agctcctcca cagtcagagt ttctcaaagg atgctggggt gggggtgggg gggtgggaca 300 ctggtttttt aggaaacatg ttgtattatg cagtagaatg catgcctgcc tggctagaat 360 aggccacgcc ccaggggtaa ccttcttctg ctatttgggg tg 402 <210> 5 <211> 402 <212> DNA <213> Artificial Sequence <220> <223> No5 <400> 5 agttcaggga ctggatccca ggtctgtcta ctcaaagcct gtgcttttaa cacctgcatt 60 ttctgcagag tacagagcac tttccaaata gcattttctt tgttgtgtcc tgtgaaaggc 120 tcagacctgg ttcttattac aaatgtcata gctgcctggc agttggagaa ttgagtgagt 180 ttgtcatcca tcttcccccc aggctcataa atatggacag tcccctttcc ccacatcaga 240 tagaatggca ttgactctaa actttaaccc ctcttatttt cacctgtgtc agagagcagt 300 tccccgagag atacagagca cctacaatcc aggatgcagg tagctgggaa gatggggttg 360 agctgagctg caggggtggg gagtgcaggg ccctgggaaa gt 402 <210> 6 <211> 402 <212> DNA <213> Artificial Sequence <220> <223> No6 <400> 6 gtattttgta gaccagcttg ggagcctgac agctatatct ggtattgccg ttttggaaaa 60 tggagttttg gatgtttaat tggtctttgc aaatctccct atgttctggt gcttgtctga 120 ctcctgcagc ctgctgagtg gacctgcatt cccactagca tctctcttag aactggtatt 180 tccaagtctc ttctgtctgt aggatgagaa aatggcgtca tatcggcctt tactcccaaa 240 ttacaaagga aacaagaccc tcatctgctc aaacaccaat tcctctcagt ctctactttg 300 gggtgcttcc acaaactagt cataatttcc ccctttgcct aaaaggaacc tcactgctgg 360 cagtatgcat caagagaaca cccgagcctg ggccaatgga ac 402 <210> 7 <211> 402 <212> DNA <213> Artificial Sequence <220> <223> No7 <400> 7 gggcgggtga ctttctctat gccctcagtt ccattggctg ggatttagtg actggagtgt 60 tgcaagcagg aaacaaaaat aaatgctctg tgtctctgat cagaaggctt gacaccctgt 120 agggctaaca cctaaacaga ttttaagcta aaggattcca ctcattcaaa gaaatgtttt 180 catccctacc tgtcctctgt agtctccttc ttccttgctg acacgcatgc ttgtggcaca 240 ccccctttcc cttcctgctg ctgctgctaa gttgcttcag tcgtgtccga ctctgtgcga 300 ccccagagac ggaagcccac caggctcccc tgtctttggg attctctagg caagaacact 360 ggagtgggtt gccatttcct tctccaatgc atgaaagtga aa 402 <210> 8 <211> 402 <212> DNA <213> Artificial Sequence <220> <223> No8 <400> 8 gggtgcagtc ataccttttc caaaccttta tgagcgtggt gtgattatcc agtcaaatct 60 aacatcactg atggccagag gtgtgtgcac cacattgcca tgtgccacga tgggcttttc 120 attctctcag ttcactctga caaagagcct tcagtgtgtc cggagctctg ctatagaggg 180 atgaatgaaa gtggatccag agtctcaagg cccatgcagt ctaatcatgg gttcttcaac 240 agaagctcat gggttgtaga atccagttat tattttccaa ctgtatccta cagttgtgaa 300 caggggttca ccgagaaggg ggtccatggt caaataagtt tgagagatac agcatcatat 360 atgctcttga tctggttagc tattgctctg tttcagacta ct 402 <210> 9 <211> 402 <212> DNA <213> Artificial Sequence <220> <223> No9 <400> 9 aagaatacct ttaagaggca ggtgaggctg tggggcattt gtgttcctgg aacagccact 60 ttgatatgtc tgtgtgcctg tgtgtctgtg tttgtgtggt gtggtttctg cctgcgtgtg 120 tgtatgtctg gatgtgtgtg gtgtgtgtgt gtgatgtgtg gcggtgtgtg ggcccccaca 180 acacagtgct tttctctcag acgtttctac agctggagtc tgtggctcct gtgtggggtc 240 caagtgactc tgatgtgcat acctccctct ttagacgctg gccagagcaa gtgtcgcctg 300 gagcgggctc aggccctgga gcaagccaag aagccccagg aggcggtgtt tgtcccggag 360 tgcaccgagg atggctcctt tacccaggta ggccttggct ac 402 <210> 10 <211> 402 <212> DNA <213> Artificial Sequence <220> <223> No10 <400> 10 atgaggtagt gataaggatt aaataagtca acacatgtga aatgcttgga acacagaaat 60 actgtataaa tatctgccat tatcaacatt agccccatct ctactctggt tctgtcactc 120 agatcttctc aagatgttac agacgatgtc acccaagaaa aagaaaatct tctgttgcac 180 ctttcctcca tgggttcttt agtttaccat tttttctagt gaaaagaacc caggtgatct 240 ttgtactgaa aattcacgta tgcgggagaa tatcaaacct cttgcagctg catttggggc 300 tgagacaggg gtcaaggtaa tagggaggag gcagaatcct tttcccccct ttgtaggctt 360 ctgtaatcca gagtgtgttg cttacgagaa cggttgaccc ct 402 <210> 11 <211> 402 <212> DNA <213> Artificial Sequence <220> <223> No11 <400> 11 agccaccact gccatctttt caatgaaaga accctgtggg cacccacaag tacagaggag 60 ggcaagatga gggtgggtgg agctccaatg ctgtgtccgt tgtattcggt ggtgacctgc 120 ctactccgac cactgctcgg tcctcctctc caatggctgc atggcccctg agcctcctct 180 cccagattgg ctggcatcat agtgctttgg agcaagttgt aggctctggg tgctcccaac 240 tgggtatctg acctccctgc agtctctggt gaatcttccc ctaagtctat cttggaaata 300 gaaggagcaa gccctgggca gtacccacag ctgctcttct gcaagggggt gtacttggga 360 ggaagggatg gatgaggcct gcttctctgg tcttcctgct gc 402 <210> 12 <211> 402 <212> DNA <213> Artificial Sequence <220> <223> No12 <400> 12 tcctatgaag gatttgcaca ctgggtgacc ttgaggccca aatgagatac tgcctcagtc 60 atggcggtgt ttggaaaatc attgtaaaga cagaaagtgg tgctttccat ttgggctgtg 120 acatgtgagt gctccttggc tctacatatg gcccatcatc tgctttgctt ttctcatggt 180 ttcctccctt ttgatgcccc aggttggttt gttttgctct gttagctggt gtttgctctg 240 ttctctgctc atcagttact caactgcact cactggcagt gctggcctcc cacattttcc 300 caggtgtcca caagttgggc taatgttgaa gactaacaag cttaaaataa tttcagattt 360 gtcttcattt ggacagagag gttgacccga agtaaaccac tg 402 <210> 13 <211> 402 <212> DNA <213> Artificial Sequence <220> <223> No13 <400> 13 ggggagagca ggggccagat catggggatc cctaagcttg tgcaaatcag catcatctcc 60 atcctgagtc cgatggcaaa tctttgaggt attttaagca ggggattgat gcccagattt 120 gtgtagaaag tgggtggaaa agggatagtg gggtgaggtg gaggaggtga gcagagtgac 180 aactattgaa agagaaagaa acagtgaccc cgaatgaagc ccaaagcctc gtttttatgt 240 actgttccca ggcagcgtgg acctgaccat ggaggcttga agaagatcaa gccgtggtgc 300 agcagggcat ctagaaccag cacctgcagg cccaagtggt cttagcccca tcacgtgtgg 360 ccttctctca ggaaagtcac gtgatcagta ggcaagcgga ct 402 <210> 14 <211> 402 <212> DNA <213> Artificial Sequence <220> <223> No14 <400> 14 ccagggatcg aacctgggtc tcctgcattg caggcagatt ctttactgtc tgagccacta 60 gggaagcctt tctttaccta gaacaatgat aaatagcgag ttgaaaaaca ccatgacaaa 120 tcaagagaga gaccatggag gagaggatca gacttaatac tttagtgtct ttcatggcac 180 cctttctcct gtgttttgaa acaaaaggct ttttcactct gcactgggcc ctacaaattg 240 tgcagttggc cttgatgagg gcatttatct aagtgctggt tagtggacca accgacatga 300 gaaatgggcc tccgggatgt gctgaccata ctttctagta tggaagacgc taagatttgc 360 cacttcttac ctccagaaca gcatacacac agacagttgg tg 402 <210> 15 <211> 402 <212> DNA <213> Artificial Sequence <220> <223> No15 <400> 15 tgttggtgga gcaggtgaag aatgggagac tagtgggaga ggacttggac aatgatttct 60 ggcagttcct gggaactttt tctccggagc gcctaggata catggccaat gaaaatttag 120 gatcataggg agttttctgc agtttactct tttcttactt ctaagagctc tgggagtgag 180 cagtgtaagg aaagtgaaag agagagtaaa aacatttgga aaactgctct cttttctgca 240 cgtgctcctg tatttgtcaa gccagtcttg gagagaagtg gaacttccaa aatacaactc 300 atagagactt cttttattgc accccccacc tcccgcccct ccactgactg cttcatagca 360 tgtgttccaa gtgaagaatg gatcacagtt attttccaga gt 402 <210> 16 <211> 5 <212> DNA <213> Artificial Sequence <220> <223> No16 <400> 16 ggaga 5 <210> 17 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> No17 <400> 17 ggggaaacag 10

Claims (9)

Single nucleotide polymorphism (SNP) in which the 201 base is A or G in the polynucleotide described in SEQ ID NO: 1, single nucleotide polymorphism in which the 201 base is A or G in the polynucleotide described in SEQ ID NO: 2, the sequence In the polynucleotide of SEQ ID NO: 3, the monobasic polymorphism of which the 201 base is A or G, in the polynucleotide of SEQ ID NO: 4, the monobasic polymorphism of the 201 base is A or G, and in the polynucleotide of SEQ ID NO: A composition for determining the loin cross-sectional area of Hanwoo, comprising an agent capable of detecting or amplifying a haplotype for loin cross-sectional area discrimination of Hanwoo of a monobasic polymorphism having a 20th base as A or C.
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 determining the loin cross-sectional area of Hanwoo.
A kit for determining the loin cross-sectional area of a Korean beef comprising the composition of claim 1.
delete delete 1) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 from the DNA of a sample isolated from Hanwoo, a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2, a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3, Amplifying a site comprising a polynucleotide consisting of a nucleotide sequence, and the SNP of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 5 or a complementary polynucleotide thereof,
The SNP is a base 201 of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, base 201 of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2, base 201 of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3 , Base 201 of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4, and base 201 of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 5; And
2) comprising the step of determining the base type of the SNP at the site containing the amplified SNP, sirloin cross-sectional area determination method.


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