KR101213217B1 - SNP Markers Associated with Meat Quantity and Beef Quality in Hanwoo - Google Patents

Abstract

The present invention provides an SNP marker useful for judging carcass weight, abdominal muscle cross-sectional area, back fat thickness, or muscle mass and meat quality of Hanwoo. In addition, the present invention provides a method for determining the quality of Hanwoo beef using the SNP marker of the present invention, and provides a kit used for the determination of the meat quality of Hanwoo. By using the SNP marker of the present invention, it is possible to determine the carcass weight, abdominal muscle area, back fat thickness or intramuscular fat degree of Hanwoo by a simple method. Can be.

Description

SNP Markers Associated with Meat Quantity and Beef Quality in Hanwoo}

The present invention relates to polynucleotides or their complementary polynucleotides useful for the early determination of carcass weight, gastrocnemius muscle area, dorsal fat thickness, or intramuscular fatity of Hanwoo.

Many studies are underway to use individual DNA information as a supplementary means to accurately select livestock with genetically superior traits.

The improvement of Hanwoo is selecting endorsement hydrogen in parallel with the contemporary and later tests, and the candidate's hydrogen was selected by evaluating the individual's growth ability in the contemporary test, and the growth and carcass performance of the descendants of these individuals, namely castrates, were tested. Thus, a dual verification system for selecting guaranteed hydrogen among candidate hydrogens is being performed. However, the traits that assess the genetic ability of an individual at the time of the current test are mainly evaluated only for growth, but it is impossible to measure meat quality data. Therefore, the accurate evaluation of the meat quality of the individual is low because the evaluation is based on parental breeders. Therefore, if the genetic marker that can estimate the genetic ability of meat is selected at the time of the current test, it will be possible to increase the speed and accuracy of the Hanwoo. After the conclusion of the Korea-US FTA negotiations, the increase in meat quality and quality improvement of Korean beef has been very important due to the cheap opening of US beef imports. In particular, in order to differentiate quality from imported beef and improve competitiveness, various measures including the use of advanced science and technology for the production of high-quality beef Han beef should be sought. The carcass price of Korean beef is evaluated by meat grade and meat grade. Meat is judged on the basis of carcass weight, backfat thickness, and gastrointestinal cross-sectional area, and meat quality is determined by intramuscular fat, meat color, fat color, texture, and maturity. Animal Product Rating Service, 2004). Intramuscular fat is closely related to the year, juiciness, and flavor that are the sensory characteristics of beef. Therefore, it is very important to increase the intramuscular fat content for high quality meat production. Genetic information on the economic performance of Korean cattle at the DNA level provides important information not only for producers, but also for breeders, for the early selection of superior cattle. This DNA information improves the accuracy of selection by applying marker-assisted selection (MAS) method for meat mass and meat quality, and saves considerable expenses and time (generation intervals) for subsequent tests. Is expected to increase.

Conventionally, single gene markers related to important meat-related traits of Hanwoo, such as carcass weight, backfat thickness, and intramuscular fatity, have been reported using a genera- tive test group of the Hanwoo Improvement Office (Cho et al., 2007; Cheong et al., 2008; Kim et al. 2009; Kim et al. 2010). However, there is no report on whether the DNA markers obtained in this study are actually applied to the Hanwoo cattle group for the visible effects, and these markers are also polygeny, one of the genetic characteristics of the target trait. Because a large number of genes are involved in the quantitative trait of, the effect of each gene on the trait is very insignificant, so a few markers have a limit to show a very small effect on the trait.

Recently, with the development of mass sequencing and genotyping technology, SNP chips that can simultaneously analyze many genes in livestock are being made, and many large chromosomal loci (SNPs) affecting traits using these large chips ), A kit, and a set of genetic marker combinations that can be provided as a supplementary means to select excellent Hanwoo cattle and cows by applying them to the Hanwoo cattle and cattle breeding populations used in the Hanwoo Improvement Program. Can be utilized as

Studies on the detection of marker genes related to meat and meat phenotype of Hanwoo have been done in pieces, and as a result, few genes have been discovered. Was none. When selecting Hanwoo individuals with excellent meat quality and meat quality, in addition to the existing selection method for estimating the breeding value (genetic qualities of the individual) using phenotype and pedigree information, the introduction of meat and meat quality DNA markers can improve the accuracy of selection. This can increase the efficiency of genetic improvement.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

To develop DNA markers involved in the expression of carcass weight, backfat thickness, intramuscular fat, and intestinal muscle area traits influencing the price fluctuations of Hanwoo beef in Hanwoo, the candidate cattle and their offspring, (Gasewoo) ) Whole-genome association analysis was performed to search for SNPs related to traits using a Sire model in which SNP effects were randomly corrected using commercially available SNP chips. As a result, we identified 27 SNP loci in the backfat thickness, 19 SNP loci in the 24-month-old carcass, 24 SNP loci in the gastrointestinal cross-sectional area, and 96 SNP loci in 26 intramuscular fat loci. The present invention has been completed by confirming that it can be used to select early.

Accordingly, it is an object of the present invention to provide a SNP marker useful for judging meat mass and quality, such as carcass weight, abdominal muscle cross-sectional area, back fat thickness, or intramuscular fat degree of Hanwoo.

It is another object of the present invention to provide a method for determining the carcass weight, gastrocnemius muscle area, back fat thickness, or intramuscular fat degree of Korean cattle using the SNP marker.

Still another object of the present invention is to provide a kit for detecting SNP markers used for determining the carcass weight, gastrocnemius muscle area, dorsal fat thickness, or intramuscular fat degree of Hanwoo.

The objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to an aspect of the present invention, the present invention provides the 61st nucleotide of SEQ ID NO: 1 sequence, the 58 nucleotide of SEQ ID NO: 2 sequence, or the 61 nucleotide of any one of SEQ ID NO: 3 to 96 sequences Polynucleotides or complementary polynucleotides comprising 8-100 contiguous nucleotides, which are useful for the determination of carcass weight, intestinal muscle cross-sectional area, backfat thickness, or intramuscular fatity of Hanwoo, are provided.

The SNP marker of the present invention is applied to bovine, most preferably to hanwoo. In the present specification, the term "Korean Cattle (Hanwoo)" refers to a reverse cattle (役 牛) of a conventional species that has been conventionally bred for transportation or farming on the Korean peninsula, which means Bos taurus coreanae. do.

As used herein, the term "nucleotide" is a deoxyribonucleotide or ribonucleotide present in single-stranded or double-stranded form and includes analogs of natural nucleotides unless otherwise specifically indicated (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990)).

In the present invention, each of the 96 SNP markers can be used to determine meat mass and meat quality such as carcass weight, abdominal muscle cross-sectional area, back fat thickness, intramuscular fat degree, and the like. It is based on high frequency.

The present invention relates to base variants of the respective SNP positions in SEQ ID NO: 1 to 96, but when such SNP base mutations are found in double stranded genomic DNA (gDNA), complementary to the nucleotide sequences described above It is also interpreted to include polynucleotide sequences. Thus, the base of the SNP position in the complementary polynucleotide sequence also becomes the complementary base.

According to another aspect of the present invention, the present invention provides a method for determining the carcass weight, gastrocnemius muscle area, back fat thickness, or intramuscular fat degree of Hanwoo, comprising the following steps: (a) Isolating nucleic acid molecules from Hanwoo Making; And (b) a single nucleotide polymorphism (SNP) corresponding to the 61st nucleotide of SEQ ID NO: 1, the 58th nucleotide of SEQ ID NO: 2, or the 61st nucleotide of any of SEQ ID NOs: 3 to 96 Identifying the base type of the nucleic acid molecule.

As used herein, the term “nucleic acid molecule” is meant to encompass DNA (gDNA and cDNA) and RNA molecules inclusively, and the nucleotides, which are the basic structural units in nucleic acid molecules, are modified from sugar or base sites, as well as natural nucleotides. Analogues also include (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990)).

In the method of the invention, the nucleic acid molecule can be obtained from various sources of beef, for example from muscle, epidermis, blood, bone, organs, and most preferably from muscle or blood.

When the starting material in the method of the present invention is gDNA, the separation of gDNA can be carried out according to conventional methods known in the art (Rogers & Bendich (1994)). When the starting material is mRNA, the total RNA is isolated by a conventional method known in the art (see Sambrook, J. et al., Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (1987); and Chomczynski, P. et al., Anal. Biochem. 162: 156 (1987)). The isolated total RNA is synthesized by cDNA using reverse transcriptase. Since the total RNA is isolated from animal cells, it has a poly-A tail at the end of the mRNA, and cDNA can be easily synthesized using oligo dT primer and reverse transcriptase using this sequence characteristic (see PNAS). USA, 85: 8998 (1988); Libert F, et al., Science, 244: 569 (1989); and Sambrook, J. et al., Molecular Cloning.A Laboratory Manual, 3rd ed.Cold Spring Harbor Press (2001) )).

In the method of the present invention, step (b) may be carried out by applying various methods known in the art used to identify specific sequences. For example, techniques applicable to the present invention include fluorescence phosphorylation hybridization (FISH), direct DNA sequencing, PFGE analysis, Southern blot analysis, single-strand conformation analysis (SSCA, Orita et al., PNAS, USA 86: 2776 (1989)), RNase protection assay (Finkelstein et al., Genomics, 7: 167 (1990)), dot blot analysis, denaturation gradient gel electrophoresis (DGGE, Wartell et al., Nucl. Acids Res. 18: 2699 (1990)), methods using proteins that recognize nucleotide mismatches (e.g., mutS protein of E. coli) (Modrich, Ann. Rev. Genet., 25: 229-253 (1991)), and Allele-specific PCR, including but not limited to.

Sequence changes result in differences in base bonds within single-stranded molecules, leading to the appearance of bands with different mobility, which SSCA detects. DGGE analysis uses a denaturing gradient gel to detect sequences that represent wild type sequences and other mobility. Other techniques generally employ probes or primers that are complementary to sequences comprising the SNPs of the invention. For example, in an RNase protection assay, a riboprobe complementary to a sequence comprising a SNP of the present invention is used. The riboprobe is hybridized with DNA or mRNA isolated from a plant, and then cleaved with an RNase A enzyme capable of detecting a mismatch. If there is a mismatch and RNase A recognizes, a smaller band is observed.

In assays using hybridization signals, probes complementary to the sequences comprising the SNPs of the invention are used. In this technique, hybridization signals of probes and target sequences are detected to directly determine whether SNP variants are present.

As used herein, the term “probe” refers to a linear oligomer having naturally occurring or modified monomers or bonds, including deoxyribonucleotides and ribonucleotides that can hybridize to a particular nucleotide sequence. Preferably, the probe is single stranded for maximum efficiency in hybridization. The probe is preferably deoxyribonucleotide. As the probe used in the present invention, a sequence perfectly complementary to the sequence including the SNP may be used, but a sequence complementarily complementary to a range that does not prevent specific hybridization may be used. It may be.

Preferably, the probe used in the present invention is a SNP nucleotide, the 61st nucleotide of SEQ ID NO: 1 sequence, the 58th nucleotide of SEQ ID NO: 2 sequence, the 61st of any sequence of SEQ ID NO: 3 to 96 A sequence capable of hybridizing to a sequence comprising 8-100 consecutive nucleotides comprising nucleotides.

More preferably, the 3'-end or the 5'-end of the probe has a base complementary to the SNP base. Generally, the stability of the duplex formed by hybridization tends to be determined by the agreement of terminal sequences, so that in a probe having a base complementary to the SNP base at the 3'-terminal or 5'-terminal, If the part is not hybridized, such a duplex can be disassembled under stringent conditions. Suitable conditions for hybridization include, but are not limited to, Nucleic Acid Hybridization, A Practical Approach, Hayes, BD, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, IRL Press, Washington, DC (1985). ≪ / RTI > The stringent condition used for hybridization can be determined by controlling the temperature, the ionic strength (buffer concentration) and the presence of a compound such as an organic solvent, and the like. This stringent condition can be determined differently depending on the sequence to be hybridized.

Step (b) of the method of the invention can be carried out by an allele-specific gene amplification method. When SNPs are applied to gene amplification methods, they are collectively referred to as single nucleotide amplified polymophism (SNAP).

According to a preferred embodiment of the invention, such gene amplification is any one of the 61st nucleotide of SEQ ID NO: 1 sequence, the 58th nucleotide of SEQ ID NO: 2 sequence, or the SEQ ID NO: 3 sequence to 96 sequence of SNP nucleotides Primer pairs designed to amplify polynucleotides containing the 61st nucleotide of one sequence are basically used.

A “primer” in the context of the present invention is a single strand of oligonucleotide, suitable conditions (the presence of four different nucleoside triphosphates and polymerases such as DNA or RNA polymerases), suitable temperatures and suitable It is meant to act as an initiation point that can initiate template-directed DNA synthesis under a buffer.

The suitable length of the primer is determined by the nature of the primer to be used, but is usually used as a length of 15 to 30 bp. The primer need not be exactly complementary to the sequence of the template, but should be complementary enough to form a hybrid-complex with the template.

Amplification techniques that can be used in the methods of the invention include PCR amplification (Miller, HI (WO 89/06700) and Davey, C. et al. (EP 329, 822)), ligase chain reaction (LCR, Wu, DY) et al., Genomics 4: 560 (1989)), polymerase ligase chain reaction (Barany, PCR Methods and Applic., 1: 5-16 (1991)), Gap-LCR (WO 90/01069), repair chain Reactions (EP 439,182), 3SR (Kwoh et al., PNAS, USA, 86: 1173 (1989)) and NASBA (US Pat. No. 5,130,238). Most preferably by PCR amplification step. Where amplification is applied, it is important to design suitable primers to identify the SNP bases of the present invention.

According to a preferred embodiment of the present invention, the present invention relates to (i) the 61st nucleotide of SEQ ID NO: 1 sequence, the 58th nucleotide of SEQ ID NO: 2 sequence, or the SEQ ID NO: 3 sequence to SEQ ID NO: 96 sequence Annealed with a sequence adjacent to the 61st nucleotide of either sequence, and (ii) the genome of the SNP using a genotyping primer designed to match the nucleotide at the 3 'end with the nucleotide at position -1 from the SNP nucleotide. Provided are methods for analyzing base types.

In a preferred embodiment of the present invention, a dideoxy adenosine triphosphate (ddATP), a dideoxy cytosine triphosphate (ddCTP), and a dideoxy guanosine triphosphate (ddGTP) labeled to show different colors of fluorescence in the amplification reaction solution, respectively. ), And dideoxythymidine triphosphate (ddTTP).

When amplification reaction was carried out by including the genotyping primer designed as described above and ddATP, ddCTP, ddGTP and ddTTP as a reaction material in the reaction solution, only one base matching the target SNP nucleotide from the 3 'end of the genotyping primer was extended ( extension), the type of the extended base can be easily identified by fluorescence. By this method the type of SNP nucleotide can be determined.

The conditions of the amplification reaction by PCR and the reagents and enzymes used can be those conventionally known in the art.

The basis for the use of the SNP marker of the present invention for determining carcass weight, abdominal muscle cross-sectional area, isofat thickness, and intramuscular fatity is based on the high frequency of the specific base at the SNP mutation site in the Korean cattle.

According to another aspect of the invention, the invention is any one of the 61st nucleotide of SEQ ID NO: 1 sequence of SNP nucleotides, the 58th nucleotide of SEQ ID NO: 2 sequence, or SEQ ID NO: 3 to 96 Provided is a kit used for determining the carcass weight, gastrocnemius muscle cross-sectional area, dorsal fat thickness, or intramuscular fat degree of Hanwoo including primer pairs designed to amplify polynucleotides including the 61st nucleotide of one sequence.

According to a preferred embodiment of the present invention, the primer pair is (i) the SNP nucleotide, the 61st nucleotide of SEQ ID NO: 1, the 58 nucleotide of SEQ ID NO: 2, or the SEQ ID NO: 3 to SEQ ID NO: A kit characterized by annealing with a sequence adjacent to the 61st nucleotide of any of the 96 sequences, and (ii) the nucleotide at the 3 'end comprises a genotyping primer that matches the nucleotide at position -1 from the SNP nucleotide. to provide.

According to another preferred embodiment of the present invention, dideoxy adenosine triphosphate (ddATP), dideoxycytosine triphosphate (ddCTP), dideoxyguanosine triphosphate, each labeled in the kit to show fluorescence of different colors (ddGTP), dideoxythymidinetriphosphate (ddTTP) is further provided.

When the kit of the present invention is subjected to a PCR amplification process, the kit of the present invention 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 heat stable DNA polymerase obtained from Pyrococcus furiosus (Pfu)), DNA polymerase cofactors and dNTPs. The kit of the present invention may be made from a number of separate packaging or compartments containing the above reagent components.

The present invention provides an SNP marker useful for judging carcass weight, abdominal muscle cross-sectional area, back fat thickness, or muscle mass and meat quality of Hanwoo. In addition, the present invention provides a method for determining the quality of Hanwoo beef using the SNP marker of the present invention, and provides a kit used for the determination of the meat quality of Hanwoo. By using the SNP marker of the present invention, it is possible to determine the carcass weight, abdominal muscle area, back fat thickness or intramuscular fat degree of Hanwoo by a simple method. Can be.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example  1: Korean beef useful SNP  Mass Excavation and Mapping

556 DNAs of candidate hydrogen and Candidate Black Cow (Geo Se-woo) were extracted from the National Agricultural Cooperative Federation, and genotyping was performed using Illumia 50k SNP chip. Among the total 55074 SNPs planted on the Illumina chip, the SNPs were selected and identified as SNPs satisfying the following conditions: (i) minor allele frequnecy (MAF)> 0.05, (ii) call rate (≠ missing genotype) > 90%, (iii) Genotype_group> 1, (iii) SNPs with known genomic positions, (iii) SNPs on autosomal chromosomes.

The SNP markers satisfying these five conditions were a total of 36,567 SNP markers (66%). The number of each chromosome was shown in the following table and the average interval between markers was 69.2 ± 66.2 kbp.

The file shown in the figure below is an example that contains 36,567 SNP marker information and the physical map (distance bp) of the Korean cattle.

Example  2: battlefield correlation analysis whole - genome association analysis Excavation of trait-related SNPs through

   (1) Single marker analysis

The effect of each SNP was corrected in an animal model with randomness, and the model equation is:

Y = Xb + Z1u + Z2q + e,

Where Y is the phenotypic vector, b is the fixed and covariate effect associated with the trait (total mean, date of birth, place, test order, carcass age), X is the relevant design matrix, and u is the multiple of the individuals. Genetic Effects (EPD), Z1 is an EPD related design matrix, q is a vector representing the additive effect of three genotypes (AA, AB, BB) of SNP, Z2 is a related design matrix, e is a residual effect.

The test statistic is LRT (likelihood ratio statistics, LRT) = -2ln ( L ₁ / L ₀ ), where L ₁ is the likelihood obtained from the model mentioned above and L ₀ does not correct the SNP effect in the model above. Likelihood values obtained from non-models.

When the statistical value is less than the significance level P = 0.001 (or -log10P> 3) set by obtaining a P value under the null hypothesis condition compared to the value obtained from a chi-square distribution table with 1 degree of freedom (or -log10P> 3), And conclusions (Meuwissen & Goddard, 2007, Genetics).

(2) Haplotype analysis

The haplotypes of two adjacent SNPs were corrected by random effects, and then the association analysis was performed in the same way as the single marker analysis (Meuwissen & Goddard, 2007, Genetics).

   (3) Clustering analysis

Unlike the previous assays based on one or two SNP genotypes in each test, several dozen clusters were grouped by genetic haplotypes of all haplotypes of the subjects tested. After classification with clusters, the model was calibrated with random effects, and correlation analysis was performed in the same way as the single marker analysis (Druet & Goerges, 2010, Genetics).

By applying the three methods of single marker analysis, haploid analysis, and clustering analysis, full-length correlation analysis is performed to determine the chromosome position where SNPs having statistical significance of P <0.001 (or -log ₁₀ P> 3) are located ( Quantitative trait loci (QTL) were selected.

Phenotype was applied to the analysis using two forms for each trait. First, the original value was used or the breeding value estimated for each individual was used as the phenotype. As a result, 19, 27, 24, and 26 SNPs were identified in carcass weight, backfat thickness, loin area and intramuscular fat map, respectively.

Tables 1 to 4 below show information on 96 SNP loci affecting carcass weight, dorsal fat thickness, abdominal muscle cross-sectional area and intramuscular fat at 24 months of age.

In Table 1 to Table 4,

^a : SNP annotations listed on Illumian bovine 54k chip

^b : SNP annotation registered in NCBI, animal QTL DB

^c : SR, single marker model; HR, haplotype marker model; CR, marker-clustering model; RES (BV), regarding residuals (BV) as dependent variable after adjusting phenotypes for fixed and covariates.

^d : This value is expressed as -log ₁₀ P and P is the probability of the test statistic under the null hypothesis.

^e : The ratio of the variance described by the SNP out of the total phenotypic variances, indicating the magnitude of the SNP effect.

Base sequences comprising 96 SNPs discovered in the present invention are shown in Tables 5 to 8.

The figure below shows a profile for an SNP associated with Hanwoo's gastrocnemius muscle area. That is, the SNP is located at the 10 Mb region of chromosome 20 which is related to the Hanwoo's gastrocnemius muscle cross-sectional area.

Example  3: SNP Marker  Used Flesh  And meat quality assessment process

First, DNA is extracted for each individual, and genotyping is performed on the meat and meat SNP panels. Each individual is assigned a coefficient according to the genotype for each SNP. For example, one SNP can have three genotypes, such as AA, AB, and BB, with each coefficient assigned to +1, 0, -1. After multiplying the effect value (e.g. βSNP1) of the corresponding trait for each SNP obtained through full-length correlation analysis with the coefficient value corresponding to the genotype, adding up all the SNP effects for the given trait, the expected value of the individual (molecular breeder value) ) Can be obtained. For example, a total of 10 SNPs involved in intramuscular fatness were identified as a result of full-length analysis, and genotypes for βSNP1, βSNP2, βSNP3, ---, βSNP10, and 10 individual SNPs, respectively, were found. A1A1, A2B2, B3B3, ---, A10A10 yieotdamyeon, expected value for intramuscular local roads of the object is _object MBV _{1 = (+ 1) β SNP1} + (0) β SNP1 + (- 1) β SNP1 --- + ( +1) β _SNP10 . Finally, MBV values are evaluated for each individual to determine whether to select.

Exemplary examples of the results of the evaluation of meat and meat quality of Hanwoo using the SNP of the present invention are shown in Tables 9 to 12 below.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Attach an electronic file to a sequence list

Claims (11)

Carcass weight, gastrocnemius muscle area, backfat thickness of Hanwoo comprising a polynucleotide consisting of 8-100 contiguous nucleotide sequences comprising the 61st nucleotide of SEQ ID NO: 1 or a complementary polynucleotide thereof A composition useful for the determination of intramuscular fat.
Method of determining carcass weight, abdominal muscle cross-sectional area, back fat thickness, or intramuscular fat degree of Hanwoo including the following steps:
(a) separating nucleic acid molecules from Hanwoo; And
(b) identifying the base type of the single nucleotide polymorphism (SNP) corresponding to the 61st nucleotide of SEQ ID NO: 1 in the isolated nucleic acid molecule.
The method of claim 2, wherein step (b) is performed by amplifying the isolated nucleic acid molecule using a primer pair prepared to amplify a polynucleotide comprising the 61st nucleotide of SEQ ID NO: 1 How to.
The method of claim 3, wherein (iii) an annealing with a sequence adjacent to the 61st nucleotide of SEQ ID NO: 1, which is an SNP nucleotide, and (ii) the nucleotide at the 3 'end is matched with a nucleotide at position -1 from the SNP nucleotide A method of analyzing the base type of SNP nucleotides using genotyping primers.
The method of claim 2, wherein the nucleic acid molecule in step (a) is obtained from muscle, epidermis, blood, bone, or organ of Hanwoo.
A kit used for determining the carcass weight, gastrocnemius muscle area, dorsal fat thickness, or intramuscular fat of Hanwoo comprising primer pairs designed to amplify polynucleotides comprising the 61st nucleotide of SEQ ID NO: 1.
7. The primer pair of claim 6, wherein the primer pair is (i) an anneal with a sequence adjacent to the 61st nucleotide of SEQ ID NO: 1, which is an SNP nucleotide, and (ii) the nucleotide at the 3 'end is nucleotide at position -1 from the SNP nucleotide. Kit comprising a genotyping primer to match with.
7. The kit of claim 6, wherein the kit further comprises a PCR amplification buffer, DNA polymerase and dNTPs.
The polynucleotide of claim 1, wherein the polynucleotide consists of 8-100 contiguous nucleotide sequences comprising the 58th nucleotide of SEQ ID NO: 2, or the 61st nucleotide of any of SEQ ID NOs: 3-96 Compositions further comprising their complementary polynucleotides.
3. The single nucleotide polymorphism (SNP) of claim 2, wherein in step (b), the 58th nucleotide of SEQ ID NO: 2 or the 61st nucleotide of any of SEQ ID NOs: 3 to 96 Further comprising the step of further confirming the base type of the isolated nucleic acid molecule.
7. The primer pair of claim 6, further comprising a pair of primers designed to amplify a polynucleotide comprising a 58th nucleotide of SEQ ID NO: 2, or a 61 nucleotide of any of SEQ ID NOs: 3 to 96 Kit comprising a.