KR20160048316A - A marker for predicting marbling score in Hanwoo and diagnosing method using the same - Google Patents

Abstract

The present invention relates to a marker for predicting a marbling score in hanwoo (Korean cattle) and a method for diagnosing the same. More particularly, the present invention relates to a marker for predicting a marbling score in hanwoo capable of detecting insertion or deletion in the base at position 258 of a polynucleotide derived from ADIPOQ gene expressed by SEQ ID NO: 1, or a marker for predicting a marbling score in hanwoo capable of detecting single base polymorphism in which the base at position 73 of a polynucleotide derived from ADIPOQ gene expressed by SEQ ID NO: 1 is C or T, a composition, a kit, and a method for predicting a marbling score in hanwoo by using the gene marker for predicting a marbling score in hanwoo. By applying the gene marker according to the present invention, a marbling score in hanwoo can be predicted in an effective and practical method. Also, based on the present invention, it is possible to select a seed bull which produces high quality of hanwoo meat.

Description

[0001] The present invention relates to a marker for predicting the marbling of Hanwoo and a method for diagnosing the marbling,

More particularly, the present invention relates to a marker and a diagnostic method thereof for predicting the insertion or deletion of polynucleotide derived from ADIPOQ gene of SEQ ID NO: 1 in the 258th base A marker, a composition, a kit, and a marker for predicting a single base polymorphism in which the 73rd base of the polynucleotide derived from ADIPOQ gene represented by SEQ ID NO: 1 is C or T can be used for Hanwoo marbling To a method for predicting the likelihood.

Conventional traditional Hanwoo marbling and meat quality measurement techniques depend on the phenotypic measurement values, and the growth and trait traits of the progeny of selected candidate sow mothers were verified and the candidate sow mowers were selected again. However, the measurement of the meat quality of Hanwoo is not only time consuming, but also is based on the breeding price of the parents. In addition, since many candidate mothers are raised through two generations, labor expenses, rearing costs, facility management expenses, and so on, can be economically burdensome to the breeder.

In order to solve such a problem, researches on gene markers capable of estimating the genetic capability of meat quality have been actively conducted recently. For example, using various DNA analysis techniques such as Random Amplified Polymorphic DNA (RAPD), Single Strand Conformational Polymorphism (SSCP), Amplified Fragment Length Polymorphism (AFLP) and Restriction Fragment Length Polymorphism (RFLP) We have screened molecular markers related to economic traits. Among these molecular markers, the most widely used molecular markers are single nucleotide polymorphism (SNP) markers. The SNP can be used as an economic trait marker of livestock if the SNP affects the phenotype and the major economic traits of the livestock are changed. A conventional technique using SNP markers is disclosed in Korean Patent No. 10-2012-0011728 (Feb. 20, 2012), which relates to a single base polymorphism marker useful for early selection of meat or meat quality of Korean beef cattle.

Adiponectin (ADIPOQ) is the most abundant protein secreted by white adipose tissue in some mammalian species, and secretion of adiponectin is inversely related to adipose tissue mass. In a recent study, adiponectin was also found in brown adipose tissue and skeletal muscle and was found to inhibit lipid biosynthesis and glucose neosynthesis. It has also been shown that adiponectin is associated with increased fatty acid oxidation and regulation of glucose uptake and insulin sensitivity. These results indicate that adiponectin plays an important role in the regulation of fat and carbohydrate metabolism.

Under such technical background, the present inventors have developed markers and diagnostic methods for predicting marbling traits of Hanwoo.

SUMMARY OF THE INVENTION It is, therefore, an object of the present invention to provide a marker for Hanwoo Marbling prediction that enables practical and rapid marbling prediction.

It is another object of the present invention to provide a composition for predicting cow's milk marbling.

Yet another object of the present invention is to provide a kit for cow ' s marbling prediction.

It is still another object of the present invention to provide a method for predicting Hanwoo marbling using a marker for Hanwoo marbling prediction.

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

In order to solve the above technical problem,

In one aspect of the present invention, a marker for prediction of Hanwoo marbling that can detect insertion or deletion of the polynucleotide derived from ADIPOQ gene of SEQ ID NO: 1 in the 258th base can be provided.

In another aspect of the present invention, a marker for Hanwoo marbling prediction can be provided which can detect a single base polymorphism wherein the 73rd base of the ADIPOQ gene-derived polynucleotide represented by SEQ ID NO: 1 is C or T.

In another aspect of the present invention, a composition for predicting Hanwoo marbling, which comprises the marker, may be provided.

In one embodiment of the present invention, the marker is a primer set of SEQ ID NOS: 2 and 3.

In another aspect of the present invention, a Hanwoo Marbling prediction kit including the composition may be provided.

According to another aspect of the present invention, there is provided a method for producing a recombinant vector comprising the steps of: (a)

(b) amplifying the polynucleotide using the genomic DNA extracted from the step (a) as a template and the primer sets of SEQ ID NOS: 2 and 3; And

(c) Analysis of the amplified result shows that the insertion of the polynucleotide represented by SEQ ID NO: 4 in the 258th nucleotide sequence of SEQ ID NO: 1 is more effective than the case in which the insertion of the polynucleotide of SEQ ID NO: A method can be provided.

According to another aspect of the present invention, there is provided a method for producing a recombinant vector comprising the steps of: (a)

(b) amplifying the polynucleotide using the genomic DNA extracted from the step (a) as a template and the primer sets of SEQ ID NOS: 2 and 3; And

(c) By analyzing the amplified result, it is possible to provide a Korean-marbling prediction method in which it is judged that the cow's marbling grade is superior to the case where the allele genotype of the 73rd sequence of SEQ ID NO: 1 is CT or CC have.

According to another aspect of the present invention, there is provided a method for producing a recombinant vector comprising the steps of: (a)

(b) amplifying the polynucleotide using the genomic DNA extracted from the step (a) as a template and the primer sets of SEQ ID NOS: 2 and 3; And

(c) analyzing the amplified result to determine whether the polynucleotide of SEQ ID NO: 4 occurs in the 258th nucleotide sequence of SEQ ID NO: 1 and the allele of the 73rd nucleotide sequence of SEQ ID NO: 1 is CT, A Hanwoo marbling prediction method in which the insertion of the polynucleotide shown in SEQ ID NO: 4 does not occur in the 258th nucleotide sequence of SEQ ID NO: 1 and the cow's marbling grade is judged to be superior to the case where the allele genotype of the 73rd nucleotide sequence is CC or TT Can be provided.

Using the marker according to the present invention, marbling of Hanwoo can be predicted by an effective and practical method, and it is possible to diagnose more rapidly than the existing technology depending on the phenotype measurement value. Based on this, it is possible to select myeongwoo which produces high quality Hanwoo meat.

1 shows an agarose gel and a primer set showing SNPs, nucleotide sequence insertion and deletion of the ADIPOQ gene according to one embodiment of the present invention.

Hereinafter, the present invention will be described in more detail.

In one aspect of the present invention, a marker for prediction of Hanwoo marbling that can detect insertion or deletion of the polynucleotide derived from ADIPOQ gene of SEQ ID NO: 1 in the 258th base can be provided.

In another aspect of the present invention, a marker for Hanwoo marbling prediction can be provided which can detect a single base polymorphism wherein the 73rd base of the ADIPOQ gene-derived polynucleotide represented by SEQ ID NO: 1 is C or T.

In the present invention, "polynucleotide" means a nucleotide polymer in which a nucleotide unit is linked in a chain by covalent bonds, and may include an analogue of a nucleotide.

In the present invention, "ADIPOQ gene" means a gene encoding adiponectin, which is known to regulate glucose level and fatty acid degradation. The ADIPOQ gene is located in chromosome 1 and has a DNA sequence of about 11,429,556 in length. The nucleotide sequence of the ADIPOQ gene can be obtained from a known database such as Blast of NCBI, and preferably includes the polynucleotide of SEQ ID NO: 1.

In the present invention, "SEQ ID NO. 1" is a polymorphic sequence comprising a polymorphic site. A polymorphic sequence means a sequence comprising a polymorphic site containing a single nucleotide polymorphism (SNP) in the polynucleotide sequence. The polynucleotide sequence may be DNA or RNA.

In the present invention, "single nucleotide polymorphism" means a genetic change or mutation showing a difference in one nucleotide sequence (A, T, G, C) in DNA base sequence.

In another aspect of the present invention, a composition for predicting Hanwoo marbling, which comprises the marker, may be provided.

In one embodiment of the present invention, the marker is a primer set of SEQ ID NOS: 2 and 3.

As used herein, the term "primer " refers to a single-stranded oligonucleotide sequence complementary to a nucleic acid strand to be copied, and may serve as a starting point for the synthesis of a primer extension product. The length and sequence of the primer should allow the synthesis of the extension product to begin. The primer preferably has a length of 15 to 30 base pairs.

In another aspect of the present invention, a Hanwoo Marbling prediction kit including the composition may be provided.

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 (e.g., Thermus aquaticus (Taq), Thermus thermophilus , Thermisflavus, Thermococcus literalis or Pyrococcus furiosus (Pfu)), DNA polymerase joins and dNTPs. The kit may be made from a number of separate packaging or compartments containing the reagent components described above.

According to another aspect of the present invention, there is provided a method for producing a recombinant vector comprising the steps of: (a)

(b) amplifying the polynucleotide using the genomic DNA extracted from the step (a) as a template and the primer sets of SEQ ID NOS: 2 and 3; And

(c) Analysis of the amplified result shows that the insertion of the polynucleotide represented by SEQ ID NO: 4 in the 258th nucleotide sequence of SEQ ID NO: 1 is more effective than the case in which the insertion of the polynucleotide of SEQ ID NO: A method can be provided.

According to another aspect of the present invention, there is provided a method for producing a recombinant vector comprising the steps of: (a)

(b) amplifying the polynucleotide using the genomic DNA extracted from the step (a) as a template and the primer sets of SEQ ID NOS: 2 and 3; And

(c) By analyzing the amplified result, it is possible to provide a Korean-marbling prediction method in which it is judged that the cow's marbling grade is superior to the case where the allele genotype of the 73rd sequence of SEQ ID NO: 1 is CT or CC have.

According to another aspect of the present invention, there is provided a method for producing a recombinant vector comprising the steps of: (a)

(b) amplifying the polynucleotide using the genomic DNA extracted from the step (a) as a template and the primer sets of SEQ ID NOS: 2 and 3; And

(c) analyzing the amplified result to determine whether the polynucleotide of SEQ ID NO: 4 occurs in the 258th nucleotide sequence of SEQ ID NO: 1 and the allele of the 73rd nucleotide sequence of SEQ ID NO: 1 is CT, A Hanwoo marbling prediction method in which the insertion of the polynucleotide represented by SEQ ID NO: 4 does not occur in the 258th nucleotide sequence of SEQ ID NO: 1 and the cow's marbling grade is judged to be superior to the case where the allele genotype of the 73rd nucleotide sequence is CC or TT Can be provided.

The genomic DNA can be extracted from various sources such as muscle, epidermis, blood, bone, organs of Hanwoo, and most preferably, it can be obtained from muscle or blood.

The genomic DNA extraction method of the present invention is carried out according to a conventional method known in the art. For this purpose, a commercial kit can be used, but the present invention is not limited thereto.

The amplification step (b) can be carried out by using a polymerase chain reaction (PCR) (including real time PCR), a ligase chain reaction, nucleic acid sequence-based amplification, a transcription- based amplification system, a strand displacement amplification or Q-replication enzyme, or any other suitable method for amplifying nucleic acid molecules known in the art to which the present invention pertains. The PCR is a method of amplifying a gene containing a target sequence from a pair of primers that specifically bind to a target sequence using a polymerase, and is the most preferable gene amplification method.

The method (c) for analyzing the genotypes may include allele-specific hybridization, fluorescence in situ hybridization (FISH), molecular beacons, SNP microarray, restriction enzyme fragment Restriction fragment length polymorphism, PCR-based methods, Flap endonuclease, primer extension, 5'-nuclease, Oligonucleotide ligation assays, size analysis, single-strand conformation analysis (SSCA), denaturing gradient gel electrophoresis (DGGE), and single-stranded conformation polymorphism. , But the present invention is not limited thereto.

Sequence changes in the genotype result in differences in base-linkage within the single-stranded molecule, resulting in different bands of mobility, and SSCA detects this band. DGGE analysis uses a denaturing gradient gel to detect sequences that represent wild type sequences and other mobility. Other techniques generally use probes or primers complementary to sequences comprising the SNPs of the invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. It should be understood, however, that these examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention.

Example  1: Seductive characterization

From the National Agricultural Cooperative Federation Hanwoo Improvement Facility, 1954 Hanwoo were randomly selected from the whole country and the Hanwoo meat samples registered in the national database were randomly selected from nine packaging facilities of Korea Animal Product Assessment Service (KAPE). Data collected from KAPE include conductor weight, silliman crossover area, backfill thickness and marble grade. After 24 hrs of slaughter, approximately 5 g of muscle samples were collected from the chest length muscle around the sixth rib.

Example  2: The genome DNA  extraction

For extraction of genomic DNA, about 1 gram of muscle sample was used. Samples were minced, the pieces placed in tubes containing extraction buffer, and genomic DNA was extracted using a commercial kit (Wizard DNA extraction kit, Promega) according to the manufacturer's guidelines.

Example  3: Amplification

The primers were designed using a DNA screening program from DNAstar versio 6.0 based on the sequence of bovine (a GenBank accession number JQ775868). To select a primer set with sufficient polymorphism in all small species, the alignment was performed with six reference sequences from GenBank with accession numbers (DQ156119, EU296533, EU313339, EU492456, EU492457 and JQ775868) G.81966790T> C, g.81966798G> A, g.81966940A> G, and g.81967160T> C (g.81965420G> C, g.81965993G> C, g.81966286C> A, g.81966690A> ) Were found. The present invention designed a primer for a portion of the genome containing the SNP by screening a SNP (g.81966377 > C) that appears to have sufficient variability for all small breeds. The forward and reverse primers are GCAGC TCTAC TTGGC ATCC (SEQ ID NO: 2, nucleotide position 81966163 - 81966184) and CTTGA ATCAG TCGTC CTTAC CC (SEQ ID NO: 3, 81966410 - 81966429), respectively.

(10 mM Tris, pH 8.3, 50 mM KCl, 0.1% Triton X-100, 1.5 mM MgCl ₂ ), 2.5 mM dNTO, 10 pmol of each primer, 50 ng of genomic DNA and One unit of Taq DNA polymerase (Gibco BRL, Grand Island, NY) was used. A total of 35 cycles were performed with heat at 95 [deg.] C for 2 min followed by 94 [deg.] C / 1 min for denaturation, 59 [deg.] C / 1 min for heat cooling and 72 [ , PT-200, Watertown, Mass.). DNA bands stained with ethidium bromide were detected via UV light.

Example  4: Detection of single nucleotide polymorphisms

DNA samples were purified using a PCR purification system and direct sequencing analysis was performed using the ABI3730 XL gene analyzer on NIAS for all samples with forward and reverse primers. The conditions of the direct sequencing method were 94 ° C / 10 sec and 60 ° C / 4 min cycles with a total volume of 10 μl (5 μl of sequencing buffer, 1.6 pM primer, DNA of 50 ng genome, 0.5 μl big- A total of 35 cycles were performed. The purification procedure for the product of the test results involved centrifugation at 7800 rpm for 45 minutes with 78% isopropanol and 70% ethanol. The sample was dried at room temperature for 2 hours, and then ion-removed formamide (10 μl) was added. The alignment of all individual sequences was carried out with the SeqMan program of DNAstar version 6.0, and the genotypes were determined according to the apex of the sequence chart. For genetic characterization of inserts, 1.5% agarose gel electrophoresis was performed and the genotype was determined based on the band pattern of DNA for fast mobility (276 bp, deletion) and slow mobility (323 bp, insert) ( 1).

Example  5: Statistical analysis

To investigate the effect of genotype on conductor traits, variance analysis was performed using a statistical analysis system (SAS, 1990) with a general linear model (GLM) procedure. The least squares mean was compared using Fisher's least significant difference test with a relative error rate of 0.05.

The advective effect of the gene is measured by different points between the solutions for the two homozygous genotypes, the dominant deviation is determined by the difference between the solutions of the heterozygous genotypes and the average of the solutions of the two homozygous genotypes . The least-squares mean and standard error for each trait were measured using the following linear model: Y = μ + G + e, where Y is the observed value of the trait, μ is the overall average for each trait, G is the fixed effect of the genotype, and e is the residual error. Allele frequency and Hardy-Weinberg equilibrium were measured using Arlenquin version 3.0. For analysis, we used Haploview's software to measure the association non-equilibrium.

Experiment result

Table 1 below provides a technical overview of the conductor traits.

Amplification yielded fragments of the 267 and 323 bp genomes. The analysis of the present invention determined the genotype according to the mobility of the DNA band pattern. That is, the fast DNA band (267 bp) and the slow DNA band (323 bp) were assigned alleles D (deletion) and I (insertion), respectively. In addition, sequencing confirmed the insertion of 56 bp between nucleotide positions 81966364 and 81966429 according to the reference sequence. The PCR analysis of the present invention did not find any animals with two insertions, and 195 birds (about 10%) had the above insert. Measurements of allele frequency for D and I were 0.844 and 0.156, respectively (Fig. 1).

The alignment of the sequences demonstrated nucleotide substitutions of 81966235 (C > T) and 81966377 (T > C) in the promoter portion of 5. The SNPs with g.81966235C> T show allelic frequencies of C (0.828) and T (0.172), ie, 68.5% for CC, 28.5% for TC and 3% for TC. Genotype frequencies for CC, CT, and TT were 47.1%, 43.1%, and 9.8%, respectively, and the frequencies of alleles of G (0.686) and A (0.314) against g.81966377T> C are shown. The SNPs with g.81966364D> I show allele frequencies of D (0.844) and I (0.156), with DD being 71.2%, DI being 26.5% and II being 2.4%. No significant deviations from the measurement of Hardy-Weinberg equilibrium were found for SNPs and insertions. Single phase analysis confirmed a significant association between insertions (g.81966364D> I) and g.81966377T> C.

The results of the association rule analysis between ADIPOQ gene and SNP are shown in Table 2 below. The genetic impact of g.81966337T> C explained significant phenotypic differences between MAR and LEA (P <0.05) and BFT and CAW (P <0.01). Animals with genotypes CC (6.054) and TC (5.944) had significantly higher MARs than animals with genotypic TT (5.130) and showed significant gene status and effects. On the other hand, the opposite pattern was observed, with animals with alleles T showing higher BFT, LEA and CAW values than animals with allele C. The analysis also confirmed the commercial and dominant effect on BFT and CAW, while LEA was only perceived as a significant additive effect. The major allele genotypes (allele C and D (deletion)) of g.8196235C> T and g.81966364D> I showed a lower MAR rating than the other alleles, g.8196235C> T) and commercial value (g.81966364D> I). Significant associations to other traits other than MAR were not found in g.8196235C> T and g.81966364D> I (Table 2).

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> RURAL DEVELOPMENT ADMINISTRATION <120> A marker for predicting marbling score in Hanwoo and diagnosing          method using the same <130> NPF-26785 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 267 <212> DNA <213> Bos taurus coreanae <400> 1 cttgaatcag tcgtccttac cctcaggtgt ggaatcagag ccacagactt cagtcagggt 60 ggaagtagga ggcgatggcg cagcctcagc agctcggtac tcatggggac aagcagcccc 120 cgccccaccg ccgtcccttc ccattggcca gcacagtgag agccagagac caatgggtag 180 acacgtgccc ctggcagctc ctttggcttg ccactccagg gaacctggtg caacccagtt 240 cgggcttggg atgccaagta gagctgc 267 <210> 2 <211> 19 <212> DNA <213> Bos taurus coreanae <400> 2 gcagctctac ttggcatcc 19 <210> 3 <211> 22 <212> DNA <213> Bos taurus coreanae <400> 3 cttgaatcag tcgtccttac cc 22 <210> 4 <211> 56 <212> DNA <213> Bos taurus coreanae <400> 4 tttggcttgc cactccaggg aacctggtgc aacccagttc gggcttggga tgccaa 56

Claims (11)

A marking marker for Hanwoo marbling prediction capable of detecting insertion or deletion of polynucleotide derived from ADIPOQ gene represented by SEQ ID NO: 1 in the 258th base. A composition for predicting Hanwoo marbling comprising the marker of claim 1. 3. The method of claim 2,
Wherein the marker is a primer set of SEQ ID NOS: 2 and 3. 3. A Hanwoo marbling prediction kit comprising the composition of claim 2 or 3. (a) extracting genomic DNA of Hanwoo;
(b) amplifying the polynucleotide using the genomic DNA extracted from the step (a) as a template and the primer sets of SEQ ID NOS: 2 and 3; And
(c) Analysis of the amplified result shows that the insertion of the polynucleotide represented by SEQ ID NO: 4 in the 258th nucleotide sequence of SEQ ID NO: 1 is more effective than the case in which the insertion of the polynucleotide of SEQ ID NO: Way. A marker for Hanwoo marbling prediction capable of detecting a single base polymorphism wherein the 73 &lt; th &gt; base of the ADIPOQ gene-derived polynucleotide represented by SEQ ID NO: 1 is C or T; A composition for predicting Hanwoo marbling comprising the marker of claim 6. 8. The method of claim 7,
Wherein the marker is a primer set of SEQ ID NOS: 2 and 3. 8. A kit for predicting Hanwoo marbling comprising the composition of claim 7 or 8. (a) extracting genomic DNA of Hanwoo;
(b) amplifying the polynucleotide using the genomic DNA extracted from the step (a) as a template and the primer sets of SEQ ID NOS: 2 and 3; And
(c) analyzing the amplified result to determine that the alleles of the 73rd sequence of SEQ ID NO: 1 are more excellent than those of CC or TT when CT is CT. (a) extracting genomic DNA of Hanwoo;
(b) amplifying the polynucleotide using the genomic DNA extracted from the step (a) as a template and the primer sets of SEQ ID NOS: 2 and 3; And
(c) analyzing the amplified result to determine whether the polynucleotide of SEQ ID NO: 4 occurs in the 258th nucleotide sequence of SEQ ID NO: 1 and the allele of the 73rd nucleotide sequence of SEQ ID NO: 1 is CT, A Hanwoo marbling prediction method in which the insertion of the polynucleotide represented by SEQ ID NO: 4 does not occur in the 258th nucleotide sequence of SEQ ID NO: 1 and the cow's marbling grade is judged to be superior to the case where the allele genotype of the 73rd nucleotide sequence is CC or TT .

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