KR20140055203A

KR20140055203A - Diagnostic method for marbling by using fattness gene in hanwoo

Info

Publication number: KR20140055203A
Application number: KR1020120121655A
Authority: KR
Inventors: 정의룡; 신성철; 백창균; 심승화; 고기한; 윤기환
Original assignee: 상지대학교산학협력단
Priority date: 2012-10-30
Filing date: 2012-10-30
Publication date: 2014-05-09

Abstract

The present invention relates to a method for diagnosing marbling using a single nucleotide polymorphism marker of a Korean beef obesity gene. Provided is a method for inspecting a gene that can be utilized to promote accuracy for the early section and entity evaluation of high quality Korean beef by excavating marbling-related molecular markers for predicting and discerning the genetic characteristics of Korean beef having excellent marbling. More specifically, in the present invention, a SNP marker according to the SNP genotype for each Korean beef entity is detected through a PCR-restriction fragment length polymorphism (PCR-RFLP) technique by using a particular SNP that exists in the exon number 2 domain of a Korean beef fat mass and obesity (FTO) gene, so that entities having AA and TT genotypes are diagnosed and discerned as entities having genetically excellent marbling than entities having the AT genotype.

Description

[0001] The present invention relates to a method for diagnosing marbling using a Hanwoo obesity gene,

The present invention relates to a method for detecting a FTO (fat mass and obesity) gene which is closely related to a marbling characteristic of Hanwoo through PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) By using the specific SNP (single nucleotide polymorphism) to identify Korean beef meat-related molecular markers, it provides a technique to diagnose and distinguish Hanwoo, which has excellent marbling, in an early stage.

Polymerase chain reaction (PCR) was first introduced by Kary Mullis in 1986. The development of the PCR method is a revolutionary technology that allows the state of the target gene to be known using a small amount of DNA. In 1953, the world of DNA molecular biology, which began with the discovery of the DNA helix structure by Watson and Crick, was a new development 20 years later with the discovery of restriction enzymes and the subsequent development of DNA cloning and sequencing methods. Recent developments in molecular genetic techniques have led to the development of gene markers at the DNA level.

In the field of genetics and breeding of livestock, the gene is used as a marker gene for breeding improvement by using DNA polymorphism. DNA polymorphisms enable the development of markers by major genes and quantitative trait loci (QTLs) that determine important economic traits and include linkage analysis, genetic maps, parenetage testing has been used as a powerful tool to analyze distingtion of breeds, pedigree analysis, genetic diversity and relationships. In particular, selective breeding techniques using DNA markers have been used as a way to enhance the hereditary capacity of livestock beyond the limits of traditional selection breeding methods. Such genetic engineering techniques include Random Amplified Polymorphic DNA (RAPD), Amplified Fragment Length Polymorphism (AFLP), Single Strand Conformational Polymorphism (SSCP), and Restriction Fragment Length Polymorphism (RFLP). Among them, the RFLP technique, which is a technique for analyzing the polymorphism of DNA fragments by restriction enzyme treatment, is excellent in reproducibility and high in efficiency, and thus is usefully used as a gene diagnosis method.

Accordingly, the present invention provides a technique for detecting a gene molecule marker closely related to a Korean beef cattle trait using the PCR-RFLP technique and utilizing it for early diagnosis of high quality Korean cattle.

At present, Korean livestock grading office uses Hanchu meat quality grade 1 +, 2, 3, 4, 5, 6, 7, and 8 by using the marbling, meat color, fat color, meat texture, and meat firmness, 1+, 1, 2 and 3 grades. Of these, marbling is one of the most influential factors in determining the meat quality grade. Marbling refers to the fat between the first and second muscles of the muscle and is sometimes referred to as the "supergroup fat", because the shape of the fat in the red meat is like frost or is like marble Marbling is an important determinant of meat quality and is closely related to the taste of meat. Therefore, in order to improve the meat quality of Hanwoo, it is important to diagnose and select an animal having excellent marbling early, and to manage it in an optimal environment. In addition, breeding improvement that can improve the quality of Hanwoo more efficiently in a short period of time Model development is needed.

Accordingly, the present invention provides a method for genetically testing a Korean beef cattle having excellent marbling using an advanced molecular breeding technique and developing a genetic test method for a cattle marbling diagnosis.

The inventors of the present invention have found that a fat mass and obesity gene present in the chromosome 18 of bovine is selected as a candidate gene related to the flesh trait of Korean beef cattle, The inventors of the present invention have invented a method for genetic testing for Hanwoo marbling diagnosis using markers. FTO gene is a gene that increases lipolytic activity in adipocytes and is known to be closely related to obesity in humans. In recent years, specific SNP of FTO in swine has been correlated with daily gain, backfat thickness, and ham weight . Therefore, in the present invention, the FTO gene is finally selected as a candidate gene related to the fleshy trait, and the PCR-RFLP analysis using the specific SNP of the FTO gene and the statistical analysis of the association with the meat quality and the fat-related traits are closely related to the marbling We have developed a genetic testing method that can be used for the early diagnosis of high-quality Korean beef with high marbling by excavating molecular markers.

More specifically, genomic DNA was isolated from the blood of Hanwoo, and amplified by PCR using a primer containing the exon 2 region of the FTO gene. Then, HpyCH4 Ⅲ (= Bst4C I , Taa I ) restriction enzymes were added to the DNA fragments, and the digested DNA fragments were electrophoresed on an agarose gel, and SNP markers were determined for each genotype. , We have developed SNP markers that are closely related to marbling and provide genetic testing methods to diagnose and discriminate genetically marbled Korean beef cattle.

(AA, AT and TT type) due to A↔T base substitution of exon 2 region of Hanwoo FTO gene (172th position in PCR amplification region: 172th place of SEQ ID NO: 1) It is possible to identify the characteristics of carcasses and to make early diagnosis and selection of high quality Korean livestock and to utilize useful genetic resources industrially. In other words, early identification and selection of individuals possessing the AA and TT genotypes of the FTO gene maximizes the efficiency and improvement of the breeding program of Korean beef breeding, Can be obtained.

The PCR-RFLP (Restriction Fragment Length Polymorphism) analysis technique used in the present invention as a state-of-the-art gene detection technique using PCR-RFLP analysis method for genomic DNA of Korean cattle is fast, And DNA marker genotypes can be more easily, quickly, and accurately determined for a large number of samples, as well as maximizing the efficiency and practicality of the selection.

1 shows the molecular markers of SNP genotypes of each individual due to the 172th A↔T single nucleotide polymorphism (SNP) in the amplified region of FTO gene exon 2 of SEQ ID NO: 1 detected by the PCR-RFLP analysis method in the present invention marker) electrophoresis photograph
2 shows the DNA marker of each SNP genotype for the 172th A↔T single nucleotide polymorphism (SNP) in the amplification region of the exon 2 FTO gene of SEQ ID NO: 1 detected by PCR-RFLP analysis in the present invention. Nucleotide Sequence Analysis Chromatogram

In order to achieve the above objects, the present invention will be described in more detail with reference to the following non-limiting examples.

Example 1: Hanwoo FTO Gene SNP Genotype detection

1. Disclosure material and DNA separation purification

A total of 310 Korean farms were selected as the public axes for the present invention. Genomic DNA was separated and purified from each test tube by the method of Miller et al. (1988). The DNA concentration was measured using a spectrophotometer, and then TE buffer (10 mM Tris-HCl, pH 7.4; 1 mM EDTA) and stored in a -20 ° C freezer to be used as a reference material.

2. PCR amplification and single base polymorphism (SNP) detection of Hanwoo FTO gene

A primer for amplifying the DNA fragment of 244 bp size shown in SEQ ID NO: 1 including exon 2 and 3 of the Hanwoo FTO gene can be obtained by referring to the nucleotide sequence information registered in GenBank registration number NC_007316.5 SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and the primer sequences used in the present invention are shown in Table 1.

Primer base sequence for PCR amplification of FTO gene Gene name Amplification region The primer sequence (5'-3 ') SEQ ID NO: FTO exon 2 Forward F-TTCCTCAAGCTCAACAGCTACC
Reverse R-ACGGTTCCTCTTTCAGGTATGG 2
3

Reaction conditions for the PCR amplification of the FTO gene were carried out using the GeneMP System 9700 (GenAmp PE Applied Biosystem, USA) under the following conditions. The reaction solution was prepared by adding 50 ng of template DNA, 0.1 μM of each primer, 250 μM of dNTP, 2 μl of 10X PCR buffer and 1 unit of Taq DNA polymerase to a 0.2 ml tube. &Lt; / RTI > The PCR reaction conditions were preliminary heating at 94 ° C for 5 minutes, followed by 35 cycles of 94 ° C for 30 seconds, 55 ° C for 20 seconds and 72 ° C for 1 minute, and finally heating at 72 ° C for 5 minutes to amplify DNA The process was terminated. PCR amplification products were electrophoresed on 2% agarose gel to verify DNA amplification success. As shown in SEQ ID NO: 1, a total of 244 bp DNA bases were detected by the direct sequencing method using the PCR amplification product of the Hanwoo FTO gene. As a result, a total of one Single nucleotide polymorphism (SNP) sites were detected. That is, the SNPs according to the A? T base substitution were detected in the 172th nucleotide of SEQ ID NO: 1 (nucleotide 17,516 of NCBI GenBank registration number NC_007316.5). As a result, amino acid substitution with arginine (CG A ) ↔ arginine (CG T ) did not occur in the present SNP region. An RFLP analysis was performed using the restriction HpyCH4 Ⅲ (= Bst4C I, Taa I) for the molecular markers (DNA marker) detected for each test subject beef object-specific SNP genotypes for the SNP detection.

3. SNP genotype marker analysis of Hanwoo FTO gene by PCR-RFLP technique

My 172nd A↔T SNP site amplification area of the detected beef FTO gene HpyCH4 Ⅲ (= Bst4C I, Taa I) restriction enzyme recognition sites ^{(5'-AC_N ↓ GT-3} ') is present by PCR-RFLP technique Molecular markers (DNA markers) corresponding to the three SNP genotypes (AA, AT and TT) of Hanwoo FTO gene were detected (FIGS. 1 and 2). RFLP analysis of Hanwoo FTO gene was performed by adding 2 units of HpyCH4 Ⅲ restriction enzyme to 10 ㎕ of PCR amplification products and reacting at 37 ℃ for more than 3 hours. The DNA fragments obtained by digesting the PCR amplification products with restriction enzymes were subjected to electrophoresis in a 2% agarose gel at about 100 volts for about 2 hours using TBE buffer (90 mM Tris-borate, 2 mM EDTA, pH 8.0) And stained with EtBr (ethidium bromide), and the SNP genotype of each test subject was determined by observing the DNA fragment pattern. In other words, in the individuals with AA homo genotype, there was no restriction enzyme recognition site, so a total of 244 bp DNA bands were detected. In the individuals with TT homo genotype, restriction enzyme recognition sites existed and 172 and 72 Two DNA bands with bp size were detected. In the case of AT heterozygotes, DNA fragments of 72, 172 and 244 bp were detected in all three DNA fragments (Fig. 1).

As a result of analysis of allele frequency and genotype frequency of 172th SNP in the amplified region of FTO gene analyzed in 310 farms, The incidence of A allele was higher than that of the T allele. The frequency of the SNP marker genotype was found to be about 47.1% (146 cases), GA genotype about 44.5% (138 cases), and AA genotype was 8.4% (26 cases). The genotype of GA was the most frequent and AA genotype was the lowest.

Example 2 DNA marker Genotype and meat quality Weight Statistical analysis of association with traits

In order to analyze the SNP genotype of the Hanwoo FTO gene detected by the present invention and the relationship between the carcass and meat quality traits related to the meat quality of Hanwoo, statistical analysis was carried out using the PROC GLM method using SAS a9.1 Package / PC program, The significance test was performed by the Duncan's Multiple Range Test (DMRT) method for differences in the least significant squares of each genotype. As a result, the genotype of the SNP region (sequence 172 in SEQ ID NO: 1) due to A↔T base substitution in the exon 2 region of the Hanwoo FTO gene was highly correlated with Hanwoo marbling (P <0.01) As shown in Table 2, individuals with AA and TT genotypes in marbling were significantly higher than those with AT genotype by 0.909 and 0.827, respectively (P <0.01).

Analysis of association between SNP genotype and fat and meat quality traits of Hanwoo FTO gene Meat and meat quality
SNP genotype P-value
AA (n = 146) AT (n = 138) TT (n = 26) Marble 6.082 + 0.194 ^a 5.173 + - 0.200 ^b 6.000 + - 0.461 ^a 0.004 Color 4.931 + 0.033 4.956 + 0.034 4.923 + 0.079 0.846 Local color 3.000 ± 0.009 2.985 ± 0.009 3.000 ± 0.022 0.539 Texture 1.068 + 0.036 1.159 + 0.037 1.076 + 0.086 0.209 Maturity 2.054 + 0.034 2.130 + 0.035 2.153 + 0.082 0.244 Backfill thickness 14.301 + - 0.614 14.797 + 0.631 13.692 ± 1.455 0.730 Abdominal muscle cross-sectional area 92.671 ± 1.132 89.739 ± 1.165 91.230 ± 2.684 0.199 In conductor 431.045 ± 6.360 432.972 + - 6.541 430.923 ± 15.071 0.976 Meat index 64.172 + 0.478 63.436 + 0.492 64.369 ± 1.135 0.506

As a result of the above results of the present invention, the SNP genotype of the 172th A↔T base substitution in the amplification region of the Hanwoo FTO gene of the present invention is a very important item in determining the meat quality of Hanwoo, The results of this study suggest that a single nucleotide polymorphism marker can be used as a genetic testing method for early diagnosis and selection of genetically marbled Korean beef cattle.

In Table 2, ^{a and b} : there is a significant difference between the different codes (P <0.05).
1, M: 100 bp DNA ladder; AA, AT, TT: each single nucleotide polymorphic marker genotype by the FTO gene SNP of the present invention; 244 bp, 172 bp, 72 bp: the size of the DNA fragment detected by RFLP analysis
In FIG. 2, AA, AT, and TT: respective DNA marker genotypes for the nucleotide sequence analysis chromatogram of the FTO gene SNP of the present invention.

<110> SANGJI UNIVERSITY INDUSTRY ACADEMIC COOPERATION FOUNDATION <120> Diagnostic method for marbling by using fattness gene in Hanwoo <160> 3 <170> Kopatentin 1.71 <210> 1 <211> 244 <212> DNA <213> Bos taurus <220> <221> gene &Lt; 222 > (1) <223> FTO (fat mass and obesity) gene of Hanwoo (Korean cattle) <220> <221> exon <222> (1) (22) <223> exon 2 <220> <221> variation <172> <223> SNP: A> T <220> <221> primer_bind <222> (1) (22) <223> Forward primer binding site <220> <221> primer_bind (222) (244) <223> Reverse primer binding site <400> 1 ttcctcaagc tcaacagcta cctgcaggta gagaccatcc aggctttgga agagcttgct 60 gccaaggaga aagccaacat cgatgccgtg ccagtgtgca taggtccaga tttccccagg 120 gttggcatgg ggtcctcctt tgacgggcac gatgagattg acatgaagaa ccgagcagcg 180 tacaacgtca ctttgttgaa tttcatggat ccccagaaga tgccatacct gaaagaggaa 240 ccgt 244 <210> 2 <211> 22 <212> DNA <213> Bos taurus <220> <221> primer_bind <222> (1) (22) <223> Forward primer sequence of FTO gene in Hanwoo <400> 2 ttcctcaagc tcaacagcta cc 22 <210> 3 <211> 22 <212> DNA <213> Bos taurus <220> <221> primer_bind <222> (1) (22) <223> Reverse primer sequence of FTO gene in Hanwoo <400> 3 acggttcctc tttcaggtat gg 22

Claims

SEQ ID NO: 2 and SEQ ID NO: 3 primer (primer) PCR by (Polymerase chain reaction; polymerase chain reaction) amplification of bovine FTO (Fat mass and obesity) HpyCH4 Ⅲ (= Bst4C I, Taa I) limited to the gene amplification product The restriction enzyme fragment polymorphism (PCR-RFLP) method comprising the step of treating the enzyme with the FTO gene sequence 172th A↔T base substitution shown in SEQ ID NO: 1, Wherein the polymorphism marker (SNP marker) is detected, and the SNP genotypes (AA, AT and TT) are analyzed to estimate the genetically highly marbled Hanwoo, and the marbling diagnosis method using the Hanwoo Obesity gene

The method according to claim 1,
2 primer of SEQ ID NO: 2 having the nucleotide sequence structure of TTCCTCAAGCTCAACAGCTACC in the 5 'to 3' direction and the reverse primer of SEQ ID NO: 3 having the nucleotide sequence structure of ACGGTTCCTCTTTCAGGTATGG in the 3 'direction in the 5' Is amplified by PCR, and the marbling diagnosis method using the Hanbu obesity gene

The method according to claim 1,
Of the three single-nucleotide polymorphism marker genotypes (AA, AT and AA) of the Hanwoo FTO gene detected by PCR-RFLP analysis, Hanwoo individuals showing AA and TT genotypes were compared with Hanwoo individuals with genetically marbling A method for diagnosing marbling using a Korean beef obesity gene