KR101700529B1 - Diagnosis method for meat quantity using the DNA marker associated with longissimus muscle area in Hanwoo - Google Patents

Diagnosis method for meat quantity using the DNA marker associated with longissimus muscle area in Hanwoo Download PDF

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KR101700529B1
KR101700529B1 KR1020150135162A KR20150135162A KR101700529B1 KR 101700529 B1 KR101700529 B1 KR 101700529B1 KR 1020150135162 A KR1020150135162 A KR 1020150135162A KR 20150135162 A KR20150135162 A KR 20150135162A KR 101700529 B1 KR101700529 B1 KR 101700529B1
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hanwoo
gene
adh1c
pcr
dna
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정의룡
신성철
김준성
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상지대학교산학협력단
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Abstract

The present invention relates to a method for diagnosing a muscle longissimus dorsi cross-sectional area by using a single nucleotide polymorphism marker of a Korean native cattle ADH1C gene. Provided is a method for inspecting a gene, in which a muscle longissimus dorsi cross-sectional area-related DNA molecular marker for early predicting and determining genetic characteristics of Korean native cattle having a large muscle longissimus dorsi cross-sectional area is discovered to be utilized in early selecting good quality Korean beef and promoting accuracy in entity evaluation. More specifically, the present invention provides a method for diagnosing a Korean native cattle quantity trait, in which a molecular marker (DNA marker) according to an SNP genotype for each Korean native cattle entity is detected through a PCR-RFLP scheme by using a specific SNP existing in an exon NO. 6 region of a Korean native cattle ADH1C gene, and entities having GG genotypes thereamong are determined to be excellent entities having genetically large muscle longissimus dorsi cross-sectional areas compared to entities having TT and TG genotypes.

Description

[0001] The present invention relates to a method for diagnosing a Korean beef cattle meat using an associative molecular cross-

The present invention relates to a method and apparatus for detecting the presence of ADH1C (alcohol dehydrogenase (ADH)), which is closely related to the longissimus muscle area trait of Korean beef cattle through PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) 1K (class I), gamma polypeptide) gene by using single nucleotide polymorphism (SNP), and by using this, it is possible to diagnose Hanwoo, Provide technology that can be done.

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 technology for detecting molecular markers closely related to the cross-sectional trait of Korean muscle of Hanwoo steer using PCR-RFLP technique and utilizing it for early detection and selection of high quality Korean milk.

  In general, conventional quantitative genetic methods for improving breeding of livestock have relied on phenotypic records of livestock. In other words, the candidates were selected for the first time, such as pedigree record and body development, and the next black sheep produced by mating with the birds was reared and slaughtered to record the carcass and meat quality traits, In order to estimate the true breeding price of livestock, the most suitable statistical model formula is devised to select the breed axis. It takes a long time to measure these phenotypic observations, The cost of labor, such as feed costs, facility investment and maintenance costs, labor costs, and excessive labor are required. Due to this very inefficient method, there has been a demand for a method of determining the weight of Korean beef cattle in order to early select and breed cattle having excellent weights in a more scientific and quick manner. With the remarkable development of genetic engineering technology, various experimental techniques at the DNA molecule level have been continuously developed. Based on the development of these experimental techniques, various DNA analysis techniques have been developed to improve the genetic characteristics and capacity of livestock, . Among these methods, PCR-RFLP (Restriction Fragment Length Polymorphism) among the analysis methods using the recently used PCR (Polymerase Chain Reaction) technique is limited to specific point mutation sites. In the presence of the enzyme recognition site, DNA polymorphism in which the lengths of the fragments produced by restriction enzymes vary according to the genotype difference of each individual can be used to determine the desirable genotype of each individual more quickly and accurately There is an advantage.

Accordingly, the present invention provides a method for early diagnosis and selection of Hanwoo which has genetically broadened cross-sectional area of stomach muscles by using a cutting-edge molecular breeding technique,

As a result, the inventors of the present invention have found that ADH1C gene located on chromosome 6 of bovine is selected as a candidate gene for flesh trait related to Hanwoo, and molecular markers closely related to the cross-sectional area of the root of Hanwoo were obtained , And invented a genetic test method for diagnosing the cross-sectional area of the abdominal muscle of Hanwoo using this.

The gene for ADH1C (alcohol dehydrogenase 1C (class I), gamma polypeptide) is a gene encoding an ethanololytic enzyme that decomposes and inhibits vitamin A, or retinol, which has an alcoholic structure. Vitamin A is present in the blood, penetrates into the cytoplasm, and expresses a gene of a specific function to accumulate fat and increase body weight. When the expression of vitamin A, which accumulates fat, is promoted, the body weight gain is expected to be high. As a result, the ADH1C gene is newly recognized as a candidate gene for early diagnosis of beef cattle. Therefore, in the present invention, the ADH1C gene is finally selected as a candidate gene related to the Korean native cattle trait, and PCR-RFLP analysis using the specific SNP of the ADH1C gene and statistical analysis on the relationship with the carcass traits related to the trait of the trait are performed. We have developed a genetic testing method that can be used for the early detection of high-quality Korean beef cattle, which has a genetically diverse cross-sectional area of the stem.

More specifically, genomic DNA is isolated from blood of Korean beef cattle, amplified by PCR using a primer containing exon 6 of the ADH1C gene, and then Sty I restriction enzyme is added to the amplified PCR product The DNA fragments were electrophoresed on agarose gel, and the DNA markers of each genotype were determined. The DNA markers closely related to the cross-sectional area of the embryo were finally identified through a statistical analysis of the correlation between the DNA markers of the Hanwoo To provide genetic testing methods for diagnosing and discriminating genetically modified Hanwoo which has a wide cross-sectional area.

Using the DNA markers (TT, TG and GG type) due to T↔G base substitution in the exon 6 region of the Hanwoo ADH1C gene (96th position in PCR amplification region: 96th position in SEQ ID NO: 1) And it can be used for the early diagnosis and selection of high - yielding Korean beef cattle and industrial application of useful genetic resources. In other words, by early diagnosis and selective breeding of the individuals having the GG genotype of the ADH1C gene, it is possible to maximize the efficiency and improvement performance of the breeding breeding program of Korean beef breeding breed, .

The PCR-RFLP (Restriction Fragment Length Polymorphism) analysis technique used in the present invention as a state-of-the-art gene inspection 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.

FIG. 1 shows the molecular markers of SNP genotypes of each individual due to the 96th T↔G single nucleotide polymorphism (SNP) in the amplification region of the exon 6 gene of SEQ ID NO: 1, which was detected by PCR-RFLP analysis in the present invention marker) electrophoresis photograph
FIG. 2 shows the DNA marker of each SNP genotype due to the 96th TEGG single nucleotide polymorphism (SNP) in the amplification region of the exon 6 gene of the Korean wild-type ADH1C 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 ADH1C SNP genotype detection of genes

1. Disclosure material and DNA separation purification

A total of 300 Hanwoo farms selected for the present invention were selected as the public service axes. 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 ADH1C gene

A primer for amplifying the 461 bp DNA fragment shown in SEQ ID NO: 1 containing the exon 6 region of the Hanwoo ADH1C gene was constructed using the Btau 4.6.1 assembly registered in NCBI GenBank, 2 and SEQ ID NO: 3 with reference to nucleotide sequence information registered in NC_007304. The primer sequences used in the present invention are shown in Table 1.

Primer base sequence for PCR amplification of ADH1C gene Gene name Amplification region The primer sequence (5'-3 ') SEQ ID NO: ADH1C exon 6 Forward F-CACCTGGAAAGCCCTTAACAAT
Reverse R-GCAACCCAGGTAACAAATAGGA 2
3

The reaction conditions for the PCR amplification of the ADH1C gene were carried out using the JAMP 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. ≪ / RTI > The PCR reaction conditions were preliminary heating at 94 ° C for 5 minutes, followed by repeating 35 cycles of 94 ° C for 30 seconds, 57 ° C for 20 seconds and 72 ° C for 30 seconds, 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 a result of analysis of the nucleotide sequence by direct sequencing using the PCR amplification product of the Hanwoo ADH1C gene, a total of 461 bp DNA bases were detected as shown in SEQ ID NO: 1, and a total of 1 Single nucleotide polymorphism (SNP) sites were detected. That is, SNPs were detected in the 96th nucleotide of SEQ ID NO: 1 (NCBI GenBank registration number Btau 4.6.1 assembly, 27,000th, 443rd nucleotide of alternate reference sequence NC_007304) base. As a result, amino acid substitution by leucine (CT T ) ↔ leucine (CT G ) was not observed in the present SNP region. PCR-RFLP analysis was performed using the restriction enzyme Sty I (5'-C ↓ CWWGG-3 ') for the detection of the DNA marker according to SNP genotype of each test subject.

3. SNP genotype marker analysis of Hanwoo ADH1C gene by PCR-RFLP technique

The presence of Sty I (5'-C ↓ CWWGG-3 ') restriction enzyme recognition site in the 96th T↔G SNP region of the amplified region of the detected Hanwoo ADH1C gene was confirmed by PCR-RFLP technique. (TT, TG, and GG) (FIG. 1 and FIG. 2). RFLP analysis of the Hanwoo ADH1C gene was performed by adding 2 units of Sty I restriction enzyme to 10 μl of PCR amplification product and then reacting at 37 ° C for at least 3 hours. The DNA fragments obtained by digesting the PCR amplification products with restriction enzymes were electrophoresed on 2% agarose gel using TBE buffer (90 mM Tris-borate, 2 mM EDTA, pH 8.0) at about 80 volts for about 2 hours After staining with EtBr (ethidium bromide), the SNP genotypes of each test individual were determined by observing the DNA fragment pattern. In other words, in the individuals with the TT homo genotype, there were two restriction enzyme recognition sites and three DNA bands of 93, 144 and 224 bp were detected. In the case of the individuals having the GG homo genotype, one restriction Two DNA bands of size 144 and 317 bp were detected in the presence of the enzyme recognition site. The DNA fragments of 93, 144, 224, and 317 bp were detected in the TG heterozygous individuals in all four DNA fragments (Fig. 1).

The frequencies of alleles and genotypes of the 96th SNP in the amplified region of the ADH1C gene analyzed in the 300 farms were compared with those of the T allele The genotype frequencies of TG genotypes were 55.67%, TG genotypes were about 34.66%, and GG genotypes were the most frequent. The frequency of T allele was higher than that of G allele, The genotype was about 9.67%, the frequency of TT genotype was highest, and the GG genotype was the lowest.

Example 2. Hanwoo ADH1C  Genomic DNA Marker Genotype and Genotype Of the trait  Association statistical analysis

In order to analyze the SNP genotypes of Hanwoo ADH1C gene and the relationship between carcass traits of Hanwoo and carcass traits detected by the present invention, statistical analysis was performed using PROC GLM method using SAS a9.2 Package / PC program. The significance test was carried out by Duncan's Multiple Range Test (DMRT) for differences in least squares of each genotype. As a result, the genotype of the SNP region (No. 96 of SEQ ID NO: 1) due to the T↔G base substitution in the exon 6 region of the Hanwoo ADH1C gene proved to be significantly related to the cross-sectional area of the abdominal muscle of Hanwoo (P <0.05). As shown in Table 2, the area of the GG genotypes in the cross-sectional area of the embryo was significantly higher than those of the TT and TG genotypes (P <0.05). In other words, it was found that individuals with GG genotypes had a mean cross - sectional area of 86.000 ± 4.526 ㎠, which was significantly higher than those with TT genotype (73.454 ± 1.364 ㎠) and TG genotype (75.560 ± 0.964 ㎠) Wide.

Analysis of association between SNP genotype and meat and meat quality traits in Hanwoo ADH1C gene characteristics ADH1C g.96T> C SNP genotype (means ± SE) p-value
TT TG GG Fresh weight / kg 549.393 + - 8.423 536.212 + - 5.956 583.333 ± 27.937 0.147 Conductor weight / kg 313.333 + - 5.379 305.515 ± 3.804 341.333 + 17.842 0.097 Conductor ratio /% 56.984 + 0.265 56.942 + 0.187 58.433 + 0.881 0.258 Back ground thickness / cm 0.715 + 0.049 0.621 + 0.034 0.433 + 0.164 0.132 Double longest cross-sectional area / ㎠ 73.454 ± 1.364 a 75.560 + 0.964 a 86,000 + 1.526 b 0.017

The results of the present invention are summarized as follows. The SNP genotype of the 96th T↔G base substitution in the amplification region of the Korean Native Cow ADH1C gene of the present invention was significantly It can be used as a genetic test method for early diagnosis and selection of Hanwoo which has genetically broadened myosin cross section using the DNA molecule marker of the present invention.

In Table 2, a and b : there is a significant difference between different codes (P <0.05)
1, M: 100 bp DNA ladder; TT, TG, GG: each single nucleotide polymorphic marker genotype by the ADH1C gene SNP of the invention; 93, 144, 224, 317 bp: Size of the DNA fragment detected by RFLP analysis
In FIG. 2, TT, TG, and GG are the DNA marker genotypes for the nucleotide sequence analysis chromatogram of the ADH1C gene SNP of the present invention

<110> SANGJI UNIVERSITY INDUSTRY ACADEMIC COOPERATION FOUNDATION &Lt; 120 > Diagnosis method for meat quantity using the DNA marker          associated with longissimus muscle area in Hanwoo <130> 2015-02 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 461 <212> DNA <213> Bos taurus <220> <221> gene &Lt; 222 > (1) .. (461) <223> ADH1C gene of Hanwoo <220> <221> intron <222> (1) (59) <223> intron 5 <220> <221> exon &Lt; 222 > (60) <223> exon 6 <220> <221> intron (320). (461) <223> intron 6 <220> <221> variation <222> (96) <223> SNP: T> G <220> <221> primer_bind <222> (1) (22) <223> Forward primer binding site <220> <221> primer_bind &Lt; 222 > (439) .. (461) <223> Reverse primer binding site <400> 1 cacctggaaa gcccttaaca atcattactt aatccatttt cacattttct gaaaacacag 60 gtcacccagg gctccacctg tgccgtgttt ggccttggag gagttggcct gtctgttatc 120 atgggctgca aagcagctgg agcagccagg atcattgcag tggacatcaa caaagacaaa 180 tttgcaaggg ccaaacaggt gggtgccacg gagtgcatca accctcagga ctacgagaaa 240 cccatcgagg aggtgctgaa agaagtgagt ggtggaggtg tagatttttc atttgaagtc 300 atcggtcggc ttgacaccat ggtatgtatt ttgaaatgtg tcaagacata agcttctgca 360 ttctagagtt ttctttaatg tggcagaata taagcattat atagaaagaa tatttttaaa 420 actatgatga ttttccattt cctatttgtt acctgggttg c 461 <210> 2 <211> 22 <212> DNA <213> Bos taurus <220> <221> primer_bind <222> (1) (22) <223> Forward primer sequeuce <400> 2 cacctggaaa gcccttaaca at 22 <210> 3 <211> 22 <212> DNA <213> Bos taurus <220> <221> primer_bind <222> (1) (22) <223> Reverse primer sequence <400> 3 gcaacccagg taacaaatag ga 22

Claims (4)

A PCR-RFLP (Restriction Polymerase Chain Reaction) method, which comprises the step of treating a fatty acid and obesity gene amplification product of Hanwoo ADH1C (Polymerase chain reaction) amplified with the primers of SEQ ID NO: 2 and SEQ ID NO: (SNP) marker of the individual Hanwoo subject to the 96th T &lt; G &gt; base substitution in the ADH1C gene amplification region shown in SEQ ID NO: 1 by the Fragment Length Polymorphism analysis method, The present invention relates to a method for predicting a genotype of a GG genotype of a Korean Hanwoo, which comprises genotyping TG, TG and GG, Korean Society of Food Science and Technology The method according to claim 1,
2 with the forward primer of SEQ ID NO: 2 having the nucleotide sequence of CACCTGGAAAGCCCTTAACAAT in the 5 'to 3' direction and the reverse primer of SEQ ID NO: 3 with the nucleotide sequence of GCAACCCAGGTAACAAATAGGA in the 5 'to 3' direction, Is amplified by PCR. &Lt; tb &gt;&lt; TABLE &gt; Columns = &lt; The method according to claim 1,
The present invention relates to a method for diagnosing Korean native wheat fat using the stigma-associated cross-sectional molecular markers of Sty I (5'-C ↓ CWWGG-3 ') restriction enzyme in the PCR-RFLP analysis using the Hanwoo ADH1C gene The method according to claim 1,
Among the three DNA molecule markers (TT, TG and GG) of the Hanwoo ADH1C gene detected by the PCR-RFLP assay of the present invention, Hanwoo individuals having GG genotypes were genetically different from Hanwoo individuals having TT and TG genotypes Wherein the method comprises predicting and predicting with a Hanwoo having a high cross-sectional area value,
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20220152605A (en) * 2021-05-10 2022-11-17 한경대학교 산학협력단 Primer sets for determining genotype of ADH1C gene related to meat of Korean native cattle and uses thereof

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* Cited by examiner, † Cited by third party
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J. Anim. Sci., Vol. 90, No. 8, pp. 2476-2483 (2012.08.) *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20220152605A (en) * 2021-05-10 2022-11-17 한경대학교 산학협력단 Primer sets for determining genotype of ADH1C gene related to meat of Korean native cattle and uses thereof
KR102634978B1 (en) * 2021-05-10 2024-02-06 한경국립대학교 산학협력단 Primer sets for determining genotype of ADH1C gene related to meat of Korean native cattle and uses thereof

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