KR101764012B1 - Diagnostic method of Hanwoo with high dressing percentage by using DNA testing - Google Patents
Diagnostic method of Hanwoo with high dressing percentage by using DNA testing Download PDFInfo
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Abstract
The present invention relates to a method for diagnosing Korean beef cattle having a high carcass percentage through genetic testing using a specific single nucleotide polymorphism marker (SNP marker) of a growth hormone secretagogue receptor (GHSR) gene in Korean cattle , The method of diagnosing a Korean beef cattle according to the present invention is not only capable of maximizing the efficiency and practicality of the selection because it is fast, simple and highly accurate, but also diagnoses and selects the highly transferred beef cattle in the beef cattle group, .
Description
The present invention relates to a growth hormone secretagogue receptor (GHSR) closely related to a Hanwoo dressing percentage trait through analysis of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) The present invention provides a technique for early identification and discrimination of genetically engineered Korean beef cattle using molecular markers associated with Korean native cattle traits using SNP markers.
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 early detection and screening of a genetic marker that is closely related to a Korean trait using the PCR-RFLP technique.
Hanwoo is a small domestic cattle species that has been adapted to the climate of Korea for a long time. As a main trait to improve the breeding characteristics of Korean beef cattle, the weight rating criteria affecting the carcass weight and carcass weight of individuals are dressing percentage, carcass weight, backfat thickness, And M. longissimus dorsi area. Among them, the carcass ratio is one of the most important factors for determining the carcass grade. 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 better grade cattle in a more scientific and rapid 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.
To date, studies on useful genes related to economic traits such as carcass and meat quality traits have been relatively inadequate compared to other livestock such as pigs. In Korea, a study on the leptin gene, which has genetic influence on the intramuscular fat level of the carcass and meat quality traits of beef cattle, has been reported (now registered as Patent No. 0282246, November 27, 2000) (Application No. 10-2005-0065732), and CPE genes (GenBank No. 10-2005-0073890) and SCD genes (Application No. 10-2005 -0088417), and the FABP gene (Application No. 10-2005-0108866), which is related to the cross-sectional area and backfat thickness of the embryo, is currently a domestic patent application as a functional gene related to the carcass and meat quality traits of Hanwoo.
The growth hormone secretagogue receptor (GHSR) gene used in the present invention is a gene involved in promoting the secretion of growth hormone and is a functional candidate gene related to the growth and the offspring of Korean cattle. Therefore, in the present invention, it is intended to provide a DNA molecule marker capable of early diagnosis of Hanwoo having high carcass percentage and its application to selection through genetic test using restriction enzyme fragment polymorphism (RFLP) molecular marker of GHSR gene involved in animal growth .
As a result, the inventors of the present invention found that a GHSR gene located on chromosome 1 of bovine cow was selected as a candidate for a trait related to the trait of Korean beef cattle, PCR-RFLP analysis method was used to develop a diagnostic method for genetically engineered Korean beef cattle.
More specifically, a genomic DNA was isolated from the blood of Korean beef cattle, and a primer containing the 3'-UTR region of the GHSR gene was prepared and amplified by PCR. Then, the amplified product of 1,025 bp was amplified NmuC I (5'-GTSAC-3 ') restriction enzyme was added and reacted at 65 ° C for 3 hours or longer. DNA fragments cut by different SNP genotypes were electrophoresed on 2% agarose gel and SNP markers of each genotype were determined . Finally, SNP markers closely related to the carcass yield were identified by statistical analysis of the relationship between SNP markers and carcass traits related to meat quality and meat quality traits in Hanwoo. Accordingly, the present invention provides a method for diagnosing and discriminating genetically engineered Korean beef cattle through such a genetic testing method.
Using the DNA genotypic markers (TT, TC and CC type) due to T↔C base substitution of the Hanwoo GHSR gene 3'-UTR region (762nd in PCR amplification region: No. 762 of SEQ ID NO: 1) It is possible to identify the characteristics of the carcass for traits and to make early diagnosis and selection of high quality Korean beef and to utilize useful genetic resources industrially. In other words, by early diagnosis and selective breeding of individuals having the TT genotype of the GHSR gene, it is possible to maximize the efficiency and improvement performance of the breeding breeding program and to improve and preserve the genetic resources of the breeding herb .
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.
1 shows the molecular markers of SNP genotypes of each individual due to the T? C single nucleotide polymorphism (SNP) located at the 762nd position in the PCR amplification region of the Korean GHSR gene of SEQ ID NO: 1 detected by the PCR-RFLP analysis method in the present invention DNA marker) electrophoresis photograph
FIG. 2 is a graph showing the relationship between the nucleotide sequence of the DNA marker of each SNP genotype due to the 762th T↔C single nucleotide polymorphism (SNP) in the PCR amplification region of the Korean GHSR gene of SEQ ID NO: 1 detected by PCR- RFLP analysis in the present invention 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 GHSR SNP genotype detection of genes
1. Disclosure material and DNA separation purification
A total of 309 Hanwoo farms were selected for the purpose of 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 nucleotide polymorphism (SNP) detection of Hanwoo GHSR gene
A primer for amplifying a DNA fragment of the size of 1,025 bp shown in SEQ ID NO: 1 containing the 3'-UTR region of the GWR gene of Hanwoo GHGH was obtained from the registered number of the Bos taurus UMD 3.1.1 reference sequence registered in NCBI GenBank 2 and SEQ ID NO: 3, respectively, with reference to the nucleotide sequence information registered in AC_000158.1. The primer sequences used in the present invention are shown in Table 1.
Reverse R-acccaatgtccaaattaaggtg
3
The reaction conditions for the PCR amplification of the GHSR gene were performed using the JEAMP 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 preliminarily heated at 94 ° C for 5 minutes, followed by 30 cycles of 94 ° C for 30 seconds, 58 ° C for 30 seconds, and 72 ° C for 1 minute, followed by heating at 72 ° C for 5 minutes. 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 1,025 bp DNA bases were detected using a direct sequencing method using a PCR amplification product of the Hanwoo GHSR gene. As a result, a total of 1 Single nucleotide polymorphism (SNP) sites were detected. That is, SNPs detected by T < + > C base substitution at base 762 of SEQ ID NO: 1 (95,799,449th nucleotide of NCBI GenBank registration number Bos taurus UMD 3.1.1 reference sequence AC_000158.1) For reference, the present SNP site is a SNP present in the 3'-UTR region, and thus is not directly involved in amino acid synthesis. PCR-RFLP analysis was performed using the NmuC I (5'-GTSA C -3 ') restriction enzyme for the detection of DNA markers according to the SNP genotype of each test subject.
3. SNP genotype marker analysis of GHSR gene in Hanwoo by PCR-RFLP
The presence of the NmuC I (5'-GTSAC-3 ') restriction enzyme recognition site in the 762nd T↔C SNP region in the amplified region of the detected Hanwoo GHSR gene revealed three types of SNP genotypes (TT , TC and CC) (FIG. 1 and FIG. 2). RFLP analysis of Hanwoo GHSR gene was performed by adding 2 units of NmuC I restriction enzyme to 10 μl of PCR amplification product and reacting at 65 ° C for more than 3 hours. The DNA fragments obtained by digesting the PCR amplification products with restriction enzymes were electrophoresed in 2% agarose gel using TBE buffer (90 mM Tris-borate, 2 mM EDTA, pH 8.0) at about 100 volt 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 CC homo genotype, one restriction enzyme recognition site was present and two DNA bands of 263 and 762 bp in size were detected. In the case of TT homo genotype, restriction enzyme recognition site was present One DNA band with a size of 1,025 bp was detected. And all of these three DNA fragments were detected in individuals with the TC hetero genotype, and DNA bands of 263, 762 and 1,025 bp were detected (FIG. 1).
The frequencies of alleles and genotypes of 762 SNPs in the amplified regions of the GHSR gene were analyzed by using the above method. The frequencies of alleles were T allele frequency The frequency of the C allele was higher than that of the T allele. The frequencies of the SNP marker genotypes were 23.61% for the TT genotype, 45.83% for the TC genotype, and 47.61% for the T allele CC genotype was about 30.55%, TC genotype was the most frequent, and TT genotype was the lowest.
Example 2. Analysis of SNP genotypes of Hanwoo GHSR gene and its relationship with meat quality and meat quality traits
In order to analyze the SNP genotypes of Hanwoo GHSR gene and the relationship between carcass and meat quality traits related to Hanwoo, we analyzed by PROC GLM method using SAS a9.2 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, SNP genotypes by T↔C base substitution in the 3'-UTR region of the Hanwoo GHSR gene detected by the present invention were proved to be significantly correlated with Hanwoo carcass ratio (P <0.05) As shown, individuals with TT genotypes were significantly higher than those with CC and TC genotypes.
As a result of the above results of the present invention, the SNP genotype due to the 762-th T? C base substitution in the amplification region of the Hanwoo GHSR gene of the present invention was significantly related to the carcass ratio The SNP maker of the present invention can be used as a genetic test method capable of early diagnosis and selection of genetically engineered Korean beef cattle.
In Table 2, a and b : there is a significant difference between different codes (P <0.05)
1, M: 100 bp DNA ladder; TT, TC, TC: each single nucleotide polymorphic marker genotype by the GHSR gene SNP of the present invention; 263, 762, and 1,025 bp: the size of the DNA fragment detected through RFLP analysis
In FIG. 2, TT, TC, and CC: the respective DNA marker genotypes for the nucleotide sequence analysis chromatograms of the GHSR gene SNPs of the present invention
<110> SANGJI UNIVERSITY INDUSTRY ACADEMIC COOPERATION FOUNDATION <120> Diagnostic method of Hanwoo with high dressing percentage using DNA testing <130> 2015-01 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 1025 <212> DNA <213> Bos taurus <220> <221> gene ≪ 222 > (1) .. (1025) <223> GHSR gene of Hanwoo <220> <221> variation <222> <223> SNP: T> C <220> <221> primer_bind <222> (1) (22) <223> Forward primer binding site <220> <221> primer_bind ≪ 222 > (1004) .. (1025) <223> Reverse primer binding site <400> 1 ataggaatgg ggaagagcaa aacatgatat ttttctgaag tcaattgtca taccacacat 60 aatcaaaaca agtccatgtc ttcaaaagag atgatggttt gctatgacat atcctgaact 120 gacgttctct ttctcattgt cttttcagct gtagtggttt ttgctttcat cctctgctgg 180 ctgcccttcc acgtaggacg gtatttattt tccaaatcct tcgagcctgg ctctgtggag 240 atcgcgcaga tcagccaata ctgcaacctc gtctctttcg tcctcttcta cctcagtgcc 300 gccatcaacc ctattctcta caacatcatg tccaagaagt accgggtggc agtgttcaaa 360 cttctgggat ttgaaccctt ctcccagagg aagctctcca ctctaaagga tgaaagttct 420 cgggcctgga cagaatctag tattaataca tgatcaagca cattgctcag taaagtcaac 480 gttcattatt ctataccaga agccatagta cagcggaact tgggaggaag tttgaaggtt 540 aattttggaa ttagggacac ataggtctga agacaattgg gagcaagaaa agatagactt 600 tgtagtgtgt gagcagtttg atttgattgc acactcatca gtgctatcat acacgatttc 660 tgcatatttc actgcctatc atttttgcat tgtgctgttg gtgctagcaa gggctctgca 720 gttcggagaa gggaggtagt gtagagaggg caattaggtc attttttact agacgaattc 780 taactctaat ttctttttca taatgagaat tttttagtaa aataataaaa aacttaaaac 840 atgactttca gaagctagca aatttcatgg ggtcccagat cagtgctgtc caggagaaat 900 ataatgcaaa gtacttaagc aatcaatata aaattgttaa tagaatttta tgctttttat 960 gttaaagtct ttaaaatctg gtttgttttt taaattttaa gttcacctta atttggacat 1020 tgggt 1025 <210> 2 <211> 22 <212> DNA <213> Bos taurus <220> <221> primer_bind <222> (1) (22) The forward primer sequence (5'-3 ') <400> 2 ataggaatgg ggaagagcaa aa 22 <210> 3 <211> 22 <212> DNA <213> Bos taurus <220> <221> primer_bind <222> (1) (22) Reverse primer sequence (5'-3 ') <400> 3 acccaatgtc caaattaagg tg 22
Claims (4)
The reverse primer of SEQ ID NO: 3 having the nucleotide sequence of ataggaatggggaagagcaaaa in the 5 'to 3' direction and the nucleotide sequence of acccaatgtccaaattaaggtg in the 5 'to 3' direction was used to amplify the GHSR gene 3'-UTR region Is amplified by PCR and is used for RFLP analysis.
In the PCR-RFLP analysis using the Hanwoo GHSR gene of the present invention, a Hanwoo diagnosing method with high carcass ratio through genetic testing characterized by using NmuC I restriction enzyme
Among the three DNA molecule markers (TT, TC and CC) of the Hanwoo GHSR gene detected by the PCR-RFLP assay of the present invention, Hanwoo individuals exhibiting TT genotypes were genetically higher than Hanwoo individuals expressing CC and TC genotypes The method of the present invention is a method for diagnosing a Korean beef cattle having a high carcass through a genetic test
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