KR100352059B1 - DNA probe to identify the Korean Cattle, Hanwoo and method for identification thereof - Google Patents

Abstract

The present invention relates to a DNA probe for Hanwoo identification, wherein the DNA probe specific to the present invention identified by RAPD-PCR analysis and DNA fingerprinting distinguishes pure Korean beef from imported beef, identifies pure Korean beef, There is an excellent effect of improving the quality.

Description

TECHNICAL FIELD [0001] The present invention relates to a DNA probe for discriminating a Hanwoo, and a method for discriminating a pure Hanwoo using the same,

The present invention relates to a DNA probe for Hanwoo identification. More particularly, the present invention relates to a DNA probe capable of distinguishing pure Korean beef from imported beef by identifying a specific DNA marker based on random amplified polymorphic DNA (RAPD) -PCR (polymerase chain reaction) analysis and DNA fingerprinting, and a pure- .

The extinction and eradication of genetic resources may reduce the worldwide genetic diversity that is genetically important for improving food resources. Developing simple genetic markers to characterize genetically different populations is needed as an efficient strategy to preserve the genetic resources.

Hanwoo is an important player in the Korean economy because it produces excellent flesh quality favored by Korean consumers. For this reason, although there is a low productivity of Korean beef cattle and low competition for imported beef in terms of price in the open market, related research has continued to improve the quality of Korean beef cattle industry and Korean beef cattle in Korean urban areas. Nevertheless, due to the long indifference to improved Hanwoo and the crossbreeding of foreign cattle to improve meat productivity, the Hanwoo remediation system is inferior to other species in uniformity and genetic homogeneity. Since the evaluation of gene parameters, which is an important factor of the base population aggregation, system and gene modification system, is uncertain, the KWU improvement system is showing stagnation nowadays. Due to recent advances in the scientific field, classical breeding programs based on statistical probabilities are gradually changing to breed improvement based on molecular basis for genetic estimation and accuracy of simple selection. From this perspective, the distinction of Korean beef from other foreign species by molecules is necessary to investigate what the molecular characteristics of Hanwoo are and how to improve it.

Since 1990, several specific DNA markers have been developed by RAPD-PCR and DNA fingerprinting to identify each bovine species. Black cattle, Mannen, H. and Tsuji, S. (1993)J. Animal Genet. (Japan) 21, Zebu cattle, Gawakisa, P. S., Kemp, S.J., and Teale, A.J. (1994)Animal Genet. 25, 89-94), German native cattle (Buitkamp, J., Zischler, H., Epplen, J. T. and Geldermann,Animal Genet. 22, 137), bos indikus and bostaurus (Bos indicusandBos taurus,Kemp, S.J. and Teale, A. J. (1994)Animal Genet. 25, 83-88), Brown Swiss, Simmental and Eringer (Glowatzki-Mullis, C., Gaillard, G., Wigger, G., and Fries, )Animal Genet. 26, 7), the Ukrainian pigments (Semyenova, S. K., Vasilyev, V.A., Stekleneve, E.P., Prosnjak, M.I., and Ryskov, A.P. (1996)25th Int. Conf. Animal Genet.(Tours, France) A038, 41), Kholmogor, Terletski, V. and Dementieva, D. (1996) Proceed. 1st Korea / Russia Joint Symp.One, 145), Holstein, Kwon, Y. M., Yim, D. S., Lee, H. J., Cho, K. K., Choi, Y. J., and Baik, M. G. (1995)Mol. Cells 5Lee, M., Y., Park, N. H., and Yang, Y. H. (1998)Korean J. Animal Genet. Breeding 2Jung, Y., and Nam, D. H. (2000), < RTI ID = 0.0 &Biotechnol. Bioprocess Eng.,5, in press). Even if some DNA markers are part of the breed, more specific markers can make breed identification more accurate.

RAPD-PCR analysis of DNA pool samples was also performed using a variety of primers in an attempt to identify new DNA markers that can distinguish Hanwoo from other WPC based on population-specific polymorphisms (Cho, BW and Han, JY (1994)Korean J. Anim. Sci.36, 263-270). However, the RAPD-PCR format is often difficult to standardize because the PCR results are very sensitive to amplification conditions and may have problems with the reproducibility of the pattern (Yu, K. F. and Pauls, K. P. (1992)Nucleic Acid Res. 20, 2606). Thus, the DNA markers identified by RAPD-PCR were used only for cloning and sequencing to find new repeat DNA sequence fragments. DNA fingerprinting (Jeffreys, A.J., Wilson, V., and Thein, S.L. (1985)) using primers in repeated DNAs or sequenced DNA fragments to reveal more accurate and reproducible molecular results,Nature 316, RAPD-PCR (Williams J. G. K., Kubelik, A. R., Livak, K. J., Rafalski, A., and Tingey, S. V. (1990)Nucleic Acid Res. 8, 6531-6535). ≪ / RTI >

Until now, polymorphic DNA analysis of Hanwoo and its application in breeding have been meaningless, but the necessity of gene modification using DNA markers has been highly emphasized along with research trends in the world these days.

The present inventors have attempted to develop new DNA fragments as probes for DNA fingerprinting using DNA samples from various individuals of various breeds, and as a result, they have been able to distinguish between hybridomas and hybrids, and furthermore, The present invention has been accomplished by separating the population-specific markers that can be used for the analysis into specific probes.

Accordingly, an object of the present invention is to provide a DNA probe capable of discriminating pure Korean cattle.

The above object of the present invention was achieved by identifying a specific DNA marker using a randomly synthesized primer by RAPD-PCR analysis and confirming the reproducibility of the DNA probe by applying DNA fingerprinting.

The configuration and operation of the present invention will be described below

1 shows the RAPD-PCR band pattern of a bovine species amplified with a random primer,

Fig. 2 shows the DNA base sequence and the designed DNA fragment of a specific marker obtained from the RAPD-PCR of Hanwoo.

FIG. 3 shows the DNA fingerprinting of 6 individual bovine DNA samples (left) and DNA pool samples (postal) using the inventive probe (AAC) ₅ and the restriction enzyme Pst I on the left and right, respectively,

FIG. 4 is a graph showing the DNA markers of bovine and bovine hybridized with Hanwoo and other species standardized as the registered phenotype using the probe (AAC) ₅ and the restriction enzyme Pst I of the present invention, respectively.

The present invention relates to a method for preparing a DNA fragment, which comprises performing DNA cloning and sequencing through RAPD-PCR analysis; Preparing a specific DNA probe of the repetitive sequence prepared above; And confirming the reproducibility of the prepared probe.

In the present invention, 281 Hanwoo, 15 foreign and 15 Asian species were used to collect blood. Imported foreign species including Holstein, Carolece, Simmental, Aberdeen Angus, and Brahman have been imported to Korea for the past two and a half decades and hybridized with Hanwoo. Two Asian species are Japanese black cattle And China Yan Bien.

Hereinafter, specific methods of the present invention will be described in detail with reference to Examples and Experimental Examples, but the scope of the present invention is not limited to these Examples and Experimental Examples.

Example 1: RAPD-PCR analysis and DNA probe preparation

Step 1: DNA sequencing by RAPD-PCR analysis

In this step, white blood cells collected from 10 mL blood samples were dissolved by SDS (sodium dodecyl sulfate) and proteinase-K, and genomic DNA was extracted by the phenol-chloroform method (Kwon, YM, Yim, DS , Lee, HJ, Cho, KK, Choi, YJ, and Baik, MG (1995) Mol. Cells 5 , 393-396). Sixty different 10-base oligonucleotide primers for RAPD-PCR analysis were provided by Bioniar (Cheongwon, Korea). All 10-mer random primers had both guanine (G) and cytosine (C) in the range of 60-70%.

The PCR amplification was carried out in the presence of 25 μl reaction (Takara, Japan), 1 × PCR buffer (50 mM KCl, 10 mM Tris-HCl, pH 8.3, 0.01% gelatin) containing 0.5 U Taq DNA polymerase, 0.2 mM each dNTP, 200 pM and 50 ng of genomic DNA template at 94 ° C for 5 minutes, followed by 94 ° C for 1 minute, 36 ° C for 2 minutes, and 72 ° C for 2 minutes. After completion of the PCR, 5 ㎕ aliquots of the reaction mixture were loaded on 1.5% agarose gel and electrophoresed at 15 V / cm for 1 hour.

The DNA band on the agarose gel was extracted using a JET sorb kit (GENOMED Inc., NC, USA) and amplified again by the PCR reaction of the same primers. The amplified PCR products were further purified with JET pure kit (GENOMED Inc., NC, USA). The DNA fragment was ligated with pGET-T vector (Promega Corporation, CA, USA) by T4 DNA ligase. The combined mixture was transferred to E. coli (JM109) (Sambrook et al ., 1989). D-thiogalacto-pyranoside (IPTG), 0.04% 5-bromo-4-chloro- pyranoside, 0.1 g of ampicillin, 0.1 mM isopropyl p- (1% tryptone, 0.5% yeast extract, 1 < RTI ID = 0.0 > % NaCl, pH 7.2), and PCR was carried out with the same primers to confirm whether or not the DNA was inserted. The nucleotide sequence of the inserted DNA was determined using a universal sequencing primer provided by Bioniar (Cheongwon, Korea).

Sixty randomized primers were used to identify specific DNA markers to distinguish Hanwoo from five foreign species (Bioni, Chungwon, Korea). Particularly, RAPD-PCR analysis was performed on the DNA pools of each aliquot together with a mixed primer of No. 4 (5'-TCGGCGATAG-3 ') and No. 6 (5'-AGCCAGCCAA-3'). A DNA band of 519 bp was found as a unique DNA marker in Hanwoo (Fig. 1).

Such useful RAPD markers may be a useful tool in itself, but their use has been limited because they are highly sensitive to amplification conditions and have low reproducibility. To overcome this difficulty in Hanwoo identification based on RAPD DNA markers, it was necessary to develop new DNA probes from RAPD markers. For this, the 519 bp DNA band identified as a specific marker of Hanwoo was first isolated, amplified again by PCR, sequenced and inserted into pGEM-T vector. The nucleotide sequence of the DNA fragment determined by the didxyxy method was as shown in Fig. Two repeat sequences of (AAC) ₅ and (GAAGA) ₂ were found in the above base sequence.

Step 2: Probe preparation and Southern analysis using DNA fingerprinting

A specific DNA fragment was prepared as a suitable primer for discriminating repetitive sequences and microsatellite as microsatellite in double strands complementary to the DNA sequence obtained as a result of cloning in this step. The synthesized single DNAs were annealed at 2 각 each, bound using T4 DNA ligase, and amplified by PCR. 50 ㎍ of DNA treated with restriction endonuclease Pst I was electrophoresed at 40 cm in 1.2% agarose gel. The probe was hybridized overnight at 37 ° C in a solution consisting of 6X SSC, 40% formamide, 5 mM EDTA, 0.25% defatted milk. Washing was carried out at 55 ° C for 30 minutes in 4X SSC, 0.1% SDS twice, 2X SSC, 0.1% SDS once. The membranes were then sensitized with an intensifying screen to Fuji Super HR-A film at -80 ° C.

The primer and the microsatellite probe were separated from the determined nucleotide sequence under the assumption that the specific marker for the Hanwoo had different DNA polymorphism. The separated DNA fragments (AAC) ₅ and (GAAGA) ₂ were synthesized with complementary strands. The DNA probes were identified to discriminate between the other woo and the Hanwoo.

As shown in Fig. 3, among the two kinds of designed probes, the repeating sequence (AAC) ₅ showed characteristic markers of 10 kb in DNA fingerprinting of Hanwoo, which is not a foreign species. As a result of examination of each individual, genetic markers such as Hanwoo could be observed in two Chinese yann biannos. Although this is known to have the same hereditary characteristics as the three wolves of Far East Asia, Hanwoo, Japanese black cattle and Chinese Yanbian, Hanwoo is closer to Yanbian in China than Japanese black cattle.

Experimental Example 1: Development probe inspection

In order to confirm the reproduced acidity in this experiment, the (AAC) ₅ probe was applied to the DNA fingerprinting of Hanwoo individuals together with Pst I restriction enzyme. 109 cattle were registered as phenotypic standards in the Korean Cattle Breeding Association, 126 cattle selected by the Korean Society for the Improvement of Animals, 20 cattle from the local farms, and 281 HanDoo including 26 cattle not confirmed by the national government agencies Were examined by DNA fingerprinting with (AAC) ₅ . As shown in Fig. 4, the cattle having the descendants of the Korean Cattle Breeding Center and the cattle having the Korean Cattle Breeding Association were identified as pure individuals having specific DNA markers. However, as shown in Table 1, 26 dogs with unknown phenotypes did not show any particular markers and most individuals from local farms were not genetically obeyed. This means that the Hanwoo in the local farm did not have distinct genetic uniformity by DNA fingerprinting using DNA markers. This is very similar to the case of Tanzanian hymen which genetically screened 61-89% from foreign cows.

(AAC) ₅ / Distribution of specific markers in Hanwoo by Pst I Resources No. of head Specific markers (10kb) has exist none NLCF-HIC ¹⁾ 109 109 0 KAIA ²⁾ 126 126 0 Local Hanwoo Farm 20 2 18 Unidentified object 26 0 26 [Note] ¹⁾ National Livestock Cooperative, Hanwoo Improvement Center ²⁾ Korea Animal Improvement Association

As described above, the present invention has the effect of providing a DNA probe for pure Korean cattle. The DNA probe specific to the present invention identified by RAPD-PCR analysis and DNA fingerprinting is pure Korean beef and imported beef And discriminating pure Hanwoo and using it as a genetic resource, it is a very useful invention in the Korean meat industry because it has an excellent effect of improving the quality of Hanwoo.

Claims (2)

A Hanwoo DNA probe having the following sequence. AACAACAACAACAAC The method of claim 1, wherein the DNA probe is used as a selection marker.