KR101557408B1 - Method and Kit for identification of Korean cattle - Google Patents

Abstract

The present invention relates to a method and kit for identification of Hanwoo, and more particularly, to a method and kit for identifying Hanwoo, and more particularly, to a method for identifying Hanwoo, The present invention relates to a method for identifying a Hanwoo object to be determined and a kit for Hanwoo object identification for implementing the method.

The method and kit for analyzing Hanwoo according to the present invention can provide a standardized integrated database on alleles by providing a multiplex PCR method and an allelic ladder that can quickly and accurately analyze a large number of alleles of a supersatellite , And can be used to identify Hanwoo individuals.

Hanwoo, object identification, super satellites, allelic ladder, multiplex PC

Description

Description: TECHNICAL FIELD The present invention relates to a method and kit for identification of a Korean beef cattle,

The present invention relates to a method and kit for identification of Hanwoo, and more particularly, to a method and kit for identifying Hanwoo, and more particularly, to a method for identifying Hanwoo, And to a kits for identification of Hanwoo to implement the method.

Recognizing the value of conservation and utilization of native livestock as a genetic resource in different parts of the world (Notter (1999) J. Anim. Sci . 77: 61-69) And the relationship between them. For the breed of cattle, studies have been performed using various genetic markers such as blood proteins, milk proteins, microsatellites, mitochondrial DNA (MacHugh et al (1998) Anim. Genet. 29: Genetics 150: 1169-1175; Edwards et al (2000) Anim. Genet. 31: 127-130; Troy et al (2001) Nature 410: 1088-1091; Hanotte et al 2000) Molecular Ecology 9: 387-396, (2002) Science 296: 336-339). The existing blood type and biochemical polymorphism analysis methods have limitations on the tissues used for analysis and blood type tests on Hanwoo have a disadvantage in not determining the correct paternal blood lineage (Glowatzki-Mullis et al., 1995 [JoC]; Microsatellite-based parentage control in cattle, Anim. Genet. 26: 7-12). In recent years, various genetic analysis methods based on DNA polymorphisms have been developed and identification has been made between cultivars and individuals (Peelman et al., 1998. Evaluation of the genetic variability of 23 bovine microsatellite markers in four Belgian cattle breeds, Anim Genet., 29: 161-167).

Microsatellite (MS) is a repetitive DNA group of about 2 to 6 nucleotides, which is a non-coding DNA sequence that is uniformly distributed throughout the genome and exhibits a very high polymorphism (Zajc I and Sampson J (1999) J Hered 90: 104-107). Variability among individuals is recognized according to the number of repeats in a particular locus (Koreth J, O'Leary JJand McGee JO (1996) J Pathol 178: 239-248.), (Polymerase Chain Reaction (PCR)) using a primer designed in the region, it is possible to observe the polymorphism in the PCR product length and to detect the DNA polymorphism. In addition, the supersatellite, like other genes, is transmitted to the offspring according to the Mendel's law of genetics and can be amplified by PCR and co-dominant when electrophoresed. In particular, since the supersatellite is small in size, it can amplify two to eight primers at the same time (Chamberlain JS et al., (1988) Nucleic Acids Res 16: 11141-11156) It can save time and money and can easily analyze their allelic genotypes. At present, about one thousand locomotives are identified in the cattle, and their characteristics and chromosomal location have been identified. (Barendse et al., 1994). A genetic linkage map of the bovine genome has been used as a means of screening and screening for supernatant markers that are highly polymorphic among varieties -235). In this process, a multiplex PCR method is used in which a plurality of primers are mixed and amplified. Thus, an efficient blood flow test and association mapping are performed. Through these analyzes, the supersatellite has been recognized as an important DNA marker for analysis of association with genetic diversity-based group and group genetic components in mammalian, fish, plant, etc., including humans (Achmann et al., 2004. Microsatellite diversity, population subdividing and gene flow in the Lipizzan horse, Gen. 35: 285-292 Ayub et al., 2003. Reconstruction of human evolutionary tree using polymorphic autosomal microsatellites. Anthropol., 122: 259-268.

The analysis of these supersatellites is widely used all over the world. In addition to academic research in Korea, it is widely used for beef traceability system, development of Hanwoo and imported meat discrimination method, but the final result values are all expressed as numbers with decimal points Data sharing with other research results is very difficult. That is, even if the same PCR product is continuously and repeatedly analyzed in the same analyzer, the size (bp) changes each time. Therefore, such data is a very inefficient system when it is integrated or managed, or when it is necessary to check the identity.

The allelic ladder designates the minimum or maximum range in which each genotype of each supersatellite can appear as a single allele number, and the peaks located within that range are unconditionally represented by a single numerical value. Which means conversion.

As a result of intensive research to develop an efficient Hanwoo identification method by accurately analyzing allele genes of supersatellite loci and standardizing them, the present inventors selected and amplified a supersatellite locus having a discriminating power in a Hanwoo group, We confirmed that standardized genotypes can be determined by applying an allelic ladder, and the present invention has been completed.

Accordingly, it is an object of the present invention to provide a method for identifying a Hanwoo individual, which selects and amplifies a supersatellite locus having discriminative power in a Hanwoo group to detect alleles and determines a standardized genotype by applying an allelic ladder.

It is also an object of the present invention to provide a kit for identifying Hanwoo using the above primer for amplification and a method comprising the allelic ladder thereof.

It is also an object of the present invention to provide a method for constructing an integrated database through an allelic ladder in which all possible alleles in each locus of a supersatellite are mixed.

In order to accomplish the above object, the present invention provides a Hanwoo identification method for selecting and amplifying a supersatellite locus having discriminative power in a Hanwoo group to detect alleles and determining a standardized genotype by applying an allelic ladder.

Preferably, (a) obtaining a nucleic acid sample to be analyzed; (b) selecting the locus of the supersatellite locus and amplifying with multiplex PCR using a primer specific to each locus of the locus satellites; (c) detecting the allele by electrophoresis of the amplified product of step (b); And (d) determining the normalized genotype of the allele of step (c) by applying an allelic ladder comprising alleles of the supersatellite locus of step (b). .

The selection of the supersatellite loci was made by considering the comprehensive amplification conditions (annealing temperature of the primer, product size, labeling fluorescent material), mixing the primers according to the concentration to prepare the reaction solution for the amplification reaction, The satellite loci are amplified by Multiplex PCR method. The amplified products were separated using an automatic DNA sequencer, and the distribution and size of the alleles of each locus were analyzed. Based on these analysis data, genetic markers were selected for identification of Korean beef cattle, ≪ / RTI >

It is desirable to use a genetic marker of at least 7 alleles and a size of amplified product of 80bp and 300bp. In particular, the preparation of the primer should be carried out in consideration of multiplex PCR conditions such as annealing temperature and amplification product size.

In one embodiment of the present invention, supermodel gene loci TGLA227, BM2113, TGLA53, ETH10, SPS115, RM011, TGLA122, INRA23, BMS4032, ETH225 and BM1824 corresponding to the above conditions were selected and all Allelic ladder mixed with possible alleles was prepared.

Selecting an allelic ladder by marker for allelic forms for TGLA227, BM2113, TGLA53, ETH10, SPS115, RM011, TGLA122, INRA23, BMS4032, ETH225 and BM1824; Amplifying the selected allelic genotype by polymerase chain reaction (PCR); Cloning the amplified PCR product and confirming the repeated base sequence; And amplifying the plasmid DNA of the confirmed allelic form by a polymerase chain reaction using a fluorescently labeled primer to obtain a PCR product for each marker and mixing the obtained PCR product have.

In the present invention, analytical DNA samples for individual identification of Hanwoo can be obtained from blood, hair roots, etc., and amplified by Multiplex PCR using the primers for each locus in the DNA sample, An allele is detected through an electrophoresis device, and an allelic ladder is applied to determine a standardized genotype.

The primer of the present invention may be one having the nucleotide sequence shown in SEQ ID NO: 1 to SEQ ID NO:

The nucleotide sequence of the nucleotide sequence of the nucleotide sequence of the nucleotide sequence of the nucleotide sequence of SEQ ID NO:

The method of analyzing Hanwoo according to the present invention provides a standardized integrated database on alleles by providing a multiplexed PCA technique and an allelic ladder that can quickly and accurately analyze a large number of alleles of a supersatellite And can be useful for identifying Hanwoo individuals.

Therefore, a kennel identification kit including an allelic ladder of a primer and a supersatellite locus specific to a supersatellite locus using the method of analyzing a Korean native cattle according to the present invention is also included within the scope of the present invention. A standardized integrated database of alleles based on the present invention can also be included in the technical idea of the present invention.

The method and kit for analyzing Hanwoo according to the present invention can provide a multiplexed PCA technique and an allelic ladder that can quickly and accurately analyze a large number of alleles of a supersagget, And can be used to identify Hanwoo entities.

Hereinafter, preferred embodiments and experimental examples are provided to facilitate understanding of the present invention. However, the following examples and experimental examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

<Example> Identification of Korean Cattle

1.1. Disclosure Animal and DNA

Blood of Hanwoo was collected from 145 breeding stocks of five brands in Gyeonggi province. Genomic DNA extraction kit (Qiagen, USA) was used for the isolation of genomic DNA from blood of Hanwoo.

1.2. Selection of ultra-satellites locus

The supersatellite loci used in this study were obtained from Applied Biosystems' Stockmarker (TM) and Roslin Institute ( http://www.projects.roslin.ac.uk/cdiv/markers.html ), and the National Center for Biotechnology Information (NCBI, National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/genome/sts/sts.cgi?uid=250746 , http://www.ncbi.nlm.nih.gov/genome/sts/sts .cgi? uid = 34781 ), and the super-satellites loci for polymorphism analysis proposed by the International Society for Animal Genetics (http://www.isag.org.uk/ISAG/all/02_PVpanels_LPCGH.doc). (Table 1).

Supersatellite locus for polymorphism analysis of cattle Locus Chromosome location (bp) Primer sequence SEQ ID NO: TGLA227 18 76 ~ 104 F CGAATTCCAAATCTGTTAATTTGCT One R ACAGACAGAAACTCAATGAAAGCA 2 BM2113 2 123 ~ 143 F GCTGCCTTCTACCAAATACCC 3 R CTTCCTGAGAGAAGCAACACC 4 TGLA53 16 154 ~ 188 F GCTTTCAGAAATAGTTTGCATTCA 5 R ATCTTCACATGATATTACAGCAGA 6 ETH10 5 212-224 F GTTCAGGACTGGCCCTGCTAACA 7 R CCTCCAGCCCACTTTCTCTTCTC 8 SPS115 15 246-260 F AAAGTGACACAACAGCTTCTCCAG 9 R AACGAGTGTCCTAGTTTGGCTGTG 10 RM011
14 95-121 F AGAAATTGCCCCAAAGAGATGTT 11 R GATCAAACCCCTGGAGGAGTC 12 TGLA122 21 137-181 F CCCTCCTCCAGGTAAATCAGC 13 R AATCACATGGCAAATAAGTACATAC 14 INRA23 3 196-222 F GAGTAGAGCTACAAGATAAACTTC 15 R TAACTACAGGGTGTTAGATGAACTC 16 BMS4032 One 90-118 F CGGACACAACTGAGCAACTC 17 R AGATGGCCAACAAACACATG 18 ETH225 9 141 ~ 159 F GATCACCTTGCCACTATTTCCT 19 R ACATGACAGCCAGCTGCTACT 20 BM1824 One 178-192 F GAGCAAGGTGTTTTTCCAATC 21 R CATTCTCCAACTGCTTCCTTG 22

1.3. Preparation of primers and labeling of fluorescent materials

 The primers used in the present invention were customized by Applied Biosystems (USA), and the names of the respective loci, the label fluorescent materials and the primer base sequences are shown in Table 1 above.

The fluorescent material was labeled only in one of the pair of amplification primers, and the fluorescent materials used in the labeling were 6-FAM (6-carboxylfluorescein), VIC, NED, PET and the like.

1.4. Multiplex PCR and MS analysis

Multiplex PCR was performed to obtain a final volume of 15 μl, 50 ng of template DNA, 2 μl of primer mixture, and dNTP concentration of 0.25 mM. In addition, 0.5 unit of Taq DNA polymerase (Zengdaks Co., Ltd.) and 1.5 10 of 10 × buffer were added and the final volume was adjusted with sterilized tertiary distilled water. The PCR conditions were denaturation at 95 ° C for 15 minutes, followed by 32 cycles of 95 ° C for 50 seconds, 56 ° C for 70 seconds, and 72 ° C for 70 seconds. And then terminated at 65 ° C for 30 minutes and then at 8 ° C.

In order to confirm the amplification status and the concentration of each PCR product, first electrophoresis was carried out on 3% agarose gel, and then, using Applied Biosystems 3130xl Genetic Analyzer (Applied Biosystems, USA) Was diluted with distilled water to about 80 times for the second electrophoresis. For fractionation by size, a 1: 9 mixture of GeneScan ™ 500 LIZ Size Standard and Hi-Di Formamide (Applied Biosystems, USA) was diluted and electrophoresed.

The allelic ladder for all supersell bodies was diluted 1: 9 with a mixture of GeneScan ™ 500 LIZ Size Standard and Hi-Di Formamide (Applied Biosystems, USA) as well as electrophoresis of PCR final products. Respectively.

After the electrophoresis, amplification size and marker type of each supersatellite locus were confirmed using GeneMapper 4.0 (Applied Biosystems, USA), and standardized results were obtained through an allelic ladder as shown in Table 2 .

Sample Super satellites Size 1 Size 2 A1 A2 Control TGLA227 88.07 14 BM2113 140.54 142.65 20 21 TGLA53 164.41 174.48 23 28 ETH10 221.96 227.74 19 22 SPS115 249.59 251.51 21 22 RM011
106.13 113.54 20 23 TGLA122 147.15 151.81 24 26 INRA23 211.62 219.32 20 24 BMS4032 122.81 23 ETH225 146.38 148.57 23 24 BM1824 189.45 14

Statistical analysis was performed on the values derived using the SAS V8.2 (USA) statistical program, and the heterozygosity (He, Heterozygosity) and polymorphic information content (PIC) .

The polymorphism index and other information for each gene locus were found as shown in Table 3 below.

Polymorphism index of supersatellite Locus
Lable
N
No. of alleles
Size (bp)
He
PIC
TGLA227
B (FAM)
351
13
76 ~ 115
0.830
0.808
BM2113
B (FAM)
339
11
123 ~ 156
0.744
0.708
TGLA53
B (FAM)
315
17
158-196
0.872
0.858
ETH10
B (FAM)
345
8
212-230
0.761
0.720
SPS115
B (FAM)
351
7
240 ~ 270
0.766
0.733
RM011
G (VIC)
318
12 95 ~ 121 0.700 0.715 TGLA122
G (VIC)
334
19
137-195
0.863
0.847
INRA23
G (VIC)
347
11
196-222
0.756
0.721
BMS4032 Y (NED)
298
11 90 ~ 118 0.625 0.632 ETH225
Y (NED)
345
8
141-170
0.651
0.609
BM1824
Y (NED)
357
7
176-200
0.656
0.600

The highest number of alleles was 19 in TGLA122 and TGLA53 was the highest in He (0.872) and PIC (0.858). These results indicate that the frequency of occurrence of alleles can be increased even though the number of alleles is large. The difference between the He and PIC values is different from that of the estimation formula due to the number and frequency of alleles .

As shown in Table 4 below, the probability of occurrence of the same individuals in the random mating group was 1.54 × 10 ^-23 when the statistical treatment was performed using the 11 satellites markers, which was 2,448,305 The statistics of the Ministry of Agriculture and Forestry), it is considered that there is no probability that the same individuals will appear.

Statistical analysis results H _e PIC PE PE _pu PI PI _half-sibs 0.747 0.722 0.999942 0.999901 1.54 x 10 ^-23 2.34 × 10 ^-15

FIG. 1 shows an electrophoresis image obtained by analyzing a product obtained by multiplex PCR on 11 locus loci of a satellite with an automatic base sequence analyzer.

FIG. 2 shows an electrophoresis image obtained by analyzing an allelic ladder for 11 locus locus loci in an automatic base sequence analyzer.

<110> HANKYONG INDUSTRY ACADEMIC COOPERATION CENTER <120> Method and Kit for identification of Korean cattle <160> 22 <170> Kopatentin 1.71 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> TGLA227 forward primer <400> 1 cgaattccaa atctgttaat ttgct 25 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> TGLA227 reverse primer <400> 2 acagacagaa actcaatgaa agca 24 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> BM2113 forward primer <400> 3 gctgccttct accaaatacc c 21 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> BM2113 reverse primer <400> 4 cttcctgaga gaagcaacac c 21 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> TGLA53 forward primer <400> 5 gctttcagaa atagtttgca ttca 24 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> TGLA53 reverse primer <400> 6 atcttcacat gatattacag caga 24 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> ETH10 forward primer <400> 7 gttcaggact ggccctgcta aca 23 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> ETH 10 reverse primer <400> 8 cctccagccc actttctctt ctc 23 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> SPS115 forward primer <400> 9 aaagtgacac aacagcttct ccag 24 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> SPS115 reverse primer <400> 10 aacgagtgtc ctagtttggc tgtg 24 <210> 11 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> RM011 forward primer <400> 11 agaaattgcc ccaaagagat gtt 23 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> RM011 reverse primer <400> 12 gatcaaaccc ctggaggagt c 21 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> TGLA122 forward primer <400> 13 ccctcctcca ggtaaatcag c 21 <210> 14 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> TGLA122 reverse primer <400> 14 aatcacatgg caaataagta catac 25 <210> 15 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> INRA23 forward primer <400> 15 gagtagagct acaagataaa cttc 24 <210> 16 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> INRA23 reverse primer <400> 16 taactacagg gtgttagatg aactc 25 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BMS4032 forward primer <400> 17 cggacacaac tgagcaactc 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BMS4032 reverse primer <400> 18 agatggccaa caaacacatg 20 <210> 19 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> ETH225 forward primer <400> 19 gatcaccttg ccactatttc ct 22 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ETH225 reverse primer <400> 20 acatgacagc cagctgctac t 21 <210> 21 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> BM1824 forward primer <400> 21 gagcaaggtg tttttccaat c 21 <210> 22 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> BM1824 reverse primer <400> 22 cattctccaa ctgcttcctt g 21

Claims (10)

(a) obtaining a nucleic acid sample to be analyzed; (b) amplifying with multiplex PCR using primers specific to the 11 locus loci of TGLA227, BM2113, TGLA53, ETH10, SPS115, RM011, TGLA122, INRA23, BMS4032, ETH225 and BM1824;  (c) detecting the allele by electrophoresis of the amplified product of step (b); And (d) determining the normalized genotype of the allele of step (c) by applying an allelic ladder consisting of alleles of the supersatellite locus of step (b). 2. The method of claim 1, wherein the locus of the supersatellite locus has an allele number of 7 or more and a size of the amplified product is 80 bp or more and 300 bp or less. delete [2] The method of claim 1, wherein the nucleic acid sample in step (a) is blood or hair roots of cow to be analyzed. The method according to claim 1, wherein the primer of step (b) is selected from primer sets consisting of the nucleotide sequences of SEQ ID NOS: 1 to 22. The method according to claim 1, wherein the allelic ladder comprises alleles of the TGLA227, BM2113, TGLA53, ETH10, SPS115, RM011, TGLA122, INRA23, BMS4032, ETH225 and BM1824 by marker. Amplifying the selected allelic genotype by polymerase chain reaction (PCR); Cloning the amplified PCR product and confirming the repeated base sequence; And amplifying the plasmid DNA of the confirmed allelic form by a polymerase chain reaction using a fluorescent substance-labeled primer to obtain a PCR product for each marker and mixing the obtained PCR product To identify Hanwoo individuals. Primers specific for 11 supersatellite loci consisting of TGLA227, BM2113, TGLA53, ETH10, SPS115, RM011, TGLA122, INRA23, BMS4032, ETH225 and BM1824; A kennel identification kit containing an allelic ladder of supersatellite loci. [Claim 7] The kit according to claim 7, wherein the primer is selected from a primer set consisting of the nucleotide sequence shown in SEQ ID NOs: 1-22. delete 8. The method of claim 7, wherein the allelic ladder comprises alleles of the TGLA227, BM2113, TGLA53, ETH10, SPS115, RM011, TGLA122, INRA23, BMS4032, ETH225 and BM1824 by marker. Amplifying the selected allelic genotype by polymerase chain reaction (PCR); Cloning the amplified PCR product and confirming the repeated base sequence; And amplifying the plasmid DNA of the confirmed allelic form by a polymerase chain reaction using a fluorescent substance-labeled primer to obtain a PCR product for each marker and mixing the obtained PCR product A kit for identifying a Hanwoo body.

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