KR101535925B1 - Microsatellite markers for identification of goats - Google Patents

Abstract

The present invention relates to a composition for identifying a chlorine object comprising all of the primer pairs specific for each of the microsatellite markers OarFCB048, OarFCB193, SRCRSP008, INRA063, BOBT24A, SRCRSP001 and DRBP1, Kits. And a method for identifying a chlorine object using the composition.

Description

{Microsatellite markers for identification of goat species}

The present invention relates to a composition for identifying a chlorine object comprising all of the primer pairs specific for each of the microsatellite markers OarFCB048, OarFCB193, SRCRSP008, INRA063, BOBT24A, SRCRSP001 and DRBP1, Kits. And a method for identifying a chlorine object using the composition.

Chlorine is believed to be the first animal to be domesticated by humans. It is one of the most important livestock for humankind and provides humans with a wide range of products, including meat, milk, leather, hair, bone and fuel. In Korea, chlorine was mainly used as a medicinal product rather than common meat, and it was used as a health supplement food mainly for boiling. In recent years, however, the consumption of chlorine has been on an increasing trend, due to the development of the food service industry and the increasing awareness of well-being and health food. However, it is not an exaggeration to say that the current breeding chlorine is almost hybrids as hybridization with exotic species has been made to improve the low productivity of conventional chlorine, such as slow growth and poor growth of the acid (Kim et al., (2002) J. Anim Sci. &Amp; Technol (Kor) 44: 171-180). Fortunately, the National Livestock Research Institute of the Rural Development Administration has collected and preserved conventional chlorine in the early 1990s to preserve pure chlorine in consideration of its external characteristics.

In addition, in recent years, as the importance of future utilization value of genetic resources has been recognized, much attention and efforts have been devoted to securing, preserving and managing resources as well as characterization and utilization of resources. In particular, molecular genetic characterization has been actively carried out to identify the genetic characteristics of resources, including genetic markers based on DNA polymorphism, genetic diversity, genetic diversity, and relationships with other cultivars (Groeneveld et al., (2010) Anim.Genet . 41: 6-31).

Microsatellite (MS) represents a repeating structure of 2 to 6 nucleotides and is widely spread in noncoding regions on eukaryotic DNA (Tautz and Renz, (1984) Nucleic Acids Res . 12: 4127-4138). In addition, polymorphism information is abundant because it represents about 10 alleles per locus in a supersampled satellite. Because of its small size, it can be easily amplified using DNA extracted from various samples such as blood, root hair, and skin. In particular, since multiplex PCR method capable of reacting and amplifying two or more supersatellite specific primers at once can be applied, it is advantageous to easily analyze alleles at low cost (Butler, 2007 Biotechniques 43: Sii-Sv).

Since the 1990s, it has been utilized as the most effective genetic marker in the analysis of genetic characteristics and relationships of animals including livestock, and related mapping (Naidoo and Chetty, (1998) Pathol. Oncol Res . 4: 310-315). Gutierrez-Gil et al. (2010) Meat Sci . 85: 721-729; Costa et al., (1996) and others, have also been used to identify genetic diversity, quantitative gene locus identification, (2012) BMC Res. Notes . 5: 479). In addition, various academic and industrial achievements have been derived through the analysis of supersatellites in Korea, along with the development of Hanwoo, importation discrimination law, and beef history management system.

Under these circumstances, the present inventors have made intensive efforts to identify the locus of supersatellite gene which can effectively identify chlorine species. As a result, it has been found that a total of seven supersatellites including OarFCB048, OarFCB193, SRCRSP008, INRA063, BOBT24A, SRCRSP001 and DRBP1, It was confirmed that chlorine being kept in Korea can be identified without the possibility of occurrence of the same species, and the present invention has been completed.

One object of the present invention is to provide a composition for identifying chlorine individuals, which comprises all of the primer pairs specific for each of the microsatellite markers OarFCB048, OarFCB193, SRCRSP008, INRA063, BOBT24A, SRCRSP001 and DRBP1.

Another object of the present invention is to provide a kit for identifying a chlorine object comprising the composition for identifying chlorine.

It is a further object of the present invention to provide a method for detecting a chromosome in a biological sample by using all the primer pairs specific to each of the template DNA of the biological sample isolated from the chlorine object to be analyzed and the supersatellite markers consisting of OarFCB048, OarFCB193, SRCRSP008, INRA063, BOBT24A, SRCRSP001 and DRBP1 PCR amplification of the chlorinomic acid.

In one aspect to achieve the above object, the present invention provides a method for identifying chlorine species, comprising all primer pairs specific for each of the microsatellite markers OarFCB048, OarFCB193, SRCRSP008, INRA063, BOBT24A, SRCRSP001 and DRBP1, ≪ / RTI >

The term "microsatellite " in the present invention means a simple sequence repeat (SSR) having a structure in which 2 to 6 nucleotide sequences are repeated. These supersatellites are known to be uniformly distributed in most eukaryotic genomes, and are mainly present in non-coding DNA.

The term "supersatellite marker" in the present specification means a polymorphism that occurs due to a mutation in the number of repetitions of the supersatellite repeat unit.

In the present invention, a set of supersatellite markers capable of identifying individual chlorine without the possibility of occurrence of the same species has been identified in consideration of the heterozygosity ( He ) and amplification product size. The supersatellite marker set of the present invention includes OarFCB048, OarFCB193, SRCRSP008, INRA063, BOBT24A, SRCRSP001, and DRBP1, and information about the supersatellite can be obtained from the NCBI database or the United Nations Food and Agriculture Organization http://www.fao.org/) to obtain the information.

Since the number of repetition units of supersatellite repeating units may vary depending on individuals or species, such a supersatellite marker is designed as a primer sequence and PCR amplification is carried out by designating a sequence unique to the genome among the base pairs of repeated portions of supersatellite , The size of the resulting product can be compared to identify the individual or species.

The term "primer " as used herein refers to a nucleic acid sequence having a short free 3 'terminal hydroxyl group, a short nucleic acid sequence capable of forming base pairs with a complementary template and serving as a starting point for template strand copy . In the present invention, the primer pair for OarFCB048 is a forward primer of SEQ ID NO: 1 and the reverse primer pair of SEQ ID NO: 2, the primer pair for OarFCB193 is a forward primer of SEQ ID NO: 3 and the reverse primer pair of SEQ ID NO: 4, The reverse primer of SEQ ID NO: 5 and the reverse primer of SEQ ID NO: 6, the primer pair of INRA63 is the forward primer of SEQ ID NO: 7, the reverse primer pair of SEQ ID NO: 8, the primer pair of BOBT24A is the forward primer of SEQ ID NO: The reverse primer pair of SEQ ID NO: 10, the primer pair of SRCRSP1, the forward primer of SEQ ID NO: 11, the reverse primer pair of SEQ ID NO: 12, and the primer pair to DRBP1 are the forward primer of SEQ ID NO: 13 and the reverse primer pair of SEQ ID NO: But is not limited to this, Any primer capable of specifically binding to a primer to produce an amplification product is included in the scope of the present invention.

The primers of the present invention can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i.e., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures. The primer of the present invention may be a sense and an antisense nucleic acid having 7 to 50 nucleotide sequences as a primer specific to each marker gene, and the primer may be an addition or deletion primer which does not change the basic properties of the primer serving as a starting point of DNA synthesis And the like.

Such primers of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using many means known in the art. Non-limiting examples of such modifications include, but are not limited to, methylation, capping, substitution of one or more natural nucleotides with one or more homologues, and modification between nucleotides, such as uncharged linkers (e.g., methylphosphonate, phosphotriester, (E.g., dodecanedioic acid, dodecanedioic acid, dodecanedioic acid, tartaric acid, tartaric acid, tartaric acid, The nucleic acid can be in the form of one or more additional covalently linked residues such as a protein such as a nuclease, a toxin, an antibody, a signal peptide, a poly-L-lysine, an intercalator such as acridine, ), Chelating agents (e.g., metals, radioactive metals, iron, oxidizing metals, etc.), and alkylating agents. In addition, the nucleic acid sequences of the present invention can also be modified using labels that can directly or indirectly provide a detectable signal. Examples of labels include radioactive isotopes, fluorescent molecules, and biotin.

The term "PCR (polymerase chain reaction)" in the present invention is a molecular biological technique for replicating and destroying a desired portion of DNA. Specifically, PCR involves a series of three steps. The first step in PCR is denaturation of the DNA, which can be separated by heating two strands of DNA. Each separated DNA serves as a template. At this time, the denaturation temperature depends on the amount and length of G + C in the DNA. The second step in PCR is annealing, in which primers for the supersatellite marker bind to the template DNA. Annealing temperature is an important factor in determining the accuracy of the reaction. If the temperature is too high, the primer will bind too weakly to the template DNA, resulting in very little amplified DNA product. If the temperature is too low, undesired DNA can be amplified because the primer binds nonspecifically, and the temperature should be appropriately controlled depending on the kind of primers used. The third step in the PCR is the elongation step. At this stage, when the primer binds to the template, a heat-resistant DNA polymerase will make a new DNA from the template DNA.

Multiplex PCR may be used when analysis is performed on several supersatellite markers as in the present invention.

In the present invention, the term "multiplex PCR" means a PCR technique in which multiple genes are simultaneously amplified, unlike a single PCR that amplifies one gene for one template DNA. In multiplex PCR, it is desirable to include multiple pairs of primers in a single PCR mixture, each of which has a size range of amplification products specific for the different DNA sequences so that the sizes do not overlap with each other. In multiplex PCR, multiple primer pairs are inserted into one tube and reacted, so that inhibition may occur between primers. Therefore, when applying to multiplex PCR, selection of primers for the genes to be amplified is important Do. In addition, since the appropriate binding temperature may be different for each of the included primers, it is necessary to optimize the binding temperature suitable for multiplex PCR so that all pairs of PCR primers included in a single PCR reaction can be effectively combined when multiplex PCR is applied.

After such PCR, the PCR amplification product can be identified by a technique that can be classified according to the size of the PCR amplification product, such as SDS-PAGE or capillary electrophoresis.

Specifically, it is possible to determine how many repeating units are contained in each allele by classifying the PCR amplification products by size and determining their sizes by electrophoresis or the like. Therefore, the genotype data and the database for recording the data of the chlorine thus verified can be separately constructed or the identified data can be compared with each other by comparing the data and the experimentally confirmed data. In particular, in the case of using the seven satellites markers of the present invention, the probability of occurrence of the same individuals was 1.88 × 10 ^-16 in the random mating group and 9.38 × 10 ^-12 in the half mating group, , And then collecting all the information about the seven super satellites' markers, it is possible to identify the object with high accuracy.

In the example of the present invention, the combination of supersatellite markers that can be effectively used for identifying individual chlorine was identified (Example 4), considering the heterozygosity ratio and the amplification product size, and a primer pair SEQ ID NOs: 1 and 2; OarFCB193: SEQ ID NOs: 3 and 4; SRCRSP8: SEQ ID NOs: 5 and 6; INRA63: SEQ ID NOs: 7 and 8; BOBT24A: SEQ ID NOs: 9 and 10; SRCRSP1: SEQ ID NOs: 11 and 12; DRBP1 : SEQ ID NOS: 13 and 14), it was possible to effectively amplify the supersatellite markers (Fig. 1). In addition, the seven species of supersatellite markers showed 1.88 × 10 ^-16 in the random mating group and 9.38 × 10 ^-12 in the half mating group, It was judged that there was no possibility of applying for the same individual when considering the in-point (Example 6).

In another aspect, the present invention provides a kit for identifying a chlorine object, comprising the composition.

The composition and chlorine individual identification are as described above.

The goat species identification kit may be in the form of a supersatellite PCR kit, preferably a multiplex PCR kit. The PCR kit includes a primer pair, a DNA polymerase, a deoxynucleotide (dNTPs), an RNAase, a DNA restriction enzyme, a DEPC-water, a test tube and a suitable container for a polynucleotide including the marker . ≪ / RTI > It may also contain a primer pair specific for the gene used as a quantitative control.

In another embodiment, the present invention is characterized in that the primer pair specific to each of the template DNA of the biological sample isolated from the chlorine target to be analyzed and the supersatellite markers consisting of OarFCB048, OarFCB193, SRCRSP008, INRA063, BOBT24A, SRCRSP001 and DRBP1 And then performing PCR amplification using the PCR product.

The supersatellite marker, primer and PCR are as described above.

The term "biological sample" in the present invention means a sample containing DNA of chlorine capable of amplifying supersatellite markers of the present invention. Such biological samples include, but are not limited to, roots of chlorine, blood, various body fluids, isolated tissues, isolated cells or saliva-like samples, and the like.

The method of the present invention can be carried out by mixing primer pairs of seven kinds of supersatellite markers of the present invention with template DNA derived from the above-mentioned chlorine entities, followed by PCR amplification.

Here, the PCR amplification process amplifies a specific site by repeating denaturation, binding, and elongation steps. In this case, it is more preferable to perform multiplex PCR in the method of the present invention. In this case, since a plurality of primer pairs are included in a single tube, it is necessary to optimize the binding temperature.

After performing the PCR, the size of the amplified product can be analyzed by electrophoresis, and the allele can be determined based on the size of the amplified product.

The seven species of supersatellite markers of the present invention have the same probability of occurrence of 1.88 × 10 ^-16 in the random mating group and 9.38 × 10 ^-12 in the half mating group, It can be useful for identifying chlorine objects.

Fig. 1 shows the results of electrophoresis of seven supersatellite markers selected based on the heterozygosity ( He ).

Hereinafter, the present invention will be described in detail. However, the following examples are for illustrating the present invention, and the scope of the present invention is not limited by the following examples.

Example 1: Disclosed animal and DNA

The blood of goat was collected from 380 traditional goat breeders and 40 outgoing goat varieties (Jannen, Sablejannen, Alpine, Boer, Nubian, Togenbug) 420 individuals were used. DNA extraction from goat blood was performed using MagExtractor (TOYOBO, Japan).

Example 2: Super-satellites collection and primer production

A total of 30 species, such as 14 species recommended by the FAO and 16 species through the National Center for Biological Information (NCBI), were collected to select the supersatellite markers that can effectively be used to identify goat species. Specific primers were prepared from Applied Biosystems (USA). During the manufacturing process, only one of the pair of primers was labeled with a fluorescent substance, and the fluorescent materials used in the labeling were FAM, VIC, NED, and PET. The name of the supersatellite, the labeled fluorescent substance and the base sequence of the primer are as shown in Table 1 below.

Primary selectivity satellites marker for chlorine polymorphism analysis and specific primers Super satellites chromosome Fluorescent marker Primer sequences (5 ' - > 3 ') SEQ ID NO: OarFCB48 17 (quantity) PET F GACTCTAGAGGATCGCAAAGAACCAG One R GAGTTAGTACAAGGATGACAAGAGGCAC 2 OarFCB193 11 (quantity) FAM F TTCATCTCAGACTGGGATTCAGAAAGGC 3 R GCTTGGAAATAACCCTCCTGCATCCC 4 SRCRSP008 Unknown NED F TGCGGTCTGGTTCTGATTTCAC 5 R CCTGCATGAGAAAGTCGATGCTTAG 6 INRA063 18 (small), 14 (positive) FAM F ATTTGCACAAGCTAAATCTAACC 7 R AAACCACAGAAATGCTTGGAAG 8 BOBT24A none FAM F GAGCAAGGGAATTCAGTGGAGC 9 R TGTATTTTACATTCAGGTCTGTGATCC 10 SRCRSP001 1 or 13 (quantity) NED F TGCAAGAAGTTTTTCCAGAGC 11 R ACCCTGGTTTCACAAAAGG 12 DRBP1 23 (small), 20 (positive) PET F ATGGTGCAGCAGCAAGGTGAGCA 13 R GGGACTCAGTCTCTCTATCTCTTTG 14 RM006 7 (small), 5 (positive) FAM F CTACAATATCTGGTCACTGGA 15 R GATCACCATATTTATGAGATGG 16 INRA172 26 (small) NED F CCAGGGCAGTAAAATGCATAACTG 17 R GGCCTTGCTAGCCTCTGCAAAC 18 ILSTS008 14 (small), 9 (positive) VIC F GAATCATGGATTTTCTGGGG 19 R TAGCAGTGAGTGAGGTTGGC 20 INRA006 3 (small), 1 (positive) NED F AGGAATATCTGTATCAACCTCAGTC 21 R CTGAGCTGGGGTGGGAGCTATAAATA 22 INRA49 Unknown VIC F TGTATTAGTTTGTGTTCTTTGGC 23 R TTGGCTTCCACAATCACACA 24 BM1258 23 (small), 20 (positive) FAM F GTATGTATTTTTCCCACCCTGC 25 R GAGTCAGACATGACTGAGCCTG 26 BM1329 6 (small), 6 (positive) NED F TTGTTTAGGCAAGTCCAAAGTC 27 R AACACCGCAGCTTCATCC 28 CSRD0247 14 (quantity) VIC F GGACTTGCCAGAACTCTGCAAT 29 R CACTGTGGTTTGTATTAGTCAGG 30 SRCRSP005 18 (quantity) NED F GGACTCTACCAACTGAGCTACAAG 31 R TGAAATGAAGCTAAAGCAATGC 32 ILST087 Unknown FAM F AGCAGACATGATGACTCAGC 33 R CTGCCTCTTTTCTTGAGAG 34 BM1818 23 (small), 20 (positive) VIC F AGCTGGGAATATAACCAAAGG 35 R AGTGCTTTCAAGGTCCATGC 36 ETH10 5 (small), 3 (positive) FAM F GTTCAGGACTGGCCCTGCTAACA 37 R CCTCCAGCCCACTTTCTCTTCTC 38 TGLA53 16 (small), 12 (positive) PET F CAGCAGACAGCTGCAAGAGTTAGC 39 R CTTTCAGAAATAGTTTGCATTCATGCAG 40 OarFCB020 2 (quantity) NED F GGAAAACCCCCATATATACCTATAC 41 R AAATGTGTTTAAGATTCCATACATGTG 42 SRCRSP024 2 (quantity) FAM F AGCAAGAAGTGTCCACTGACAG 43 R TCTAGGTCCATCTGTTGTTATTGC 44 ILSTS029 3 (small), 1 (positive) PET F TGTTTTGATGGAACACAGCC 45 R TGGATTTAGACCAGGGTTGG 46 SRCRSP023 Unknown FAM F TGAACGGGTAAAGATGTG 47 R TGTTTTTAATGGCTGAGTAG 48 ILST019 Unknown VIC F AGGGACCTCATGTAGAAGC 49 R ACTTTTGGACCCTGTAGTGC 50 INRA023 3 (small) NED F GAGTAGAGC6TACAAGATAAACTTC 51 R TAACTACAGGGTGTTAGATGAACT 52 INRA185 18 (small) PET F TTACTGGGGAGACATACCAAGC 53 R CCCACACTAGGGAACCCC 54 INRA005 10 (quantity) VIC F TTCAGGCATACCCTACACCACATG 55 R AAATATTAGCCAACTGAAAACTGGG 56 BM6444 2 (small), 2 (positive) PET F CTCTGGGTACAACACTGAGTCC 57 R TAGAGAGTTTCCCTGTCCATCC 58 MCM0527 5 (quantity) VIC F GTCCATTGCCTCAAATCAATTC 59 R AAACCACTTGACTACTCCCCAA 60

Example  3: PCR  Amplification and electrophoresis

The PCR reaction was carried out using AmpliTaq Gold 360 Master Mix (Applied Biosystems, USA), 1 μl of template DNA (10 ng), 1 μl of primer mixture and distilled water to make a final volume of 20 μl. The PCR conditions were pre-denaturation at 95 ° C for 15 minutes, followed by 35 cycles of 95 ° C for 30 seconds, 55 ° C to 60 ° C for 90 seconds, and 72 ° C for 90 seconds. And then terminated at 72 ° C for 40 minutes and then at 8 ° C. To fractionate the PCR products by size, Hi-Di Formamide and GeneScan ™ 500 LIZ Size Standard (Applied Biosystems, USA) were diluted 1: 9, denatured at 95 ° C for 10 minutes, and then analyzed with ABI PRISM 3130XL Genetic Analyzer (Applied Biosystems, USA ) Was used for electrophoresis. After the electrophoresis, amplification size and marker type of each locus of each supersatellite were confirmed using GeneMapper version 4.0 software (Applied Biosystems, USA).

Example 4: Diversity index analysis of supersatellite

( He , Heterozygosity) and Polymorphic Information Content ( PIC ) were calculated using Cervus version 2.0 (Marshall et al (1998) Mol. Ecol . 7: 639-655), and the results are shown in Table 2.

Position Number of alleles N Size range (bp) He PIC SRCRSP001 14 409 131-137 0.682 0.628 SRCRSP008 13 393 211-245 0.766 0.739 OarFCB193 12 391 114-134 0.794 0.770 BOBT24A 11 412 140 ~ 168 0.652 0.626 INRA063 6 410 171 ~ 189 0.694 0.644 OarFCB48 10 406 147-171 0.798 0.767 DRBP1 11 400 113 ~ 139 0.586 0.543 BM1258 13 407 98 to 128 0.754 0.715 BM1329 9 399 166 ~ 182 0.751 0.713 BM1818 10 405 254 to 268 0.349 0.336 BM6444 25 386 128-184 0.876 0.863 CSRD0247 9 403 221-245 0.654 0.614 ETH10 3 402 205 ~ 209 0.226 0.212 ILSTS008 7 419 172 ~ 184 0.496 0.466 ILSTS019 8 405 140 ~ 156 0.620 0.548 ILSTS029 10 416 151-185 0.688 0.641 ILSTS087 10 400 136 ~ 158 0.745 0.716 INRA005 5 408 161 to 181 0.452 0.403 INRA006 11 415 105 ~ 125 0.693 0.642 INRA023 15 410 81 ~ 107 0.755 0.728 INRA049 8 412 138 ~ 156 0.566 0.475 INRA172 8 419 139 ~ 153 0.731 0.698 INRA185 5 411 159-175 0.385 0.355 MCM0527 7 395 154-170 0.627 0.568 OarFCB020 9 401 93 to 107 0.552 0.481 RM006 14 414 104 to 146 0.658 0.631 SRCRSP005 11 401 161 to 181 0.659 0.628 SRCRSP023 11 405 81 ~ 107 0.800 0.774 SRCRSP024 13 412 142-170 0.609 0.532 TGLA53 7 415 114-130 0.639 0.578

The inventors of the present invention found that among the super satellites exhibiting the above results, seven of the 30 super satellites (OarFCB048, OarFCB193, SRCRSP008, INRA063, BOBT24A) were selected in consideration of the order of He, the size of the amplification product, , SRCRSP001, DRBP1) were selected.

Example 5: Multiplex PCR

In Example 4, a primer pair specific for seven selected satellites (OarFCB048, OarFCB193, SRCRSP008, INRA063, BOBT24A, SRCRSP001, DRBP1) selected in consideration of He value, amplification product size, Were used to perform multiplex PCR. The PCR reaction was carried out using AmpliTaq Gold 360 Master Mix (Applied Biosystems, USA), 1 μl of template DNA (10 ng), 6.8 μl of primer mixture, and distilled water to make a final volume of 20 μl. The PCR conditions were pre-denaturation at 95 ° C for 15 minutes, followed by 35 cycles of 95 ° C for 30 seconds, 56 ° C for 90 seconds, and 72 ° C for 90 seconds. And then terminated at 72 ° C for 40 minutes and then at 8 ° C. The PCR products were diluted 1: 9 with Hi-Di Formamide and GeneScan ™ 500 LIZ Size Standard (Applied Biosystems, USA), and electrophoresis was performed using ABI PRISM 3130XL Genetic Analyzer (Applied Biosystems, USA) Respectively. After the electrophoresis, amplification size and marker type of each locus of each supersatellite were identified using GeneMapper version 4.0 software (Applied Biosystems, USA), and the results are shown in FIG.

Example 6: Statistical analysis

Weir and Hill (2002, Annu. Rev. Genet. 36: 721-750) Estimated F-statistics (Fst, Fit, Fis) are available from FSTAT version 2.9.3 (Goudet (2001) Available from http: // www2. unil.ch/popgen/softwares/fstat.htm). Based on the estimated Fst and Fis values, the probability of identification (PI) and the probability of identification of half-sibs (PIhalf-sibs) CALC version 1.0 (Ayres and Overall (2004) Molecular Ecology Notes 4: 315-318). The results are shown in Table 3 below.

Selected by Random mating group ( PI ) PI _half-sibs ( PI _half-sibs ) Typical mating group ( PI _sibs ) Total 1.88 × 10 ^-16 9.38 × 10 ^-12 7.14 x 10 ^-5

As a result, the probability of occurrence of the same species was 1.88 × 10 ^-16 in the random mating group and 9.38 × 10 ^-12 in the semi-sibling mating group when the final selected seven satellites marker was used based on the He value. Since the number of chlorine in Korea is currently more than 250,000 (Ministry of Food, Agriculture, Forestry and Livestock, and other livestock statistics in 2011), these estimates were not considered to be the same.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Microsatellite markers for identification of goats <130> PA130987KR <160> 60 <170> Kopatentin 2.0 <210> 1 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Primer for OarFCB48 (forward) <400> 1 gactctagag gatcgcaaag aaccag 26 <210> 2 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Primer for OarFCB48 (reverse) <400> 2 gagttagtac aaggatgaca agaggcac 28 <210> 3 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Primer for OarFCB193 (forward) <400> 3 ttcatctcag actgggattc agaaaggc 28 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Primer for OarFCB193 (reverse) <400> 4 gcttggaaat aaccctcctg catccc 26 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer for SRCRSP008 (Forward) <400> 5 tgcggtctgg ttctgatttc ac 22 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer for SRCRSP008 (reverse) <400> 6 cctgcatgag aaagtcgatg cttag 25 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer for INRA063 (forward) <400> 7 atttgcacaa gctaaatcta acc 23 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer for INRA063 (reverse) <400> 8 aaaccacaga aatgcttgga ag 22 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer for BOBT24A (forward) <400> 9 gagcaaggga attcagtgga gc 22 <210> 10 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer for BOBT24A (reverse) <400> 10 tgtattttac attcaggtct gtgatcc 27 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer for SRCRSP001 (forward) <400> 11 tgcaagaagt ttttccagag c 21 <210> 12 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer for SRCRSP001 (reverse) <400> 12 accctggttt cacaaaagg 19 <210> 13 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer for DRBP1 (forward) <400> 13 atggtgcagc agcaaggtga gca 23 <210> 14 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer for DRBP1 (reverse) <400> 14 gggactcagt ctctctatct ctttg 25 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer for RM006 (forward) <400> 15 ctacaatatc tggtcactgg a 21 <210> 16 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer for RM006 (reverse) <400> 16 gatcaccata tttatgagat gg 22 <210> 17 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer for INRA172 (forward) <400> 17 ccagggcagt aaaatgcata actg 24 <210> 18 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer for INRA172 (reverse) <400> 18 ggccttgcta gcctctgcaa ac 22 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for ILSTS008 (forward) <400> 19 gaatcatgga ttttctgggg 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for ILSTS008 (reverse) <400> 20 tagcagtgag tgaggttggc 20 <210> 21 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer for INRA006 (forward) <400> 21 aggaatatct gtatcaacct cagtc 25 <210> 22 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Primer for INRA006 (reverse) <400> 22 ctgagctggg gtgggagcta taaata 26 <210> 23 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer for INRA49 (forward) <400> 23 tgtattagtt tgtgttcttt ggc 23 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for INRA49 (reverse) <400> 24 ttggcttcca caatcacaca 20 <210> 25 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer for BM1258 (forward) <400> 25 gtatgtattt ttcccaccct gc 22 <210> 26 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer for BM1258 (reverse) <400> 26 gagtcagaca tgactgagcc tg 22 <210> 27 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer for BM1329 (forward) <400> 27 ttgtttaggc aagtccaaag tc 22 <210> 28 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer for BM1329 (reverse) <400> 28 aacaccgcag cttcatcc 18 <210> 29 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer for CSRD0247 (forward) <400> 29 ggacttgcca gaactctgca at 22 <210> 30 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer for CSRD0247 (reverse) <400> 30 cactgtggtt tgtattagtc agg 23 <210> 31 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer for SRCRSP005 (forward) <400> 31 ggactctacc aactgagcta caag 24 <210> 32 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer for SRCRSP005 (reverse) <400> 32 tgaaatgaag ctaaagcaat gc 22 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for ILST087 (forward) <400> 33 agcagacatg atgactcagc 20 <210> 34 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer for ILST087 (reverse) <400> 34 ctgcctcttt tcttgagag 19 <210> 35 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer for BM1818 (forward) <400> 35 agctgggaat ataaccaaag g 21 <210> 36 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for BM1818 (reverse) <400> 36 agtgctttca aggtccatgc 20 <210> 37 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer for ETH10 (forward) <400> 37 gttcaggact ggccctgcta aca 23 <210> 38 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer for ETH10 (reverse) <400> 38 cctccagccc actttctctt ctc 23 <210> 39 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer for TGLA53 (forward) <400> 39 cagcagacag ctgcaagagt tagc 24 <210> 40 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Primer for TGLA53 (reverse) <400> 40 ctttcagaaa tagtttgcat tcatgcag 28 <210> 41 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer for OarFCB020 (forward) <400> 41 ggaaaacccc catatatacc tatac 25 <210> 42 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer for OarFCB020 (reverse) <400> 42 aaatgtgttt aagattccat acatgtg 27 <210> 43 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer for SRCRSP024 (forward) <400> 43 agcaagaagt gtccactgac ag 22 <210> 44 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer for SRCRSP024 (reverse) <400> 44 tctaggtcca tctgttgtta ttgc 24 <210> 45 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for ILSTS029 (forward) <400> 45 tgttttgatg gaacacagcc 20 <210> 46 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for ILSTS029 (reverse) <400> 46 tggatttaga ccagggttgg 20 <210> 47 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer for SRCRSP023 (forward) <400> 47 tgaacgggta aagatgtg 18 <210> 48 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for SRCRSP023 (reverse) <400> 48 tgtttttaat ggctgagtag 20 <210> 49 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer for ILST019 (forward) <400> 49 agggacctca tgtagaagc 19 <210> 50 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for ILST019 (reverse) <400> 50 acttttggac cctgtagtgc 20 <210> 51 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer for INRA023 (forward) <400> 51 gagtagagct acaagataaa cttc 24 <210> 52 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer for INRA023 (reverse) <400> 52 taactacagg gtgttagatg aact 24 <210> 53 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer for INRA185 (forward) <400> 53 ttactgggga gacataccaa gc 22 <210> 54 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer for INRA185 (reverse) <400> 54 cccacactag ggaacccc 18 <210> 55 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer for INRA005 (forward) <400> 55 ttcaggcata ccctacacca catg 24 <210> 56 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer for INRA005 (reverse) <400> 56 aaatattagc caactgaaaa ctggg 25 <210> 57 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer for BM6444 (forward) <400> 57 ctctgggtac aacactgagt cc 22 <210> 58 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer for BM6444 (reverse) <400> 58 tagagagttt ccctgtccat cc 22 <210> 59 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer for MCM0527 (forward) <400> 59 gtccattgcc tcaaatcaat tc 22 <210> 60 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer for MCM0527 (reverse) <400> 60 aaaccacttg actactcccc aa 22

Claims (6)

delete A forward primer of SEQ ID NO: 1 and a reverse primer pair of SEQ ID NO: 2, a forward primer of SEQ ID NO: 3 which is a primer pair to OarFCB193, a reverse primer pair of SEQ ID NO: 4, a SRCRSP008 And a reverse primer pair of SEQ ID NO: 6, a forward primer of SEQ ID NO: 7 and a reverse primer pair of SEQ ID NO: 8, which are primer pairs for INRA063 and SEQ ID NO: 9, respectively, which are primer pairs for BOBT24A A forward primer of SEQ ID NO: 10, a forward primer of SEQ ID NO: 11, a reverse primer pair of SEQ ID NO: 12 and a forward primer of SEQ ID NO: 13, which is a primer pair to DRBP1, and a forward primer of SEQ ID NO: A pair of reverse primers, Honesty.
A kit for identifying chlorine objects, comprising the composition of claim 2.
A forward primer of SEQ ID NO: 1 and a reverse primer pair of SEQ ID NO: 2, which are primer pairs for OarFCB048 which is a microsatellite marker, and a primer pair of SEQ ID NO: 2, which is a primer pair for OarFCB193, 3 and the reverse primer pair of SEQ ID NO: 4, the forward primer of SEQ ID NO: 5 and the reverse primer pair of SEQ ID NO: 6, the forward primer of SEQ ID NO: 7 and the primer pair of SEQ ID NO: The forward primer of SEQ ID NO: 9 and the reverse primer pair of SEQ ID NO: 10, the forward primer of SEQ ID NO: 11 being the primer pair to SRCRSP001, the reverse primer pair of SEQ ID NO: 12 and the reverse primer pair of DRBP1 Forward primer of SEQ ID NO: 13 and primer pair How to identify chlorine object comprises the step of PCR amplification using all the pairs of the reverse primer of the number 14.
5. The method of claim 4, wherein the PCR is multiplex PCR.
5. The method of claim 4, wherein the method further comprises analyzing the size of the amplified product to determine the genotype.

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