KR20190053704A - Method for identification of Baekwoo breed using single nucleotide polymorphism markers - Google Patents

Abstract

The present invention relates to an SNP marker capable of identifying a white cattle breed, a composition for identifying a white cattle breed including the SNP markers, a kit for identifying a white cattle breed including the composition, and a method for identifying a white cattle breed using the kit or the composition. By using the nine SNP markers of the present invention, it is possible to easily identify the white cattle breed, which can be utilized variously in the fields of agriculture and food production through the systematic and scientific establishment of livestock genetic resources.

Description

[Technical Field] The present invention relates to a method for identification of Baekwoo breeds using single nucleotide polymorphism markers,

More particularly, the present invention relates to SNP markers for identification of varieties of Baekwoo, a composition or kit containing the same, and a method for identifying Baekwu varieties using the composition or kit will be.

In the past, the search for Korean Hanwoo was diversified because it had various searches such as brown, black, white, semicolon, pale color and variegation. However, due to the unification and search unification of the title by the Japanese Government General in 1916, And the diversity of genetic resources was lost. Recently, the importance of diversity of genetic resources has been emphasized, and the restoration and propagation studies of white wax among native breeds have been progressing actively. Baekwoo has been separated from Hanwoo, a native species, This study will be a basic research for the enhancement of the value. However, the distinction of the varieties of Baekwoo is limited only by the expression of the qualitative trait called white, which is a unique search.

In addition, as the improvement of livestock around the world is proceeding in accordance with the preferences of each country, the genetic diversity of the varieties is decreasing rapidly and it is estimated that 20% of the animal varieties listed in the Food and Agriculture Organization (FAO) The breed is in danger of extinction. It is necessary to study the characteristics of systematic and scientific pedigree establishment because the conservation and management of genetic resources are carried out in preparation for the loss of these resources.

As such, in recent years, the importance of future utilization value of genetic resources has been recognized, and we are paying much attention and efforts to acquire, preserve and manage resources, and to evaluate and utilize the characteristics. 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).

Under these circumstances, the present inventors have made intensive researches to discover SNP markers that can effectively identify Baekwoo variety. As a result, it has been confirmed that Baekwoo variety can be identified when SNP markers specific to Baekwoo variety are used, .

It is an object of the present invention to provide a single nucleotide polymorphism (SNP) marker for identification of a white horse variety, comprising at least one polynucleotide comprising a single base polymorphism (SNP) present on the 29 th chromosome of bovine.

It is another object of the present invention to provide a composition for identifying a white wax variety, which comprises a preparation capable of detecting or amplifying the SNP markers for identifying the white wax variety.

It is still another object of the present invention to provide a kit for identifying a cattle breed including the above composition.

It is still another object of the present invention to provide a microarray for screening a varieties of Baekwoo including SNP markers for identification of Baekwoo variety.

It is still another object of the present invention to provide a method for amplifying SNP markers comprising the steps of: (a) amplifying polymorphic sites of SNP markers for hybridization of the Baekwoo variety from DNA of a sample isolated from an individual or hybridizing with probes; And (b) determining the base of the amplified or hybridized polymorphic site.

This will be described in detail as follows. On the other hand, each description and embodiment disclosed in the present invention can be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present invention fall within the scope of the present invention. Further, the scope of the present invention is not limited by the detailed description described below.

(A) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, wherein the 21st base is G or A, and 5 to 41 consecutive A polynucleotide consisting of a base; (b) a polynucleotide consisting of 5 to 41 consecutive bases, wherein the 21st base is G or A in the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2, and the 21st base; (c) a polynucleotide consisting of 5 to 41 consecutive bases, wherein the 21st base is G or A in the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3, and the 21st base; (d) a polynucleotide consisting of 5 to 41 consecutive bases, wherein the 21st base is G or A in the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4, and the 21st base; (e) a polynucleotide consisting of 5 to 41 consecutive bases, wherein the 21st base is G or A in the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 5, and the 21st base; (f) a polynucleotide consisting of 5 to 41 consecutive bases, wherein the 21st base is C or T in the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 6, and the 21st base; (g) a polynucleotide consisting of 5 to 41 consecutive bases, wherein the 21st base is A or C in the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 7, and the 21st base; (h) a polynucleotide consisting of 5 to 41 consecutive bases, wherein the 21st base is C or T in the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 8, and the 21st base; (i) a polynucleotide consisting of 5 to 41 consecutive bases, wherein the 21st base is G or A in the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 9, and the 21st base; And (j) at least one polynucleotide selected from the group consisting of polynucleotides complementary to any of the polynucleotides of (a) to (i), wherein the polynucleotide is a single nucleotide polymorphism do.

The SNP marker is a SNP marker containing a SNP located on the 29th chromosome of cattle, for identifying a cattle breed. The polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 may comprise rs42125585 which is the SNP, the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2 may comprise rs42125591 which is the SNP, The polynucleotide comprising the nucleotide sequence of SEQ ID NO: 4 may comprise rs42125833 which is the SNP, and the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4 may comprise the SNP rs109461720 and the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: The polynucleotide comprising the nucleotide sequence of SEQ ID NO: 6 may comprise rs109447299 which is the SNP, and the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 7 may comprise rs42164846 which is the SNP And the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 8 may comprise SNP rs42160000 Said polynucleotide comprising the nucleotide sequence of SEQ ID NO: 9 may comprise the SNP rs137353829.

The SNP markers for identifying the varieties of cattle may include a polynucleotide consisting of 5 to 41 consecutive bases comprising specifically the 21st base of one or more polynucleotides selected from the group consisting of SEQ ID NOS: And more specifically, a polynucleotide consisting of 5 to 21 consecutive bases. However, if the SNP site of the present invention is included, the number of consecutive bases contained in the SNP marker can be increased without limitation based on the 29th chromosomal sequence information of known bovines.

The term " Baekwoo variety " of the present invention is a mutant species of Korean native cattle having albinism, a pigment deficiency. In the natural state, the probability of birth of white birds is only about one millionth. The search for such white pearl is not based on white genes appearing in exotic varieties such as white pearl, but it is white according to the variation of yellow pear, which is our original varietal, Have.

The identification of the above varieties may be to identify varieties of domestic varieties of domestic varieties, such as Korean beef cattle, beef cattle, sheep cattle, sheep cattle, and white cattle, and may specifically identify cattle breeds of Hanwoo and Cheongwoo.

The term " SNP marker " of the present invention means a single base polymorphism allele base pair on the DNA sequence used to identify an individual or species. Because SNPs are relatively frequent and stable and distributed throughout the genome, and because of the genetic diversity of individuals, SNP markers can serve as indicators of genetic proximity among individuals. SNP markers generally involve phenotypic changes associated with single nucleotide polymorphisms but may or may not be. In the case of the SNP marker of the present invention, the amino acid sequence or the phenotype of the individual such as the varieties of Baekwoo can be different.

The term " polymorphism " of the present invention refers to a case where two or more alleles exist in one locus. Of the polymorphic sites, only a single base is different depending on the individual, Polymorphism. Preferred polymorphic markers have two or more alleles with a frequency of occurrence of 1% or more, more preferably 5% or 10% or more in the selected population.

The term " allele " of the present invention refers to various types of a gene existing on the same locus of a homologous chromosome. Alleles are also used to represent polymorphisms, for example, SNPs have two kinds of bialles.

According to one embodiment of the present invention, a total of 70 native Korean beef cattle, 22 Korean beef cattle and 48 Korean cattle, were analyzed to identify 9 SNPs that can accurately distinguish Hanwoo variety group and Baekwoo variety group , And it was confirmed that the Hanwoo and Baekwoo cultivars were distinctly distinguished through the DNA chip using the SNP (Table 2). Therefore, when the genotype of the SNP marker of the present invention is amplified and analyzed, and the genotype of the SNP marker is read, it is expected that the genus of Baekwoo can be selected. The SNP marker was first identified by the present inventors .

In another aspect of the present invention, there is provided a composition for identifying a Baekwoo variety, which comprises a preparation capable of detecting or amplifying SNP markers for identifying Baekwoo variety. Specifically, the identification of the Baekwoo variety may be to identify Baekwoo from the group consisting of Hanwoo and Baekwoo, but the present invention is not limited thereto.

The term " agent capable of detecting or amplifying a marker " of the present invention means a preparation capable of specifically recognizing SNPs contained in the SNP markers for screening of the Baekwoo varieties and amplifying the SNPs, May be a probe capable of specifically binding to a polymorphic site containing a SNP, a polynucleotide comprising the SNP marker, or a primer capable of specifically amplifying a complementary polynucleotide of the polynucleotide.

The term " probe " of the present invention refers to a nucleic acid fragment such as RNA or DNA corresponding to a few bases or a few hundred bases, which can be specifically bound to mRNA, and is labeled The presence or absence of a specific mRNA can be confirmed. The probe can be produced in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like.

In the present invention, a probe used to recognize and bind to a SNP marker includes a sequence complementary to a polynucleotide sequence including a SNP, and may be a DNA, RNA, or DNA-RNA hybrid form . Further, fluorescent markers, radiation markers, and the like can be additionally attached to the 5 'or 3' ends of the probe so as to be visually recognizable.

The term " primer " of the present invention means a base sequence having a short free 3 'hydroxyl group and can form base pairs with a complementary template, It means a short sequence functioning as a point. The primers used in the present invention for the amplification of SNP markers can be amplified by PCR using appropriate conditions in suitable buffers (for example, 4 different nucleoside triphosphates and polymerase such as DNA, RNA polymerase or reverse transcriptase) Stranded oligonucleotide that can serve as a starting point for the directed DNA synthesis. The appropriate length of the primer may vary depending on the purpose of use, but it may be generally used in a size of 15 to 30 nucleotides. The primer sequence is not necessarily completely complementary to the polynucleotide comprising the SNP marker or its complementary polynucleotide, and can be used if it is sufficiently complementary to hybridize.

The primers can also be modified, for example, by methylation, capping, substitution of nucleotides or modifications between nucleotides, such as uncharged linkers (e.g., methylphosphonate, phosphotriester, phosphoramidate, Carbamates, etc.) or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).

In another aspect of the present invention, there is provided a kit for identifying a cattle breed including the above composition. Specifically, the kit may be, but not limited to, a Reverse Transcription Polymerase Chain Reaction (RT-PCR) kit or a kit for DNA analysis (eg, a DNA chip).

The kit of the present invention can identify the Baekwoo variety by confirming the base of the SNP provided by the present invention using the above composition by amplification or by confirming the expression level of mRNA. As a specific example, the kit provided in the present invention may be a kit containing essential elements necessary for carrying out RT-PCR.

For example, in addition to the respective primer pairs specific for the SNPs, RT-PCR kits may also include test tubes or other appropriate containers, reaction buffers (varying in pH and magnesium concentration), deoxynucleotides (dNTPs) , Enzymes such as Taq polymerase and reverse transcriptase, DNase, RNAse inhibitors, DEPC-water, sterile water, and the like. It may also contain a primer pair specific for the gene used as a quantitative control.

As another example, the kit of the present invention may be a DNA chip kit for identification of a cattle breed including essential elements necessary for carrying out a DNA chip.

The term " DNA chip " of the present invention means one of DNA microarrays capable of confirming each base of hundreds of thousands of DNAs at a time.

The DNA chip kit is formed by attaching nucleic acid species to a glass surface, which is generally not larger than a flat solid support plate, typically a slide for a microscope, in a gridded array. The nucleic acid is uniformly arranged on the chip surface, It is a tool that enables multiple parallel hybridization reactions between the nucleic acid on the chip and the complementary nucleic acid contained in the treated solution on the chip surface.

According to still another aspect of the present invention, there is provided a microarray for identifying Baekwoo cultivar comprising SNP markers for identifying Baekwoo variety.

Specifically, the microarray comprises: (a) a polynucleotide consisting of 5 to 41 consecutive bases comprising the 21st base, wherein the 21st base is G or A in the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1; ; (b) a polynucleotide consisting of 5 to 41 consecutive bases, wherein the 21st base is G or A in the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2, and the 21st base; (c) a polynucleotide consisting of 5 to 41 consecutive bases, wherein the 21st base is G or A in the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3, and the 21st base; (d) a polynucleotide consisting of 5 to 41 consecutive bases, wherein the 21st base is G or A in the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4, and the 21st base; (e) a polynucleotide consisting of 5 to 41 consecutive bases, wherein the 21st base is G or A in the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 5, and the 21st base; (f) a polynucleotide consisting of 5 to 41 consecutive bases, wherein the 21st base is C or T in the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 6, and the 21st base; (g) a polynucleotide consisting of 5 to 41 consecutive bases, wherein the 21st base is A or C in the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 7, and the 21st base; (h) a polynucleotide consisting of 5 to 41 consecutive bases, wherein the 21st base is C or T in the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 8, and the 21st base; (i) a polynucleotide consisting of 5 to 41 consecutive bases, wherein the 21st base is G or A in the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 9, and the 21st base; And (j) a polynucleotide complementary to any one of the polynucleotides of (a) to (i).

The microarray may comprise DNA or RNA polynucleotides. The microarray comprises a conventional microarray except that the polynucleotide of the present invention is contained in the probe polynucleotide.

Methods for producing microarrays by immobilizing probe polynucleotides on a substrate are well known in the art. The probe polynucleotide means a polynucleotide capable of hybridizing, and means an oligonucleotide capable of binding to the complementary strand of the nucleic acid in a sequence-specific manner. The probe of the present invention is an allele-specific probe in which a polymorphic site exists in a nucleic acid fragment derived from two members of the same species and hybridizes to a DNA fragment derived from one member but does not hybridize to a fragment derived from another member . In this case, the hybridization conditions show a significant difference in the intensity of hybridization between alleles, and should be sufficiently stringent to hybridize to only one of the alleles. This can lead to good hybridization differences between different allelic forms. The probe of the present invention can be used for identification of alleles and identification of the allele. The determination method includes detection methods based on hybridization of nucleic acids such as Southern blot, and may be provided in a form pre-bonded to a substrate of a DNA chip in a method using a DNA chip. The hybridization can usually be performed under stringent conditions, for example, a salt concentration of 1 M or less and a temperature of 25 ° C or higher. For example, conditions of 5x SSPE (750 mM NaCl, 50 mM Na Phosphate, 5 mM EDTA, pH 7.4) and 25-30 < 0 > C may be suitable for allele-specific probe hybridization.

The process of immobilizing the probe polynucleotide associated with the Baekwoo breed identification of the present invention on a substrate can also be easily carried out using this conventional technique. In addition, hybridization of nucleic acids on a microarray and detection of hybridization results are well known in the art. The detection can be accomplished, for example, by labeling the nucleic acid sample with a labeling substance capable of generating a detectable signal comprising a fluorescent material, such as Cy3 and Cy5, and then hybridizing on the microarray and generating The hybridization result can be detected.

(A) amplifying or hybridizing a polymorphic site of a SNP marker of the present invention for identifying a Baekwoo cultivar of the present invention from DNA of a sample isolated from an individual; And (b) determining the base of the amplified or hybridized polymorphic site. At this time, DNA of the separated sample can be obtained from a sample isolated from an individual.

As described above, the SNP markers for identification of the cattle breeds provided in the present invention include respective SNPs contained in the polynucleotides of SEQ ID NOS: 1 to 9,

When the 21st nucleotide in the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 is A; A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2 has the 21st base of A; The polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3 has the 21st base of A; The polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4 has the 21st base of A; The polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 5 has the 21st base of A; The polynucleotide comprising the nucleotide sequence of SEQ ID NO: 6 has the 21st base T; The polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 7 is C; The polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 8 has the 21st base T; Or the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 9 is A at position 21, it can be determined that the polynucleotide is a Baekwoo variety.

Therefore, by determining the base of each SNP, it is possible to identify whether or not the individual including the SNP is a Baekwu variety.

The term " individual " of the present invention refers to native cattle which is a target to be identified as Baekwoo cultivar. By analyzing the genotype of the SNP marker using the sample obtained from the native cattle, it can be identified as Baekwu cultivar. Such samples include, but are not limited to, roots of native bovine, blood, various body fluids, isolated tissues, isolated cells or saliva-like samples, and the like. In one example, the sample may be blood of native cattle.

The step of amplifying the polynucleotide from the DNA sample of step (a) may be any method known to those skilled in the art. For example, the target nucleic acid can be obtained by PCR amplification and purification thereof. Other ligase chain reaction (LCR) (Wu and allace, Genomics 4, 560 (1989), Landegren et al., Science 241, 1077 (1988)), transcription amplification (Kwoh et al., Proc. Natl. Acad. USA, 86, 173 (1989)) and self-sustained sequence replication (Guatelli et al., Proc. Natl. Acad. Sci. USA 87, 874 (1990)) and nucleic acid based sequence amplification (NASBA).

Methods for determining the base of the amplified SNP marker region in step (b) of the above method include sequencing analysis, hybridization by microarray, allele specific PCR, dynamic allele specific hybridization PCR-SSCP, PCR-RFLP analysis, HRM analysis or TaqMan technique, SNPlex platform (Applied Biosystems), mass spectrometry (eg, Sequenom's MassARRAY system), mini-sequencing (mini-sequencing) Sequencing methods, Bio-Plex systems (BioRad), CEQ and SNPstream systems (Beckman), Molecular Inversion probe array techniques (e.g. Affymetrix GeneChip), and BeadArray Technologies (e.g. Illumina GoldenGate and Infinium analysis) But the present invention is not particularly limited thereto. One or more alleles in the SNP marker can be identified by the methods described above or other methods available to those skilled in the art to which the invention pertains. The base at such a mutation site can be determined preferably through a DNA chip.

The TaqMan method comprises the steps of (1) designing and preparing a primer and a TaqMan probe to amplify a desired DNA fragment; (2) labeling probes of different alleles with FAM dyes and VIC dyes (Applied Biosystems); (3) Using the DNA as a template

Performing PCR using the primer and the probe; (4) after completion of the PCR reaction, analyzing and confirming the TaqMan assay plate with a nucleic acid analyzer; And (5) determining the base of the polynucleotides of step (1) from the analysis results.

The sequencing analysis can be performed using a conventional method for nucleotide sequencing, and can be performed using an automated gene analyzer. In addition, allele-specific PCR means a PCR method in which a DNA fragment in which the mutation is located is amplified with a primer set including a primer designed with the base at the 3 'end at which the mutation site is located.

The principle of the above method is that, for example, when a specific base is substituted by A to G, an opposite primer capable of amplifying a primer containing the A as a 3 'terminal base and a DNA fragment of an appropriate size is designed, When the base at the mutation position is A, the amplification reaction is normally performed and a band at a desired position is observed. When the base is substituted with G, the primer can be complementarily bound to the template DNA, 3 'terminus does not perform complementary binding so that the amplification reaction can not be performed properly. DASH can be performed by a conventional method, preferably by a method such as Prince et al.

PCR extension analysis is performed by first amplifying a DNA fragment containing a base in which a single nucleotide polymorphism is located into a pair of primers, inactivating all the nucleotides added to the reaction by dephosphorylation, and adding a specific extension primer, dNTP mixture , Digoxinucleotide, reaction buffer, and DNA polymerase to perform primer extension reaction. At this time, the extension primer has the 3 'end immediately adjacent to the 5' direction of the base where the mutation site is located, and the nucleic acid having the same base as the didyoxynucleotide is excluded in the dNTP mixture, and the didyoxynucleotide has a mutation ≪ / RTI > For example, when dGTP, dCTP and TTP mixture and ddATP are added to the reaction in the presence of substitution from A to G, the primer is extended by the DNA polymerase in the base in which the substitution has occurred, The primer extension reaction is terminated by ddATP at the position where the base first appears. If the substitution has not occurred, the extension reaction is terminated at the position, so that it is possible to discriminate the kind of base showing the mutation by comparing the length of the extended primer.

At this time, as a detection method, when the extension primer or the didyxin nucleotide is fluorescently labeled, the mutation is detected by detecting fluorescence using a gene analyzer (for example, Model 3700 manufactured by ABI Co., Ltd.) used for general nucleotide sequence determination And when the unlabeled extension primer and the didyxin nucleotide are used, the genetic variation of the SNP can be detected by measuring the molecular weight using MALDITOF (matrix assisted laser desorption ionization-time of flight) technique.

By using the nine kinds of SNP markers of the present invention, it is possible to easily identify the varieties of Baekwoo, and it can be utilized variously in the fields of agriculture and food production through systematic and scientific establishment of the genetic resources of livestock.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. However, these examples and experimental examples are intended to illustrate the present invention, and the scope of the present invention is not limited to these examples and experimental examples.

Example  One: Traditional  Isolation of DNA from peripheral blood

In order to select single nucleotide polymorphism (SNP) markers for the selection of Baekwoo varieties, a total of 70 native bovine DNA were isolated from 22 Baekwoo varieties and 48 Hanwoo varieties.

Specifically, blood was collected from a total of 70 domestic cattle, 22 cattle and 48 Korean cattle. 300ul of collected blood and 900ul of cell lysis solution were mixed. The mixed sample was allowed to stand at room temperature for 10 minutes and centrifuged at 15000 g for 20 minutes. After centrifugation, the supernatant was discarded and 300 ul of Nuclei lysis solution was added to the pellet attached to the bottom. Then, 100 μl of Protein precipitation solution was added and mixed for 20 seconds, followed by centrifugation at 15000 g for 3 minutes. After centrifugation, the resulting supernatant was transferred to a new tube and mixed with 300 μl of isopropanol. The tube was centrifuged at 15,000 g for 1 minute, and the resulting supernatant was discarded and 300 ul of 70% ethanol was added thereto, followed by centrifugation at 15,000 g for 1 minute.

After centrifugation, the ethanol was removed, and the pellet that had settled on the bottom was naturally dried. After drying, 30ul of tertiary distilled water was added and the DNA was stored in a constant temperature water bath at 65 ° C for 1 hour and stored at -20 ° C.

Example  2: Sequence analysis using Bovine HD SNP chip

DNA of Baekwoo and Hanwoo were analyzed in order to analyze the nucleotide sequences of SNP markers existing only in Baekwoo cultivars.

Specifically, SNP genotyping of the DNA of Hanwoo and Baekwoo obtained in Example 1 was performed using Illumina Bovine HD 777K SNP chip. GenomeStudio® software was used to check the genotype cluster image to remove errors.

The raw data extracted from the report tool in GenomeStudio® software is composed of 70 small two-sided samples in the width and length matrix format and 777,000 SNP genotype data in the vertical direction. SNPs, mitochondrial SNPs and Y chromosome SNPs without chromosome information were deleted in the raw data. Because mitochondrial SNP and Y chromosome SNPs are not homologous chromosomes, genetic analysis based on homologous chromosomes is impossible. In addition, SNPs with genotyping rate of 98% or less in each SNP were deleted because of the large number of missing values. The minor allele frequency (MAF) of the minor allele frequency was calculated, and the genetic diversity was less than 0.005, so statistical analysis was impossible. The X chromosome The number of chromosomes in cow and hydrogen was different and was not suitable for genetic analysis.

Then, the MAF difference (absolute difference between Hanwoo and Baekwoo) of each SNP was calculated, and the statistical significance (P-value) of MAF difference was calculated through Fisher's Exact Test (Genotype). SNPs with 100% difference in MAF difference were selected based on marker selection criteria.

Using the SNP genotyping, SNP genotypic diversity and frequency in the chromosomes of 70 native cattle, 22 cattle and 48 Korean cattle, were used to identify genetic differences according to the cultivars in two native cattle (Paek and Hanwoo) Respectively.

Nine single nucleotide polymorphic markers (rs42125585, rs42125591, rs42125833, rs109461720, rs134735704, rs109447299, rs42164846, rs42160000 and rs137353829) showing genetic differences were selected and the information on them is shown in Table 1.

Table 2 shows the allele genotypes of each marker or the varieties of Hanwoo.

Specifically, only adenine (A) appears in the case of Baeku and only guanine (G) in the case of Hanwoo in rs42125585, rs42125591, rs42125833, rs109461720, rs134735704 and rs137353829. In addition, in rs109447299 and rs42160000, bacon appears only in thymine (T) and in cows in cytosine (C). In rs42164846, only the cytosine (C) appeared in the white wax and only the adenine (A) appeared in the Korean wax, and the breed specific allele was identified to distinguish the white wax and Hanwoo variety.

Through the SNP markers, it was found that Baekwoo and Hanwoo varieties could be detected with high accuracy.

It is to be understood that both the foregoing general description and the following detailed description of the present invention are exemplary and explanatory and are intended to provide further explanation of the invention as claimed. More variations are possible within a range that does not. Accordingly, the present invention may be embodied in other ways than those specifically described and illustrated herein, and it may be understood by those skilled in the art.

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Method for identification of Baekwoo breed using single          nucleotide polymorphism markers <130> KPA171051-KR <160> 9 <170> KoPatentin 3.0 <210> 1 <211> 41 <212> DNA <213> Bos taurus <400> 1 ggtagaacct agagattgtc rtatacagtt acataagtca g 41 <210> 2 <211> 41 <212> DNA <213> Bos taurus <400> 2 cgatgcagtc ttctggaaga rtttcgtttt ggctctgagg t 41 <210> 3 <211> 41 <212> DNA <213> Bos taurus <400> 3 atgtgtgtgt agagagagtc rgatgaccaa agcttgtgta t 41 <210> 4 <211> 41 <212> DNA <213> Bos taurus <400> 4 caactgaagc cacttggcac rctcacatgt tgattgtgtg a 41 <210> 5 <211> 41 <212> DNA <213> Bos taurus <400> 5 tattttgtaa aattcttatt rtcctgggga aaacgtcata t 41 <210> 6 <211> 41 <212> DNA <213> Bos taurus <400> 6 aagagtcgga caccacttag ygactaaaca acaacaaaac g 41 <210> 7 <211> 41 <212> DNA <213> Bos taurus <400> 7 cttgtagttt gaagtaaata mtccttatat attcttacgt c 41 <210> 8 <211> 41 <212> DNA <213> Bos taurus <400> 8 acctctccct caagcttttg ytaacactgc tttgaattat t 41 <210> 9 <211> 41 <212> DNA <213> Bos taurus <400> 9 tatggctttc ccaaacctct rcaaagtgga gatattgcta t 41

Claims (9)

(a) a polynucleotide consisting of 5 to 41 consecutive bases, wherein the 21st base is G or A in the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, and the 21st base;
(b) a polynucleotide consisting of 5 to 41 consecutive bases, wherein the 21st base is G or A in the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2, and the 21st base;
(c) a polynucleotide consisting of 5 to 41 consecutive bases, wherein the 21st base is G or A in the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3, and the 21st base;
(d) a polynucleotide consisting of 5 to 41 consecutive bases, wherein the 21st base is G or A in the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4, and the 21st base;
(e) a polynucleotide consisting of 5 to 41 consecutive bases, wherein the 21st base is G or A in the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 5, and the 21st base;
(f) a polynucleotide consisting of 5 to 41 consecutive bases, wherein the 21st base is C or T in the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 6, and the 21st base;
(g) a polynucleotide consisting of 5 to 41 consecutive bases, wherein the 21st base is A or C in the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 7, and the 21st base;
(h) a polynucleotide consisting of 5 to 41 consecutive bases, wherein the 21st base is C or T in the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 8, and the 21st base;
(i) a polynucleotide consisting of 5 to 41 consecutive bases, wherein the 21st base is G or A in the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 9, and the 21st base; And
(j) a polynucleotide complementary to any one of the polynucleotides of (a) to (i)
Wherein the polynucleotide comprises at least one polynucleotide selected from the group consisting of: &lt; RTI ID = 0.0 &gt; a &lt; / RTI &gt; single nucleotide polymorphism (SNP) marker.
A composition for identifying a bauxite variant, which comprises a preparation capable of detecting or amplifying a SNP marker for identifying a bauxite variant of claim 1.
3. The composition according to claim 2, wherein the identification of the varieties of Baekwu is to identify Baekwoo from the group consisting of Hanwoo and Baekwoo.
A kit for identifying white birch varieties comprising the composition of claim 2.
The kit according to claim 4, wherein the kit is an RT-PCR kit or a DNA chip kit.
A microarray for identifying a white woof variety, comprising the SNP marker for identifying the white woof variety according to claim 1.
(a) amplifying or hybridizing a polymorphic site of SNP markers for identifying the Baekwoo variety of claim 1 from a DNA of a sample separated from the individual; And
(b) determining the base of the amplified or hybridized polymorphic site.
8. The polynucleotide according to claim 7, wherein the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 is A at position 21; A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2 has the 21st base of A; The polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3 has the 21st base of A; The polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4 has the 21st base of A; The polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 5 has the 21st base of A; The polynucleotide comprising the nucleotide sequence of SEQ ID NO: 6 has the 21st base T; The polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 7 is C; The polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 8 has the 21st base T; Or a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 9 is A at the 21st base, it is judged to be a Baekwoo variety.
The method of claim 7, wherein the base crystals of step (b) are selected from the group consisting of sequence analysis, microarray hybridization, allele specific PCR, dynamic allele-specific hybridization (DASH) Wherein the amplification is performed by at least one method selected from the group consisting of PCR extension analysis, PCR-single strand conformation polymorphism (PCR-SSCP), PCR-restriction fragment length polymorphism (PCR-RFLP) and TagMan technique.