KR101821554B1 - Method for identification of Chikso breed genotype using microsatellite markers - Google Patents

Abstract

The present invention relates to a composition including a primer specific to the TGLA-325 used as a microsatellite marker to identify one of the native dark brown cattle, Jeju black cattle, and Korean cattle, a kit including the composition for the same to identify one of the native dark brown cattle, Jeju black cattle, and Korean cattle, and a method using the composition or kit for the same to identify one of the native dark brown cattle, Jeju black cattle, and Korean cattle. According to the present invention, the microsatellite marker of four species are used to easily identify the native dark brown cattle, Jeju black cattle, or Korean cattle, thereby enabling various utilization in agricultural and food production fields through the systematic and scientific establishment of the genetic resources of livestock.

Description

[0001] The present invention relates to a method for identifying a gene varieties of Chesoso using a supersatellite marker,

More particularly, the present invention relates to a method for identifying a gene varieties of ChowSo using a supersatellite marker, and more particularly, to a method for identifying a gene varieties of ChowSo using a supersatellite marker, A kit for identifying one of the native soybean fowl, the Jeju black cattle and the Korean cattle containing the composition, and a method for identifying one of the native cattle fox cattle, Jeju cattle and Korean cattle using the composition or kit .

In recent years, as the importance of future utilization value of genetic resources has been recognized, many attention and efforts have been devoted to securing, preserving and managing resources as well as evaluating and utilizing 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).

Microsatellite (MS) represents a repeating structure of 2 to 6 nucleotide sequences and is widely spread in the non-coding region on the eukaryotic DNA. In addition, polymorphism information is abundant because it represents about 10 alleles per locus in a supersampling body. 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., 2002), the genetic diversity of a variety or population, identification of quantitative gene loci, , (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.

The search for Korean Hanwoo had various searches such as brown, black, white, semicolon, pale color, and variegation. However, in 1916, the Japanese Government General decided to unify and seek unification of the title, The diversity of genetic resources was lost. Recently, importance of diversity of genetic resources has emerged, and studies on the restoration and propagation of Jeju black cattle, black cattle and black cattle have been actively carried out, but the classification method of species has a limit that can only depend on searching and body shape.

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. In order to prevent the loss of these resources with the vulnerable varieties, conservation and management of the genetic resources are carried out by the regulatory agencies. Therefore, studies on the characteristics for systematic and scientific breeding are needed.

Under these circumstances, the inventors of the present invention have made extensive efforts to find a supersatellite marker capable of effectively identifying native species, and as a result, it has been confirmed that it is possible to identify native species using TGLA325, which is a supersatellite marker, Completed.

It is an object of the present invention to provide a composition for identifying one of native wheat bran, Jeju bran and Korean beef containing a primer specific to the supersatellite marker TGLA325.

It is another object of the present invention to provide a kit for identifying one of domestic cattle fowl, Jeju cattle and Korean cattle containing the composition.

It is another object of the present invention to provide a method of identifying one of the native soybean fowls, Jeju black fowls and Korean fowls using the composition or kit.

As a result of various studies to find a supersatellite marker capable of effectively identifying native species, the present inventors have found that when using INRA30, TGLA325, UMN0905 or UMN0929, which are four supersatellite markers, And Hanwoo were identified. Particularly, when TGLA325 is used among the four supersatellite markers, the three species of native species can be individually identified, and the three remaining locus markers can be used to identify the three native species. It is possible to identify them more clearly.

Techniques for identifying individuals or species using supersatellite markers have already been known, but techniques for identifying native species using four supersatellite markers INRA30, TGLA325, UMN0905, or UMN0929 have not been developed so far, It was first developed by the inventor.

In one aspect to achieve the above object, the present invention provides a composition for identifying one of domestic wheat bran, Jeju bran and Korean beef containing a primer specific to TGLA325 as a microsatellite marker.

The term "microsatellite " of the present invention means a simple sequence repeat (SSR) having a structure in which two to six 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, PCR was performed using the respective primers corresponding to the supersatellite marker, and the size and specificity of the amplified products of the supersatellite marker, , And a set of supersampling markers capable of identifying Jeju black beef or Hanwoo varieties. The supersatellite marker set of the present invention includes TGLA325, and optionally can further include IRNA30, UMN905, or UMN0929. Information about the super-satellite can be obtained from the National Center for Biological Information (NCBI) database or the United Nations Food and Agriculture Organization (http: //www.fao/org/). For example, the supergalactic marker TGLA325 is known as Accession No: NW_003104788.1 of NCBI, the supergalactic marker IRNA30 is known as Accession No: NW_003104789.1 of NCBI, and the supergalactic marker UMN905 is known as Accession No: AF483748.1, and the supersatellite marker UMN0929 is known as GenBank Accession No: AF483749.1.

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 " of the present invention refers to a nucleic acid sequence having a short free 3 'terminal hydroxyl group, a short nucleic acid which can form a base pair with a complementary template and serves as a starting point for template strand copying ≪ / RTI >

In the present invention, as long as the primer can specifically bind to the supersatellite marker to produce an amplification product, it includes a base pair consisting of the nucleotide sequences of SEQ ID NOS: 3 and 4, which is specific to TGLA325 And optionally a base pair consisting of the nucleotide sequences of SEQ ID NOS: 1 and 2 specific to INRA30, a base pair consisting of nucleotide sequences of SEQ ID NOS: 5 and 6 specific to UMN0905, and a nucleotide sequence of SEQ ID NOS: 7 and 8 specific to UMN0929 A base pair constructed, and the like.

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 acting 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 " native native cow " of the present invention means a cow which has been inhabited since ancient times in Korea.

According to one embodiment of the present invention, when a primer capable of amplifying TGLA325, which is a supersatellite marker, is used, it is possible to identify native TGLA, Jeju beef or Korean beef, respectively. In addition to TGLA325, other supersatellite markers INRA30, It was confirmed that primers capable of specifically amplifying UMN0905, UMN0929 and the like can be more clearly discriminated from native wheat bran, Jeju bran or Korean wheat (Table 2).

In another aspect, the present invention provides a kit for identifying one of a homemade beef cattle, a Jeju beef cattle, and a Korean beef cattle including the above composition.

The kit of the present invention comprises a primer specific to TGLA325 which is a supersatellite marker. The primers can be used to identify native species, but not limited to, one or more other components suitable for amplifying TGLA325, a supersaturse marker, using the primers. At this time, a kit that can be used may be a PCR kit.

As a specific example, the indigenous cattle identification kit of the present invention may be a PCR kit. The kit may contain a DNA polymerase, a deoxy nucleotide (dNTP), an RNAase, a DNA restriction enzyme, DEPC-water, a test tube and a suitable container in addition to the TGLA325 specific primer. It may also contain a primer pair specific for the gene used as a quantitative control.

In addition to the primers specific to TGLA325, the PCR kit may further include a primer specific for INRA30, a primer specific for UMN0905, and a primer specific for UMN0929, either singly or in combination.

In yet another aspect, the present invention provides a method of identifying one of native soybean fowl, Jeju black cattle, and Korean cattle using the composition or kit.

Specifically, the method for identifying one of the native soybean fodder, the Jeju black fodder and the Korean native fodder provided in the present invention comprises the steps of: (a) obtaining genomic DNA from a biological sample of a native fish selected from the group consisting of fowl, ; And (b) performing a PCR reaction using the obtained genomic DNA and a primer specific to TGLA325.

The term "biological sample " of the present invention means a sample containing native genomic DNA capable of amplifying the supersatellite marker of the present invention. Such biological 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 biological sample may be blood of native cattle.

PCR was performed using the genomic DNA obtained from the biological sample separated from the conventional PCR and a primer specific to TGLA325, and the result was analyzed to confirm what the native species was.

As an example, when the number of amplification products obtained by carrying out the PCR reaction is 8, the native species can be identified as a feline; If the number of amplification products obtained by performing the PCR reaction is 7, the native bovine can be identified as Jeju black beef; When the number of amplification products obtained by performing the PCR reaction is 5, the native species can be identified as Hanwoo.

As another example, when an amplification product of 106, 112 and 122 bp in size is detected in the amplification product obtained by performing the PCR reaction, the native bovine can be identified as Bovine; If an amplification product of 102 and 118 bp in size is detected in the amplification product obtained by performing the PCR reaction, the native bovine can be identified as Jeju black beef; When an amplification product of 110 bp in size is detected in the amplification product obtained by performing the PCR reaction, the native species can be identified as Hanwoo.

On the other hand, the identification method may further include a step of performing the PCR reaction using primers specific for INRA30, primers specific for UMN0905, or primers specific for UMN0929, singly or in combination.

PCR was performed using the genomic DNA obtained from the biological sample separated from the conventional PCR and a primer specific for INRA30, a primer specific for UMN0905, or a primer specific for UMN0929, and the result was shown to be specific for TGLA325 By analyzing the result of performing the PCR reaction using the primer, it is possible to confirm what the native species is.

As an example, when the number of amplification products obtained by carrying out the PCR reaction specific to UMN0905 is 6, the native species can be identified as Hanwoo; If the number of amplification products obtained by carrying out the PCR reaction specific to UMN0929 is 9, it is possible to identify the locally produced bovine.

As another example, when an amplification product of 169 bp in size is detected in the amplification product obtained by performing the PCR reaction specific for INRA30, the native bovine can be identified as Jeju black beef; When amplification product of size 159 and 163 bp is detected in the amplification product obtained by carrying out a PCR reaction specific to UMN0905 or amplification product of size 195 bp is detected in the amplification product obtained by performing PCR reaction specific to UMN0929, Can be identified as Hanwoo; If a 191 bp amplification product is detected in the amplification product obtained by carrying out a PCR reaction specific to UMN0929, the native bovine can be identified as Bovine.

Thus, PCR-specific PCR reactions for TGLA325 and UMN0929 can more effectively distinguish fungi from native cattle; PCR reactions specific to TGLA325 and INRA30 can more effectively identify Jeju black cattle among native cattle; When PCR reactions specific to TGLA325, UMN0905 and UMN0929 are performed, Hanwoo can be more effectively identified among native species.

Using the four supersatellite markers of the present invention, it is possible to easily identify native cattle cattle, Jeju black cattle, or Korean cattle, and can be used in a variety of agricultural and food production fields through the systematic and scientific establishment of herd genetic resources There will be.

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

Example  1: For identification of domestic cattle Super satellites Marker  Screening and Super satellites Marker  Production of amplification primer

INRA30 (DXS8), TGLA325 (DXYS3), UMN0905 and UMN0929 were selected as supersatellite markers that can be used to identify major native cattle beef cattle, Korean beef cattle or Jeju beef cattle, and the following primer base pairs (Table 1). At this time, one sequence of each prepared primer base pair was labeled with a fluorescent substance FAM.

Supersatellite marker-specific primer base pair for identification of native species Locus Primer sequence (F: forward primer,
R: Reverse primer) A fluorescent tag (fluorescent tag) Repeating sequence
shape
(type of repeat) Super satellites
Left-handed
size
range
(size range of loci, bp) The binding temperature
(Anneal Temperature, ° C) INRA30 (DXS8) F: 5'-ATGCAAATGTGCTACATCACCTAT-3 '
(SEQ ID NO: 1)
R: 5'-TGGCCCAACTCTCACATCCAGATC-3 '
(SEQ ID NO: 2) FAM (TG) ₁₃ 163-169 60 TGLA325 (DXYS3) F: 5'-GGGCACTTTACTCTCTGAACAAATC-3 '
(SEQ ID NO: 3)
R: 5'-GCTGACAGTCTATTTCCAGAAGGTA-3 '
(SEQ ID NO: 4) FAM (CA) ₁₇ 98-122 56 UMN0905 F: 5'-ATCAACCGTGGTAGCTCTAA-3 '
(SEQ ID NO: 5)
R: 5'-CTAGAATGTAAACCAGCTGC-3 '
(SEQ ID NO: 6) FAM (CA) ₁₆ 159-169 61 UMN0929 F: 5'-ACCAGCTGATACACAAGTGC-3 '
(SEQ ID NO: 7)
R: 5'-GGTCAGAGAATGAAACAGAG-3 '
(SEQ ID NO: 8) FAM (CA) ₁₉ 175-201 61

Example  2: Genomic DNA of native bovine Super satellites Marker  analysis

First, blood was obtained from a total of 132 dogs, 60 dogs, 25 dogs, or 47 cattle dogs, and genomic DNA was isolated from the obtained blood using a genomic DNA extraction kit.

Next, using the genomic DNAs obtained above as templates, PCR amplification was carried out using the respective primers prepared in Example 1 to obtain respective amplified DNA fragments. The DNA fragment thus obtained was applied to an automatic sequencer (Applied Biosystems, USA), and the results were analyzed using GeneMapper version 4.0 (Applied Biosystems, USA) and Genotyper software (Table 2).

(Bp) and the number of alleles Super satellites
Marker The Jeju Black Sheep Hanwoo Size of allele fragment (bp) The size of the specific allele gene fragment (bp) Size of allele fragment (bp) The size of the specific allele gene fragment (bp) Size of allele fragment (bp) The size of the specific allele gene fragment (bp) INRA30 163, 165, 167 165, 167 169 163, 165, 167 TGLA325 98, 100, 104, 116, 120 106, 112, 122 98, 100, 104, 116, 120 102, 118 98, 100, 104, 120 110 UMN0905 161, 165, 167, 169 161, 165, 167, 169 161, 165, 167, 169 159, 163 UMN0929 175, 177, 181, 187, 189, 193, 197, 201 191 175, 177, 187, 189, 193, 197, 201 175, 177, 181, 187, 193, 197 195

As shown in Table 2, the size of the allele gene fragment amplified with INRA30 is 163 to 169 bp, the size of the allele gene amplified with TGLA325 is 98 to 120 bp, The size of the gene fragment was 161 to 169 bp and the size of the allele gene amplified in UMN0929 was 175 to 201 bp.

In addition, the number of alleles specifically amplified according to the supersatellite marker is 3 (INRA30) to 9 (UMN0929) in the case of beef cattle, and 3 (INRA30) to 7 (TGLA325, UMN0929) In case of Hanwoo, 3 (INRA30) to 7 (UMN0929) were confirmed. In addition, the number of alleles of the supersatellite markers amplified specifically in one native cattle was 4 in the case of cattle, 3 in cattle of Jeju, and 4 in case of Hanwoo.

In particular, in identifying the three species, TGLA325 amplifies a different number of allele fragments in each of three species of native species (8 females, 7 black females and 5 Korean females), a specific allele Since the number of fragments was also different (3, 3, 4, 6, and 1, respectively), the TGLA325 was the most important supersatellite marker.

Thus, by analyzing the total number of alleles amplified in the four supersatellite markers, the size of alleles to be amplified, and the number of alleles specifically amplified in each marker, Hanwoo could be identified.

Example  3: Genomic DNA of native bovine Supercell Marker  Diversity index analysis

Each of the amplified DNA fragments obtained in Example 2 was applied to Cervus version 2.0 (version 3.0.3 The University of Edinburgh), and the diversity index of the supersatellite marker was analyzed (Table 3). The number of alleles (N _A ) and the number of samples (N _S ) were used as the main data used in the analysis. The diversity index to be analyzed was an expired heterozygosity , Hexp), observed heterozygosity (Hobs), and polymorphic information content (PIC).

Expectation and observed heterozygosity rate and diversity information index for four native varieties of four satellite markers
Super satellites marker Chikso Jeju Black Hanwoo N _A N _S Hobs Hexp PIC N _A N _S Hobs Hexp PIC N _A N _S Hobs Hexp PIC INRA30 3 60 0.083 0.112 0.106 3 47 0.128 0.123 0.118 3 25 0.560 0.513 0.395 TGLA325 8 60 0.800 0.770 0.729 7 47 0.319 0.309 0.297 5 25 0.760 0.757 0.698 UMN0905 4 60 0.433 0.639 0.577 4 47 0.426 0.575 0.490 6 25 0.360 0.691 0.623 UMN0929 9 60 0.847 0.791 0.755 7 47 0.750 0.662 0.603 7 25 0.760 0.741 0.683 Average 6 60 0.541 0.578 0.542 5.3 47 0.406 0.417 0.377 5.3 25 0.610 0.676 0.600

As shown in Table 3, the average value of the three diversity indices is the highest in Korean beef cattle, while the lowest in Jeju beef cattle. Therefore, relatively high genetic diversity Respectively.

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 Chikso breed genotype using          microsatellite markers <130> KPA160385-KR <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 atgcaaatgt gctacatcac ctat 24 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 tggcccaact ctcacatcca gatc 24 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 gggcacttta ctctctgaac aaatc 25 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 gctgacagtc tatttccaga aggta 25 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 atcaaccgtg gtagctctaa 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 ctagaatgta aaccagctgc 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 accagctgat acacaagtgc 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 ggtcagagaa tgaaacagag 20

Claims (16)

A composition for discriminating among varieties of domestic cattle consisting of cattle, Jeju black cattle and Korean cattle, which contains a primer specific to TGLA325, which is a microsatellite marker, such as cattle, Jeju black cattle and Korean cattle.
The method according to claim 1,
Wherein the primer comprises the nucleotide sequence of SEQ ID NOS: 3 and 4.
delete The method according to claim 1,
Wherein said composition further comprises a primer selected from the group consisting of a primer specific for INRA30, a primer specific for UMN0905, a primer specific for UMN0929, and combinations thereof. Composition.
5. The method of claim 4,
The primers specific for INRA30 are composed of the nucleotide sequences of SEQ ID NOS: 1 and 2; The primers specific for UMN0905 consist of the nucleotide sequences of SEQ ID NOS: 5 and 6; Wherein the primers specific for UMN0929 are comprised of the nucleotide sequences of SEQ ID NOS: 7 and 8.
A kit for identifying one of domestic cattle, cattle, and Korean cattle comprising the composition of any one of claims 1, 2, 4, and 5.
The method according to claim 6,
Wherein the kit is a PCR kit.
(a) obtaining genomic DNA from a biological sample of native cattle selected from the group consisting of cattle, Jeju cattle and Korean beef; And
(b) carrying out a PCR reaction using the genomic DNA obtained and a primer specific to TGLA325, wherein the strain is selected from the group consisting of Bacillus subtilis, Jeju black beef and Korean beef, How to identify.
9. The method of claim 8,
Wherein the biological sample is a hair follicle, blood, various body fluids, isolated tissue, isolated cells or saliva.
9. The method of claim 8,
Wherein the primer comprises the nucleotide sequence of SEQ ID NOS: 3 and 4.
9. The method of claim 8,
If the number of amplification products obtained by carrying out the PCR reaction is 8, identify the native species as feline; If the number of amplification products is seven, identify the native cattle as Jeju black cattle; And identifying the native species as Hanwoo when the number of amplification products is five.
9. The method of claim 8,
Identifying amplified products of 106, 112 and 122 bp in the amplification products obtained by performing the PCR reaction as follicles; If said amplification products detect amplicons of the size of 102 and 118 bp, identify said native species as Jeju black beef; And identifying said native species as Hanwoo when an amplification product of 110 bp in size is detected in said amplification product.
9. The method of claim 8,
Further comprising the step of performing the PCR reaction using primers specific for INRA30, primers specific for UMN0905, or primers specific for UMN0929, alone or in combination.
14. The method of claim 13,
The primers specific for INRA30 are composed of the nucleotide sequences of SEQ ID NOS: 1 and 2; The primers specific for UMN0905 consist of the nucleotide sequences of SEQ ID NOS: 5 and 6; Wherein the primers specific for UMN0929 are comprised of the nucleotide sequences of SEQ ID NOS: 7 and 8.
14. The method of claim 13,
If the number of amplification products obtained from the PCR performed using the primer specific to UMN0905 is 6, the locus is identified as Hanwoo; Wherein said native species is identified as feline when the number of amplification products obtained from the PCR performed using the primer specific for UMN0929 is nine.
14. The method of claim 13,
If the 169 bp amplification product is detected in the amplification product obtained by the PCR performed using the INRA30-specific primer, the native bovine is identified as Jeju black beef; In the amplification product obtained from PCR performed using the primer specific to UMN0905, amplification products of 159 and 163 bp in size were detected or PCR products were carried out using primers specific for UMN0929, amplification of 195 bp in size Identifies the native cattle as cow when the product is detected; Wherein said native bovine is identified as a null when an amplification product of 191 bp in size is detected in an amplification product obtained from a PCR carried out using a primer specific for UMN0929.