JP2002209581A - Method for identifying breed of bovine - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for efficiently identifying a bread of bovine serving as beef using commercial beef as specimen. SOLUTION: This method for identifying beef is based on the finding of a single nucleotide polymorphism that permits easily discriminating black-hair cattle and Holstein, which need to be identified as beef cattle, with the single nucleotide polymorphism being utilized as gene probe or primer for the gene amplification.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for discriminating cattle breeds.

[0002]

2. Description of the Related Art The types of beef distributed in the domestic market are:
It is roughly divided into beef meat, dairy cow meat and other beef meat. Japanese beef includes Japanese Black, Japanese Brown, and Hornless Japanese,
The dairy cows include dairy cows (Holstein and Jersey breeds), dairy cows and Japanese beef, and crossbreeds of dairy cows and foreign breeds. Other cattle include the exclusive species of meat of foreign cattle and the cross species of exclusive meat of foreign cattle and Japanese beef.

[0003] In Japan, marbled beef containing so-called "sashi" is a representative of luxury goods. For example, Japanese black meat, known as the finest beef, is characterized by the fineness of its muscle fibers and good quality sashimi. Since the transaction price of carcasses is determined by the condition of the sashi, the added value of Japanese black cattle, which has the quality that sashi can easily enter, increases, and there are various Japanese beef brands. The price of carcasses of Holstein and other dairy cows is about half the price of Wagyu, and it is unlikely to marble, and although it has a unique smell of dairy beef, the number of dairy beef shipped exceeds that of Wagyu. The percentage of slaughter is 47.
2%, dairy cows 51.7%, other cows 1.1% (meat distribution statistics published by the Ministry of Agriculture, Forestry and Fisheries, Ministry of Agriculture, Forestry and Fisheries, published November 27, 2000, from November 2000) .

[0004] On the other hand, beef distribution systems include biological distribution,
Although it is composed of four stages: slaughter, wholesale and retail, there are no standards or guarantees that directly link slaughter organisms to retail meat. Therefore, it is difficult for general consumers to know information on the breed of beef that was the basis of meat sold in retail stores such as supermarkets,
It is also difficult to determine the varieties from carcasses and meat processed into cuts and minced meat. That is, under the current distribution system, a general consumer cannot deny the possibility of being made to purchase an unintended product different from a desired product. Furthermore, even if a product is replaced during the distribution process, for example, Japanese black meat of a specific brand is replaced with another beef, it is difficult to scientifically verify it.

In view of this, attention has been paid to DNA that is the most accurate as individual information on living organisms, and an attempt has been made to develop an inspection method for identifying varieties and production areas using DNA extracted from meat as a material, but is still in practical use. Has not been reached.

[0006]

The problem to be solved by the present invention is a method for efficiently discriminating the breed of beef on which beef is based on actually sold beef as a material, in particular,
It is an object of the present invention to provide a method for discriminating whether or not beef is derived from Holstein breeds, which has a large production amount and may be confused with consumers by Japanese black beef and the like.

[0007]

It is considered that the evolution of organisms was brought about by mutations in the base sequence of DNA, which is the essence of genes. Variations in varieties are also considered to be the result of accumulation of such base sequence changes.Therefore, varieties to be identified can be used as varieties identification markers if the base sequence of genomic DNA differs even by one base. Become.

[0008] Single nucleotide polymorphism (Single Nuc
leotide Polymorphism (hereinafter also referred to as “SNP”), a marker to be used for identification of varieties.
Since the range of NA can be narrowed, it is considered that even meat that has been frozen or refrigerated and preserved since slaughter can be tested as long as DNA can be extracted. Therefore, in the case of a test relating to a gene targeting meat, it is preferable to use SNP as a test marker.

[0009] As a method of searching for SNP, RAPD-PCR proposed by Williams et al.
(Random Amplified Polymorphic DNA Polymerase Chai
n Reaction) method. This method has also been applied to the discrimination of animal and plant species, and also to forensic individual discrimination.

The present inventor has sought to solve the above problems.
The gene was RAP between Holstein and Japanese Black
As a result of analysis by D-PCR, SNPs capable of distinguishing cattle breeds were found.

Hereinafter, the search for the SNP of the cow will be described. RAPD-PCR method [Williams et al., Nucleic acid
ic Acids Res., 18 (22), 6531-6535 (1990)]
Gene amplification in which an oligonucleotide primer having a random sequence around 0 is used as a PCR primer, the primer is annealed to a template DNA under low-temperature conditions that cause primer mismatch, and then the DNA is amplified according to a normal PCR method. Is the law. In this gene amplification method, the SNP
Is present, DNA replication from the primer binding portion is not induced, and as a result, a difference appears in the band pattern of the agarose gel electrophoresis image of the PCR product in comparison with the case where no SNP is present. Therefore, RAP
When a difference is found in the band pattern by the D-PCR method, it is possible to specify that the SNP is present in a portion where the used primer is bound.

However, since the DNA fragment which is the amplification product of RAPD-PCR has both ends replaced by the nucleotide sequence of the PCR primer, it is necessary to determine the presence or absence of SNP and to determine the exact nucleotide sequence around SNP. In addition, it is necessary to examine the nucleotide sequence of the corresponding site on the genomic DNA. In this case, since the nucleotide sequence other than the primer portion can be analyzed from the RAPD-PCR product, for example, a nucleotide chain having a nucleotide sequence other than the primer portion is used as a probe, and a DNA fragment having homology with the nucleotide chain is screened from a genomic DNA library. Then, by analyzing the base sequence of the DNA, the base sequence of the corresponding site can be confirmed.

The preparation of a probe for selecting such a nucleotide sequence analysis technique can be performed according to a conventional method.
That is, an RAPD-PCR product, specifically, a band to be used as a probe is selected from bands obtained by electrophoresis, DNA is extracted, and this is extracted with a gene transfer vector [for example, pT7Bluescript vector (Novagen
And the like), and thereby a suitable host (for example,
Competent E. coli) is transformed and cloned. From the thus obtained gene integration vector of a large number of clones, only the integrated DNA is cut out with a DNA restriction enzyme, and when a complementary strand is synthesized using this DNA fragment as a template, a detection label, for example, α
By adding a radioactive label such as- ³² P-dCTP to the complementary strand, such a labeled DNA fragment can be used as a desired probe. Since the DNA fragment in the genomic DNA library to which the probe is bound is a DNA fragment containing the corresponding nucleotide sequence, for example, the peripheral nucleotide sequence is changed by genomic walking from a known sequence region (probe portion) to the outside. Decryptable, RAPD-PCR
The nucleotide sequence of the SNP portion to which the primer has bound can be determined.

Actual Search for SNP in Cattle SNP Search by RAPD-PCR The present inventors investigated the SNP in Holstein and Japanese Black cattle.
In order to find out, SNP search was performed by the method exemplified above using seven Holstein breeds (two males and five females) and five Japanese Black breeds (two males and three females).

First, leukocytes of the bovine peripheral blood were centrifuged, and proteinase K and SDS were added thereto.
After digestion at ℃, genomic DNA is extracted and purified by phenol / chloroform extraction and ethanol precipitation.
The genomic DNA was used as a gene sample.

Next, RAPD-PCR was performed using each of the obtained genomic DNAs as a template and 420 kinds of 10-mer lantam primers (synthesis of Lifetech Oriental) as PCR primers. The PCR reaction solution was 20 ng genomic DNA, 1 × ExTaq buffer, 4 d
NTP (0.4 mM), 10-mer random primer (5
0pmol), ExTaq DNA polymerase (Takara)
(1 U) and adjusted to a total volume of 25 μl with sterile water. P
The CR reaction was performed at 94 ° C./1 after heat denaturation at 94 ° C./5 minutes.
The reaction was carried out for 45 cycles with each cycle consisting of 1 minute, 42 ° C./1 minute and 72 ° C./3 minutes, and an additional extension reaction was performed at 72 ° C. for 7 minutes. The thermal cycler is PE Bio
PJ2000 from systems was used. After the reaction, PC
The R reaction solution was subjected to 1% agarose gel electrophoresis, and a primer showing a different electrophoresis pattern between the Holstein species and the Japanese Black species was searched. As a result, primers having the nucleotide sequences represented by SEQ ID NO: 1 and SEQ ID NO: 2 (hereinafter referred to as primers) , K-1 respectively,
When K-2 primer was used for RAPD-PCR, a band specific to Japanese Black appeared (FIG. 1:
The left uses K-1 primer and the right uses K-2 primer. Each of the bands indicated by arrows is a band that appears specifically in Japanese black cattle. Lanes 1 to 7 show electrophoretic images of Holstein species, lanes 8 to 12 show Japanese black cattle, and M shows an electrophoretic image of a 200 bp ladder marker).

[0017] A variety-specific RAPD-PCR product
Roning and analysis of nucleotide sequence Next, a band specific to this Japanese black species was cut out from an agarose gel, and the band was analyzed using a QIAquick Gel Extraction Kit (QIAGEN
DNA was extracted. The extracted DNA fragment is transferred to a PC
PP by R-Script Amp Cloning Kit (Stratagene)
CR-Script AmpSK (+) cloning vector (Stratagene)
E. coli XL10-Gold Kan ultracompetent cells
(Stratagene) and cultured overnight at 37 ° C. on an LB-agar plate containing the antibiotic ampicillin. After completion of the culture, colonies were formed on the plate,
A colony of ampicillin-resistant Escherichia coli (ampicillin-resistant) transformed with the above plasmid was further added to an LB culture solution containing ampicillin, and shake-cultured at 37 ° C. for 12 hours to grow ampicillin-resistant Escherichia coli. . The QIAGEN Plasmid Mini Kit (QIAGEN) was obtained from the ampicillin-resistant E. coli clone thus obtained.
The plasmid was extracted and purified by the method described above, and the nucleotide sequence of the inserted DNA fragment was analyzed.

The sequencing reaction solution used in the analysis of the nucleotide sequence was composed of plasmid DNA (500 ng), 8
μl BigDye Terminator Cycle Sequencing Ready React
ionMix (PE Biosystems), pPCR-Script AmpSK (+)
3.2 pmol of a sequencing primer (T-7 or T-3) to be annealed outside the multiple cloning site of a cloning vector (Stratagene) was adjusted to a total volume of 20 μl with sterile water. This is carried out at 96 ° C./10
Second cycle, 50 ° C / 5 seconds, 60 ° C / 4 minutes as one cycle,
Reaction was performed for 25 cycles (The thermal cycler was PE B
Use GeneAmp PCR System2400 from iosystems). The PCR reaction product labeled with the terminator region is CENTRI-S
After gel filtration and purification using EP columns (PE Biosystems), and drying with an evaporator, 4 μl of loading buffer [Form
amide: Blue Dextran (50mg / ml in 25mM EDT
A) = 5: 1], and heat denatured at 95 ° C. for 2 minutes.
Immediately cool on ice and use the auto sequencer (ABI PRISM 377
The nucleotide sequence was automatically analyzed by DNA Sequencer (PE Biosystems).

Analysis of SNP Base Sequence Next, a bovine BAC library (RPCI-42 BOVINE BAC LIBRARY: Roswell Park Canc.) Was used as a probe with a DNA fragment cloned from the aforementioned RAPD-PCR band.
er Inst., NY), screening was carried out according to the attached manual.
One clone was obtained using a clone and a probe derived from the K-2 primer. Using the sequencing primers shown in SEQ ID NOs: 3 and 5, each clone was analyzed for the portion where the K-1 and K-2 primers bind and the nucleotide sequence of the subsequent clone from the clone by the method described in the preceding section. The approximate nucleotide sequence around the primer binding portion was determined. As a result, a portion where the nucleotide sequence of the portion to which each of K-1 and K-2 binds was different from the nucleotide sequence of the primer was observed.

In order to confirm whether this difference was due to SNP, Holstein Species 10 owned by the Animal Genetics Research Institute, Inc.
QIAamp D from 7 blood samples of 109 Japanese black cats
For DNA extracted with NA Blood Kit (QIAGEN),
A region around the portion to which the K-1 primer binds (hereinafter referred to as K
-1 region), the primers of SEQ ID NOs: 3 and 4 were used, and the region surrounding the portion to which the K-2 primer binds (hereinafter also referred to as the K-2 region) was SEQ ID NO: 5 PCR was carried out using primers Nos. 6 and 6 with the respective regions interposed between the primers. Then, the gene amplification product was subjected to direct sequence analysis using the above-mentioned primers (SEQ ID NOS: 3 to 6) as sequencing primers, and the base sequence on the genomic DNA of the portion where the K-1 and K-2 primers bind was obtained. It was determined. As a result, the K-1 primer was bound at two sites,
One SNP was found at the binding site of the two primers, and could be classified into A, B, C, and D and E allele types in the K-1 region (FIG. 2).

Determination of nucleotide sequence around SNP From the nucleotide sequence information obtained by the direct sequence described above, PCR primers of SEQ ID NOs: 7 and 8 in the K-1 region, and SEQ ID NOs: 9 and 10 in the K-2 region are sandwiched by the SNP. Genomic DNA of Holstein and Japanese Black
Was performed. The PCR reaction solution contains genomic DNA (2
0 ng), 1 × PCR buffer, 4dNTP (0.2 mM),
The primers (1 set: 25 pmol) and AmpliTaq DNA polymerase (PEBiosystems) (0.5 U) were used, and the total volume was adjusted to 25 μl with sterile water. The PCR reaction was performed at 94 ° C /
After heat denaturation for 5 minutes, 94 ° C / 1 minute, 54 ° C / 1 minute and 7 minutes
After 30 cycles of reaction at 2 ° C./1 minute as one cycle, 7
An additional extension reaction was performed at 2 ° C. for 10 minutes. As a thermal cycler, PC707 manufactured by Astex was used.

The DNA fragment of the PCR product is pT7 Bluescri
After insertion into the pt vector, it was introduced into E. coli JM109 (Toyobo) to obtain a clone. Colony PCR was performed on each clone to confirm that the target DNA fragment was correctly inserted. The PCR reaction solution is 1
× PCR buffer, 4dNTP (0.2 mM), 25 pmol each of U-19 and R-20 primers, AmpliTaq DNA
A polymerase (PE Biosystems) (0.5 U) was used, and the total volume was adjusted to 25 μl with sterile water. A portion of Escherichia coli was added to this PCR reaction solution by immersing a toothpick having its tip in contact with a single white colony formed on a plate, and the E. coli DNA was used as a PCR template.

For those in which insertion of the DNA fragment was confirmed, the reaction product of this colony PCR was subjected to QIAquick PCR.
Purification was performed using a purification kit (QIAGEN), and an autosequencer (ABI PRISM 377 DNA Sequencer: PE Biosystem) was prepared using primers of SEQ ID NOS: 11 and 12 for the K-1 region and SEQ ID NOs: 13 and 14 for the K-2 region.
s company), the nucleotide sequence is analyzed, and SEQ ID NO: 15 (allele type A), 16 (allele type B) and 17 (allele type C) (above, K-1 region) and SEQ ID NO: 18 (allele type D) And 19 (allele type E) (above, K
-2 region). Analysis of this nucleotide sequence revealed six new SNPs in the K-1 region (a total of eight SNPs as the K-1 region), all of which were on the same allele [third. Figure: The base sequence of alleles C and E, the part common to A and D is "-"
The base is shown in the SNP part. The portion surrounded by a square indicates a SNP found by direct sequence analysis. Note that <C> of allele C is <T>
The allele B is the one that has been replaced by the above.
It was found that there were two specific regions (K-1 region and K-2 region) that could be used for discrimination of varieties using SNP as an index.

The eight SNPs in the K-1 region are 3
Species alleles (SEQ ID NOs: 15, 16 and 17), and one SNP in the K-2 region was found to be found in two alleles (SEQ ID NOs: 18 and 19). became.

That is, in the nucleotide sequence represented by SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17, the SNP in the K-1 region is 130th, 142nd, 154th, 157th, 195th, 195th, 280th, It was found to be present at bases 339 and 345.

The SNP in the K-2 region is represented by SEQ ID NO: 1.
In the base sequence selected from the base sequence represented by SEQ ID NO: 8 or SEQ ID NO: 19, it was revealed that the base sequence exists at the 235th base.

Based on these findings, which allele combination was observed in the K-1 region and / or the K-2 region in the test cow gene sample was examined using the SNP in these specific regions as an index. The present inventor has found that it is possible to discriminate the breeds of the test cows by doing this.

The present invention is, firstly, an invention which provides nucleotide chains corresponding to the K-1 region and the K-2 region, including SNPs, which serve as a basis for discriminating between breeds of test cattle. Specifically, the present invention relates to a continuous base sequence selected from the base sequences represented by SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17,
157th, 195th, 280th, 339th and 345th nucleotides containing at least one base selected from the group consisting of and having 10 to 360 nucleotides (nucleotide chain of K-1 region) ), And a nucleotide sequence comprising the 235th base in the continuous base sequence selected from the base sequences represented by SEQ ID NO: 18 or SEQ ID NO: 19 and having 10 to 360 bases (K-2 region) Nucleotide chain).

[0029] The nucleotide sequences of these nucleotide chains are target sites of genes in the present invention for identifying cattle breeds. Therefore, it is a base sequence to be used as a basis when designing a gene probe or a primer for gene amplification in a method for identifying a breed of cattle described below.

Here, the reason why the minimum number of nucleotides in the nucleotide chains of the K-1 region and the K-2 region was set to 10 is that if the number of nucleotides is less than 10, the nucleotide chain containing the SNP functions as a nucleotide chain. This is because it is hard to be actualized. For example, a probe based on the nucleotide sequence of the nucleotide chain of the K-1 or K-2 region having a base number of less than 10 has reduced hybridization accuracy with respect to the corresponding base sequence, and also has a similar base number. A primer for gene amplification based on a nucleotide chain having a number of less than 10 tends to have a reduced function of amplifying a desired region.

In the present invention, SEQ ID NOS: 15 to 1
The base sequence shown in FIG. 9 is one of the complementary strands of the double-stranded DNA, and unless otherwise specified, the other complementary strand is also shown at the same time.

Next, the present invention relates to the aforementioned K-1 region and K-region.
The present invention provides a method for identifying cattle breeds according to the present invention, which utilizes two nucleotide nucleotide chains to discriminate cattle breeds. Specifically, the present invention relates information on the polymorphic bases in the bovine gene sample and the breed of the test cow, and in a breed identification method of a cow for identifying the breed of the test cow, the information on the polymorphic bases, The present invention provides a method for identifying a breed of a cow (hereinafter, also referred to as the present identification method), which is information represented by the following and / or.

Information on which of the nucleic acid contained in the gene sample has a base sequence among the base sequences containing the polymorphic base represented by SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17 .

Information on which of the nucleotide sequences of the nucleic acid contained in the gene sample contains the polymorphic base represented by SEQ ID NO: 18 or SEQ ID NO: 19

[0035]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, embodiments of the present invention, particularly, the present discrimination method will be described. This discrimination method is a method of discriminating the allele type in a gene sample of a test cow and discriminating the breed of the test cow from the allele type.

Here, the gene sample preferably refers to a bovine somatic cell gene (specifically, DNA) or a sample containing the same as a main component. The preferred gene sample was the bovine somatic cell gene because of the germ cell gene,
This is because only one paternal or maternal allele type can be determined in one germ cell, and it is difficult to quickly and accurately determine the allele type of an individual. The collection site of the gene sample is not particularly limited, and can be collected, for example, from muscle tissue, subcutaneous tissue, peripheral blood leukocytes, hair root cells, and the like. In addition, the collection target is not only live cows,
The meat may be on the market. In view of the fact that the main embodiment of the present discrimination method is the discrimination of commercially available meat varieties, it is extremely important that the target of collecting a gene sample in the present discrimination method can be meat. The preparation of a gene sample from the target sample is
Usually, it can be carried out according to a known DNA extraction / purification method. It is also possible to prepare a gene sample using a commercially available DNA extraction / purification kit.
Extraction kit (QIAGEN's QIAamp DNA Mini Kit)
Etc.) can also be used].

In the present discrimination method, the allele type of the test cow can be determined using the above-mentioned SNP in the K-1 region and / or K-2 region as an index. Specifically, the determination of the allele type can be made based on the information shown below and / or.

Information on which of the nucleic acid contained in the gene sample has a base sequence among the base sequences containing the polymorphic bases represented by SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17 (Allele type discrimination information regarding the K-1 region).

Information on which nucleic acid is contained in the nucleic acid contained in the gene sample among the nucleotide sequences containing the polymorphic base represented by SEQ ID NO: 18 or SEQ ID NO: 19 (K-2 Allele type discrimination information for the region).

The above information and the above information can be obtained by using a method which is susceptible to the presence of SNP. Specifically, SSCP (Single-strand
conformation polymorphism) method, RFLP (restricti
on fragment length polymorphism) method and the like. The most typical and preferred means is K-
It is possible to hybridize with a nucleotide chain containing a polymorphic base in the 1 region or the K-2 region, and has a base corresponding to the polymorphic base at its 3 ′ end, and has 10 to 10 bases.
A method of using a gene amplification product obtained by amplifying a gene of a test cow using 45, preferably 15 to 40 nucleotide chains as one of a pair of gene amplification primers as discrimination information of a desired allele type. Can be mentioned.

PCR which is a typical method of gene amplification
In the reaction, the primer binds to the single-stranded DNA serving as a template, and a complementary strand is synthesized by a DNA polymerase by an elongation reaction generated from the 3 ′ end of the primer for gene amplification. If the DNA sequence is different from that of the template, the elongation reaction by the DNA polymerase is not started because the bond between the primer and the template is incomplete. On the other hand, when the 3 ′ end of the primer used matches the nucleotide sequence of the SNP in the allele possessed by the target individual, the PCR product is detected as a band by electrophoresis. Therefore, the presence or absence of a specific band in an agarose gel electrophoresis image of a gene amplification product obtained by a gene amplification method using a gene amplification primer having a base corresponding to a SNP at the 3 ′ end, for example, It is possible to easily determine the allele type of the gene sample.

That is, the K-1 region of the gene of the test cow can hybridize with the nucleotide chain of the nucleotide sequence represented by SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17, and its 3 ′ Polymorphic bases (terminals 130, 142, 154 in the nucleotide sequence represented by SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17)
157th, 195th, 280th, 339th or 345th bases), and the above-described gene amplification using a nucleotide chain having 10 to 45 bases, preferably 15 to 40 bases, having a base corresponding to The product was analyzed, and the K-2 region was capable of hybridizing with the nucleotide chain of the nucleotide sequence represented by SEQ ID NO: 18 or SEQ ID NO: 19, and having a polymorphic base (SEQ ID NO: 18 or the nucleotide sequence having the nucleotide number corresponding to 235 in the nucleotide sequence represented by SEQ ID NO: 19) and having a nucleotide number of 10 to 45, preferably 15 to 40, By performing the analysis, it is possible to easily determine the allele type of the test cow.

Here, that it is possible to hybridize with a specific nucleotide chain means that the polymorphic base at the 3 ′ end of the nucleotide chain is correctly recognized (the 3 ′ end is a base complementary to the template DNA). In this case, the base at the 3 'end hybridizes with the template DNA, but does not hybridize when it is a non-complementary base).
Hybridization is possible under conditions according to a gene amplification method such as the CR method.

The conditions for the gene amplification reaction can be appropriately selected according to the type of the gene amplification method used. For example, when the PCR method is used as a gene amplification method, a temperature lower than the Tm value of the nucleotide chain by 2 to 4 ° C. (about 60 to 62 ° C.) may be set in a normal PCR reaction solution. The annealing temperature in a normal PCR reaction is determined by the Tm of the nucleotide chain.
Generally, it is set at a temperature lower by about 5 to 10 ° C. than the value.

The Tm value is represented by the following formula: Tm = (A + T) × 2 + (C + G) × 4 + 2 (A, T, C, and G represent the number of bases of each type in a nucleotide chain). This is a guideline value.

When the set temperature is lower than the Tm value of the nucleotide chain by more than 4 ° C., the recognition of the 3 ′ end of the nucleotide chain becomes weak, and a 3 ′ base nucleotide chain not complementary to the template DNA is used as a PCR primer. Even if used,
On the contrary, if the set temperature is higher than the temperature 2 ° C. lower than the Tm value of the nucleotide chain, the annealing between the base at the 3 ′ end of the nucleotide chain and the template DNA becomes uncertain. However, even if the base at the 3 'end of the nucleotide chain and the corresponding base in the template DNA are complementary, there is a tendency for the PCR reaction not to be performed in many cases.

The number of nucleotides in the nucleotide chain is 10 to 45, preferably 15 to 40. If the number of nucleotides is less than 10, a nucleotide sequence complementary to the nucleotide sequence will stochastically appear on the template DNA. Since it is assumed that there are a plurality of sites, the accuracy of hybridization as a primer for gene amplification to the target portion of the template DNA is low,
If it exceeds 5, the possibility that an extension reaction will occur ignoring the 3'-terminal SNP is frequently recognized.

When SNPs are present in a pair of alleles located at a set of homologous loci and the frequency of possession of each allele is biased depending on the variety, the variety is identified by determining the type of allele possessed. In addition, when alleles containing SNPs are present at different loci, the combination of allele types associated with different loci allows more accurate and more diverse breeds of test cows. It is possible to discriminate from various viewpoints.

As described above, the present discrimination method uses SNs at different loci of the K-1 region and the K-2 region, respectively.
It is an invention that has been completed by finding different allele types based on P, and it is possible to distinguish test animal breeds accurately and from various viewpoints.

According to the present identification method, the breed of the test cow can be identified from the following viewpoints. It is possible to determine whether or not the test cow is of the Holstein breed. The above-mentioned SNPs were found between Japanese Black and Holstein species, and in particular, in the Holstein species, it is clear that characteristic allele patterns are shown in the K-1 region and the K-2 region. (Described later). Because there is concern that consumers may confuse Holstein meat with Japanese Black meat in the market, it is not possible to clearly distinguish whether or not meat is Holstein meat. It is very useful.

It is possible to determine whether or not the test cow is a Japanese Black cattle. In the K-1 region, several specific allelic patterns were observed in Japanese black cats (described below). Therefore, it has been clarified that the present discrimination method can discriminate whether or not the meat is Japanese Black.

It is also possible to discriminate other aspects. By combining the SNPs of the K-1 region and the K-2 region found in the present invention with information on other SNPs, it is possible to discriminate even more diverse breeds of cattle (Holstein and Japanese bulls). Identification of species hybrids, etc.).

As described above, according to the present discrimination method, it is possible to discriminate the breed of the test cow, preferably, whether or not the test cow is of the Holstein breed, even at the meat stage.
Using this identification method to identify the cattle breed on which it is based from meat that is being retailed can be expected to have the effect of protecting consumers as well as deterring fraud that may occur during the distribution process. . In addition, this identification method, as one of the means to identify cloned cattle, which is expected to increase in the future, artificial inseminated cattle with sashimi of Japanese black breeds raised overseas, and even cloned cattle, There is technical application value.

[0054]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the examples do not limit the technical scope of the present invention. Of the allele type pattern used in this appraisal method
Examination The allele types in the blood samples of 107 Holsteins and 109 Japanese Blacks owned by the Animal Genetics Research Institute, Inc. were examined in the K-1 region and the K-2 region.

The allele type B observed in the K-1 region is allele type C except for one SNP (cytosine at position 154 in SEQ ID NO: 16) in the portion to which the K-1 primer binds. They had the same nucleotide sequence. Therefore, when allele type B was included in allele type C and the combination of alleles possessed was examined in all Holstein and Japanese Black individuals, the distribution as shown in Table 1 was shown.

[0056]

[Table 1] Table 1 ──────────────────────────────────── Allele type Holstein type Japanese black hair Species ──────────────────────────────────── AA / DD 94 (87.9%) 6 (5 AA / DE 3 (2.8%) 10 (9.2%) AA / EE 01 (0.9%) CC / DD 0 15 (13.8%) CC / EE 01 (0 CC / DE 0 12 (11.0%) AC / DD 4 (3.7%) 23 (21.1%) AC / EE 06 (5.5%) AC / DE 0 25 (22 Unsatisfactory 6 (5.6%) 10 (9.2%) ────────────────────────────── ────── As a result, more than 80% of Holsteins Since the allele type is AA / DD and the combination of other alleles is hardly seen, the allele type is AA / D
It was clear that D indicates that the test cow was of the Holstein breed with very high accuracy. In addition, when the K-1 region was AC or CC, it became clear that the test cow had a strong tendency to be Japanese Black.

As described above, by analyzing the combination of allele types in the K-1 and K-2 regions, it is possible to discriminate breeds such as whether the test cow is a Holstein breed or a Japanese black breed. It became clear.

Next, without analyzing the base sequence, the PC
An attempt was made to construct the best method that can easily analyze allele types by the R method. Examination of the best mode of the present discrimination method Regarding the K-1 region, the 195th S in SEQ ID NOS: 15 and 17 (SEQ ID NO: 16 was included in 17)
PCR was performed by synthesizing oligonucleotides having the nucleotide sequence shown in SEQ ID NO: 22 and SEQ ID NO: 22 paired with SEQ ID NOS: 20 and 21, which were designed so that NP would be at the 3 'end, and using them as primers for gene amplification. Using genomic DNA extracted from white blood cells of Holstein and Japanese Black cattle prepared according to a conventional method as a template DNA, a PCR reaction was carried out to amplify the gene.

The PCR reaction solution was composed of genomic DNA (20 n
g) 1 × PCR buffer, 4dNTP (0.2 mM), primers (25 pmol: 1 set), AmpliTaq GOLD DNA
Polymerase (PE Biosystems) (0.5 U)
The total volume was adjusted to 25 μl with sterile water. The PCR reaction was performed at 94 ° C /
After heat denaturation for 10 minutes, 94 ° C / 30 seconds, 62 ° C / 30 seconds,
72 ° C./1 minute is one cycle, and after 30 cycles of reaction,
An additional extension reaction was performed at 72 ° C. for 7 minutes. The thermal cycler is a GeneAmp PCR System from PE Biosystems.
2400 was used. As a result, in each individual, as shown in FIG. 4 (a), the allele type of the K-1 region was able to be determined, which was consistent with the determination result of the allele type by direct sequencing.

The K-2 region is shown in SEQ ID NOS: 23 and 24 designed to have the SNP at position 235 of SEQ ID NOS: 18 and 19 at the 3 'end, and SEQ ID NO: 5 paired with these. The oligonucleotides having the nucleotide sequences were synthesized, and PCR using these as primers for gene amplification was carried out using the above bovine genomic DNA as a template DN.
As A, a PCR reaction was performed to amplify the gene.

The composition of the PCR reaction solution and the reaction conditions were the same as those for the P-1 region of the K-1 region except that the annealing temperature was 60 ° C. when the primer sets were SEQ ID NOS: 23 and 5.
Same as CR. As a result, as shown in FIG. 4 (b), the allele type of the K-2 region was able to be determined, which coincided with the determination result of the allele type by direct sequencing.

It is to be noted that in FIG. 4, the sample from which a band appears has the allele. M indicates an electrophoretic image of a 200 bp ladder marker.

Table 2 shows the allele types in each lane of the electrophoresis shown in FIG.

[0064]

[Table 2] Table 2 ──────────────────────────────────── Lane No. Allele type (K- 1 / K-2) ──────────────────────────────────── 1 CC / EE 2 CC / EE 3 AC / DE 4 AC / DE 5 AA / DD 6 AA / DD ── The present invention relates to the nucleotide chain (SEQ ID NOS: 20 and 21, and
SEQ ID NOS: 23 and 24). In addition, the present invention relates to a nucleotide set in which these SNPs are located at the 3 ′ end, and a primer set that forms a pair as a primer for gene amplification (SEQ ID NO: A set of nucleotide chains represented by 22, and
A set of nucleotide chains represented by SEQ ID NO: 5 paired with primers having the nucleotide sequences represented by SEQ ID NOS: 23 and 24).

The present invention also relates to a nucleotide chain represented by SEQ ID NOS: 20 and 21 and / or SEQ ID NOS: 23 and 2
Using the nucleotide chain represented by No. 4 as one of a pair of primers for gene amplification, and using the gene amplification product obtained by amplifying the gene of the test cow as information on polymorphic bases in the gene sample of the cow, A method for identifying a breed of a test cow by associating information on a polymorphic base in a gene sample with a breed of the test cow is provided.

Arrays made from DNA extracted from meat
Approximately 0.1 g of a piece of meat is cut out from beef sold in a supermarket and analyzed using the QIAamp DNA Mini Kit (QIAGEN).
Was used to extract DNA according to the attached instructions. The extracted DNA was colorimetrically determined, dissolved in TE to a final concentration of 20 ng / μl, and stored at 4 ° C.

By PCR using the primers for gene amplification represented by SEQ ID NOS: 5 and 20 to 24, the allele types of the K-1 and K-2 regions of each meat sample were determined to obtain 3
% Agarose gel was electrophoresed and analyzed based on the presence or absence of a band. The breed of cattle on which the beef is based is unknown because some breeds are not specified, but when the allele type was determined to be Holstein or other, the third
The results shown in the table were obtained.

[0068]

[Table 3]

From the above results, it was clarified that it is possible to identify the breed as the basis of the beef, such as whether or not the commercially available beef is Holstein beef, by the present identification method.

[0070]

According to the present invention, there is provided a method for efficiently discriminating beef varieties based on beef from actually sold beef as a material. There is provided a method for determining whether or not beef is derived from Holstein breeds, which may be confused with the breed and the like by consumers.

[0071]

[Sequence List] SEQUENCE LISTING <110> BML, INC. <120> Method for judging breed of bovine <130> PBM57 <140> <141> <160> 24 <170> PatentIn Ver. 2.1 <210> 1 <211> 10 <212> DNA <213> bovine <400> 1 gtcttgcgga 10 <210> 2 <211> 10 <212> DNA <213> bovine <400> 2 tcgccgcaaa 10 <210> 3 <211> 20 <212> DNA < 213> bovine <400> 3 tctgtcaggg aagttgcaga 20 <210> 4 <211> 20 <212> DNA <213> bovine <400> 4 tcttcggggg cagtgagaat 20 <210> 5 <211> 20 <212> DNA <213> bovine < 400> 5 tcacccctcg ttgtctcaaa 20 <210> 6 <211> 19 <212> DNA <213> bovine <400> 6 ctccaggtca acggcattc 19 <210> 7 <211> 24 <212> DNA <213> bovine <400> 7 ataaaggcct cttgaactct tgga 24 <210> 8 <211> 24 <212> DNA <213> bovine <400> 8 gtctcaagta ctctaacatt ccac 24 <210> 9 <211> 25 <212> DNA <213> bovine <400> 9 ggagctaaac tcggtggcaa gcttc 25 <210> 10 <211> 25 <212> DNA <213> bovine <400> 10 tctggcattg cttgtgatac ccctg 25 <210> 11 <211> 20 <212> DNA <213> bovine <400> 11 cttgaactct tggagccgtg 20 <210 > 12 <211> 20 <212> DNA <213> bovine <400 > 12 ctctaacatt ccacttacgg 20 <210> 13 <211> 20 <212> DNA <213> bovine <400> 13 gcttcagcag gtgtgttaca 20 <210> 14 <211> 20 <212> DNA <213> bovine <400> 14 ccctgagctc ctgagctgtg 20 <210> 15 <211> 360 <212> DNA <213> bovine <400> 15 gtgtttcctg gttagcgtaa tctttttaat attttgtgtg gttacaagtg attcacatgt 60 tttccaaatg ttctgtcagg gaagttgcag aaaatagttg agattgttct cgtgtgtcct 120 tcccctgctc tcccgaagca ctgtgctcag gtctgcgtga cgccagacca gaggcctaac 180 ccggagctga ccacattcat aggcactttg gtgtggtttt ggtctctggt gtatagaatt 240 ctgtgtaatt gtatcacttg catagattca agtaaccact gccactgtca ggattagaac 300 attctcactg cccccgaaga accctcctca tgctgcccac tgggcgtcac accctgaacc 360 <210> 16 <211> 360 <212> DNA <213> bovine <400> 16 gtgtttcctg gttagcgtaa tctttttaat attttgtgtg gttacaagtg attcacatgt 60 tttccaaatg ttctgtcagg gaagttgcag aaaatagttg agattgttct cgtgtgtcct 120 tcccctgctg tcccgaagca cggtgctcag gtctgcatga cgccagacca gaggcctaac 180 ccggagctga ccacgttcat aggcactttg gtgtggtttt ggtctctggt gtatagaatt 240 ctgtgtaatt gtatcact tg catagattca agtaaccacc gccactgtca ggattagaac 300 attctcactg cccccgaaga accctcctca tgctgccccc tgggtgtcac accctgaacc 360 <210> 17 <211> 360 <212> DNA <213> bovine <400> 17 gtgtttcctg gttagcgtaa tctttttaat attttgtgtg gttacaagtg attcacatgt 60 tttccaaatg ttctgtcagg gaagttgcag aaaatagttg agattgttct cgtgtgtcct 120 tcccctgctg tcccgaagca cggtgctcag gtccgcatga cgccagacca gaggcctaac 180 ccggagctga ccacgttcat aggcactttg gtgtggtttt ggtctctggt gtatagaatt 240 ctgtgtaatt gtatcacttg catagattca agtaaccacc gccactgtca ggattagaac 300 attctcactg cccccgaaga accctcctca tgctgccccc tgggtgtcac accctgaacc 360 <210> 18 <211> 360 <212> DNA <213> bovine <400> 18 gccactcaca gctgcacggg gaccccgcga cccacaccct cacccctcgt tgtctcaaac 60 atgatatgca gttcctgctc cagaagccca ggcccaatgt catagttgaa gaatctggtt 120 cttgtgccag ctctcttcgg taccattttt caatttcaga aagattaaaa cagacagcca 180 ctgaagccgg tcatgtatct gctttagaaa aacagttaca gcactaattt gcggcagaaa 240 tcttcagatc cattcagaag gtgcaaaaga tgaaaaaaaa aaaaaagatg ctgctatatc 300 acgtgtgaa t gccgttgacc tggagtcatt tcttatactg tgtgaaggga agtcagtgta 360 <210> 19 <211> 360 <212> DNA <213> bovine <400> 19 gccactcaca gctgcacggg gaccccgcga cccacaccct cacccctcgt tgtctcaaac 60 atgatatgca gttcctgctc cagaagccca ggcccaatgt catagttgaa gaatctggtt 120 cttgtgccag ctctcttcgg taccattttt caatttcaga aagattaaaa cagacagcca 180 ctgaagccgg tcatgtatct gctttagaaa aacagttaca gcactaattt gcggtagaaa 240 tcttcagatc cattcagaag gtgcaaaaga tgaaaaaaaa aaaaaagatg ctgctatatc 300 acgtgtgaat gccgttgacc tggagtcatt tcttatactg tgtgaaggga agtcagtg 360 <210> ac <210> ac <210> ac <210> g 21 <212> DNA <213> bovine <400> 21 cacaccaaag tgcctatgaa c 21 <210> 22 <211> 24 <212> DNA <213> bovine <400> 22 gtgtggttac aagtgattca catg 24 <210> 23 <211> 23 < 212> DNA <213> bovine <400> 23 ctgaatggat ctgaagattt ctg 23 <210> 24 <211> 23 <212> DNA <213> bovine <400> 24 ctgaatggat ctgaagattt cta 23

[0072]

[Brief description of the drawings]

FIG. 1. RAPD-P with K-1 and K-2 primers
It is the figure which showed the migration pattern of CR.

FIG. 2: S detected by K-1 and K-2 primers
It is the figure which showed NP and allele type.

FIG. 3 shows genomic nucleotide sequences of K-1 and K-2 regions and S
It is the figure which showed NP.

FIG. 4 is a drawing showing the results of allele analysis by PCR using allele-specific primers.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Tsuyoshi Saito 1361-1 Matoba, Kawagoe City, Saitama Prefecture Within BML Research Institute, Inc. Hara Odakura 1 Animal Genetics Research Institute affiliated with Japan Livestock Technology Association (72) Inventor Koji Tahara 1361-1, Matoba, Kawagoe-shi, Saitama Prefecture Within BML Research Institute, Inc. (72) Inventor Hajif Yamazaki Saitama 1361-1 Kawagoe-shi Matoba FLM Co., Ltd. F-term (reference) 4B024 AA10 AA11 CA20 HA11 4B063 QA01 QA13 QA18 QQ02 QQ42 QR08 QR62 QS25

Claims (11)

[Claims] 1. A continuous base sequence selected from the base sequences represented by SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17, 130, 142, 154, 1
A nucleotide chain comprising at least one base selected from the group consisting of the 57th, 195th, 280th, 339th and 345th bases, and having 10 to 360 bases. 2. A continuous nucleotide sequence selected from the nucleotide sequences represented by SEQ ID NO: 18 or SEQ ID NO: 19,
A nucleotide chain containing the 35th base and having 10 to 360 bases. 3. It is possible to hybridize with the nucleotide chain of the nucleotide sequence represented by SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17, and to have a polymorphic base (SEQ ID NO: 15, SEQ ID NO: No. 130, No. 142, No. 1 in the base sequence represented by No. 16 or SEQ ID NO: 17
54th, 157th, 195th, 280th, 33rd
A nucleotide chain having 10 to 45 bases and having a base corresponding to the ninth or 345th base). 4. The nucleotide chain according to claim 3, which is a nucleotide chain of the nucleotide sequence represented by SEQ ID NO: 20 or SEQ ID NO: 21. 5. A primer set for gene amplification comprising a nucleotide chain according to claim 4 and a nucleotide chain having the nucleotide sequence represented by SEQ ID NO: 22. 6. It is possible to hybridize with the nucleotide chain of the nucleotide sequence represented by SEQ ID NO: 18 or SEQ ID NO: 19, and to have a polymorphic base at the 3 ′ end (SEQ ID NO: 18 or SEQ ID NO: 19). Base number corresponding to 235th base in the base sequence
45 nucleotide chains. 7. The nucleotide chain according to claim 6, which is a nucleotide chain of the nucleotide sequence represented by SEQ ID NO: 23 or SEQ ID NO: 24. 8. A nucleotide sequence comprising the nucleotide chain according to claim 7 and the nucleotide sequence represented by SEQ ID NO: 5.
Primer set for gene amplification. 9. A method for identifying a breed of a test cattle by associating the information on the polymorphic base in the gene sample of the cattle with the breed of the test cattle, wherein the information on the polymorphic base is as follows: Is information represented by
Cattle breed identification method. Information on which of the nucleic acid contained in the gene sample has a base sequence among the base sequences containing the polymorphic base represented by SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17. Information on which of the nucleotide sequences of the nucleic acid contained in the gene sample contains the polymorphic base represented by SEQ ID NO: 18 or SEQ ID NO: 19. 10. A gene obtained by amplifying a test cow gene using the nucleotide chain of the nucleotide sequence of claim 3 and / or the nucleotide chain of claim 6 as one of a pair of primers for gene amplification. Using the amplified product as information on polymorphic bases in a bovine gene sample,
The method for identifying breeds of cattle according to claim 9. 11. The method of claim 9 or 10, wherein the discrimination of the breed of the test cow includes discrimination as to whether or not the test cow is a Holstein breed.