JP3048149B1 - Rice varieties identification method - Google Patents

Abstract

Abstract: [PROBLEMS] To enable rice varieties to be identified even in the case of rice supplied in the restaurant industry, etc., rice is targeted, and varieties of closely related varieties are not affected by the influence of denaturation of ingredients due to rice cooking. To provide a method that can accurately and simply identify rice varieties. SOLUTION: DNA extracted from cooked rice is used as template DNA, and PC is used in the presence of 8 to 25-mer random primer.
A method for identifying rice varieties, characterized in that the amplified DNA is amplified by the R method and the resulting amplified DNA polymorphism is identified.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for identifying rice varieties.
The present invention relates to a method for identifying a variety of raw rice based on a polymorphism of DNA obtained by amplification by a CR method.

[0002]

2. Description of the Related Art Conventionally, rice varieties have been identified by comparing fertility, rice plant type, rice grain type, enzyme polymorphism and the like when crossed. These conventional techniques are effective for discriminating between Indica rice and Japonica rice, and for discriminating between Indica rice and Japonica rice, which are distantly related. On the other hand, it is not enough to identify closely related rice, and it is not possible to use it for distinguishing between closely related good-tasting rice developed recently with "Koshihikari" as the center. was there. For example, Hitomebore, a good-tasting rice comparable to Koshihikari, was bred by crossing Koshihikari again with Hoshi, a child of Koshihikari and Kimine. However, identification is difficult with the above-mentioned conventional technology.

[0003] With the enforcement of the Food Law, it has been required to indicate the variety, place of origin, and year of production of the milled rice on the milled rice packaging. These indications can be one of the indicators for cultivar identification, but it is extremely difficult to identify closely related varieties of good-tasting rice if there is no information on plants, paddy, or brown rice. .

[0004] Recently, it is necessary to specify varieties of rice supplied as cooked rice in convenience stores, commercial rice cooking services, the restaurant industry, and the like. However, since gelatinization of starch and denaturation of proteins due to rice cooking occur, it is impossible to extract DNA or identify varieties by conventional techniques.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to produce a sample of cooked rice without being influenced by the above-described effects of the denaturation of components caused by cooking, and to accurately determine even a closely related variety.
Moreover, it is an object of the present invention to provide a method capable of identifying rice varieties by a simple method.

[0006] Cooked rice contains DNA derived from plants, paddy, brown rice, and the like, and since this DNA varies depending on the rice variety, it is considered to be an index for variety identification.
However, as described above, since rice is subjected to starch gelatinization and protein denaturation during the rice cooking process, it is extremely difficult to extract DNA from cooked rice as compared with polished rice.

[0007]

Under these circumstances, the present inventors have studied a method for extracting DNA from cooked rice, and a method for extracting DNA from rice.
The purification method of NA was examined. As a result, they have found that highly purified DNA can be extracted in high yield by causing a specific surfactant or enzyme to act on cooked rice.
The extracted and purified DNA has specific properties that differ depending on rice varieties. Therefore, the RAPD method (random amplified polymormor) can be used without confirming its sequence.
Based on the phic DNA method), it was clarified that varieties can be identified only by amplifying using specific primers by a PCR method and analyzing the amplified products by electrophoresis. The present invention has been completed based on such findings.

According to the first aspect of the present invention, rice rice
After the gelatinized starch is decomposed and removed by the action of DNA, the DNA extracted from the cooked rice is used as a template DNA and amplified by a PCR method in the presence of random primers of 8 to 25 mer, and the resulting polymorphism of the amplified DNA is determined. This is a method of identifying rice varieties characterized by identification.

The present invention according to claim 2, wherein the cooked rice is one grain
The method according to claim 1, wherein the rice is cooked rice .

The present invention according to claim 3 is characterized in that the amylase is
The method according to claim 1 or 2 , which is a thermostable amylase having an optimum temperature of 60 ° C or higher, and treating the amylase at a temperature of 50 to 80 ° C.

[0011] The present invention according to claim 4 is a method according to the present invention, wherein the random primer comprises B1 and SEQ ID NO: 2 in SEQ ID NO: 1 in the sequence listing.
B18 described in SEQ ID NO: 3, M2CG described in SEQ ID NO: 3, Q16 described in SEQ ID NO: 4, S13 described in SEQ ID NO: 5, T8 described in SEQ ID NO: 6, D3 described in SEQ ID NO: 7, described in SEQ ID NO: 8 1 selected from the group consisting of M2 of SEQ ID NO: 9, M11 of SEQ ID NO: 9 and T16 of SEQ ID NO: 10
2. The method according to claim 1, wherein the method is a species or two or more species.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. In the method of the present invention, DNA obtained from cooked rice is used as a material for identifying varieties. Here, the cooked rice refers to cooked rice obtained by cooking polished rice with a normal electric rice cooker, gas cooker, steam cooking system, or the like. As a means for extracting and purifying DNA from cooked rice, known methods can be employed. However, since the obtained DNA is used for identifying rice varieties, the DNA can be obtained in high yield. It is necessary to extract without decomposition. That is, it is necessary to extract the DNA while suppressing the enzyme activity so that the DNA is not degraded by the DNA degrading enzyme. Furthermore, in order to extract DNA in high yield,
It is effective to expose DNA by decomposing and removing polysaccharides such as starch, which is a main component of cooked rice, and proteins.

For this purpose, it is preferable to use amylase for extraction and purification from cooked rice. In addition,
Use cetyl trimethylan
Monium bromide (hereinafter sometimes abbreviated as CTAB)
You. ) Ru also effective der be used. CTAB is a type of surfactant and has an action of suppressing the activity of a DNA degrading enzyme. That is, CTAB has the effect of making gelatinized starch easier to extract in the form of micelles and binding to proteins as an amphoteric medium to elute into the extract.

[0014] The conditions for using CTAB were examined.
DNA can be effectively extracted from cooked rice by adding a 20-fold volume, preferably a 3- to 10-fold volume of a 0.2 to 10% concentration, preferably a 0.2 to 6% concentration CTAB solution, preferably an aqueous solution. Became clear. More preferably, a 0.5 to 4% aqueous solution of CTAB is added in an amount of 2 to 8 times the volume of cooked rice. In addition, if necessary,
A DNase inhibitor such as ethylenediaminetetraacetic acid (hereinafter sometimes abbreviated as EDTA) or a pH stable buffer such as Tris-HCl buffer may be used in combination. In addition,
CTAB has been used to extract the DNA of polished rice, but there is no example of applying it to cooked rice in which starch gelatinization and protein denaturation have occurred.
It was not known at all under what conditions it should be used.

Further, in extracting DNA from cooked rice, the use of amylase and / or protease degrades starch and / or protein to lower molecular weight and elutes it into the extract, so that the target DNA is It is exposed and easy to extract.

Here, amylase is a generic term for starch-degrading enzymes, α-amylase which indiscriminately cuts the middle of starch molecules, β-amylase which sequentially cuts β-maltose from the non-reducing end of starch, and non-starch. There is glucoamylase that sequentially cuts glucose from the reducing end. In the present invention, any of these can be used,
It is preferable to use a thermostable amylase for the following reasons.

The thermostable amylase used in the present invention is an amylase having an optimum temperature of 60 ° C. or higher. An ordinary enzyme has an optimum temperature of about 30 to 45 ° C., and a thermostable enzyme having an optimum temperature of 60 ° C. or higher in comparison with this has a shorter reaction time by increasing the reaction temperature, There are advantages that the reaction yield can be increased and the progress of other enzyme reactions can be suppressed.

When amylase is used, 100 to 10
It is preferable to add 0.01 to 0.1 volume, preferably 0.01 to 0.05 volume, to the DNA extract as a 0.1 to 5 mg / mL aqueous solution of 00U / mg amylase. After the addition, the starch is decomposed by an enzyme reaction at a reaction temperature of 30 to 90 ° C., more preferably 60 to 70 ° C., for at least 20 minutes, usually about 30 to 90 minutes. When a thermostable amylase is selected as the amylase, the reaction can be carried out at 50 to 80 ° C., which is higher than the gelatinization temperature of rice starch, so that particularly good decomposition efficiency can be obtained.

Next, protease is a general term for proteolytic enzymes, and examples thereof include pepsin, trypsin, and chymotrypsin. In the present invention, various proteases can be used, but those having high enzyme activity even in the presence of a surfactant, that is, enzymes having high reactivity and properties such as high cleavage efficiency are preferable, for example, It is preferable to use an enzyme (trade name: Protease K, manufactured by Worthington Biochemical Co.) produced by Tritirachium album . This enzyme has a wide reaction specificity, cleaves at the carboxyl group side of aromatic amino acids, hydrophobic amino acids, aliphatic amino acids, etc., and further has sodium lauryl sulfate (hereinafter sometimes abbreviated as SDS), agarose, acrylamide. Has the advantage of expressing the activity even in the presence of By causing protease to act on cooked rice, proteins in the cooked rice can be decomposed and subdivided. Furthermore, since DNAse is also a protein, it is degraded by the action of the enzyme, and D
NA decomposition can be prevented.

When a protease is used, 10 to 10
As a 5-50 mg / mL aqueous solution of 00 U / mg protease, 0.01-0.1 volume, preferably 0.02-0.05 volume, is added to the DNA extract,
More preferably at 25 to 40 ° C for 10 minutes or more, usually 3
The protein is decomposed by reacting for about 0 to 120 minutes.
In addition, a surfactant such as SDS or a pH stabilizer such as a Tris-HCl buffer may be used in combination in order to promote the solubilization of the protein and the efficiency of the enzymatic action.

In addition, after amylase or protease is allowed to act on cooked rice, CTAB is added and extracted.
DNA can be extracted more efficiently. CTA
After extracting DNA from cooked rice with high efficiency by using B or enzymatic treatment, purification is carried out by a conventional method in order to remove impurities such as protein and its degradation products, starch and its degradation products, lipids and polyphenols. In this case, the DNA can be purified by applying phenol treatment, chloroform treatment, ethyl alcohol treatment, etc., alone or in combination.

Next, in the present invention, DNA extracted and purified from cooked rice as described above is used as a template,
PCR reaction (polyme
rasechain reaction). Here, the PCR reaction refers to denaturation (dissociation of the double-stranded DNA into single-stranded DNA at about 94 ° C.), binding (binding of a DNA fragment called a primer to the dissociated single-stranded DNA, and a partial ), Elongation (the DNA strand is extended by the action of the heat-resistant DNA polymerase from the double-stranded binding primer portion, and each single-stranded DNA is duplicated into a double-stranded DNA.)
Is a reaction that amplifies the original DNA about 1,000,000 times by repeating the above three steps a plurality of times, usually 20 to 50 times. In addition, the template DNA refers to the PCR
The original DNA that is amplified in the reaction.

The primer used in the present invention is PC
From the viewpoint of the amplification rate of the DNA in R and the possibility of discriminating the variety by the amplified DNA, random primers of 8 to 25 mer, more preferably 10 to 12 mer, are most suitable. When the primer is a DNA fragment shorter than the octamer, it binds non-specifically to many portions of rice DNA, so that the sum of the amplified bands is too large and the discrimination and reproducibility are reduced. On the other hand, a fragment larger than a 25-mer is inappropriate because the specificity becomes too high, the number of amplified DNAs decreases, and the discriminability decreases.

Specific examples of the primer used in the present invention include primer B1 described in SEQ ID NOS: 1 to 10 in the sequence listing;
B18, M2CG, Q16, S13, T8, D3, M
2, M11 and T16, of which one or two
It is preferred to use a combination of more than one species. The sequences of these primers have been selected or synthesized by the present inventors in order to select those that can more accurately identify varieties. In addition, primers B1, B18,
M2CG, Q16, S13, and T8 (see the nucleotide sequences described in SEQ ID NOs: 1 to 6 in the sequence listing) were selected by the method described in Examples. In addition, primer D
3, M2, M11 and T16 (SEQ ID NOs: 7 to
10) are selected based on the reproducibility of the band pattern amplified by PCR and the clarity of the discrimination band.

[0025] In the present invention, PCR is performed using the extracted DN.
Except for using A as a template and the above-mentioned primer, it may be carried out according to a conventional method. For example, 10 to 50 ng of template DNA, 0.5 to 10 ng of primer, 0.5 to 5 U of DNA polymerase, 50
~ 500 μM deoxyribonucleotide triphosphate mixed solution, 1-5 μM magnesium chloride and 10-50 m
Mix M Tris-HCl buffer (pH 8). Then
The mixture is denatured at 90-98 ° C., 35-65.
C. binding, 60-80 ° C. elongation
The DNA is amplified by repeating about 50 cycles. DNA
As the amplifying device, a commercially available product can be used. For example, PC-700 manufactured by Astec Co., Ltd., Takara Shuzo Co., Ltd., thermal cycler MP, or the like can be used.

In the present invention, the thus amplified D
Analyze NA polymorphisms. That is, for the DNA amplified by the PCR, the variety of the raw rice is identified based on the polymorphism. Polymorphism refers to the presence of diversity in traits and morphology among normal individuals in the same species. In the present invention, DNA extracted and amplified from various types of rice (rice cooked rice) has a molecular weight and a base sequence of DNA. Refers to the emergence of diversity.

Specifically, the DNA amplified by the PCR method is separated by a molecular weight distribution using a conventional method such as agarose gel electrophoresis or acrylamide gel electrophoresis, stained with ethidium chloride, etc. This is a method of taking a picture by irradiating. The band of each sample shown in the obtained electrophoresis photograph is compared with the band of the sample in which the cultivar is known in advance.
It is possible to judge which kind it belongs to.

[0028] In this manner, the present invention, amylase
Since DNA can be extracted from cooked rice with high yield and high purity by applying zea , this DNA can be amplified by the PCR method and the amplified DNA can be analyzed to accurately identify varieties. It is possible. Therefore, it is possible to identify varieties using DNA extracted from a sample of only one piece of cooked rice.

[0029]

[Preparation of cooked rice sample]

"Koshihikari" produced in 1994 (obtained from Hokuriku Agricultural Test),
"Hitmebore", "Akitakomachi", "Sasanishiki",
"Nipponbare", "Hinohikari", "Mutsu Homare", "Kinuhikari" (obtained from Agricultural Research Center), "Yukihikari", "Kirara 397" (obtained from Hokkaido Agricultural Trial and Hokkaido Agricultural Trial) Was used as a sample. These varieties are the top 10 varieties in Japan in 1994. In addition, sterilized cooked rice manufactured by Sato Food Industry Co., Ltd. was purchased and used as commercially available cooked rice.

Each rice sample is taken one by one into a plastic tube (assist, 1.5 mL capacity), and 35 μl of deionized water is taken.
L, stand on a stand, soak for 1 hour, cook rice in an open state with an electric rice cooker (RC-183, manufactured by Toshiba Corp., add 75 mL of deionized water to the outer pot) for about 15 minutes,
Steamed for a minute to make a cooked rice sample.

[Extraction of DNA] DNA was extracted by the following three methods. (1) Enzymatic method (using amylase and protease)
A sample of each sampled rice is placed in a microtube, and a Tris / hydrochloric acid buffer (100 mM, pH 8.0, 100 mM Na
(Containing Cl) was added and pulverized with a pellet mixer. Then, 5 μL of a thermostable amylase (manufactured by Sigma, α-amylase, 790 U / mg solid, 1 mg / mL) was added.
The mixture was added and reacted at 60 ° C. for 1 hour.

Further, protease K derived from Tritirachium album (manufactured by Worthington Biochemical Co., Ltd., 20 m
g / mL) and reacted at 37 ° C. for 2 hours. After the enzyme reaction, ethanol 1 cooled to -20 ° C
After adding mL, the mixture was allowed to stand at −20 ° C. for 15 minutes, and then centrifuged (15000 G, 4 ° C., 15 minutes, the same applies hereinafter) with a microcentrifuge to obtain a precipitate.

This precipitate was mixed with 300 μL of TE solution (10
mM Tris / HCl, pH 8.0, 1 mM EDTA)
, 400 μL of phenol was added, and the DNA was extracted with a rotary stirrer for 30 minutes. Then, centrifugation (150
00G, 4 ° C, 15 minutes) to collect the supernatant, add an equal amount of PCI (phenol / chloroform / isoamyl alcohol: 25/24/1), extract for 30 minutes, and centrifuge (15000G, 4 ° C). , 15 minutes), and 6 mL of 5M NaCl was added to the supernatant obtained. Then, 400 μL of cooled ethanol was added, and the precipitate obtained by centrifugation was washed twice with 70% ethanol, and the final precipitate was washed with 40 μL.
Was dissolved in a 10-fold diluted TE solution to prepare a DNA sample solution.

(2) CTAB Method One grain of cooked rice was placed in a 1.5 mL eppendorf tube, and 150 μL of a 2% CTAB solution at 55 ° C. and then 150 μL of a 1% CTAB solution were added thereto. Was ground. Thereafter, the DNA was extracted at 55 ° C. for 30 minutes, and then subjected to chloroform / isoamyl alcohol treatment.
Precipitation buffer (1% CTAB, 20 mM Tris-HCl,
An equal volume of 10 mM EDTA (pH 8) was overlaid, and allowed to stand at 4 ° C. overnight to precipitate DNA.

Next, 400 μL of 1M NaCl was added to dissolve the DNA, and precipitated with isopropanol, 70%
After washing with ethanol, it was dissolved in a Tris / EDTA buffer (TE solution) to obtain a template DNA sample solution.

(3) Method using a commercially available kit A commercially available DNA extraction kit (manufactured by Nippon Gene Co., Ltd.)
DNA was extracted from one grain of cooked rice using ISOPLANT). That is, one grain of cooked rice is put into an Eppendorf tube, and Solution I (manufactured by Nippon Gene Co., Ltd.) is 300
After adding and crushing μL, 150 μL of Solution II (manufactured by Nippon Gene Co., Ltd.) was added, and DNA was extracted at 50 ° C. for 30 minutes. Then, Solution III (manufactured by Nippon Gene Co., Ltd.) was added, and the mixture was inverted and mixed for 1 minute.

Then, after leaving still in an ice bath for 15 minutes, the DNA precipitate obtained by centrifugation (15000 G, 4 ° C., 15 minutes) was dissolved in 150 μL of 0.1 times TE solution, and treated with phenol / chloroform. , After ethanol treatment,
It was dissolved in 40 μL of 0.1 times TE solution to obtain a template DNA sample solution.

(4) Results and Discussion DN from one grain of cooked rice of 10 test varieties such as "Koshihikari"
As for the amount of A extracted, the above (1) enzymatic method, (2) CTA
Table 1 shows the results of various extractions by the method B and (3) the method using a commercially available kit.

[0039]

[Table 1]

Also, the varieties "Nihonbare" and "Kirara 397"
For the cooked rice sample of (1) to (3), the extracted DNA spectroscopy (220
３２０320 nm) are shown in FIGS. 1 and 2, respectively.

The following can be seen from the results shown in Table 1, FIG. 1 and FIG. First, as a whole, it was (1) extraction by the enzymatic method that sufficient template DNA could be obtained in terms of quantity and quality for all 10 varieties.

When comparing the amount of extracted DNA, out of the 10 varieties tested, 8 varieties excluding "Hitomebore" and "Kinuhikari" showed the highest amount of extraction in the case of the enzyme method. In the remaining two varieties, the amount extracted by the method using a commercial kit was the largest. However, the absorbance at 220 nm of the extracted DNA obtained by the method using the commercial kit is higher than the measured value of the DNA obtained by other methods (see FIGS. 1 and 2). It was clear that carbohydrates etc. were not sufficiently removed.

When the CTAB method was used, the amount of DNA extracted was low in all varieties (see Table 1).
Is apparent from FIG. 1 and FIG.
The purity of NA was sufficient. This result shows that D
When extracting DNA from cooked rice compared to NA extraction,
It is conceivable that starch gelatinized by cooking rice, partially decomposed polysaccharides or oligosaccharides, denatured proteins, or the like hinder extraction.

As described above, by performing extraction while decomposing gelatinized starch or denatured protein using amylase or protease , the amount required for several rounds of PCR from one grain of rice is obtained.
It was also found that highly pure DNA could be efficiently extracted and purified. Also, even if the CTAB method is used,
Although the DNA amount was insufficient, it was clear that the purity of the extracted DNA was sufficiently maintained.

Example 2 [Amplification of DNA by PCR] Amplification of DNA by PCR was performed as follows. Using the DNA prepared from cooked rice by the above three methods as a template, the DNA was amplified according to a conventional method. Taq Polymerase manufactured by Toyobo Co., Ltd. was used as a DNA polymerase, and random primers (10-mer) (5 types) and 1
M2CG obtained by polymerizing two bases (CG) to the zero-mer M2 was used.

The composition of the reaction solution was determined according to the instruction manual of Operon, using 25 ng of the template DNA and the primer 3.0.
ng, 1 U of polymerase, 100 μM dNTP (deoxyribonucleotide triphosphate mixed solution) 1.0 μM
L, 2.0 μL of 2.5 mM MgCl ₂ solution, 10-fold dilution buffer (12 mM Tris-HCl, pH 8.3, 60 m
(MKCl) 2.5 μL, sterile water 10.0 μL, and the total volume of the reaction system was 20.7 μL. DNA amplification is 9
45 minutes under conditions of 4 ° C. for 1 minute, 36 ° C. for 1 minute, and 72 ° C. for 2 minutes.
Cycled. PCR includes Astec equipment (P
C-700, 0.5 μL block) was used.

[Electrophoresis of DNA] The electrophoresis of the DNA amplified by PCR was performed as follows. After completion of the PCR, a loading buffer (30% glycerin, bromophenol blue 0.1%) was added to the reaction solution.
(25%, sterile water 69.75%)
After removing mineral oil with L of chloroform, 15
Add μL to agarose gel (Sawada Technology, Ul.
tra-pure Agarose, 2%), and using a long electrophoresis submarine gel electrophoresis apparatus (manufactured by Nippon Aido).
Run for 180 minutes at V. DNA staining and electrophoresis pattern imaging were performed according to a conventional method. As a DNA molecular weight marker, Marker 4 (φX174 phage, Hae III) manufactured by Wako Pure Chemical Industries, Ltd. was used.

[0048] The electrophoresis pattern of the DNA amplification product obtained by extracting from "Koshihikari" by each of the above-mentioned methods (1) to (3) and the extraction and amplification from commercially available cooked rice by the enzymatic method of (1). FIG. 3 shows the electrophoresis pattern of the DNA. For comparison, FIG. 3 also shows the results of amplified DNA derived from polished rice (variety: Nipponbare, Koshihikari).

As is apparent from FIG. 3, when the method using the commercially available kit was applied, amplification by PCR was not carried out, possibly due to insufficient purification as described above, and no DNA band appeared (lane in FIG. 3). 3). On the other hand, the enzymatic method and C
In the case of the TAB method, amplification by PCR was carried out smoothly, and the discrimination band of “Koshihikari” for “Nipponbare” clearly appeared (lanes 4 and 5 in FIG. 3). Furthermore, in the case of DNA extracted from commercially available sterile cooked rice by the enzymatic method, PC
R was performed smoothly, and the discrimination band clearly appeared (lane 6 in FIG. 3).

From this, it can be seen that amylase and protease
In by observing the migration patterns of DNA by RAPD method using the extracted from rice DNA processing, can be accurately identified varieties rice, and the method,
It was shown that the method can be applied to not only experimental-scale cooked rice samples but also commercially available cooked rice that has been cooked and distributed.

Example 3 (Mixed rice test of two varieties of rice) Samples of "Koshihikari" marked with magic ink and "Nipponbare" (unmarked) were each taken in a pudding cup.
Six times the volume of water was added and rice was cooked in an electric kettle. After the rice was cooked, DNA was extracted from the cooked rice divided into two groups according to the presence or absence of a mark by the enzymatic method of Example 1, and PCR and electrophoresis were performed using the DNA. In addition, the rice was obtained by cooking the above two varieties of rice alone, and the same test was conducted.

As apparent from FIG. 4, after mixing and cooking “Nipponbare” and “Koshihikari”, the DNA migration pattern of each sample of “Nipponbare” and “Koshihikari” extracted and separated (lanes 2 to 4 respectively) And lanes 6 to 8) are the same as the DNA migration patterns (lanes 1 and 5) when each variety was cooked independently and extracted in the same manner. From this result, the DNA of rice obtained by mixing and cooking a plurality of types of rice of different varieties remained in each rice grain without eluting and moving during cooking, and the method of the present invention provided It became clear that even in the mixed sample, it was possible to identify which kind of rice the sample was made of.

Example 4 (Selection of primers) Using the same sample rice as in Example 1, DNA of cooked rice was extracted by an enzymatic method, and PCR was performed using the extracted DNA as a template in the presence of various primers. The electrophoresis patterns of the amplified DNAs were compared. Among these, B1, B18 and M2 were used as primers having good reproducibility and discrimination.
CG, Q16, S13 and T8 (SEQ ID NO: 1 in the sequence listing)
6) were selected. DN extracted from one grain of cooked rice
Reproducibility of RAPD method using A as a template and clarification of identification 2
Considering the points, there is a band with a clear difference (+),
Table 2 shows the results of the evaluation in two categories of no (-). In addition, PCR using each primer
The results of electrophoresis of the amplification products are shown in FIGS.

[0054]

[Table 2]

[Footnote to Table 2] +: With discrimination band-: Without discrimination band The numerical value of the discrimination band indicates the molecular weight (kbp).

From the results in Table 2 and FIGS.
By using six types of primers B1, B18, M2CG, Q16, S13, and T8,
It has been found that all 0 domestic varieties can be identified.

Example 5 (Relationship Between DNA Extraction Conditions and PCR Conditions) Koshihikari was used as a sample rice, put in a plastic tube one by one in the same manner as in Example 1, and cooked in an electric kettle. Using this sample cooked rice, under the conditions shown in Table 3, D
NA was extracted, and the obtained DNA was used as a template DNA in equal volumes (10 μL) at a time under the same conditions as in Example 1.
CR was performed. The results are shown in Table 3 together with the extraction conditions.

[0058]

[Table 3]

As shown in Table 3, the CTAB concentration was set at 0.
When the concentration was too low as 1% (No. 1), when the amylase concentration was too low (Nos. 2 and 3), and when the CTAB concentration was too high as 15% (No. 5), the DNA was all used.
Was not sufficiently extracted, and the DNA was not amplified by PCR. On the other hand, when the 2% CTAB solution was added twice (No. 4), the DNA extraction amount, PCR
In both cases, good results were obtained. From this, when extracting DNA from cooked rice, simply use Amilla
It is not possible to sufficiently identify the target variety only by adding zeolites or proteases , and it is necessary to specify these use conditions in order to effectively identify the variety.
In addition, No. As in 6, the method of adding amylase and protease and treating and then extracting with CTAB also showed extremely good results.

[0060]

According to the method of the present invention, even in cooked and processed rice samples, DNA is extracted after enzymatic treatment with amylase or protease , amplified by PCR using appropriate primers, and the polymorphism is analyzed. By identifying, it became possible to identify rice varieties.

The enzyme treatment for DNA extraction is carried out by using DN.
Carbohydrates and proteins can be decomposed and solubilized and removed while suppressing the action of A-degrading enzyme, and the DNA extraction effect can be enhanced. Genes are peculiar to varieties and do not change depending on the place of origin or storage. It is a technology that can be identified without receiving it, and can respond to new needs in the industry.

Further, since it is possible to identify varieties at the stage of cooked rice, it is possible to verify the identity of the product of raw rice in the processing field such as consigned rice cooking, and to check the raw materials of cooked rice sold at convenience stores and supermarkets. It is possible to discriminate rice varieties, judge the raw rice of cooked rice in the restaurant and restaurant industries, and determine the varieties of imported cooked rice.

The cultivar determination of rice according to the conventional method has been performed based on various information such as cross fertility of rice, plant type, rice grain type, and enzyme polymorphism. However, according to the present invention, it is possible to identify rice varieties based on only the genetic information of the rice, and the effect is extremely large.
In addition, since the method of identification is relatively easy and the experimental equipment used is relatively inexpensive, the fact that on-site surveys can be easily performed at each stage of distribution, use, and consumption of cooked rice is a major feature. I can say.

[Brief description of the drawings]

Fig. 1 DNA extracted from one rice cultivar “Nipponbare”
3 is a graph showing a spectrum of the sample.

[Explanation of symbols]

The solid line, the dotted line, and the dashed line in the graph show the spectra obtained by the enzyme method, the CTAB method, and the commercial kit method, respectively.

FIG. 2 is a graph showing a spectroscopic spectrum of DNA extracted from one rice cultivar “Kirara 397”.

[Explanation of symbols]

The solid line, the dotted line, and the dashed line in the graph show the spectra obtained by the enzyme method, the CTAB method, and the commercial kit method, respectively.

[Fig. 3] PC of each DNA obtained from rice varieties "Nipponbare" and "Koshihikari" by various extraction methods from polished rice and cooked rice
It is a photograph which shows the electrophoresis pattern of R amplification product.

[Explanation of symbols]

M is a molecular weight marker, and the numerical values of lanes 1 to 6 are, in order, amplification products of extracted DNA of "Nipponbare" (polished rice), amplification products of extracted DNA of "Koshihikari" (polished rice), and a commercial kit method of "Koshihikari" (rice rice). Of Koshihikari (rice cooked rice) by CTAB method
The results of the amplification product of NA, the amplification product of extracted DNA of “Koshihikari” (rice cooked rice) by the enzymatic method, and the amplification product of the extracted DNA of commercially available cooked rice by the enzyme method are shown.

FIG. 4 is a photograph showing an electrophoresis pattern of a PCR-amplified product of DNA extracted from cooked rice obtained by cooking two types of rice varieties alone or in combination.

[Explanation of symbols]

M is a molecular weight marker, 1 is “Nihonbare” (alone cooked rice),
2-4 are "Nipponbare" (isolated after mixing and cooking with "Koshihikari"), 5 is "Koshihikari" (single rice cooking), 6-8
Indicates the results of each amplification product of "Koshihikari" (isolated after mixing and cooking with "Nipponbare").

FIG. 5: Various types of cooked rice D amplified using primer B1
It is a photograph which shows the electrophoresis pattern of NA.

FIG. 6 is a photograph showing an electrophoresis pattern of various cooked rice DNAs amplified using a primer B18.

FIG. 7 is a photograph showing an electrophoresis pattern of various cooked rice DNAs amplified using the primer M2CG.

FIG. 8 is a photograph showing an electrophoresis pattern of various cooked rice DNAs amplified using primer Q16.

FIG. 9 is a photograph showing an electrophoresis pattern of various cooked rice DNAs amplified using the primer S13.

FIG. 10 is a photograph showing an electrophoresis pattern of various cooked rice DNAs amplified using a primer T16.

[Explanation of symbols]

Lanes 1 to 10 are respectively “Koshihikari”,
"Hitmebore", "Akitakomachi", "Sasanishiki",
The results of "Nipponbare", "Hinohikari", "Yukihikari", "Kirara 397", "Mutsu Homare", and "Kinuhikari" are shown. M represents a DNA molecular weight marker.

Continued on the front page (73) Patent holder 399008313 Hiroshi Okadome 2-11, Azuma, Tsukuba-shi, Ibaraki Prefecture 804 Building 402 No. 402 (72) Inventor Kenichi Otsubo 4227-10 Ami, Ami-cho, Inashiki-gun, Ibaraki Prefecture (72) Inventor Sumiko Nakamura 52-13 Hoyodai, Kashizaki-cho, Inashiki-gun, Ibaraki-ken Bldg. 402 (72) Inventor Shinji Kawasaki 3-5-2-665-1 Namiki, Tsukuba, Ibaraki Prefecture (56) References Japan Society for Food Science and Technology, Vol. 46, No. 3 (1999. Mar. 15) p. 117-122 Food Research Institute Research Report, No. 62 (1998) p. 9-12 Journal of Japan Society for Food Science and Technology, Vol. 44, No. 5 (1997) p. 386-390 Research Result Information Hokkaido Agriculture, Vol. 1996 (1997) p. 38-39 Breeding Journal, Vol. 48, separate volume 2 (1998) p. 115 Breeding Journal, Vol. 48, Appendix 1 (1998) p. 36 Japan Society for Food Science and Technology, Vol. 46, No. 4 (1999. Apr. 15) p. 262 −267 (58) Fields surveyed (Int. Cl. ⁷ , DB name) C12Q 1/68 C12N 15/09-15/11 G01N 33/10 BIOSIS (DIALOG) JICST file (JOIS) WPI (DIALOG)

Claims (4)

(57) [Claims] 1. A gelatinized starch obtained by reacting amylase on cooked rice.
After decomposing and removing the DNA, the DNA extracted from the cooked rice is used as template DN.
A. A method for identifying rice varieties, wherein A is amplified by a PCR method in the presence of random primers of 8 to 25 mer, and the resulting polymorphism of the amplified DNA is identified. 2. The cooked rice is one grain of cooked rice.
Way . 3. The amylase is a thermostable amylase having an optimum temperature of 60 ° C. or higher, and 50 to 50
The method according to claim 1 or 2, wherein the treatment is performed at a temperature of 80 ° C. 4. The random primer comprises B1 described in SEQ ID NO: 1, B18 described in SEQ ID NO: 2, M2CG described in SEQ ID NO: 3, Q16 described in SEQ ID NO: 4, and Q16 described in SEQ ID NO: 5 in the sequence listing. S13, T8 described in SEQ ID NO: 6, D3 described in SEQ ID NO: 7, M2 described in SEQ ID NO: 8, M11 described in SEQ ID NO: 9 and T1 described in SEQ ID NO: 10
The method according to claim 1, wherein the method is one or more selected from the group consisting of: