JP2003135082A - Method for determining whether mixed varieties exist in grain or not and mixed varieties - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for simply, rapidly and accurately determining whether grains of other varieties are mixed in good-quality grains such as rice called as 'Koshihikari' or not and the mixed varieties and to provide a kit used therefor. SOLUTION: This method for determining the varieties of grains by DNA is carried out by determining whether grains of other varieties are mixed in good-quality grains or not by using a multiplex PCR method using DNA extracted from the grains or processed products thereof as a template. In the method, a pairing primer group for identification of negative band by which identification band is not expressed in a target varieties and selectively expressed in only mixed varieties is used. This kit for identifying DNA varieties of grains comprises a pairing primer group for identification of negative band by which is not expressed in a target varieties and selectively expressed in only mixed varieties.

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 presence / absence of mixed varieties in grains and mixed varieties. For example, regarding a DNA variety determination method for determining the presence or absence of mixed rice in good-tasting rice and the variety thereof, more specifically, a method of detecting DNA extracted from grains of rice, wheat, corn, barley or the like or processed products thereof Multiplex PCR as template and in the presence of suitable pairing primers
By identifying the mixed varieties based on the band pattern of the amplified DNA.

[0002]

2. Description of the Related Art Conventionally, the price of grains is determined according to the processability and taste of the grains, and the higher the quality of the grains, the higher the price. For example, in rice, “Koshihikari”, which is the top four cultivars in Japan in 2000,
The good-tasting rice represented by "Hitomebore", "Hinohikari" and "Akitakomachi" is traded at a high price in response to consumers' preference for good-tasting rice. But in part,
While mixing rice other than these good-tasting varieties, fraudulently-mixed rice that is sold with a false label has been a problem. Under the revised JAS Law that will be enforced from April 2001, it is obligatory to display the variety of rice, the place of origin, and the year of production in the packaging of rice. The development of technology to do so is needed.

Conventionally, grain varieties such as rice have been identified by the grass type of plants, the grain type of grains, the enzyme polymorphisms of leaves and rice, and the like. However, in the current situation where there are many closely related strains, such as “Koshihikari” and its related strains in Japan, which account for over 70% of the total strains in Japan, it is not possible to discriminate the presence or absence of mixed varieties by the above method. It is possible. The present inventors have developed a technique for discriminating varieties by DNA, such as RAPD method and STS primer method, and have been published by Japan Food Science and Engineering Society Vol. 46 No. 3 (p.117-122), Patent No. 3048149. Specification and JP 2001-95
No. 589. However, with these techniques, although it is possible to clearly identify rice varieties among single varieties, when the identification band overlaps with, for example, "Koshihikari", the difference in the number of identification bands expressed is smaller than that of "Koshihikari". It was difficult to detect about 30 to 30% of varieties of rice or when a plurality of varieties of rice were mixed.

Further, in judging the presence or absence of mixed rice with 3 to 4 kinds of primer sets, even if the DNA band peculiar to mixed rice does not appear, the probability that the variety is a target variety such as good-tasting rice is high. 88-94% (= 1- (0.
5) It is only ^3-4 ), and it cannot be said that the probability is high enough. Further, even if a band peculiar to mixed rice appears, it is often necessary to identify a mixed rice variety. Therefore, the information is insufficient for identifying the mixed-rice variety only by the appearance of the identification band that should not appear in the target variety, and further PCR needs to be performed for the identification of the mixed-rice variety. Therefore, it has been impossible to simply, quickly, and accurately determine the presence or absence of mixed varieties of grains and the mixed varieties with the known techniques up to now.

[0005]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for determining whether or not a good quality grain such as "Koshihikari" is mixed with other varieties and the mixed varieties simply, quickly and accurately. I am trying.

[0006]

Means for Solving the Problems As a result of intensive studies to achieve the above object, the present inventors have found that when performing PCR amplification using a DNA extracted from a grain sample as a template, it appears in a target high-quality variety. First, multiplex PCR of the first order was performed using a pair of paired primers for identifying negative bands, which appeared uniquely only in other cultivars, and depending on the band pattern expressed, the second pair of paired primers selectively used the paired primers. DNA amplified by performing multiplex PCR
It was confirmed that the grain sample was a target variety by detecting the polymorphism of varieties, and that it was possible to easily and quickly and accurately identify the presence or absence of variety mixture and the mixed variety. The present invention has been achieved based on the above.

The present invention according to claim 1 provides a multiplex PCR method for determining whether or not the grains of other varieties are mixed with the grains, using the DNA extracted from the grains or processed products thereof as a template. A method for discriminating DNA varieties of cereals, characterized by using a pair of negative band discrimination primers that selectively expresses only in mixed varieties when the discrimination is not performed in target varieties.

The present invention according to claim 2 is the method for discriminating DNA varieties of cereals according to claim 1, wherein when the discrimination band is not expressed even when the pair of negative band discrimination primers is used The second multiplex PCR method is performed by using a primer consisting of a pair of matching primers for identifying positive bands specifically expressed in and / or a pair of matching primers for identifying negative bands not specifically expressed only in the target variety. , Expressed D
It is a method for discriminating DNA varieties of cereals, characterized in that the target varieties are confirmed by the NA band pattern.

In the present invention according to claim 3, the cereal is any one of rice, wheat, corn or barley.
Alternatively, it is the method for determining the variety of cereals described in 2. The present invention according to claim 4 provides the method for determining the DNA variety of cereals according to claim 3, wherein the rice of the target variety is a rice with good eating quality. The present invention according to claim 5 is the method for determining a DNA variety of cereals according to claim 4, wherein the good-tasting rice is any one of "Koshihikari", "Hitomebore", "Akitakomachi", and "Hinohikari".

[0010] The present invention according to claim 6 is a method for discriminating DNA varieties of cereals according to claim 4 or 5, wherein when a discrimination band is expressed using a pair of negative band discrimination primers, one sample rice each Primers consisting of paired primer groups for identifying positive bands that are specifically expressed only in the target variety and / or paired primer groups for identifying negative bands that are not specifically expressed only in the target variety, using DNA extracted from each grain as a template The expressed DN was subjected to the second multiplex PCR method using
This is a method of identifying a mixed product mixed with the target product by the A band pattern.

According to the present invention of claim 7, the pairing primers used in the multiplex PCR method are SEQ ID NOS: 1 and 2, 5 and 6, 9 and 10, 13 and 14, 17 and 18, 21 and 22, 25 and 2
6, 29 and 30, 33 and 34, 37 and 3
8, 41 and 42, 45 and 46, 49 and 5
0, 53 and 54, 57 and 58, 61 and 6
2, 65 and 66, 69 and 70, 73 and 7
4, 77 and 78, 81 and 82, 85 and 8
6, 89 and 90, 93 and 94, paired primers A6F30 and A6R30, A7F30 and A7R30, A52F30 and A52R30, B
1F30 and B1R30, B7F30 and B7R3
0, B18F30 and B18R30, B43F30 and B43R30, D4F30 and D4R30, E2
2F30 and E22R30, E30F30 and E3
0R30, F6F30 and F6R30, G4F30 and G4R30, G22F30 and G22R30, G
28F30 and G28R30, J6F30 and J6
R30, M2CGF30 and M2CGR30, M11
F30 and M11R30, P3F30 and P3R3
0, P5F30 and P5R30, Q16F30 and Q16R30, S13F30 and S13R30, T8
F30 and T8R30, T16F30 and T16R
30, WK9F30 and WK9R30, 3 '
The D of cereals according to claim 1 or 2, wherein two or more kinds of primers selected from the group consisting of pairing primers consisting of 13 to 29 bases obtained by excising 1 to 17 bases on the side are used.
This is a method for determining the NA product type.

In the present invention according to claim 8, the pairing primers used in the multiplex PCR method are SEQ ID NOs: 3 and 4, 7 and 8, 11 and 12, 15 and 16, 19 and 20, 23 and 24, 27 and 28, 31 and 32, 35 and 36, 39 and 4
0, 43 and 44, 47 and 48, 51 and 5
2, 55 and 56, 59 and 60, 63 and 6
4, 67 and 68, 71 and 72, 75 and 7
6, 79 and 80, 83 and 84, 87 and 8
8, 91 and 92, 95 and 96 paired primers A6F21 and A6R22, A7F19 and A7R16, A52F29 and A52R21, B
1F25 and B1R20, B7F22 and B7R1
7, B18F15 and B18R21, B43F17 and B43R18, D4F23 and D4R24, E2
2F20 and E22R21, E30F28 and E3
0R24, F6F25 and F6R22, G4F18 and G4R24, G22F27 and G22R23, G
28F17 and G28R28, J6F18 and J6
R20, M2CGF16 and M2CGR15, M11
F20 and M11R20, P3F20 and P3R1
5, P5F20 and P5R25, Q16F25 and Q16R20, S13F25 and S13R24, T8
F22 and T8R25, T16F24 and T16R
The method of discriminating DNA varieties of cereals according to claim 1 or 2, wherein two or more kinds of primers selected from the group consisting of 26, WK9F20 and WK9R20 are used.

In the present invention according to claim 9, the good-tasting rice is "Koshihikari", and B43, G22, M11 and W are used.
A primer set and / or A6, B7, E30, F composed of four types of paired primers consisting of K9
6, G4, M2CG, S13, T8, T16 and WK
7. A multiplex PCR method using a pairing primer set for negative band discrimination consisting of three or more pairing primers selected from the group consisting of 9.
The method for discriminating DNA varieties of cereals according to any one of 1.

According to the tenth aspect of the present invention, the good-tasting rice is "Hitomebore," which is A6, B7, F6, G4, P.
3, S13, T8, and T16, and a negative-band identifying pairing primer set consisting of three or more pairing primers and / or B7, G
7. A primer set composed of 5 types of paired primers of 4, G22, P5 and WK9 is used.
The method for discriminating DNA varieties of cereals according to any one of 1.

In the present invention according to claim 11, the good-tasting rice is "Akitakomachi", which is A6, B7, E30, F.
6, G4, G22, M2CG, P3, S13 and T1
Negative band discrimination pairing primer set consisting of 3 or more pairing primers selected from the group consisting of 6 and / or 4 types of A6, B7, M2CG and P3 or 4 types of WK9, B43, M11 and G22 The method for discriminating DNA varieties of cereals according to any one of claims 1 to 6, wherein a primer set composed of paired primers is used.

[0016] In the present invention according to claim 12, the good-tasting rice is "Hinohikari", which is A6, B7, B43, E2.
2, E30, F6, G4, G28, S13, T8, and T16, and a negative band identification paired primer set consisting of three or more paired primers and / or B43, F6, G28, and T16.
4 types or B43, G22, G28, P5 and W
The method for discriminating DNA varieties of cereals according to any one of claims 1 to 6, wherein a primer set composed of 5 types of paired primers of K9 is used.

According to the present invention of claim 13, the sample rice is cooked rice, and the DNA of cereals according to any one of claims 1 to 12.
This is a method for determining the variety. The present invention according to claim 14 is the method for determining a DNA variety of cereals according to any one of claims 1 to 12, characterized in that the mixed sample rice is assayed one by one.

The present invention according to claim 15 includes a pair of primers for negative band discrimination, which does not express the discrimination band in the target variety but selectively expresses only in the mixed variety, and the cereal DNA variety. It is a discrimination kit. The present invention according to claim 16 is a paired primer group for positive band discrimination, which is specifically expressed only in a target variety, and /
Alternatively, the present invention is a kit for determining a DNA variety of cereals, which comprises a primer consisting of a paired primer group for identifying a negative band that is not specifically expressed only in a target variety.

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below. The grain in the present invention refers to seeds of rice plants, and includes, for example, rice, wheat, corn, barley, sorghum, and the processed products thereof. The sample rice means milled rice, brown rice, milled rice flour, brown rice flour, cooked rice, rice cake, and the like.

In the present invention, the target varieties are varieties that are the mainstream among cereals, for example, "Koshihikari" for rice,
Meaning good-tasting rice such as "Hitomebore", "Akitakomachi", "Sasanishi", etc., for barley "Ichibanboshi", "Sanshu", "Large HK64", and for corn, "Honeybandam", Means "Peter Corn", "Waxy Corn", etc. In addition to using the grain itself as a sample, these grains are, if necessary, suitable crushing equipment, for example, an ultracentrifugal crusher (manufactured by Lecce), a cyclone mill (manufactured by Yudy), a millsa (manufactured by Iwatani),
Crush in a mortar etc. and use as powder.

[0021] For extraction of genomic DNA from cereal samples,
A known DNA extraction method can be applied, and examples thereof include a phenol extraction method, a cetyltrimethylammonium bromide (CTAB) method, and an alkali SDS method.
In the present invention, as shown in Examples below, CTA
It is preferable to use the method B. For example, if the sample is CTAB
Solution (0.1M Tris-HCl, 2mM ethylenediaminetetraacetic acid (EDTA) disodium, 1.4M NaC
1 (pH 8.0)) is added, the mixture is stirred, placed in a thermostatic bath, the CTAB solution is added again, and the mixture is allowed to stand for a predetermined time for extraction to obtain a genomic DNA.

When the sample is cooked rice, rice cake, etc., as described in Japanese Patent No. 3048149, the powder sample is treated with a thermostable amylase and then extracted by an enzymatic method for extracting a genomic gene. You can also After extraction of the DNA, a purification step such as chloroform / isoamyl alcohol treatment, isopropanol precipitation, deproteinization with phenol / chloroform, ethanol precipitation and the like may be added if necessary. Among these steps, chloroform / isoamyl alcohol treatment is preferable. This treatment is performed, for example, by adding chloroform / isoamyl alcohol (24: 1) to the DNA extract, stirring and centrifuging the mixture, and adding the DNA precipitation solution to the supernatant to obtain the DNA.
DNA precipitate obtained by centrifugation is extracted with 1 M NaCl, washed with isopropyl alcohol and ethanol, and precipitated, and then dissolved in TE buffer solution. Obtainable.

Then, in the presence of random primers,
P using the genomic DNA obtained by the above operation as a template
Perform CR method. This is the next PC among genomic DNA
This is carried out in order to amplify a base sequence having discriminability among varieties which is the basis for designing a pairing primer for R. Here, the PCR method in the present invention refers to a chain reaction of DNA replication by a DNA polymerase. That is, in the presence of a thermostable DNA polymerase and a primer in the template DNA, a high temperature treatment process (denaturation) at about 90 to 96 ° C, a primer / DNA binding process (annealing) at about 30 to 75 ° C, and a temperature at about 70 to 75 ° C. The above-mentioned DNA replication process (extension) is repeated for 20 to 60 cycles to increase (amplify) about 1,000,000 times to 1 billion times. Further, the multiplex PCR method means a PCR method performed by combining a plurality of paired primers, and in this method, primer dimers may be generated, identification bands may interfere with each other, or overlapping may occur. Multiple paired primers (8 ~
30-mer) is selected and used. As a result, the number of times of PCR and the number of times of electrophoresis / staining can be reduced.

The pairing primers are a pair of primers capable of amplifying a nucleotide sequence showing distinctiveness between varieties, and as described above, PCR is carried out from the DNA obtained from the above cereal sample based on the RAPD method. It is designed based on the nucleotide sequence of the band portion that is distinctive among varieties obtained by the method. That is, using the genomic DNA of the target cereal as a template, the nucleotide sequence of the DNA obtained as an identification band by the PCR method performed in the presence of random primers was determined, and 15 to 29 of the forward and reverse sides of the random primer portion were determined. It refers to a primer used as a pair of primers and used in the PCR method for identifying a variety together with the template DNA of the target cereal. By selecting a pairing primer, among the plural positions where the random primer binds to the template DNA, in the PCR method performed for the type identification, it selectively binds only to the base sequence part which is an identification band useful for identification. Will be done.

The random primer in the present invention means
When the genomic DNA extracted from a grain sample is used as a template DNA for amplification by the PCR method, it is complementary to the genomic DNA separated into single strands to form a double-stranded structure, and the initiation of template DNA replication. Refers to the point primer. Usually, it is a nucleotide of 8 to 50 mer, adenine (A),
It is a synthetic primer in which thymine (T), guanine (G), and cytosine (C) are bound to a random sequence, such as a commercially available 10-mer random primer (Operon) or a DNA oligomer set ( 12-mer)
(Manufactured by Wako Pure Chemical Industries, Ltd.) and the like.

Subsequently, the amplified DNA obtained by PCR is electrophoresed. This is to detect a band in the amplification product, which is a base sequence for designing a target pairing primer and which is a base sequence showing distinctiveness between varieties. The term "electrophoresis" as used herein means that when DNA amplified by the PCR method is attracted to a direct current charge in an agarose or polyacrylamide gel and moves, it is separated by the difference in the molecular weight of the DNA and is banded by ethidium bromide. This is an operation in which a difference in amplified DNA is detected as a band by staining.

A pair of paired primers is prepared in the following manner from the bands showing the discrimination between the varieties thus detected. That is, the band in which the distinctiveness appears is cut out from the gel to extract and collect DNA, which is incorporated into Escherichia coli and propagated. Then, the plasmid is extracted by the alkaline miniprep method or the like, and this is used as a template DN.
It is amplified by the PCR method designated as A and the nucleotide sequence is determined by a DNA automatic sequencer. From the determined nucleotide sequence,
Design pairing primers. In the PCR method using the random primer, the site to which the random primer is bound in the DNA derived from the grain sample, which is the template DNA, should have the same or complementary (homologous) sequence to the random primer. . In other words, DN with a high variety discrimination extracted from this template DNA
The A base sequence (which is the base of the pairing primer in the PCR method) has a sequence identical or homologous to the random primer at both ends. Therefore,
From each of the forward side and the reverse side of this highly discriminating DNA base sequence, a pairing primer having an appropriate sequence and length and useful for determining the variety of a grain can be designed.

The pairing primers in the present invention preferably have the same sequence or homologous sequence. The size of the pairing primer is preferably 10
It is necessary to be within the range of -40 bases, more preferably 13-29 bases. When the amount of the primer exceeds this range, the binding property to the template DNA and the dissociation after the binding are deteriorated, the discrimination band is not detected even after the PCR, and the variety discrimination becomes difficult, which is not preferable. In addition, if it is less than this range, mismatches due to non-specific binding etc. occur and the frequency of expression of bands other than the identification band increases, making it difficult to identify varieties and simplifying PCR by combining multiple primers. It is not preferable because speeding up becomes difficult.

As a result of performing the above-mentioned PCR, the present inventors have obtained several types of bands that show distinctiveness among various varieties.
Table 1 shows the relationship between the identification band and variety identification in rice.

[0030]

[Table 1] Table 1 (1) +: Indicates that a discrimination band is expressed in electrophoresis after PCR. -: Indicates that the identification band is not expressed in electrophoresis after PCR.

[0031]

[Table 2] Table 1 (Part 2) +: Indicates that a discrimination band is expressed in electrophoresis after PCR. -: Indicates that the identification band is not expressed in electrophoresis after PCR.

[0032]

[Table 3] Table 1 (Part 3) +: Indicates that a discrimination band is expressed in electrophoresis after PCR. -: Indicates that the identification band is not expressed in electrophoresis after PCR.

[0033]

[Table 4] Table 1 (Part 4) +: Indicates that a discrimination band is expressed in electrophoresis after PCR. -: Indicates that the identification band is not expressed in electrophoresis after PCR.

Table 2 shows the relationship between the discrimination band in corn and barley and variety discrimination.

[0035]

[Table 5] Table 2 (Part 1) +: Indicates that a discrimination band is expressed in electrophoresis after PCR. -: Indicates that the identification band is not expressed in electrophoresis after PCR.

[0036]

[Table 6] Table 2 (Part 2) +: Indicates that a discrimination band is expressed in electrophoresis after PCR. -: Indicates that the identification band is not expressed in electrophoresis after PCR.

Various paired primers were obtained from each band. (1) Discrimination band A7 (0.7 kbp) This band is obtained by amplifying DNA extracted from varieties such as "Koshihikari" and "Akitakomachi", but it is specific to "Nipponhare" and "Morning light". Not seen in.
From this A7, a pair of primers A7F30 (SEQ ID NO: 5 in Sequence Listing) and A7R30 (SEQ ID NO: 6 in Sequence Listing)
Designed. Subsequently, a predetermined number of bases were excised from the 3'side of the above primer to give A, which is the pairing primer of the present invention.
7F19 (SEQ ID NO: 7 in the sequence listing) and A7R16 (SEQ ID NO: 8 in the sequence listing) were obtained.

(2) Discrimination band B43 (0.9 kbp) This band can be obtained by amplifying DNA extracted from "Koshihikari", "Hitomebore" and "Sasanishi". Not seen in "light". From this B43, a pair of primers B43F30
(SEQ ID NO: 25 of Sequence Listing) and B43R30 (SEQ ID NO: 26 of Sequence Listing) were designed. Then, a predetermined number of bases were excised from the 3'side of the above primer, and B43F17 (SEQ ID NO: 27 in the sequence listing), which is the pairing primer of the present invention.
And B43R18 (SEQ ID NO: 28 in the sequence listing) were obtained.

(3) Identification band E30 (0.85 kb)
p) This band is obtained by amplifying DNA extracted from cultivars such as "Hitomebore" and "Mutsuhomare", but not found in "Koshihikari" or "Akitakomachi". From this E30, a pair of primers E30F30
(SEQ ID NO: 37 of Sequence Listing) and E30R30 (SEQ ID NO: 38 of Sequence Listing) were designed. Subsequently, the base was excised from the above-mentioned primer to obtain E, which is the pairing primer of the present invention.
30F28 (SEQ ID NO: 39 of the sequence listing) and E30R2
4 (SEQ ID NO: 40 in the sequence listing) was obtained.

(4) Discrimination band J6 (0.9 kbp) This band was obtained by amplifying DNA extracted from "Koshihikari" and "Kirara 397", but not seen in "Sasanishi". From this J6, a pair of primers J6F30 (SEQ ID NO: 57 in the Sequence Listing) and J6
R30 (SEQ ID NO: 58 in the sequence listing) was designed. continue,
The bases were excised from the above primers to obtain J6F18 (SEQ ID NO: 59 in the sequence listing) and J6R20 (SEQ ID NO: 60 in the sequence listing), which are pairing primers of the present invention.

(5) Identification band M2CG (1.2 kb
p) This band was obtained by amplifying DNA extracted from "Hitomebore" and "Nihonbare" by adding two residues to a 10-mer random primer, but not seen in "Koshihikari" and "Kinuhikari". Absent. From this M2CG, a pair of primers M2CGF30 (SEQ ID NO: 61 in Sequence Listing) and M2CGR30 (SEQ ID NO: 6 in Sequence Listing)
2) was designed. Subsequently, the base was excised from the above-mentioned primer, and M2CGF16, which is the pairing primer of the present invention.
(SEQ ID NO: 63 of the sequence listing) and M2CGR15 (SEQ ID NO: 64 of the sequence listing) were obtained.

(6) Discrimination band S13 (1.8 kbp) This band is obtained by amplifying DNA extracted from "Kirara 397" and "Hoshinoyume", but in "Nihonbare" and "Morning light". Not seen specifically. This S1
3 to one pair of primers S13F30 (SEQ ID NO: 81 in the sequence listing) and S13R30 (SEQ ID NO: 8 in the sequence listing)
2) was designed. Subsequently, the base was excised from the above-mentioned primer to obtain S13F25 which is the pairing primer of the present invention.
(SEQ ID NO: 83 of the sequence listing) and S13R24 (SEQ ID NO: 84 of the sequence listing) were obtained.

(7) Discrimination band WK9 (1.6 kbp) This band can be obtained by amplifying DNA extracted from "Hitomebore" or "Akitakomachi", but not seen in "Koshihikari" or "Sasanishiki". This WK
From 9, a pair of primers WK9F30 (SEQ ID NO: 93 in the sequence listing) and WK9R30 (SEQ ID NO: 9 in the sequence listing)
4) was designed. Subsequently, the base was excised from the above-mentioned primer to obtain WK9F20 which is the pairing primer of the present invention.
(SEQ ID NO: 95 of Sequence Listing) and WK9R20 (SEQ ID NO: 96 of Sequence Listing) were obtained.

Although the above-mentioned primers were designed based on rice, most of them can be used for identifying corn, barley and the like belonging to the same grass family. Excision of bases to obtain the pairing primer of the present invention means amplification of template DNA extracted from a grain sample, and
Excise 1 to 17 bases on the 3'side of the pair of primers,
It refers to 13 to 29 bases. The base may be excised by a conventional method using a suitable restriction enzyme.

Specifically, a pair of primers A6
F30 (SEQ ID NO: 1 in Sequence Listing) and A6R30 (SEQ ID NO: 2 in Sequence Listing), A7F30 (SEQ ID NO: 5 in Sequence Listing)
And A7R30 (SEQ ID NO: 6 in the sequence listing), A52F3
0 (SEQ ID NO: 9 in the sequence listing) and A52R30 (SEQ ID NO: 10 in the sequence listing), B1F30 (SEQ ID NO: 1 in the sequence listing)
3) and B1R30 (SEQ ID NO: 14 of the sequence listing), B7
F30 (SEQ ID NO: 17 of Sequence Listing) and B7R30 (SEQ ID NO: 18 of Sequence Listing), B18F30 (SEQ ID NO: 21 of Sequence Listing) and B18R30 (SEQ ID NO: 2 of Sequence Listing)
2), B43F30 (SEQ ID NO: 25 in the sequence listing) and B
43R30 (SEQ ID NO: 26 of Sequence Listing), D4F30 (SEQ ID NO: 29 of Sequence Listing) and D4R30 (SEQ ID NO: 30 of Sequence Listing), E22F30 (SEQ ID NO: 33 of Sequence Listing) and E22R30 (SEQ ID NO: 34 of Sequence Listing), E30F
30 (SEQ ID NO: 37 of the sequence listing) and E30R30 (SEQ ID NO: 38 of the sequence listing), F6F30 (SEQ ID NO: 41 of the sequence listing) and F6R30 (SEQ ID NO: 42 of the sequence listing), G
4F30 (SEQ ID NO: 45 of the sequence listing) and G4R30
(SEQ ID NO: 46 of Sequence Listing), G22F30 (SEQ ID NO: 49 of Sequence Listing) and G22R30 (SEQ ID NO: 5 of Sequence Listing)
0), G28F30 (SEQ ID NO: 53 in the sequence listing) and G
28R30 (SEQ ID NO: 54 of Sequence Listing), J6F30 (SEQ ID NO: 57 of Sequence Listing) and J6R30 (SEQ ID NO: 58 of Sequence Listing), M2CGF30 (SEQ ID NO: 61 of Sequence Listing)
And M2CGR30 (SEQ ID NO: 62 in the sequence listing), M1
1F30 (SEQ ID NO: 65 of Sequence Listing) and M11R30
(SEQ ID NO: 66 of Sequence Listing), P3F30 (SEQ ID NO: 69 of Sequence Listing) and P3R30 (SEQ ID NO: 7 of Sequence Listing)
0), P5F30 (SEQ ID NO: 73 of the sequence listing) and P5
R30 (SEQ ID NO: 74 of Sequence Listing), Q16F30 (SEQ ID NO: 77 of Sequence Listing) and Q16R30 (SEQ ID NO: 78 of Sequence Listing), S13F30 (SEQ ID NO: 81 of Sequence Listing) and S13R30 (SEQ ID NO: 82 of Sequence Listing), T8F3
0 (SEQ ID NO: 85 of Sequence Listing) and T8R30 (SEQ ID NO: 86 of Sequence Listing), T16F30 (SEQ ID NO: 8 of Sequence Listing)
9) and T16R30 (SEQ ID NO: 90 in the sequence listing), W
K9F30 (SEQ ID NO: 93 in the sequence listing) and WK9R3
2 or more selected from the group consisting of 13 to 29 bases obtained by removing 1 to 17 bases on the 3 ′ side from 0 (SEQ ID NO: 94 in the sequence listing) by the above method Can be used.

That is, as the pairing primer, A6
F21 (SEQ ID NO: 3 in Sequence Listing) and A6R22 (SEQ ID NO: 4 in Sequence Listing), A7F19 (SEQ ID NO: 7 in Sequence Listing)
And A7R16 (SEQ ID NO: 8 in Sequence Listing), A52F2
9 (SEQ ID NO: 11 of the sequence listing) and A52R21 (SEQ ID NO: 12 of the sequence listing), B1F25 (SEQ ID NO: 1 of the sequence listing)
5) and B1R20 (SEQ ID NO: 16 of the sequence listing), B7
F22 (SEQ ID NO: 19 in Sequence Listing) and B7R17 (SEQ ID NO: 20 in Sequence Listing), B18F15 (SEQ ID NO: 23 in Sequence Listing) and B18R21 (SEQ ID NO: 2 in Sequence Listing)
4), B43F17 (SEQ ID NO: 27 in the sequence listing) and B
43R18 (SEQ ID NO: 28 of Sequence Listing), D4F23 (SEQ ID NO: 31 of Sequence Listing) and D4R21 (SEQ ID NO: 32 of Sequence Listing), E22F20 (SEQ ID NO: 35 of Sequence Listing) and E22R21 (SEQ ID NO: 36 of Sequence Listing), E30F
28 (SEQ ID NO: 39 of the sequence listing) and E30R24 (SEQ ID NO: 40 of the sequence listing), F6F25 (SEQ ID NO: 43 of the sequence listing) and F6R22 (SEQ ID NO: 44 of the sequence listing), G
4F18 (SEQ ID NO: 47 of Sequence Listing) and G4R24
(SEQ ID NO: 48 of Sequence Listing), G22F27 (SEQ ID NO: 51 of Sequence Listing) and G22R23 (SEQ ID NO: 5 of Sequence Listing)
2), G28F17 (SEQ ID NO: 55 of the sequence listing) and G
28R28 (SEQ ID NO: 56 of Sequence Listing), J6F18 (SEQ ID NO: 59 of Sequence Listing) and J6R20 (SEQ ID NO: 60 of Sequence Listing), M2CGF16 (SEQ ID NO: 63 of Sequence Listing)
And M2CGR15 (SEQ ID NO: 64 in the sequence listing), M1
1F20 (SEQ ID NO: 67 in the Sequence Listing) and M11R20
(SEQ ID NO: 68 of Sequence Listing), P3F20 (SEQ ID NO: 71 of Sequence Listing) and P3R15 (SEQ ID NO: 7 of Sequence Listing)
2), P5F20 (SEQ ID NO: 75 in the sequence listing) and P5
R25 (SEQ ID NO: 76 of Sequence Listing), Q16F25 (SEQ ID NO: 79 of Sequence Listing) and Q16R20 (SEQ ID NO: 80 of Sequence Listing), S13F25 (SEQ ID NO: 83 of Sequence Listing) and S13R24 (SEQ ID NO: 84 of Sequence Listing), T8F2
2 (SEQ ID NO: 87 of the sequence listing) and T8R25 (SEQ ID NO: 88 of the sequence listing), T16F24 (SEQ ID NO: 9 of the sequence listing)
1) and T16R26 (SEQ ID NO: 92 in the sequence listing), W
K9F20 (SEQ ID NO: 95 in the sequence listing) and WK9R2
2 selected from the group consisting of 0 (SEQ ID NO: 96 in the sequence listing)
Oligonucleotides consisting of species or combinations of more can be used.

The pairing primer used in the present invention is not limited to the above-mentioned pairing primer, but a pairing primer which can be used in terms of discriminating property, melting temperature (Tm) and the like is newly designed. It can also be used.
Here, Tm is the temperature at which the two strands of DNA are separated into their respective single strands, and the annealing temperature for PCR is usually suitable near the Tm of the primer used. In the present invention, when the paired primer is used in combination,
Select and use paired primers with similar Tm,
By selecting an appropriate annealing temperature close to Tm, the discrimination band obtained when each paired primer is used alone can be obtained by PCR once or several times.

Specifically, the T of the pairing primer used
The difference between the average value of m and the Tm of each paired primer is 15 ° C.
It is preferable that the temperature is within (± 15 ° C.) and the annealing temperature of PCR is also within the range. It is not preferable to perform the PCR with the annealing temperature lower than the average value of Tm of the paired primer by 15 ° C. or more, because the discrimination band may appear even in a variety that originally does not show the identification band. On the other hand, when PCR is performed at a temperature higher than the average value of Tm by 15 ° C. or more, the originally expressed discrimination band may disappear, which is not preferable.

In the present invention, a multiplex PCR is carried out with the template DNA previously extracted using a pairing primer. By selecting an appropriate pairing primer, P
In CR, it selectively binds only to a nucleotide sequence site that is a discrimination band useful for discrimination of varieties. As a result, it becomes possible to discriminate the grain which is the object of the present invention. In the present invention, it is possible to mix and use one kind or a combination of two or more kinds of the paired primers.
The mixed use of paired primers in the present invention means that a plurality of selected paired primers are combined with P
In CR, it means used together in the same reaction. In the conventional RAPD method, it is difficult to use a plurality of primers at the same time because a large number of common bands are expressed in addition to the identification band. In the present invention, by using an appropriate combination of paired primers (primer set), only the identification band corresponding to each primer is expressed by electrophoresis, so that mixed use is possible.

The prepared paired primers cannot be mixed and used in a suitable combination and blending ratio while paying attention to the fact that mutual primer dimer formation does not occur between the primers and the identification bands do not overlap. It is necessary to select By mixing and using various suitable paired primers, it is not necessary to perform PCR for each primer, and many varieties can be identified accurately and easily with one PCR. , Can save a lot of time and money.

In the present invention, the positive band specifically expressed only in the target cultivar is a specific band pattern expressed only in the target cultivar and indicates a pattern unique to the target cultivar. For example, "Koshihikari" as the target variety and WK9, M11, B as the pairing primer
WK if the grain sample is "Koshihikari" when the paired primer set consisting of 43 and G22 is used.
The discrimination band of 9 is not expressed, but the three discrimination bands of M11, B43 and G22 are expressed. This identification band pattern is not shown in other varieties, and is a band pattern unique to "Koshihikari" which is the target variety. In addition, the pairing primer set for positive band identification that is specifically expressed only in the target variety or the pairing primer set for negative band identification that is not expressed only in the target variety is a primer that has a band pattern unique to the target variety as described above. It is a set.

In the above case, each rate at which the grain sample can be estimated to be the target variety "Koshihikari" will be examined. That is, if the expression probability of each discrimination band is assumed to be 0.5 of expression / non-expression, the probability that a certain cultivar other than "Koshihikari" happens to coincide with the target cultivar "Koshihikari" in all four discrimination bands. Is (0.5) ⁴
Therefore, it becomes 0.0625. From this,
When all four types of band patterns of an unknown cereal sample match the target variety, it can be estimated that the same variety as the target variety with a probability of 90% or more.

The negative band in which the discrimination band is not expressed in the target variety indicates a band pattern in which the discrimination band is expressed in other varieties but is not specifically expressed in the target variety. For example, "Koshihikari" as the target variety, WK9F20 (SEQ ID NO: 95 in the sequence listing) and WK9R20 (SEQ ID NO: 96 in the sequence listing), F6F25 (SEQ ID NO: 43 in the sequence listing) and F6R22 (SEQ ID NO: in the sequence listing) as the pairing primers. 44), B7F22
(Sequence No. 19 of Sequence Listing) and B7R17 (Sequence No. 20 of Sequence Listing), A6F21 (Sequence No. 3 of Sequence Listing) and A6R22 (Sequence No. 4 of Sequence Listing) were used. In this case, if the grain sample is “Koshihikari”, the amplified DNA band after PCR is not expressed at all. On the other hand, when the cereal sample is another cultivar, one of the discrimination bands is expressed, that is, when the other cultivar is "Kirara 397", the discrimination band WK
In the case of 9, "Kinuhikari", the identification bands WK9 and B7, and in the case of "Mutsuhomare", the identification band B7 is expressed.

In the above, when the first PCR was carried out using the pairing primer for identifying the negative band, the identification band was not expressed, and the other pairing primers shown in Tables 1 and 2 were used. If the identification band pattern expressed when the second PCR is performed also matches the target variety “Koshihikari”, the grain sample is determined to have a very high probability of being “Koshihikari”. For example,
In the target variety, there are four types of pairing primer groups for identifying negative bands in which no identifying band is expressed, and positive band specifically expressing in the target variety or pairing primer groups for identifying negative band not expressing only in the target variety. , In the former, no identification band was expressed,
When the identification band patterns match in the latter case, the probability P that the cereal sample is not “Koshihikari” is (1/2) ⁴ ×
(1/2) ⁴ = 0.0039, which is 0.01 or less.

Therefore, the discrimination band is not expressed in the target cultivar, the discrimination band is not expressed at all by PCR using the pair of negative band discrimination primer pairs, and is not expressed only in the positive band specifically expressed in the target cultivar or in the target cultivar. When the discrimination band patterns by the pair of negative band discrimination primers match, the grain sample can be estimated to be “Koshihikari” with a probability of 99.9% or more.

Next, in the case where the grain sample is one in which the target variety is mixed with another variety, a method for identifying the mixed variety will be described. For example, in the case of a mixed rice of "Mutsuhomare" and "Koshihikari", which are rice varieties, four types of WK9, M11, G22, and B43 are used as a pair of positive band-identifying primer pairs specifically expressed in the target varieties. In the comparison between single varieties by using the paired primer set of, in M11 and G22 where the discrimination band is expressed in “Koshihikari”, the discrimination band is not expressed in “Mutsuhomare”. It is possible.

However, "Mutsuhomare" is replaced by "Koshihikari"
If a small amount of rice is mixed in
In No. 2, it is impossible to detect mixed rice because the negative nature of "Mutsuhomare" in which the identification band is not expressed is hidden in the positive band of "Koshihikari" which is expressed in large amounts. Therefore, in the identification of mixed varieties in mixed rice, there is a need to distinguish between single varieties.
The pairing primer group described in Japanese Patent Publication No. 95589 is not sufficient, and a pair of negative primer pairing primers for identifying a negative band in which a discriminating band does not newly appear in the target varieties but a discriminating band appears in other varieties is required. .

As described above, the present invention provides a method for discriminating DNA varieties of cereals and a kit used in the method. The kit is characterized in that it contains a pair of paired primers for discriminating negative bands, which does not express discrimination bands in target varieties but selectively expresses only in mixed varieties. Preferable examples include a kit containing a pair of matching primers for discriminating positive bands that are specifically expressed only in the target variety and / or a pair of pairing primers for identifying negative bands that are not specifically expressed only in the target variety. As the pairing primer group contained in the kit of the present invention, the above-mentioned various primer groups can be used. Furthermore, the kit of the present invention may contain reagents used in the PCR method, for example, DNA polymerase, a reaction buffer, and the like.

[0059]

The present invention will be described in detail below with reference to examples, but the present invention is not limited thereto. Example 1 (Example of detection of mixed rice in "Koshihikari" by a negative band DNA group for identification) "Koshihikari", "Hitomebore", "Hinohikari" which are the top 20 cultivars in Japan in 1999
"Akita Komachi", "Kirara 397", "Kinuhikari", "Hoshi no Yume", "Haenuki", "Mutsuhomare", "Nippon Hare", "Sasanishi", "Tsugaru Roman", "Hanaechizen", "Yumetsukushi", "Hatsushima", "Morning light", "Moonlight", "Aichi no Kaori",
Brown rice of "Festival" and "Akiho" was used as a sample, and it was milled to a yield of 90% using a test rice mill (Rice Pal VP31T manufactured by Yamamoto Seisakusho). The polished rice sample was crushed using an ultracentrifugal crusher manufactured by Lecce, and 6 g of the sample was extracted with genomic DNA by the CTAB method. That is, 2% CT at 70 ° C
AB solution (0.1M Tris-HCl, 2mM ethylenediaminetetraacetic acid (EDTA) disodium, 1.4M N
6 ml of aCl, pH 8.0) was added and stirred, then placed in a thermostat bath at 55 ° C., and then the above CTAB solution (1% solution) 6
DNA was extracted for 30 minutes by adding ml.

Then, after adding chloroform / isoamyl alcohol (24: 1) to the DNA extract and stirring,
Centrifuge to remove the supernatant and remove the DNA precipitate (1% CTA
B, 20 mM Tris-HCl, 10 mM EDTA, pH
8.0) was added and the mixture was allowed to stand at 4 ° C. overnight to precipitate DNA. Next, from the DNA precipitate obtained by centrifugation, 1M
After extraction with NaCl, washing with isopropyl alcohol and ethanol, and precipitation, TE buffer (10 mM
Tris-hydrochloric acid, 1 mM EDTA, pH 8.0) 200
It was dissolved in μl to prepare a DNA sample solution. The composition of the PCR reaction solution was 11.74 μl of sterilized water, 0.2 μl of polymerase (Taq polymerase (5 U / μl), manufactured by Takara Shuzo Co., Ltd.),
Reaction buffer (12 mM Tris-HCl, 60 mM KC
l, pH 8.3) 2.5 μl, MgCl ₂ 2.0 μl,
Template DNA 200 ng / 1 μl, dNTPs (100
μM) 1 μl was mixed to prepare WK9F2 of SEQ ID NOs: 95 and 96 as a pairing primer used for PCR.
0 and WK9R20 0.6 μl each, SEQ ID NOS: 43 and 44 F6F25 and F6R22 0.5 μl each, SEQ ID NOS: 19 and 20 B7F22 and B7R17 0.5 μl each, and SEQ ID NOS: 3 and 4 A6F21 and A6R22 0.4 μl each 22.44μ in total
It was set to l.

A PCR thermal cycler MP manufactured by Takara Shuzo Co., Ltd. was used as a reaction apparatus, and denaturation (94 ° C., 1 minute), annealing (62 ° C., 1 minute) and extension (72 ° C., 2 minutes) were performed.
The reaction was performed for 5 cycles. The amplified DNA was electrophoresed using Mupid II manufactured by Cosmo Bio Co., Ltd., electrophoresed on a 2% agarose gel for 40 minutes, and the band pattern was detected by ultraviolet irradiation after staining with ethidium bromide. The detection results are shown in Table 3 and FIG.

As a result, in the case of 19 varieties other than "Koshihikari", all positive bands were expressed,
It became clear that mixed rice could be detected. On the other hand, Japanese Patent Laid-Open No.
In Example 10 of 001-95589, M2
In the case of using three kinds of primers of CG, WK9 and B7 together, both "Koshihikari" and "Hatsushimo" do not express the identification band, and therefore "Hatsushimo" cannot be detected even if mixed with "Koshihikari". From this, it is clear that the method of the present invention is excellent.

[0063]

[Table 7] Table 3. PC by combining 4 types of primers
R result (+: Discrimination band is expressed-: Discrimination band is not expressed)

Example 2 (Example of discrimination using a negative / positive band DNA group for discrimination) The top 20 cultivars in Japan in 1999, that is, "Koshihikari", "Hitomebore", "Hinohikari", "Akitakomachi", "Kirara" 397 ”,“ Kinuhikari ”,“ Hoshi no Yume ”,“ Haenuki ”,“ Mutsuhomare ”,“ Nihonbare ”,“ Sasanishi ”,“ Tsugaru Roman ”,“ Hana Echizen ”,“ Yume Tsukushi ”,“ Hatsushima ”,“ Morning ” "Light of the moon", "light of the moon", "Kaori of Aichi",
The brown rice of "Festival" and "Akiho" was milled to 90% yield using a test rice mill (Yamamoto Seisakusho, Rice Pal VP31T). One grain each of these milled rice was crushed in a mortar to prepare a sample, and genomic DNA was extracted with a DNA extraction kit, ISOPLANT II.

The reaction solution composition of PCR was 10.4 μm of sterilized water.
l, polymerase (Takara Shuzo Co., Ltd., Taq polymerase
(5 U / μl) 0.2 μl, reaction buffer (12 mM
Tris-HCl, 60 mM KCl, pH 8.3) 2.5μ
l, MgCl ₂ 2.0 μl, template DNA 200 ng /
μl was prepared by mixing 1 μl and 1 μl of dNTPs (100 μM), and as a pairing primer used for PCR, 0.6 μl each of primers WK9F20 and WK9R20 of SEQ ID NOs: 95 and 96, primers M11F20 and M11R20 of SEQ ID NOs: 67 and 68, respectively. 0.3μ
1, primer G22F27 of SEQ ID NOs: 51 and 52
And G22R23 0.3 μl each, SEQ ID NOS: 27 and 28 primers B43F17 and B43R18 0.1 μl each, SEQ ID NOS 71 and 72 primer P3
F20 and P3R15 0.3 μl each 20.3 μm in total
It was set to l.

A PCR thermal cycler MP manufactured by Takara Shuzo Co., Ltd. was used as a reaction apparatus, and 35 cycles of denaturation (94 ° C., 1 minute), annealing (62 ° C., 1 minute) and extension (72 ° C., 2 minutes) were performed. Let The electrophoresis of the amplified DNA was carried out by using Mupid II manufactured by Cosmo Bio Co., Ltd., which was run on a 2% agarose gel for 40 minutes, and the band pattern was detected by UV irradiation after staining with ethidium bromide. The detection results are shown in Table 4 and FIG.

[0067]

[Table 8] Table 4. PC with a combination of 5 types of primers
R result (+: Discrimination band is expressed-: Discrimination band is not expressed)

As a result, the four kinds of paired primer sets of M11, G22, B43 and P3 used in this example were used in combination with the four kinds of primers WK9, F6, A6 and B7 used in Example 1. By doing so, it is possible to identify all of the top 20 cultivars except for “Hoshino Yume” and “Akiho”. Therefore, it was shown that the combination of Example 1 and Example 2 enables most of the identification of mixed rice in addition to the determination of the presence or absence of mixed rice for “Koshihikari”.

Example 3 (Example of Detection of Mixed Rice by Three Types of Negative Band DNA for Discrimination) The top 20 cultivars in Japan in 1999, "Koshihikari", "Hitomebore", "Hinohikari",
"Akita Komachi", "Kirara 397", "Kinuhikari", "Hoshi no Yume", "Haenuki", "Mutsuhomare", "Nippon Hare", "Sasanishi", "Tsugaru Roman", "Hanaechizen", "Yumetsukushi", "Hatsushima", "Morning light", "Moonlight", "Aichi no Kaori",
Brown rice of "Festival" and "Akiho" were used as samples, and template DNA was prepared from polished rice powder in the same manner as in Example 1. Assuming that a small amount of other varieties is mixed with Koshihikari, 80% of the DNA of "Koshihikari" and 20% of the DNA of other varieties are used.
% Of the primers S13F25 and S13R24 of SEQ ID NOS: 83 and 84, respectively, and 0.08
μl, primer E30F2 of SEQ ID NOs: 39 and 40
8 and E30R24 0.05 μl each, primers M2CGF16 and M2CGR of SEQ ID NOs: 63 and 64
22 PCR was performed by adding 1.0 μl of each. The results are shown in Table 5.
And shown in FIG.

[0070]

[Table 9] Table 5. PCR results of rice polishing using 3 types of primers in combination (+: Discrimination band is expressed-: Discrimination band is not expressed)

As is clear from Table 5, "Koshihikari"
In the case of the mixed rice discrimination primer set to, among the top 20 varieties, "Akitakomachi", "Kinuhikari", "Tsugaru Roman", "Dream Tsukushi", "Morning light", "Matsuharu",
All except "Moonlight" have a positive band and are useful for detecting mixed rice against "Koshihikari".
When these three types of primers are used in combination with the nine types of primer sets shown in Examples 1 and 2, it is possible to identify “Hoshinoyume” and “Akiho” that could not be identified in Example 2 and identify mixed rice. Is effective for. In addition, JP
Example 2 M2CG and S1 of 001-95589
In the case of PCR of 3 only, as is clear from Table 3,
It is possible to distinguish "Koshihikari" from "Nihonbare" and "Kirara 397", but "Akita Komachi", "Kinuhikari",
"Sasanishi", "Tsugaru Romance", "Dream Tsukushi",
Similar to "Koshihikari", 8 varieties of "Morning light", "Moonlight", and "Festival" do not produce any identification band, so they cannot be detected even if they are mixed with rice. Is clearly inferior in.

Example 4 (Example of Detection of Mixed Rice and Identification of Mixed Rice Varieties in Cooked Rice Samples by Using Negative Band Group and Negative / Positive Band Group in Combination) “Koshihikari”, “Hitomebore”, “Hinohikari”,
20 kinds of brown rice including top 10 cultivars of "Akitakomachi", "Kirara 397", "Kinuhikari", "Hoshinoyume", "Haenuki", "Mutsuhomare" and "Nihonbare" are used as samples, and a test rice milling machine is used. (Yamamoto Seisakusho, Rice Pal VP31T) was used to refine the yield to 90%. A plastic tube (assist,
(1.5 ml volume), add 35 μl of deionized water, stand on the stand and soak for 1 hour, then open the electric rice cooker (Toshiba, RC-183, add 75 ml of deionized water to the outer pot)
The rice was cooked for 15 minutes and steamed for 15 minutes to prepare a cooked rice sample. Extraction of DNA from this sample was performed as follows.

A sample of cooked rice was taken in a microtube, and Tris / hydrochloric acid buffer solution (100 mM, pH 8.0, 1
300 μl (containing 00 mM NaCl) was added, and the mixture was pulverized with a pellet mixer. Next, thermostable amylase (manufactured by Sigma, α-amylase, 790 U / mg solid,
5 μl of 1 mg / ml) was added and reacted at 60 ° C. for 1 hour. Then Tritirachi ( Arbutum)
Protease K derived from um album (manufactured by ONCO, 20 m
(g / ml) was added, and the mixture was reacted at 37 ° C. for 2 hours. After completion of the enzymatic reaction, ethanol 1 cooled to -20 ° C
After adding ml, and leaving still at -20 degreeC for 15 minutes, centrifugation (15000 G, 4 degreeC, 15 minutes, the same below) was performed with the microcentrifuge, and the precipitation was obtained. This precipitate is added to 300 μl of T
E (10 mM Tris / hydrochloric acid, pH 8.0, 1 mM E
It was dissolved in DTA), 400 μl of phenol was added, and DNA was extracted with a rotary stirrer for 30 minutes. Then, centrifugation is performed to collect the supernatant, and an equal volume of PCI (phenol / chloroform / isoamyl alcohol: 25/24/1)
Was added and extracted for 30 minutes, followed by centrifugation to obtain a supernatant. To this supernatant, 6 ml of 5M NaCl was added, 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 4 μm.
It was dissolved in 0 μl of 10-fold diluted TE to obtain a DNA sample solution. PCR was performed as follows.

Amplification of DNA by PCR was performed using the DNA prepared from cooked rice by the above-mentioned method as a template. DNA
Taq polymerase manufactured by Toyobo Co., Ltd. was used as a polymerase, and the reaction solution composition of PCR was as follows: sterile water 10 μl, polymerase 0.2 μl, reaction buffer 2.5 μl, MgC
l ₂ 2.0 μl, template DNA 3 μl, dNTPs (2.
5 mM) 1 μl and mixed with primers S13F25 and S13R24 of SEQ ID NOs: 83 and 84 0.1 μl each
l, 0.2 μl each of the primers F6F25 and F6R22 of SEQ ID NOS: 43 and 44, 0.1 μl each of the primers A6F21 and A6R22 of SEQ ID NOS: 3 and 4, and the primers P3F20 and P of SEQ ID NOS: 71 and 72
0.25 μl each of 3R15 was 20.0 μl in total. As a reaction device, PCR Thermal Cycler MP manufactured by Takara Shuzo Co., Ltd. is used, and denaturation (94 ° C., 1 minute) and annealing (60
The reaction was carried out for 45 cycles under the conditions of 1 ° C., 1 minute) and extension (72 ° C., 2 minutes). The electrophoresis and staining of the DNA amplified by PCR were performed in the same manner as in Example 1. The results of PCR are shown in Table 6.

[0075]

[Table 10] Table 6. Results of PCR with 4 kinds of primers (+: Discrimination band is expressed-: Discrimination band is not expressed)

As shown in Table 6, since only four varieties of "Hitomebore", "Akitakomachi" and "Sasanishiki" are negative in all four discrimination bands, these three varieties have other 17 types. When any one of the varieties was mixed with rice, the identification band was positively expressed by the first PCR, and it was shown that the mixed rice can be reliably detected.

Following the above-mentioned first PCR in which the discrimination bands of the target varieties (in this case, "Hitomebore" and "Akitakomachi") were all negative, a second PCR was carried out. In the second PCR, 0.5 μl each of primers WK9F20 and WK9R20 of SEQ ID NOS: 95 and 96,
0.05 μl each of primers E30F28 and E30R24 of SEQ ID NOS: 39 and 40, SEQ ID NOS: 51 and 5
2 primers G22F27 and G22R23 0.
2 μl, primer P5F2 of SEQ ID NOs: 75 and 76
0 and P5R25 0.3 μl each, and primers B43F17 and B43R18 of SEQ ID NOs: 27 and 28 0.25 μl each were used, except that the first PCR was performed in the same manner as in this Example. DNA amplified by secondary PCR
The results of electrophoresis and staining are shown in Table 7 and FIG.

[0078]

[Table 11] Table 7. Secondary P with 5 kinds of primers
CR result (+: Discrimination band is expressed-: Discrimination band is not expressed)

From the results of the second PCR, it was shown that it was possible to discriminate the three cultivars "Hitomebore", "Akitakomachi" and "Sasanishi" which were all negative in the first PCR. In addition, by adding 17 varieties in which a positive band was expressed in the first PCR and comparing Table 6 and Table 7 together, it was shown that it was possible to identify all the top 20 cultivars. Therefore, the presence or absence of mixed rice for the target varieties "Hitomebore" or "Akitakomachi" is possible in 17 varieties other than these 3 varieties by the first PCR in which these are negative bands. Identification was possible by secondary PCR. In addition, the primary PC
It was shown that even in the case of 17 varieties in which a positive band appeared in R, all of them could be identified by adding the results of the secondary PCR using one grain of cooked rice sample.

Example 5 (Example of detection of mixed rice and identification of mixed rice varieties by combined use of negative band DNA group) Top 20 cultivars in Japan in 2000, ie, "Koshihikari", "Hitomebore", "Hinohikari",
"Akita Komachi", "Kirara 397", "Kinuhikari", "Hoshi no Yume", "Haenuki", "Mutsuhomare", "Nippon Hare", "Sasanishi", "Tsugaru Roman", "Hanaechizen", "Yumetsukushi", "Hatsushima", "Morning light", "Moonlight", "Aichi no Kaori",
Using 20 kinds of brown rice, "Festival" and "Akiho" as samples,
Whitening, grinding, genomic DNA extraction and PCR were carried out in the same manner as in Example 1.

The reaction solution composition of PCR was 10 μl of sterilized water,
Polymerase (Takara Shuzo Co., Ltd., Taq polymerase (5U
/ Μl)) 0.2 μl, reaction buffer (12 mM Tris-HCl, 60 mM KCl, pH 8.3) 2.5 μl,
2.0 μl of MgCl ₂ , 25 ng of template DNA (1 μm
l), dNTPs (100 μM) 1 μl are mixed, and PC
As an oligonucleotide primer used for R, B43, G for detecting mixed rice against "Hinohikari"
28, F6, T16 primer set, A6, E30, F for detecting mixed rice against "Akitakomachi"
6, G22, P3 primer sets were prepared and the primers B43F17 and B4 of SEQ ID NOs: 23 and 24 were prepared.
3 R18 0.05 μl each, SEQ ID NOS: 47 and 48 primers G28F17 and G28R28 0.4 μ each
l, 0.3 μl each of primers F6F25 and F6R22 of SEQ ID NOS: 35 and 36, SEQ ID NOS: 81 and 82
Each of the primers T16F30 and T16R30 0.4
The total of μl was 19.0 μl. In addition, the primers A6F21 and A6R22 of SEQ ID NOS: 3 and 4, respectively, 0.5
μl, primer P3F20 of SEQ ID NOs: 71 and 72
And P3R15 2 μl each, SEQ ID NOs: 39 and 40 primers E30F28 and E30R24 0.3 μ each
l, 0.3 μl each of primers F6F25 and F6R22 of SEQ ID NOS: 43 and 44, SEQ ID NOS: 51 and 52
G22F27 and G22R23 0.3 each
The total volume was 23.5 μl.

A PCR thermal cycler MP manufactured by Takara Shuzo Co., Ltd. was used as a reaction apparatus, and 35 cycles of denaturation (94 ° C., 1 minute), annealing (61 ° C., 1 minute) and extension (72 ° C., 2 minutes) were performed. Let The electrophoresis and staining of the amplified DNA were performed in the same manner as in Example 1. The detection results are shown in Table 8.

[0083]

[Table 12] Table 8. PCR results for small amount of mixed rice with mixed rice detection primer set (+: Discrimination band is expressed-: Discrimination band is not expressed)

From the results shown in Table 8, for both "Hinohikari" and "Akitakomachi", a group of identification DNAs which did not appear in the target varieties but appeared only in other varieties by each negative primer set was used. It became clear that mixed rice of all the other top 20 varieties can be detected. Furthermore, when the PCR results using the 9 types of primers in Table 8 are combined, it is possible to distinguish all of the top 20 cultivars for planting from each other, and in the case of mixed rice expressing a positive band with the negative primer set, D
It was shown that it is possible to identify mixed rice varieties by extracting NA as template DNA and assaying with the above 9 kinds of primers.

Example 6 (Detection of Variety Mixing in Barley and Discrimination of Mixed Variety) Three kinds of barley, that is, seeds of "Sanshu", "Ichibanboshi" and "Large HK64", were yielded by a grinding type malting machine. She was 60% refined. These barleys were crushed by an ultracentrifugal crusher manufactured by Lecce to obtain powder samples. 6 g of these powder samples were weighed, DNA was extracted and purified by the CTAB method in the same manner as in Example 1, and template DN was used.
It was set to A.

The composition of the reaction solution for PCR was 0.2 (Taka polymerase (5 U / μl), manufactured by Takara Shuzo Co., Ltd.).
μl, reaction buffer (12 mM Tris-HCl, 60 mM
KCl, pH 8.3) 2.5 μl, MgCl ₂ 2.0
μl, template DNA 200 ng / 1 μl, dNTPs (1
00 μM) 1 μl was mixed and used as pairing primers for PCR in the first PCR, SEQ ID NOs: 59 and 60.
0.6 μl each of J6F18 and J6R20, 0.5 μ each of A7F19 and A7R16 of SEQ ID NOS: 7 and 8
1, B7F22 and B7R of SEQ ID NOs: 19 and 20
17 0.5 μl each and E3 of SEQ ID NOs: 39 and 40
0F28 and E30R24 0.4 μl each of 4 types of paired primers were used, and sterilized water was added to make a total of 20.7 μl.
It was set to l. In the second PCR, 0.5 μl each of G4F18 and G4R24 of SEQ ID NOs: 47 and 48, 0.5 μl each of F6F25 and F6R22 of SEQ ID NOs: 43 and 44, and M2CGF16 of SEQ ID NOs: 63 and 64.
And M2CGR15 0.5 μl each, SEQ ID NOS: 95 and 96 WK9F20 and WK9R20 0.4 μl each
The total amount of the reaction solution was set to 20.7 μl by using the four types of paired primers.

A PCR thermal cycler MP manufactured by Takara Shuzo Co., Ltd. was used as a reaction apparatus, and 35 cycles of denaturation (94 ° C., 1 minute), annealing (62 ° C., 1 minute) and extension (72 ° C., 2 minutes) were reacted. Let The amplified DNA was electrophoresed using Mupid II manufactured by Cosmo Bio Co., Ltd., electrophoresed on a 2% agarose gel for 40 minutes, and the band pattern was detected by ultraviolet irradiation after staining with ethidium bromide. The detection results are shown in Table 9. As a result, in the first PCR for detecting a mixture of varieties, only "Ichibanboshi" does not express the identification band in all four types of paired primers, whereas "Sanshu" and "Ohkei HK64" are J6. Since a discrimination band is expressed in, it can be detected when these varieties are mixed with "Ichibanboshi". Furthermore, as a result of the second PCR for judging the mixed varieties, in the case of "Sanshu", all were negative, whereas in the case of "Ichibanboshi", M2CG, "Great system HK"
In the case of "64", the discrimination band is expressed in the case of G4 and M2CG, and therefore, together with the result of the first PCR,
It was possible to discriminate between "Sanshu" and "large system HK64."

[0088]

[Table 13] Table 9. Results of detection of mixed varieties of barley and determination of mixed varieties by PCR (+: Discrimination band is expressed-: Discrimination band is not expressed)

Example 7 (Example of detection of mixed varieties and discrimination of mixed varieties in corn) Three varieties of corn, that is, "Peter corn",
The seeds of "honey bantam" and "waxy corn" were freeze-dried by an Islay freeze dryer FD200, and then pulverized by an Iwatani coffee mill to obtain a powder sample. 6 g of these powder samples were weighed, DNA was extracted and purified by the CTAB method in the same manner as in Example 1,
The template DNA was used.

The PCR reaction solution composition was sterile water 11.74.
μl, polymerase (Takara Shuzo Co., Ltd., Taq polymerase
(5 U / μl)) 0.2 μl, reaction buffer (12 mM
Tris-HCl, 60 mM KCl, pH 8.3) 2.5
μl, MgCl ₂ 2.0 μl, template DNA 200 ng
/ 1 μl, dNTPs (100 μM) 1 μl,
As pairing primers used for PCR, in the first PCR, 0.6 μl each of A7F19 and A7R16 of SEQ ID NOS: 7 and 8, 0.5 μl each of E30F28 and E30R24 of SEQ ID NOS: 39 and 40, SEQ ID NOS: 43 and 4 were used.
0.5 μl each of F6F25 and F6R22 of 4 and S13F25 and S13R2 of SEQ ID NOs: 83 and 84
4 A total of 22.44 μl was obtained using 0.4 μl of each of the 4 matched primers.

In the second PCR, 0.5 μl each of A6F21 and A6R22 of SEQ ID NOs: 3 and 4, 0.5 μl each of G4F18 and G4R24 of SEQ ID NOs: 47 and 48, and J6F18 of SEQ ID NOs: 59 and 60.
And J6R20 0.4 μl each, SEQ ID NOs: 63 and 6
0.3 μl each of 4 M2CGF16 and M2CGR15
20.7 μl using 4 pairs of matching primers
And As a reactor, Astec Co., Ltd. temperature control system PC700 is used, and denaturation (94 ℃, 1
Min), annealing (62 ° C, 1 min), extension (72 ° C,
2 minutes) was reacted for 35 cycles. Electrophoresis of amplified DNA was performed using Cosmo Bio Co., Ltd. Mupid II.
After running 40% agarose gel for 40 minutes, the band pattern was detected by UV irradiation after ethidium bromide staining. The detection results are shown in Table 10.

[0092]

[Table 14] Table 10. Results of detection of mixed varieties of corn and determination of mixed varieties by two-step PCR (+: Discrimination band is expressed-: Discrimination band is not expressed)

As a result, the first P
In CR, "honey bantam" and "Peter Conne" did not express the discriminating band in all four paired primers, whereas "waxy corn" produced A
In the case of 7, E30, F6, and S13, the identification band is expressed, and therefore, it can be detected when "waxy corn" is mixed with "honey bantam" or "peter corn". Furthermore, as a result of the second PCR for judging mixed varieties, in the case of "honey bantam", A6 and M2C
Since G, J6 in the case of "Peter corn" and A6, G4, J6, and M2CG in the case of "waxy corn" express the identification band, these three varieties are also combined with the results of the first PCR. Could be determined.

Example 8 (Definition of Range of Primer Length) Two types of sample brown rice, "Koshihikari" and "Hoshinoyume" were used. Rice mill for testing (Yamamoto Seisakusho, rice pal
VP31T) was used to refine the yield to 90%. Each milled rice sample was crushed using an ultracentrifugal crusher manufactured by Lecce, and No. 6
Genomic DNA was extracted from g by the CTAB method. The concentration of each sample DNA was adjusted so that the DNA concentration measured by the ultraviolet absorption method (260 nm) was about 400 μg / ml, and the sample was subjected to PCR.

As an oligonucleotide primer used for PCR, a long pairing primer having a sequence of 40 residues of SEQ ID NOs: 97 and 98 in the sequence listing, SEQ ID NO: 83.
(25-mer) and SEQ ID NO: 84 (24-mer), and 3 types of pairing primer, that is, SEQ ID NO: 99 (12-mer) and SEQ ID NO: 100 (12-mer). Each was used alone.

DNA polymerase is Ta, manufactured by Toyobo Co., Ltd.
q polymerase (5 U / μl) was used. The composition of the PCR reaction solution was 10 μl of sterilized water, 0.2 μl of polymerase,
2.5 μl of reaction buffer, 2.0 μl of MgCl ₂ , template DNA 3 μl, dNTPs (2.5 mM) 1.0 μl were mixed, and each paired primer 1.0 μl (total 2.0 μl
The total of 1) was 20.7 μl. As a reaction device, Takara Shuzo Co., Ltd., PCR Thermal Cycler MP is used, and denaturation (94 ° C, 1 minute), annealing (38 ° C, 1 minute),
The reaction was carried out for 45 cycles under the condition of extension (72 ° C., 2 minutes).

The amplified DNA was electrophoresed using Mupid II manufactured by Cosmo Bio Co., Ltd., electrophoresed on a 2% agarose gel for 40 minutes, and the band pattern was detected by UV irradiation after staining with ethidium bromide. As a result, no band appeared in the case of a 40-residue long pairing primer, and a large number of bands appeared in the case of a 12-residue short pairing primer. Was unsuitable for. In contrast, 25-mer and 24
In the case of the paired primer consisting of a monomer, the 1.8 kbp discrimination band appeared in "Hoshinoyume" and not in "Koshihikari", and it was possible to identify the variety.

Example 9 (Example of identification of cooked rice samples of foreign rice) "Koshihikari" (Japan), "One dish" (Korea), "Bannishiki" (China), "Amaroo" (Australia),
10 types of brown rice for each of 7 types of "Tamaki" (US), "Khao Dok Mali 105" (Thailand), and "Motoboy" (Vietnam)
Yield 90 g of rice with a Ket Science Research Institute's pallet, and polish each rice sample into a plastic tube (assist, 1.5 ml volume) and deionized water 3
After adding 5 μl and standing on the stand and immersing for 1 hour, cook the rice in an open state with an electric rice cooker (Toshiba, RC-183, add 75 ml of deionized water to the outer pot) for 15 minutes, steam for 15 minutes, and cooked rice sample And Extraction of DNA was performed as follows.

Samples One grain of cooked rice was placed in a microtube, and Tris / hydrochloric acid buffer solution (100 mM, pH 8.0, 1,
300 μl (containing 00 mM NaCl) was added, and the mixture was pulverized with a pellet mixer. Next, thermostable amylase (manufactured by Sigma, α-amylase, 790 U / mg solid,
5 μl of 1 mg / ml) was added and reacted at 60 ° C. for 1 hour. Then Torichirachiumu album (Tritirachium
Protease K from album (Onco, 20mg
/ Ml) was added and reacted at 37 ° C. for 2 hours.
After completion of the enzymatic reaction, 1 ml of ethanol cooled to -20 ° C
Was added, and the mixture was allowed to stand at −20 ° C. for 15 minutes, and then centrifuged (15000 G, 4 ° C., 15 minutes, the same applies below) with a microcentrifuge to obtain a precipitate. This precipitate is added to 300 μl of TE
(10 mM Tris / hydrochloric acid, pH 8.0, 1 mM ED
It was dissolved in TA), 400 μl of phenol was added, and DNA was extracted with a rotary stirrer for 30 minutes. Then, centrifugation is performed to collect the supernatant, an equal amount of PCI (phenol / chloroform / isoamyl alcohol: 25/24/1) is added, the mixture is extracted for 30 minutes, and the supernatant obtained by centrifugation is mixed with 5M.
Add 6 ml of NaCl and chilled ethanol 400μ
1 was added and the precipitate obtained by centrifugation was washed twice with 70% ethanol, and the final precipitate was dissolved in 40 μl of 10-fold diluted TE to prepare a DNA sample solution. PCR was performed as follows.

Amplification of DNA was performed using the DNA prepared from polished rice by the above-mentioned method as a template. Taq polymerase manufactured by Toyobo Co., Ltd. is used as a DNA polymerase, and P
The composition of the reaction solution of CR was as follows: sterile water 10 μl, polymerase 0.2 μl, reaction buffer 2.5 μl, MgCl ₂ 2.
0 μl, template DNA 3 μl, dNTPs (2.5 mM)
1.0 μl was mixed and 0.25 μl each of primers WK9F20 and WK9R20 of SEQ ID NOs: 95 and 96,
SEQ ID NOS: 7 and 8 primers A7F19 and A7
0.25 μl each of R16, 0.25 μ each of primers S13F25 and S13R24 of SEQ ID NOs: 83 and 84
1, 0.25 μl each of primers J6F18 and J6R20 of SEQ ID NOs: 59 and 60, and a total of 20.7 μl. As a reaction apparatus, a PCR thermal cycler MP manufactured by Takara Shuzo Co., Ltd. is used, and denaturation (94 ° C., 1 minute), annealing (60 ° C., 1 minute) and extension (72 ° C., 2 minutes) are performed under conditions of 45.
Cycle reaction was performed. The electrophoresis of the DNA amplified by PCR was performed in the same manner as in Example 1. Table 11 shows the results of PCR using DNA extracted from cooked rice of the top 7 cultivars as a template.

As is apparent from the table, it was shown that the method of the present invention can be applied to the case of cooked rice samples prepared from foreign rice. Pairing primers WKA9 and S13
For use only, "Bannishiki" and "Motoboy"
It was impossible to identify the two varieties, “Amaroo”, “Tamaki”, and “Khaodokmari 105”. On the other hand, by adding two types of pairing primers A7 and J6, it was possible to identify the above-mentioned two varieties and the above-mentioned three varieties, and it was possible to identify all the seven varieties used.

[0102]

[Table 15] Table 11. PCR result of 7 kinds of cooked rice with 4 kinds of pairing primers

Example 10 (Usefulness of Other Primer Groups) As sample brown rice, "Koshihikari", "Hinohikari", "Akitakomachi", "Kinuhikari", "Haenuki", "Nihonbare", "Sasanishi" were used for pairing. As a primer
B1F25 and B1R2 of SEQ ID NOs: 15 and 16
0, E22F20 and E2 of SEQ ID NOs: 35 and 36
2R21, Q16F25 and Q16R20 of SEQ ID NOs: 79 and 80, T8F22 of SEQ ID NOs: 87 and 88
And T8R25, B18F of SEQ ID NOs: 23 and 24
PCR and electrophoresis were performed in the same manner as in Example 1 except that 15 and B18R21 were used. The test results are shown in Table 12. As is clear from the table, these paired primers allowed all discrimination among the 7 varieties.

[0104]

[Table 16] Table 12. PCR results of 7 types of milled rice with various paired primers

[0105]

INDUSTRIAL APPLICABILITY According to the present invention, the fact that the grains of other varieties are mixed with the grains of rice, wheat, corn, barley, etc. is determined by the multiplex PCR using the DNA extracted from the grains as a template. It is possible to discriminate by using, and it is possible to identify the varieties of the mixed grain. That is,
By the first PCR, the presence / absence of mixed products can be determined easily and quickly, and even if a small amount is mixed with other products, it can be determined. Furthermore, the mixed PCR can be identified by the secondary PCR.

In particular, in the present invention, since PCR is carried out using a pairing primer having a high discriminating property, bands other than the target discriminating band disappear, and for example, the target is "Koshihikari".
Accurate variety discrimination is possible even in the case of closely related species such as strains. Moreover, in the present invention, since PCR is performed by the multiplex method in which paired primers are mixed and used together, the number of PCRs and the number of electrophoresis / staining can be reduced, and the type discrimination can be performed in a short time and at a low level. Can be implemented at cost.

[0107]

[Sequence list]                   SEQUENCE LISTING <110> National Food Research Institute <120> Presence / absence of mixed varieties in grains and method for distinguishing mixed varieties <130> P141294K <160> 100 <210> 1 <211> 30 <212> DNA <213> Artificial Sequence <400> 1 ccagctgaac gcctgtacta caagaattaa 30 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <400> 2 ccagctgtac gtcttcccca gcgccggcgg 30 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <400> 3 ccagctgtac gcctgtacta c 21 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <400> 4 ccagctgtac gtcttcccca gc 22 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <400> 5 tgcctcgcac cagaaatagt ataatcccaa 30 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <400> 6 tgcctcgcac catgaggtgt ggccgagtac 30 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <400> 7 tgcctcgcac cagaaatag 19 <210> 8 <211> 16 <212> DNA <213> Artificial Sequence <400> 8 tgcctcgcac catgag 16 <210> 9 <211> 30 <212> DNA <213> Artificial Sequence <400> 9 cttgtcatgt gtttatcatt tgactattta 30 <210> 10 <211> 30 <212> DNA <213> Artificial Sequence <400> 10 cttgtcatgt gtagtgcggc ttgttttctg 30 <210> 11 <211> 29 <212> DNA <213> Artificial Sequence <400> 11 cttgtcatgt gtttatcatt tgactattta 30 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <400> 12 cttgtcatgt gtagtccggc t 21 <210> 13 <211> 30 <212> DNA <213> Artificial Sequence <400> 13 gtttcgctcc tacagtaatt aaggggctat 30 <210> 14 <211> 30 <212> DNA <213> Artificial Sequence <400> 14 gtttcgctcc catgcaatct gcaaaagttt 30 <210> 15 <211> 25 <212> DNA <213> Artificial Sequence <400> 15 gtttcgctcc tacagtaatt aaggg 25 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <400> 16 gtttcgctcc catgcaatct 20 <210> 17 <211> 30 <212> DNA <213> Artificial Sequence <400> 17 caggtgtggg ttacaaggat gacccttggg 30 <210> 18 <211> 30 <212> DNA <213> Artificial Sequence <400> 18 caggtgtggg ttcacggcct tgattaataa 30 <210> 19 <211> 22 <212> DNA <213> Artificial Sequence <400> 19 caggtgtggg ttacaaggat ga 22 <210> 20 <211> 17 <212> DNA <213> Artificial Sequence <400> 20 caggtgtggg ttcacgg 17 <210> 21 <211> 30 <212> DNA <213> Artificial Sequence <400> 21 ccacagcagt gcttcatgtc atgtagaata 30 <210> 22 <211> 30 <212> DNA <213> Artificial Sequence <400> 22 ccacagcagt tcaaatacac caggaatttc 30 <210> 23 <211> 15 <212> DNA <213> Artificial Sequence <400> 23 ccacagcagt gcttc 15 <210> 24 <211> 21 <212> DNA <213> Artificial Sequence <400> 24 ccacagcagt gcttcatgtc a 21 <210> 25 <211> 30 <212> DNA <213> Artificial Sequence <400> 25 tggccggcat gactcacata cccaacatat 30 <210> 26 <211> 30 <212> DNA <213> Artificial Sequence <400> 26 actggccggc atcaagacca accaatttgg 30 <210> 27 <211> 17 <212> DNA <213> Artificial Sequence <400> 27 tggccggcat gactcac 17 <210> 28 <211> 18 <212> DNA <213> Artificial Sequence <400> 28 actggccggc atcaagac 18 <210> 29 <211> 30 <212> DNA <213> Artificial Sequence <400> 29 gtggatctga attcactcaa ctatttgtac 30 <210> 30 <211> 30 <212> DNA <213> Artificial Sequence <400> 30 gtggatctga atcacagatg acattatagg 30 <210> 31 <211> 23 <212> DNA <213> Artificial Sequence <400> 31 gtggatctga attcactcaa cta 23 <210> 32 <211> 21 <212> DNA <213> Artificial Sequence <400> 32 gtggatctga atcacagatg a 21 <210> 33 <211> 30 <212> DNA <213> Artificial Sequence <400> 33 ggaatggaac cgaagtggag ctattccctg 30 <210> 34 <211> 30 <212> DNA <213> Artificial Sequence <400> 34 ggaatggaac cgccgtaaac ttgaatgcta 30 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <400> 35 ggaatggaac cgaagtggag 20 <210> 36 <211> 21 <212> DNA <213> Artificial Sequence <400> 36 ggaatggaac cgccgtaaac t 21 <210> 37 <211> 30 <212> DNA <213> Artificial Sequence <400> 37 tacctggttg atgtatacag atctggttat 30 <210> 38 <211> 30 <212> DNA <213> Artificial Sequence <400> 38 atccctcgat ccctctagca ttatatcctc 30 <210> 39 <211> 28 <212> DNA <213> Artificial Sequence <400> 39 tacctggttg atgtatacag atctggtt 28 <210> 40 <211> 24 <212> DNA <213> Artificial Sequence <400> 40 atccctcgat ccctctagca ttat 24 <210> 41 <211> 30 <212> DNA <213> Artificial Sequence <400> 41 accactccat atatatcatc caaagttcta 30 <210> 42 <211> 30 <212> DNA <213> Artificial Sequence <400> 42 accactccat atcaccacaa ggcgtttagg 30 <210> 43 <211> 25 <212> DNA <213> Artificial Sequence <400> 43 accactccat atatatcatc caaag 25 <210> 44 <211> 22 <212> DNA <213> Artificial Sequence <400> 44 accactccat atcaccacaa gg 22 <210> 45 <211> 30 <212> DNA <213> Artificial Sequence <400> 45 gagaccgata tgcgattcgc ggcattggac 30 <210> 46 <211> 30 <212> DNA <213> Artificial Sequence <400> 46 gtggtgttta gatccagaga cttaacttta 30 <210> 47 <211> 18 <212> DNA <213> Artificial Sequence <400> 47 gagaccgata tgcgattc 18 <210> 48 <211> 24 <212> DNA <213> Artificial Sequence <400> 48 gtggtgttta gatccagaga ctta 24 <210> 49 <211> 30 <212> DNA <213> Artificial Sequence <400> 49 ctcactcaaa tttacagtgc attttcttgt 30 <210> 50 <211> 30 <212> DNA <213> Artificial Sequence <400> 50 agggccatga tacaagactc tgttctgtag 30 <210> 51 <211> 27 <212> DNA <213> Artificial Sequence <400> 51 ctcactcaaa tttacagtgc attttct 27 <210> 52 <211> 23 <212> DNA <213> Artificial Sequence <400> 52 agggccatga tacaagactc tgt 23 <210> 53 <211> 30 <212> DNA <213> Artificial Sequence <400> 53 ggccgtcgtt ctgcgatggt ctccaagaat 30 <210> 54 <211> 30 <212> DNA <213> Artificial Sequence <400> 54 ggagaatccc acagtaagtt tttctttgtt 30 <210> 55 <211> 17 <212> DNA <213> Artificial Sequence <400> 55 ggcggtcgtt ctgcgat 17 <210> 56 <211> 28 <212> DNA <213> Artificial Sequence <400> 56 ggagaatccc acagtaagtt tttctttg 28 <210> 57 <211> 30 <212> DNA <213> Artificial Sequence <400> 57gtcggagtgg tcagaccggg ctagcttttg 30 <210> 58 <211> 30 <212> DNA <213> Artificial Sequence <400> 58 gtcggagtgg atggagtagc ggtgggtgtg 30 <210> 59 <211> 18 <212> DNA <213> Artificial Sequence <400> 59 gtcggagtgg tcagaccg 18 <210> 60 <211> 20 <212> DNA <213> Artificial Sequence <400> 60 gtcggagtgg atggagtagc 20 <210> 61 <211> 30 <212> DNA <213> Artificial Sequence <400> 61 acaacgcctc cgatgatcga accatatctt 30 <210> 62 <211> 30 <212> DNA <213> Artificial Sequence <400> 62 acaacgcctc cgacaacaag attttctcct 30 <210> 63 <211> 16 <212> DNA <213> Artificial Sequence <400> 63 acaacgcctc cgatga 16 <210> 64 <211> 15 <212> DNA <213> Artificial Sequence <400> 64 acaacgcctc cgaca 15 <210> 65 <211> 30 <212> DNA <213> Artificial Sequence <400> 65 gtccactgtg accacaacat ttcttccagc 30 <210> 66 <211> 30 <212> DNA <213> Artificial Sequence <400> 66 gtccactgtg gggattgttc cataaaagat 30 <210> 67 <211> 20 <212> DNA <213> Artificial Sequence <400> 67 gtccactgtg accacaacat 20 <210> 68 <211> 20 <212> DNA <213> Artificial Sequence <400> 68 gtccactgtg gggattgttt 20 <210> 69 <211> 30 <212> DNA <213> Artificial Sequence <400> 69 aacgggccaa aaacggaggt cgtatggagc 30 <210> 70 <211> 30 <212> DNA <213> Artificial Sequence <400> 70 aacgggccaa cgcagccatt aaagagaaat 30 <210> 71 <211> 20 <212> DNA <213> Artificial Sequence <400> 71 aacgggccaa aaacggaggt 20 <210> 72 <211> 15 <212> DNA <213> Artificial Sequence <400> 72 aacgggccaa cgcag 15 <210> 73 <211> 30 <212> DNA <213> Artificial Sequence <400> 73 acaacggtcc gtccttgctt aggaaaaggc 30 <210> 74 <211> 30 <212> DNA <213> Artificial Sequence <400> 74 acaacggtcc aacagatact tttgaaaaac 30 <210> 75 <211> 20 <212> DNA <213> Artificial Sequence <400> 75 acaacggtcc gtccttgctt 20 <210> 76 <211> 25 <212> DNA <213> Artificial Sequence <400> 76 acaacggtcc aacagatact tttga 25 <210> 77 <211> 30 <212> DNA <213> Artificial Sequence <400> 77 agtgcagcca ttatatagga ctaacaagga 30 <210> 78 <211> 30 <212> DNA <213> Artificial Sequence <400> 78 agtgcagcca aaccagaaga aagccatgtt 30 <210> 79 <211> 25 <212> DNA <213> Artificial Sequence <400> 79 agtgcagcca ttatatagga ctaac 25 <210> 80 <211> 20 <212> DNA <213> Artificial Sequence <400> 80 agtgcagcca aaccagaaga 20 <210> 81 <211> 30 <212> DNA <213> Artificial Sequence <400> 81 gtcgttcctg tggttaggac agggtcgcaa 30 <210> 82 <211> 30 <212> DNA <213> Artificial Sequence <400> 82 gtcgttcctg ctggtgtctc agatcgttcg 30 <210> 83 <211> 25 <212> DNA <213> Artificial Sequence <400> 83 gtcgttcctg tggttaggac agggt 25 <210> 84 <211> 24 <212> DNA <213> Artificial Sequence <400> 84 gtcgttcctg ctggtgtctc agat 24 <210> 85 <211> 30 <212> DNA <213> Artificial Sequence <400> 85 aacggcgaca taaaataagt tgttacatgt 30 <210> 86 <211> 30 <212> DNA <213> Artificial Sequence <400> 86 aacggcgaca gtggcatgct cgatgacgac 30 <210> 87 <211> 22 <212> DNA <213> Artificial Sequence <400> 87 aacggcgaca taaaataagt tg 22 <210> 88 <211> 25 <212> DNA <213> Artificial Sequence <400> 88 aacggcgaca gtggcatgct cgatg 25 <210> 89 <211> 30 <212> DNA <213> Artificial Sequence <400> 89 ggtgaacgct gtagttggaa tataagtata 30 <210> 90 <211> 30 <212> DNA <213> Artificial Sequence <400> 90 ggtgaacgct cagatttaaa tataattagt 30 <210> 91 <211> 24 <212> DNA <213> Artificial Sequence <400> 91 ggtgaacgct gtagttggaa tata 24 <210> 92 <211> 26 <212> DNA <213> Artificial Sequence <400> 92 ggtgaacgct cagatttaaa tataat 26 <210> 93 <211> 30 <212> DNA <213> Artificial Sequence <400> 93 ccgcagttag atgcaccatt agaattgctt 30 <210> 94 <211> 30 <212> DNA <213> Artificial Sequence <400> 94 ccgcagttag atcaagtggc aaggttccat 30 <210> 95 <211> 20 <212> DNA <213> Artificial Sequence <400> 95 ccgcagttag atgcaccatt 20 <210> 96 <211> 20 <212> DNA <213> Artificial Sequence <400> 96 ccgcagttag atcaagtggc 20 <210> 97 <211> 40 <212> DNA <213> Artificial Sequence <400> 97 gtcgttcctg tggttaggac agggtcgcaa attcagtctt 40 <210> 98 <211> 40 <212> DNA <213> Artificial Sequence <400> 98 gtcgttcctg ctggtgtctc agatcgttcg ggggcttgcg 40 <210> 99 <211> 12 <212> DNA <213> Artificial Sequence <400> 99 gtcgttcctg tg 12 <210> 100 <211> 12 <212> DNA <213> Artificial Sequence <400> 100 gtcgttcctg ct 12

[Brief description of drawings]

FIG. 1 is a migration photograph showing the results of PCR for detecting mixed rice in “Koshihikari” using a negative band DNA for discrimination.

[Explanation of symbols]

M is a molecular weight marker, 1 is “Koshihikari”, 2 is “Hitomebore”, 3 is “Hinohikari”, 4 is “Akitakomachi”, 5 is “Kirara 397”, 6 is “Kinuhikari”, 7
Is "Hoshi no Yume", 8 is "Haenuki", 9 is "Mutsuhomare", 10 is "Nihonbare", 11 is "Sasanishi", 12
Is "Tsugaru Roman", 13 is "Hana Echizen", 14 is "Dream Tsukushi", 15 is "Hatsushima", 16 is "Morning light", 17 is "Moonlight", 18 is "Aichi no Kaori",
19 indicates "Festival clear" and 20 indicates "Akiho".

FIG. 2 is a migration photograph showing the result of PCR for detecting mixed rice in “Koshihikari” by the negative band / positive band DNA group for discrimination.

[Explanation of symbols]

M is a molecular weight marker, 1 is “Koshihikari”, 2 is “Hitomebore”, 3 is “Hinohikari”, 4 is “Akitakomachi”, 5 is “Kirara 397”, 6 is “Kinuhikari”, 7
Is "Hoshi no Yume", 8 is "Haenuki", 9 is "Mutsuhomare", 10 is "Nihonbare", 11 is "Sasanishi", 12
Is "Tsugaru Roman", 13 is "Hana Echizen", 14 is "Dream Tsukushi", 15 is "Hatsushima", 16 is "Morning light", 17 is "Moonlight", 18 is "Aichi no Kaori",
19 indicates "Festival clear" and 20 indicates "Akiho".

FIG. 3 is a migration photograph showing the result of PCR for detecting mixed rice in “Koshihikari” using a negative band DNA for discrimination.

[Explanation of symbols]

M is a molecular weight marker, 1 is “Koshihikari”, 2 is “Hitomebore”, 3 is “Hinohikari”, 4 is “Akitakomachi”, 5 is “Kirara 397”, 6 is “Kinuhikari”, 7
Is "Hoshi no Yume", 8 is "Haenuki", 9 is "Mutsuhomare", 10 is "Nihonbare", 11 is "Sasanishi", 12
Is "Tsugaru Roman", 13 is "Hana Echizen", 14 is "Dream Tsukushi", 15 is "Hatsushima", 16 is "Morning light", 17 is "Moonlight", 18 is "Aichi no Kaori",
19 indicates "Festival clear" and 20 indicates "Akiho".

FIG. 4 is an electrophoretic photograph showing the results of secondary PCR with 5 types of primers.

[Explanation of symbols]

M is a molecular weight marker, 1 is “Koshihikari”, 2 is “Hitomebore”, 3 is “Hinohikari”, 4 is “Akitakomachi”, 5 is “Kirara 397”, 6 is “Kinuhikari”, 7
Is "Hoshi no Yume", 8 is "Haenuki", 9 is "Mutsuhomare", 10 is "Nihonbare", 11 is "Sasanishi", 12
Is "Tsugaru Roman", 13 is "Hana Echizen", 14 is "Dream Tsukushi", 15 is "Hatsushima", 16 is "Morning light", 17 is "Moonlight", 18 is "Aichi no Kaori",
19 indicates "Festival clear" and 20 indicates "Akiho".

Front page continuation (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 33/566 C12N 15/00 ZNAA (71) Applicant 302019245 Takara Bio Co., Ltd. 3-4-1, Seta, Otsu, Shiga Prefecture (72) Kenichi Otsubo 4227 Ami, Ami-cho, Inashiki-gun, Ibaraki Prefecture 10 (72) Sumiko Nakamura 52-13 Hoyodai, Kukizaki-cho, Inashiki-gun, Ibaraki Prefecture (72) Inventor Takeshi Miyamura 4-chome, Seta 3-chome, Otsu City, Shiga Prefecture No. 1 in Takara Bio Co., Ltd. (72) Inventor Satoshi Unmo 3-4 Seta, Otsu City, Shiga Prefecture No. 1 in Takara Bio Co., Ltd. (72) Ikunobu Kato 3-4-1 Seta, Otsu City, Shiga Prefecture Takara Bio Co., Ltd. F-term (reference) 2B030 AA02 AB03 AD20 CA06 CB03 2B121 CB09 CB25 CB46 EA01 FA04 4B024 AA11 CA02 CA09 HA12 4B063 QA01 QA18 QA20 QQ04 QQ42 QR40 QR62 QS14 QS25 QS36 QS39 QX01

Claims (16)

[Claims] 1. A DN extracted from the grain or a processed product thereof, to determine whether or not the grain is mixed with grains of other varieties.
In the discrimination using the multiplex PCR method using A as a template, the target varieties are characterized by using a pair of negative band discrimination primers that do not express discrimination bands and selectively express only in mixed varieties. Cereal DNA
Variety identification method. 2. The method for discriminating DNA varieties of cereals according to claim 1, wherein the discrimination band is not expressed even when the pair of negative band discrimination primers is used,
Second-order multiplex PCR using a primer consisting of a pair of paired primers for identifying positive bands specifically expressed only in the target variety and / or a pair of paired primers for identifying negative bands not specifically expressed only in the target variety A method for discriminating DNA varieties of cereals, characterized by performing the method and confirming that the varieties are target varieties based on the expressed DNA band pattern. 3. The grain D according to claim 1 or 2, wherein the grain is rice, wheat, corn or barley.
NA type discrimination method. 4. The method for discriminating DNA varieties of cereals according to claim 3, wherein the target varieties of rice are good-tasting rice. 5. The method for discriminating DNA varieties of cereals according to claim 4, wherein the good-tasting rice is any one of “Koshihikari”, “Hitomebore”, “Akitakomachi”, and “Hinohikari”. 6. The method for discriminating DNA varieties of cereals according to claim 4 or 5, wherein a discrimination band is expressed using a pair of negative band discrimination primers.
Using the DNA extracted from each grain of sample rice as a template, a pair of positive band-identifying pairing primers specifically expressed only in the target cultivar and / or a pair of negative band identifying primers not specifically expressed only in the target cultivar A method of performing a second multiplex PCR method using a primer consisting of a group, and identifying a mixed variety mixed with the target variety based on the expressed DNA band pattern. 7. A pairing primer used in the multiplex PCR method is represented by SEQ ID NOs: 1 and 2, 5 and 6, 9 and 10, 13 and 14, 17 and 18, of the sequence listing.
21 and 22, 25 and 26, 29 and 30, 3
3 and 34, 37 and 38, 41 and 42, 45
And 46, 49 and 50, 53 and 54, 57 and 58, 61 and 62, 65 and 66, 69 and 70, 73 and 74, 77 and 78, 81 and 82, 85 and 86, 89 and 90, 93 and 9
A6F30 and A6R which are the pairing primers described in 4.
30, A7F30 and A7R30, A52F30 and A52R30, B1F30 and B1R30, B7F
30 and B7R30, B18F30 and B18R3
0, B43F30 and B43R30, D4F30 and D4R30, E22F30 and E22R30, E3
0F30 and E30R30, F6F30 and F6R
30, G4F30 and G4R30, G22F30 and G22R30, G28F30 and G28R30, J
6F30 and J6R30, M2CGF30 and M2
CGR30, M11F30 and M11R30, P3F
30 and P3R30, P5F30 and P5R30,
Q16F30 and Q16R30, S13F30 and S13R30, T8F30 and T8R30, T16F
30 and T16R30, WK9F30 and WK9R
30 with 1 to 17 bases on the 3'side excised 1
The method for discriminating DNA varieties of cereals according to claim 1 or 2, wherein two or more kinds of primers selected from the group consisting of pairing primers composed of 3 to 29 bases are used. 8. The pairing primers used in the multiplex PCR method are SEQ ID NOS: 3 and 4, 7 and 8, 11 and 12, 15 and 16, 19 and 2 in the sequence listing.
0, 23 and 24, 27 and 28, 31 and 3
2, 35 and 36, 39 and 40, 43 and 4
4, 47 and 48, 51 and 52, 55 and 5
6, 59 and 60, 63 and 64, 67 and 6
8, 71 and 72, 75 and 76, 79 and 8
0, 83 and 84, 87 and 88, 91 and 9
A6F which is the pairing primer described in 2, 95 and 96.
21 and A6R22, A7F19 and A7R16,
A52F29 and A52R21, B1F25 and B
1R20, B7F22 and B7R17, B18F15
And B18R21, B43F17 and B43R1
8, D4F23 and D4R21, E22F20 and E22R21, E30F28 and E30R24, F6
F25 and F6R22, G4F18 and G4R2
4, G22F27 and G22R23, G28F17 and G28R28, J6F18 and J6R20, M2
CGF16 and M2CGR15, M11F20 and M11R20, P3F20 and P3R15, P5F2
0 and P5R25, Q16F25 and Q16R2
0, S13F25 and S13R24, T8F22 and T8R25, T16F24 and T16R26, WK
2 selected from the group consisting of 9F20 and WK9R20
The method for discriminating DNA varieties of cereals according to claim 1, wherein at least one kind of primer is used. 9. The good-tasting rice is “Koshihikari”, and B
43, G22, M11 and WK9, and a primer set composed of four types of paired primers and / or A6, B7, E30, F6, G4, M2CG, S1.
7. The multiplex PCR method using a pairing primer set for negative band discrimination consisting of three or more pairing primers selected from the group consisting of 3, T8, T16 and WK9, according to claim 1. A method for discriminating DNA varieties of cereals. 10. The good-tasting rice is “Hitomebore”,
A6, B7, F6, G4, P3, S13, T8 and T
Negative band discrimination pairing primer set consisting of three or more pairing primers selected from the group consisting of 16 and / or B7, G4, G22, P5 and WK
The D of the cereal grain according to any one of claims 1 to 6, wherein a primer set composed of 5 kinds of paired primers of 9 is used.
NA type discrimination method. 11. The good-tasting rice is “Akitakomachi”, which is A6, B7, E30, F6, G4, G22, M2C.
Negative band identifying pairing primer set consisting of three or more pairing primers selected from the group consisting of G, P3, S13 and T16 and / or A6, B
4, M2CG and P3 or WK9, B4
7. The method for discriminating DNA varieties of cereals according to claim 1, wherein a primer set composed of four types of pairing primers of 3, M11 and G22 is used. 12. The good-tasting rice is “Hinohikari”,
A6, B7, B43, E22, E30, F6, G4, G
No. 28, S13, T8, and T16, and a negative-band identifying pairing primer set and / or B43 consisting of three or more pairing primers selected from the group consisting of
Four types of F6, G28 and T16 or B43, G2
7. A primer set composed of 5 types of paired primers of 2, G28, P5 and WK9 is used.
The method for discriminating DNA varieties of cereals according to any one of 1. 13. The method for discriminating DNA varieties of cereals according to claim 1, wherein the sample rice is cooked rice. 14. The method for discriminating DNA varieties of cereals according to claim 1, wherein each grain of the mixed sample rice is assayed. 15. The target variety does not express a discrimination band,
A grain DN comprising a pair of primer pairs for identifying a negative band selectively expressed only in a mixed variety
A type discrimination kit. 16. A primer comprising a paired primer group for positive band discrimination specifically expressed only in a target variety and / or a paired primer group for negative band discrimination not specifically expressed only in a target variety. Kit for determining DNA variety of cereals.