JP6145315B2 - Determination method of Koshihikari from Niigata Prefecture and primer set used for it - Google Patents

Description

  The present invention relates to a method for determining Koshihikari produced in Niigata Prefecture, a primer set used therefor, and the like.

  Since Koshihikari from Niigata Prefecture is traded at a price approximately 20% higher than Koshihikari from other prefectures and other main varieties, there are no end to the production area disguise and variety disguise. Therefore, there is a need for a method for easily distinguishing Koshihikari from Niigata Prefecture from rice from other prefectures and other varieties.

  As a method of identifying Koshihikari produced in Niigata Prefecture, an identification method utilizing the fact that Koshihikari produced in Niigata Prefecture has been switched to “Koshihikari Niigata BL”, which is resistant to disease, and identification using differences between the production areas of Koshihikari The law (Patent Document 2) has been reported.

JP 2004-141079 A JP2011-193799A

  However, with regard to the above-described prior art, (1) it is necessary to determine that it is Koshihikari before determining whether it is produced in Niigata Prefecture, or (2) there is a mixture of other varieties or Koshihikari from other prefectures. There are problems such as being unable to determine correctly when there is, and (3) taking a relatively long time for DNA amplification and its confirmation because of using the PCR method. Therefore, there is a demand for the development of a method that can easily and specifically determine Koshihikari from Niigata Prefecture.

  As a result of intensive studies, the present inventors have developed a method that can easily and specifically determine Koshihikari produced in Niigata Prefecture by using a specific site in the first chromosome of a constituent variety of Koshihikari produced in Niigata Prefecture as a marker. Succeeded. The inventors of the present invention have also succeeded in developing a primer set used in the method, thereby completing the present invention.

That is, the present invention is as follows.
[1] A primer set composed of two or more primers capable of detecting the presence or absence of insertion of the first target mPing found in the first chromosome of a constituent variety of Koshihikari produced in Niigata Prefecture.
[2] The primer set of [1], wherein the primer set is a LAMP primer set.
[3] The primer set is a primer consisting of the base sequence of SEQ ID NO: 5, a primer consisting of the base sequence of SEQ ID NO: 6, a primer consisting of the base sequence of SEQ ID NO: 7, a primer consisting of the base sequence of SEQ ID NO: 8, The primer set of [2], comprising a primer comprising the nucleotide sequence of SEQ ID NO: 10 and a primer comprising the nucleotide sequence of SEQ ID NO: 10.
[4] A kit comprising the following primer set (1) and one or more primer sets selected from the group consisting of (2), (3), (4) and (5) below:
(1) a first primer set comprising two or more primers capable of detecting the presence or absence of insertion of the first target mPing found in the first chromosome of a constituent variety of Koshihikari from Niigata Prefecture; and (2) Niigata A second primer set composed of two or more primers capable of detecting the presence or absence of insertion of the second target mPing not found in chromosome 8 of the constituent Koshihikari prefecture varieties;
(3) A third primer set comprising two or more primers capable of detecting the presence or absence of insertion of a third target mPing that is not found in chromosome 8 of a constituent variety of Koshihikari produced in Niigata Prefecture;
(4) a fourth primer set comprising two or more primers capable of detecting varieties other than Koshihikari; and (5) a fifth primer comprising two or more primers capable of specifically detecting Koshihikari. Primer set.
[5] (1) The first primer set includes a primer consisting of the base sequence of SEQ ID NO: 5, a primer consisting of the base sequence of SEQ ID NO: 6, a primer consisting of the base sequence of SEQ ID NO: 7, and a base sequence of SEQ ID NO: 8. A primer consisting of the base sequence of SEQ ID NO: 9, and a primer consisting of the base sequence of SEQ ID NO: 10,
(2) The second primer set is a primer consisting of the base sequence of SEQ ID NO: 11, a primer consisting of the base sequence of SEQ ID NO: 12, a primer consisting of the base sequence of SEQ ID NO: 13, a primer consisting of the base sequence of SEQ ID NO: 14, Composed of a primer consisting of the base sequence of SEQ ID NO: 15 and a primer consisting of the base sequence of SEQ ID NO: 16,
(3) The third primer set is a primer consisting of the base sequence of SEQ ID NO: 17, a primer consisting of the base sequence of SEQ ID NO: 18, a primer consisting of the base sequence of SEQ ID NO: 19, a primer consisting of the base sequence of SEQ ID NO: 20, Composed of a primer consisting of the base sequence of SEQ ID NO: 21 and a primer consisting of the base sequence of SEQ ID NO: 22,
(4) The fourth primer set is a primer consisting of the base sequence of SEQ ID NO: 23, a primer consisting of the base sequence of SEQ ID NO: 24, a primer consisting of the base sequence of SEQ ID NO: 25, a primer consisting of the base sequence of SEQ ID NO: 26, Composed of a primer consisting of the base sequence of SEQ ID NO: 27 and a primer consisting of the base sequence of SEQ ID NO: 28,
(5) the fifth primer set is a primer consisting of the base sequence of SEQ ID NO: 29, a primer consisting of the base sequence of SEQ ID NO: 30, a primer consisting of the base sequence of SEQ ID NO: 31, a primer consisting of the base sequence of SEQ ID NO: 32, The kit according to [4], comprising a primer consisting of the base sequence of SEQ ID NO: 33 and a primer consisting of the base sequence of SEQ ID NO: 34.
[6] The first target by an amplification reaction using at least a primer set composed of two or more primers capable of detecting the presence or absence of insertion of the first target mPing found in the first chromosome of the constituent cultivar of Koshihikari produced in Niigata Prefecture A method for discriminating rice production areas or varieties, including confirming the presence or absence of mPing insertion.
[7] In an amplification reaction using the primer sets of (1), (2) and (3) below, the results of (I), (II) and (III) below are confirmed. The discrimination method of [6], which is a method for judging Koshihikari:
(1) A first primer set composed of two or more primers capable of detecting the presence or absence of insertion of the first target mPing found in the first chromosome of a constituent variety of Koshihikari produced in Niigata Prefecture;
(2) a second primer set comprising two or more primers capable of detecting the presence or absence of insertion of a second target mPing not found in chromosome 8 of a constituent variety of Koshihikari from Niigata Prefecture; and (3) A third primer set consisting of two or more primers capable of detecting the presence or absence of insertion of a third target mPing not found in chromosome 8 of a constituent variety of Koshihikari from Niigata Prefecture;
(I) Confirming that the first target mPing has been inserted by an amplification reaction using the first primer set;
(II) Confirm that the second target mPing is not inserted by an amplification reaction using the second primer set; and (III) Insert the third target mPing by an amplification reaction using the third primer set. Make sure that it is not.
[8] In the amplification reaction using the primer sets of (1), (2) and (3) below, the results of (IV) and (V) below are confirmed, and Koshihikari produced in Niigata Prefecture The discrimination method of [6], which is a method for judging that there is contamination of Koshihikari from Japan or rice of other varieties:
(1) A first primer set composed of two or more primers capable of detecting the presence or absence of insertion of the first target mPing found in the first chromosome of a constituent variety of Koshihikari produced in Niigata Prefecture;
(2) a second primer set comprising two or more primers capable of detecting the presence or absence of insertion of a second target mPing not found in chromosome 8 of a constituent variety of Koshihikari from Niigata Prefecture; and (3) A third primer set consisting of two or more primers capable of detecting the presence or absence of insertion of a third target mPing not found in chromosome 8 of a constituent variety of Koshihikari from Niigata Prefecture;
(IV) confirming that the first target mPing is inserted by an amplification reaction using the first primer set; and (V) using one or both of the second primer set and the third primer set. Confirm that the second target mPing or the third target mPing is inserted by an amplification reaction.
[9] In the amplification reaction using the primer sets of (1), (4) and (5) below, the results of (VI), (VII) and (VIII) below are confirmed, except for Koshihikari Discrimination method of [6], which is a method of judging rice of a variety:
(1) A first primer set composed of two or more primers capable of detecting the presence or absence of insertion of the first target mPing found in the first chromosome of a constituent variety of Koshihikari produced in Niigata Prefecture;
(4) a fourth primer set composed of two or more primers capable of detecting rice of varieties other than Koshihikari; and (5) a second primer composed of two or more primers capable of specifically detecting Koshihikari. 5 primer sets;
(VI) confirming that the first target mPing is not inserted by an amplification reaction using the first primer set;
(VII) Confirm that the rice is of a variety other than Koshihikari by an amplification reaction using the fourth primer set; and (VIII) Confirm that it is not Koshihikari by an amplification reaction using the fifth primer set. .
[10] Niigata Prefecture characterized by confirming the following results (IX), (X) and (XI) in an amplification reaction using the primer sets of (1), (4) and (5): Discrimination method of [6], which is a method of judging Koshihikari from other prefectures:
(1) A first primer set composed of two or more primers capable of detecting the presence or absence of insertion of the first target mPing found in the first chromosome of a constituent variety of Koshihikari produced in Niigata Prefecture;
(4) a fourth primer set composed of two or more primers capable of detecting rice of varieties other than Koshihikari; and (5) a second primer composed of two or more primers capable of specifically detecting Koshihikari. 5 primer sets;
(IX) Confirming that the first target mPing is not inserted by an amplification reaction using the first primer set;
(X) Confirm that it is not a rice of a variety other than Koshihikari by an amplification reaction using the fourth primer set; and (XI) Confirm that it is Koshihikari by an amplification reaction using the fifth primer set. .
[11] In an amplification reaction using the primer set of (1), (4) and (5) below, the results of (XII), (XIII) and (XIV) below are confirmed, except for Koshihikari The discrimination method according to [6], which is a method for judging that it is a mixture of rice of a variety and Koshihikari from prefectures other than Niigata prefecture:
(1) A first primer set composed of two or more primers capable of detecting the presence or absence of insertion of the first target mPing found in the first chromosome of a constituent variety of Koshihikari produced in Niigata Prefecture;
(4) a fourth primer set composed of two or more primers capable of detecting rice of varieties other than Koshihikari; and (5) a second primer composed of two or more primers capable of specifically detecting Koshihikari. 5 primer sets;
(XII) confirming that the first target mPing is not inserted by an amplification reaction using the first primer set;
(XIII) Confirm that the rice is of a variety other than Koshihikari by an amplification reaction using the fourth primer set; and (XIV) Confirm that it is Koshihikari by an amplification reaction using the fifth primer set. about.

  In the present invention, it is possible to determine the presence of Koshihikari produced in Niigata Prefecture and the presence or absence of contamination from other prefectures and other varieties only by performing an amplification reaction with a specific primer set. Further, the determination can be made within one hour by using the LAMP method.

FIG. 1 shows the first target mPing inserted in the first chromosome of the blast resistance line (BL) constituting Niigata Koshihikari in Nipponbare lacking the first target mPing. It is a figure which shows the base sequence (sequence number 1) around a corresponding site. The base sequence of SEQ ID NO: 1 consists of nucleotide residues 28461789 to 28463785 of chromosome 1 obtained from Os-Nipponbare-Reference-IRGSP-1.0 (IRGSP-1.0). The underlined portion indicates a target site (TAA) capable of inserting mPing (a site that can be inserted when assuming that the first target mPing is inserted in Nipponbare). FIG. 2 shows the first target mPing insertion site in the first chromosome of the constituent varieties of Koshihikari produced in Niigata Prefecture (the site consisting of the underlined nucleotides from 1001 to 1430 in the base sequence of SEQ ID NO: 2) and its It is a figure which shows an adjacent part (each 1 kb). FIG. 3 is a diagram showing corresponding sites in Nihonbare (second target mPing and its adjacent sites, SEQ ID NO: 3) in relation to the second target mPing that is not found in chromosome 8 of the constituent variety of Koshihikari from Niigata Prefecture. It is. The underlined portion indicates the base sequence of the second target mPing. FIG. 4 is a diagram showing a corresponding site in Nihonbare (third target mPing and its adjacent site, SEQ ID NO: 4) in relation to the third target mPing that is not found in chromosome 8 of the constituent variety of Koshihikari from Niigata Prefecture. It is. The underlined portion indicates the base sequence of the third target mPing. FIG. 5 is a diagram showing an outline of various regions necessary for designing a LAMP primer set. FIG. 6 is a diagram showing an outline of two or more primers for detecting the presence or absence of mPing insertion. FIG. 7 is a diagram showing a determination example using the fluorescence color development of the LAMP reaction solution. (A) Niigata Prefecture Koshihikari, (B) Tochigi Prefecture Koshihikari, (C) Miyagi Prefecture Hitomebore, (D) Niigata Prefecture Koshihikari and Tochigi Prefecture Koshihikari, (E) Niigata Prefecture Koshihikari and Miyagi Prefecture Mixed sample of Hitomeboru from Japan, (F) Mixed sample of Koshihikari from Tochigi Prefecture and Hitomeboro from Miyagi Prefecture. 1: NK1 primer set, 2: NK2 primer set, 3: NK3 primer set, 4: NK4 primer set, 5: NK5 primer set.

  The present invention provides a method for discriminating rice production areas or varieties. The method of the present invention comprises an amplification reaction using at least a primer set composed of two or more primers capable of detecting the presence or absence of insertion of the first target mPing found in the first chromosome of a constituent variety of Koshihikari produced in Niigata Prefecture, Including the presence or absence of insertion of the first target mPing.

  mPing is one type of transposon. mPing is a 430 bp polynucleotide that replicates the TAA or TTA of the insertion site in the genome and has both ends of the insertion site, so if there is an insertion (430 bp + 3) bp longer (eg see below) ).

  Koshihikari produced in Niigata Prefecture is composed of a plurality of blast disease resistant varieties (Blast resistance Line: BL). According to the present invention, by using a DNA marker specific for such a variety, Koshihikari produced in Niigata Prefecture since 2005 when it was switched to BL (eg, all produced in 2005-2012) is specifically detected. it can.

  The amplification reaction is not particularly limited as long as a test by amplification of DNA using two or more primers is possible, and examples thereof include an amplification reaction under isothermal conditions and an amplification reaction under non-isothermal conditions. Examples of the amplification reaction under isothermal conditions include, for example, the LAMP (Loop-Mediated Isometric Amplification) method, the ICAN (Isothermal and Chimeric Primer-initiated Amplified of Nucleic Acids Amplified, Amplified Amplified, Amplified Acid, Amplified of Amplified Acid, Amplified Amplified Acid, Amplified Amplified Acid, Amplified Amplified Acid, Amplified Amplified Acid. (Amplification) method. An example of the amplification reaction under non-isothermal conditions is a PCR method. In the present invention, the amplification reaction is preferably an amplification reaction under isothermal conditions, more preferably the LAMP method.

  In the method of the present invention, the sample subjected to the amplification reaction is a sample containing or possibly containing genomic DNA derived from rice or a processed product thereof. The rice may be either brown rice or polished rice. Rice can also be either domestic rice or foreign rice. Examples of processed rice products include cooked rice, processed rice, miso, rice confectionery, rice flour, packaging rice, rice noodles, brown rice tea, rice flour bread, rice flour noodles, sake, and beer.

Examples of the primer set used in the method of the present invention include the following:
(1) a primer set composed of two or more primers capable of detecting the presence or absence of insertion of the first target mPing found in the first chromosome of a constituent variety of Koshihikari from Niigata Prefecture; and (2) of Koshihikari from Niigata Prefecture; A primer set composed of two or more primers capable of detecting the presence or absence of insertion of the second target mPing not found in the chromosome 8 of the constituent variety;
(3) A primer set composed of two or more primers capable of detecting the presence or absence of insertion of a third target mPing not found in chromosome 8 of a constituent variety of Koshihikari produced in Niigata Prefecture;
(4) a primer set composed of two or more primers capable of detecting varieties other than Koshihikari; and (5) a primer set composed of two or more primers capable of specifically detecting Koshihikari.

  The number of primers included in each primer set varies depending on the amplification reaction employed, but is, for example, 2 to 6.

  The number of nucleotide residues constituting the primer (the nucleotide residue of the complementary portion that anneals to the target site) is not particularly limited as long as the primer can anneal to the target site and specifically amplify the target product. It may be 16 or more, 17 or more, 18 or more, or 20 or more. The number of nucleotide residues constituting the primer may be, for example, 50 or less, 40 or less, or 30 or less.

  The primer may be labeled with a substance such as a fluorescent substance in order to easily detect the amplification product. Examples of such fluorescent materials include 6-FAM, VIC, NED, and PET. Of course, the primer need not necessarily be labeled with such a substance. For example, the difference in size of amplification products may be confirmed by gel electrophoresis. In the detection by the LAMP method, the presence or absence of amplification can be confirmed by visually confirming the white turbidity of magnesium pyrophosphate, which is a byproduct of the amplification reaction. In such a case, it is not necessary to label the primer with a substance such as a fluorescent substance.

In a preferred embodiment, the primer set is a LAMP primer set. The LAMP primer set is composed of i) to iv) below (see also FIG. 5).
i) FIP (Forward Inner Primer): designed to have an F2 region complementary to the F2c region of the target DNA at the 3 ′ end and the same sequence as the F1c region of the target DNA at the 5 ′ end;
ii) F3 primer: designed to have an F3 region complementary to the F3c region of the target DNA;
iii) BIP (Backward Inner Primer): designed to have a B2 region complementary to the B2c region of the target DNA at the 3 ′ end and the same sequence as the B1c region of the target DNA at the 5 ′ end;
iv) B3 primer: designed to have a B3 region complementary to the B3c region of the target DNA.

Preferably, the LAMP primer set may further include the following loop primers (see also FIG. 5).
v) LF (Loop Primer F): designed to have a complementary sequence in the part between the F1 and F2 regions;
vi) LB (Loop Primer B): designed to have a complementary sequence in the portion between the B1 and B2 regions;
The relationship between the target DNA and the F1, F2, F3, F1c, F2c, F3c, B1, B2, B3, B1c, B2c, and B3c regions and details of the LAMP method are described, for example, on the Eiken Chemical website. This can be confirmed by referring to the principle of the LAMP method.

  Hereinafter, the specific primer set used suitably by the method of this invention is explained in full detail.

(1) Primer set composed of two or more primers capable of detecting the presence or absence of the insertion of the first target mPing found in the first chromosome of the Koshihikari varieties produced in Niigata Prefecture. The first target mPing inserted into the chromosome is not inserted into the corresponding site in the first chromosome of Nipponbare, which is the standard variety of Japanese rice.
For such a first target mPing, as the periphery of the corresponding site in Nipponbare (the whole genome sequence has been decrypted and published), which is a Japanese rice standard variety, Os-Nipponbare-Reference-IRGSP-1.0 (IRGSP-1. 0) (http://rapdb.dna.affrc.go.jp), the nucleotide sequence (SEQ ID NO: 1) consisting of nucleotide residues 28461789 to 28463785 of the first chromosome can be exemplified (FIG. 1). ). The location of the first target mPing inserted into the first chromosome of the Koshihikari varieties produced in Niigata Prefecture is the 1000th nucleotide residue and the 1001st nucleotide residue in the nucleotide sequence of SEQ ID NO: 1 derived from Nipponbare. (Between the nucleotide residue 28462788 and the nucleotide residue 28462789 on the first chromosome) (FIG. 1).
On the other hand, the 1st target mPing inserted in the 1st chromosome of the constituent variety of Koshihikari produced in Niigata Prefecture has the base sequence of SEQ ID NO: 2 (FIG. 2). In the base sequence of SEQ ID NO: 2, the site consisting of nucleotide residues 1001 to 1430 is an mPing insertion region, and the region consisting of nucleotide residues 1 to 1000 and 1431 to 2430 in the base sequence of SEQ ID NO: 2 is , Which are identical to the region consisting of nucleotide residues 1-1000th and 1001-2000th in the base sequence of SEQ ID NO: 1, respectively.

  In the base sequence of SEQ ID NO: 2, the insertion region of the first target mPing is a region consisting of the 1001 to 1430th nucleotide residues (FIG. 2). Therefore, as two or more primers for detecting the presence or absence of insertion of the first target mPing, the first primer having a base sequence corresponding to the upstream site (sense strand or antisense strand) of this region, and Combination of second primers having a base sequence corresponding to the downstream site (antisense strand or sense strand) (by detecting the presence or absence of mPing insertion by obtaining amplification products of different sizes depending on the presence or absence of mPing insertion) First primer having a base sequence corresponding to an upstream site (sense strand or antisense strand) of this region, or a downstream site (antisense strand or sense strand) of this region, and this region A combination of second primers (bases for amplification) having a base sequence corresponding to (sense strand or antisense strand) No makes it possible to use a combination) of the primers capable of detecting the presence or absence of insertion of mPing (see Figure 6).

  In the present invention, it is preferable to use a combination of primers that can detect the presence or absence of mPing insertion depending on the presence or absence of amplification. It is not known that the constituent varieties of Koshihikari from Niigata have an mPing insertion at the above site. In addition, there is no report of genome base sequences having mPing at the above sites for other varieties of rice. Therefore, the above combination of primers that can detect the presence or absence of mPing insertion based on the presence or absence of amplification is the motivation to produce by first elucidating that the constituent varieties of Koshihikari from Niigata Prefecture have mPing insertion at the site. In the absence of such recognition, there is no motivation to create such a combination of primers.

Specifically, as a primer set for the first target mPing, a combination of primers composed of the following primer pairs (eg, primers for PCR), and three or more primer sets including such primer combinations (eg, primers) , A primer set for LAMP using at least four primers):
1a) a first primer having at least a partial base sequence of the 1st to 1000th base sequences in the base sequence of SEQ ID NO: 1 or an equivalent base sequence thereof, and the 1431st to 2430th bases of the base sequence of SEQ ID NO: 1 A combination of second primers having a base sequence of at least a part of the sequence or a sequence complementary to the equivalent base sequence;
1b) a first primer having at least a partial base sequence of the 1st to 1000th base sequences in the base sequence of SEQ ID NO: 1 or a complementary sequence thereof, and 1431 to 2430 in the base sequence of SEQ ID NO: 1 A combination of second primers having at least a part of the base sequence or the equivalent base sequence thereof;
1c) a first primer having at least a partial base sequence of the 1st to 1000th base sequences in the base sequence of SEQ ID NO: 1 or an equivalent base sequence thereof, and the 1001st to 1430th bases of the base sequence of SEQ ID NO: 1 A combination of second primers having a base sequence of at least a part of the sequence or a sequence complementary to the equivalent base sequence;
1d) a first primer having at least a partial base sequence of the 1st to 1000th base sequences in the base sequence of SEQ ID NO: 1 or a complementary sequence thereof, and 1001-1430 in the base sequence of SEQ ID NO: 1 A combination of second primers having at least a part of the base sequence or the equivalent base sequence thereof;
1e) a first primer having at least a part of the base sequence of the 1001-1430th base sequence in the base sequence of SEQ ID NO: 1 or an equivalent base sequence thereof, and the 1431st-2430th bases in the base sequence of SEQ ID NO: 1 A combination of second primers having a base sequence of at least a part of the sequence or a sequence complementary to the equivalent base sequence;
1f) a first primer having at least a partial base sequence of the 1001 to 1430th base sequences in the base sequence of SEQ ID NO: 1 or a complementary sequence thereof, and 1431 to 2430 in the base sequence of SEQ ID NO: 1 A second primer combination having at least a part of the base sequence of the second base sequence or an equivalent base sequence thereof.

Preferably, the primer set for the first target mPing is a LAMP primer set. More specifically, such a LAMP primer set is composed of a combination of four primers (FIP, F3, BIP, B3) including at least the following two primers:
1a ′) At least a part of the base sequence of the 1st to 1000th base sequence in the base sequence of SEQ ID NO: 1 or an equivalent base sequence thereof as F1c region or F2 region in FIP, or as F3 region (preferably A first primer having (as F2 region in FIP), and at least a part of the base sequence of nucleotides 1431 to 2430 in the base sequence of SEQ ID NO: 1 or a complementary sequence equivalent thereto, B1c in BIP A combination of four primers comprising a second primer having a region or B2 region or as a B3 region (preferably as a B2 region in BIP);
1b ′) At least a part of the base sequence of the 1st to 1000th base sequence in the base sequence of SEQ ID NO: 1 or a complementary sequence thereof equivalent to the F1c region or F2 region in FIP or as the F3 region ( The first primer having (preferably as the F2 region in FIP), and at least a part of the base sequence of the 1431st to 2430th base sequences in the base sequence of SEQ ID NO: 1 or an equivalent base sequence thereof, B1c in BIP A combination of four primers comprising a second primer having a region or B2 region or as a B3 region (preferably as a B2 region in BIP);
1c ′) At least a part of the base sequence of the 1-1000th base sequence in the base sequence of SEQ ID NO: 1 or an equivalent base sequence thereof as the F1c region or F2 region in FIP, or as the F3 region (preferably A first primer having (as F2 region in FIP), and at least a part of the base sequence of positions 1001 to 1430 in the base sequence of SEQ ID NO: 1 or a complementary sequence equivalent thereto, B1c in BIP A combination of four primers comprising a second primer having a region or B2 region or as a B3 region (preferably as a B2 region in BIP);
1d ′) At least a part of the 1st to 1000th nucleotide sequences in the nucleotide sequence of SEQ ID NO: 1 or a complementary sequence thereof equivalent to the F1c region or F2 region in FIP or as the F3 region ( Preferably, the first primer having (as the F2 region in FIP) and at least a part of the base sequence of the 1001st to 1430th base sequences in the base sequence of SEQ ID NO: 1 or an equivalent base sequence thereof is represented by B1c in BIP A combination of four primers comprising a second primer having a region or B2 region or as a B3 region (preferably as a B2 region in BIP);
1e ′) At least a part of the base sequence of positions 1001 to 1430 in the base sequence of SEQ ID NO: 1 or an equivalent base sequence thereof as the F1c region or F2 region in FIP, or as the F3 region (preferably A first primer having (as F2 region in FIP), and at least a part of the base sequence of nucleotides 1431 to 2430 in the base sequence of SEQ ID NO: 1 or a complementary sequence equivalent thereto, B1c in BIP A combination of four primers comprising a second primer having a region or B2 region or as a B3 region (preferably as a B2 region in BIP);
1f ′) At least a part of the base sequence of positions 1001 to 1430 in the base sequence of SEQ ID NO: 1 or a complementary sequence equivalent thereto is used as the F1c region or F2 region in FIP, or as the F3 region ( The first primer having (preferably as the F2 region in FIP), and at least a part of the base sequence of the 1431st to 2430th base sequences in the base sequence of SEQ ID NO: 1 or an equivalent base sequence thereof, B1c in BIP A combination of four primers comprising a second primer having a region or B2 region or as a B3 region (preferably as a B2 region in BIP).
In addition to the four primer combinations 1a ′) to 1f ′), two loop primers (LF and LB) may be used in combination.

More preferably, the LAMP primer set for the first target mPing comprises a primer consisting of the base sequence of SEQ ID NO: 5, a primer consisting of the base sequence of SEQ ID NO: 6, a primer consisting of the base sequence of SEQ ID NO: 7, and Includes primers consisting of a base sequence.
Most preferably, the LAMP primer set for the first target mPing further comprises a primer consisting of the base sequence of SEQ ID NO: 9 and the base sequence of SEQ ID NO: 10, corresponding to the loop primer. Therefore, in the present invention, the NK1 primer set described in the examples is preferably used.

(2) A primer set composed of two or more primers that can detect the presence or absence of the insertion of the second target mPing not found in chromosome 8 of the constituent varieties of Koshihikari from Niigata Prefecture. The second target mPing not found in chromosome 8 is 8th when it is not found in chromosome 8 of the constituent varieties of Koshihikari from Niigata Prefecture, but is based on Nipponbare, the standard variety of Japanese rice. This refers to mPing found second from the 5 ′ end of the chromosome. More specifically, the region including such second target mPing and its adjacent site in Nipponbare is nucleotide residues 4711970 to 4714399 of Nipponbare chromosome 8 available from the above database IRGSP-1.0. (SEQ ID NO: 3) (FIG. 3). The nucleotide sequence of SEQ ID NO: 3 shown in FIG. 3 is the second target mPing in Nipponbare (the portion consisting of the nucleotides from the 1st to 1430th nucleotides underlined in the nucleotide sequence of SEQ ID NO: 3) and its adjacent site (each 1 kb).

  In the base sequence of SEQ ID NO: 3, the insertion region of the second target mPing is a region consisting of the 1001 to 1430th nucleotide residues (FIG. 3). Therefore, as two or more primers for detecting the presence or absence of insertion of the second target mPing, the first primer having a base sequence corresponding to the upstream site (sense strand or antisense strand) of this region, and Combination of second primers having a base sequence corresponding to the downstream site (antisense strand or sense strand) (by detecting the presence or absence of mPing insertion by obtaining amplification products of different sizes depending on the presence or absence of mPing insertion) First primer having a base sequence corresponding to an upstream site (sense strand or antisense strand) of this region, or a downstream site (antisense strand or sense strand) of this region, and this region A combination of second primers (bases for amplification) having a base sequence corresponding to (sense strand or antisense strand) No makes it possible to use a combination) of the primers capable of detecting the presence or absence of insertion of mPing (see Figure 6).

Specifically, as a primer set for the second target mPing, a combination of primers composed of the following primer pairs (eg, primers for PCR), and three or more primer sets (eg, a combination of such primers) , A primer set for LAMP using at least four primers):
2a) a first primer having at least a partial base sequence of the 1st to 1000th base sequences in the base sequence of SEQ ID NO: 3 or an equivalent base sequence thereof, and the 1431st to 2430th bases of the base sequence of SEQ ID NO: 3 A combination of second primers having a base sequence of at least a part of the sequence or a sequence complementary to the equivalent base sequence;
2b) a first primer having at least a partial base sequence of the 1st to 1000th base sequence in the base sequence of SEQ ID NO: 3 or a complementary sequence thereof, and 1431 to 2430 in the base sequence of SEQ ID NO: 3 A combination of second primers having at least a part of the base sequence or the equivalent base sequence thereof;
2c) a first primer having at least a partial base sequence of the 1st to 1000th base sequences in the base sequence of SEQ ID NO: 3 or an equivalent base sequence thereof, and the 1001st to 1430th bases of the base sequence of SEQ ID NO: 3 A combination of second primers having a base sequence of at least a part of the sequence or a sequence complementary to the equivalent base sequence;
2d) a first primer having at least a partial base sequence of the 1st to 1000th base sequences in the base sequence of SEQ ID NO: 3 or a complementary sequence thereof, and 1001-1430 in the base sequence of SEQ ID NO: 3 A combination of second primers having at least a part of the base sequence or the equivalent base sequence thereof;
2e) a first primer having at least a part of the base sequence of the 1001 to 1430th base sequence in the base sequence of SEQ ID NO: 3 or an equivalent base sequence thereof, and the 1431st to 2430th bases of the base sequence of SEQ ID NO: 3 A combination of second primers having a base sequence of at least a part of the sequence or a sequence complementary to the equivalent base sequence;
2f) a first primer having at least a partial base sequence of the 1001 to 1430th base sequence in the base sequence of SEQ ID NO: 3 or a complementary sequence thereof, and 1431 to 2430 in the base sequence of SEQ ID NO: 3 A second primer combination having at least a part of the base sequence of the second base sequence or an equivalent base sequence thereof.

Preferably, the primer set for the second target mPing is a LAMP primer set. More specifically, such a LAMP primer set is composed of a combination of four primers (FIP, F3, BIP, B3) including at least the following two primers:
2a ′) a base sequence of at least a part of the 1st to 1000th base sequence in the base sequence of SEQ ID NO: 3 or an equivalent base sequence thereof as an F1c region or F2 region in FIP, or as an F3 region (preferably A first primer having (as F2 region in FIP), and at least a part of the nucleotide sequence from 1431 to 2430 in the nucleotide sequence of SEQ ID NO: 3 or a complementary sequence of the equivalent nucleotide sequence, B1c in BIP A combination of four primers comprising a second primer having a region or B2 region or as a B3 region (preferably as a B2 region in BIP);
2b ′) At least a part of the base sequence of the 1st to 1000th base sequences in the base sequence of SEQ ID NO: 3 or a complementary sequence thereof equivalent to the F1c region or F2 region in FIP, or as the F3 region ( The first primer having (preferably as the F2 region in FIP), and at least a part of the base sequence of the 1431st to 2430th base sequences in the base sequence of SEQ ID NO: 3 or an equivalent base sequence thereof, B1c in BIP A combination of four primers comprising a second primer having a region or B2 region or as a B3 region (preferably as a B2 region in BIP);
2c ′) at least a part of the 1st to 1000th nucleotide sequences in the nucleotide sequence of SEQ ID NO: 3 or an equivalent nucleotide sequence thereof as F1c region or F2 region in FIP, or as F3 region (preferably A first primer having (as F2 region in FIP), and at least a part of the base sequence of positions 1001 to 1430 in the base sequence of SEQ ID NO: 3 or a complementary sequence equivalent thereto, B1c in BIP A combination of four primers comprising a second primer having a region or B2 region or as a B3 region (preferably as a B2 region in BIP);
2d ′) At least a part of the base sequence of the 1st to 1000th base sequence in the base sequence of SEQ ID NO: 3 or a complementary sequence thereof equivalent to the F1c region or F2 region in FIP or as the F3 region ( Preferably, the first primer having (as F2 region in FIP) and at least a part of the base sequence of the 1001st to 1430th base sequences in the base sequence of SEQ ID NO: 3 or an equivalent base sequence thereof is represented by B1c in BIP A combination of four primers comprising a second primer having a region or B2 region or as a B3 region (preferably as a B2 region in BIP);
2e ′) At least a part of the base sequence of positions 1001 to 1430 in the base sequence of SEQ ID NO: 3 or an equivalent base sequence thereof is used as the F1c region or F2 region in FIP, or as the F3 region (preferably A first primer having (as F2 region in FIP), and at least a part of the nucleotide sequence from 1431 to 2430 in the nucleotide sequence of SEQ ID NO: 3 or a complementary sequence of the equivalent nucleotide sequence, B1c in BIP A combination of four primers comprising a second primer having a region or B2 region or as a B3 region (preferably as a B2 region in BIP);
2f ′) At least a part of the base sequence of positions 1001 to 1430 in the base sequence of SEQ ID NO: 3 or a complementary sequence thereof equivalent to the F1c region or F2 region in FIP or as the F3 region ( The first primer having (preferably as the F2 region in FIP), and at least a part of the base sequence of the 1431st to 2430th base sequences in the base sequence of SEQ ID NO: 3 or an equivalent base sequence thereof, B1c in BIP A combination of four primers comprising a second primer having a region or B2 region or as a B3 region (preferably as a B2 region in BIP).
In addition to the four primer combinations 2a ′) to 2f ′), two loop primers (LF and LB) may be used in combination.

More preferably, the LAMP primer set for the second target mPing is a primer consisting of the base sequence of SEQ ID NO: 11, a primer consisting of the base sequence of SEQ ID NO: 12, a primer consisting of the base sequence of SEQ ID NO: 13, and Includes primers consisting of a base sequence.
Most preferably, the LAMP primer set for the second target mPing further comprises a primer consisting of the base sequence of SEQ ID NO: 15 and the base sequence of SEQ ID NO: 16 corresponding to the loop primer. Therefore, in the present invention, the NK2 primer set described in the examples is preferably used.

(3) Primer set consisting of two or more primers that can detect the presence or absence of the insertion of the third target mPing not found in chromosome 8 of the constituent varieties of Koshihikari from Niigata Prefecture. The third target mPing not found in chromosome 8 is not found in chromosome 8 of the constituent varieties of Koshihikari from Niigata Prefecture, but it is The mPing found first from the 5 ′ end of the chromosome. More specifically, the region including such third target mPing and its adjacent site in Nipponbare is nucleotide residues 1018672-1021101 of Nipponbare Chromosome 8 available from the above database IRGSP-1.0. (SEQ ID NO: 4) (FIG. 4). The base sequence of SEQ ID NO: 4 shown in FIG. 4 is the third target mPing in Nipponbare (the part consisting of the nucleotides of the 1st to 1430th nucleotides underlined in the base sequence of SEQ ID NO: 4) and its adjacent parts (respectively 1 kb).

  In the base sequence of SEQ ID NO: 4, the insertion region of the third target mPing is a region consisting of the 1001 to 1430th nucleotide residues (FIG. 4). Therefore, as two or more primers for detecting the presence or absence of insertion of the third target mPing, the first primer having a base sequence corresponding to the upstream site (sense strand or antisense strand) of this region, and Combination of second primers having a base sequence corresponding to the downstream site (antisense strand or sense strand) (by detecting the presence or absence of mPing insertion by obtaining amplification products of different sizes depending on the presence or absence of mPing insertion) First primer having a base sequence corresponding to an upstream site (sense strand or antisense strand) of this region, or a downstream site (antisense strand or sense strand) of this region, and this region A combination of second primers (bases for amplification) having a base sequence corresponding to (sense strand or antisense strand) No makes it possible to use a combination) of the primers capable of detecting the presence or absence of insertion of mPing (see Figure 6).

Specifically, as a primer set for the fourth target mPing, a combination of primers composed of the following primer pairs (eg, primers for PCR), and three or more primer sets (eg, a combination of such primers) , A primer set for LAMP using at least four primers):
3a) a first primer having at least a partial base sequence of the 1st to 1000th base sequences in the base sequence of SEQ ID NO: 4 or an equivalent base sequence thereof, and the 1431st to 2430th bases of the base sequence of SEQ ID NO: 4 A combination of second primers having a base sequence of at least a part of the sequence or a sequence complementary to the equivalent base sequence;
3b) a first primer having at least a partial base sequence of the 1st to 1000th base sequence in the base sequence of SEQ ID NO: 4 or a complementary sequence thereof, and 1431 to 2430 in the base sequence of SEQ ID NO: 3 A combination of second primers having at least a part of the base sequence or the equivalent base sequence thereof;
3c) a first primer having at least a partial base sequence of the 1st to 1000th base sequences in the base sequence of SEQ ID NO: 4 or an equivalent base sequence thereof, and the 1001st to 1430th bases of the base sequence of SEQ ID NO: 4 A combination of second primers having a base sequence of at least a part of the sequence or a sequence complementary to the equivalent base sequence;
3d) a first primer having at least a partial base sequence of the 1st to 1000th base sequences in the base sequence of SEQ ID NO: 4 or a complementary sequence thereof, and 1001-1430 in the base sequence of SEQ ID NO: 4 A combination of second primers having at least a part of the base sequence or the equivalent base sequence thereof;
3e) a first primer having at least a partial base sequence of the 1001 to 1430th base sequence in the base sequence of SEQ ID NO: 4 or an equivalent base sequence thereof, and the 1431st to 2430th bases in the base sequence of SEQ ID NO: 3 A combination of second primers having a base sequence of at least a part of the sequence or a sequence complementary to the equivalent base sequence;
3f) a first primer having at least a partial base sequence of the 1001-1430th base sequence in the base sequence of SEQ ID NO: 4 or a complementary sequence thereof, and 1431-2430 in the base sequence of SEQ ID NO: 4 A second primer combination having at least a part of the base sequence of the second base sequence or an equivalent base sequence thereof.

Preferably, the primer set for the third target mPing is a LAMP primer set. More specifically, such a LAMP primer set is composed of a combination of four primers (FIP, F3, BIP, B3) including at least the following two primers:
3a ′) At least a part of the base sequence of the 1st to 1000th base sequence in the base sequence of SEQ ID NO: 4 or an equivalent base sequence thereof as F1c region or F2 region in FIP, or as F3 region (preferably A first primer having (as an F2 region in FIP), and at least a part of the base sequence of nucleotides 1431 to 2430 in the base sequence of SEQ ID NO: 4 or a complementary sequence of the equivalent base sequence, B1c in BIP A combination of four primers comprising a second primer having a region or B2 region or as a B3 region (preferably as a B2 region in BIP);
3b ′) At least a part of the 1st to 1000th nucleotide sequences in the nucleotide sequence of SEQ ID NO: 4 or a complementary sequence thereof equivalent to the F1c region or F2 region in FIP or as the F3 region ( Preferably, the first primer having (as the F2 region in FIP) and at least a part of the base sequence of the 1431st to 2430th base sequences in the base sequence of SEQ ID NO: 4 or an equivalent base sequence thereof are represented by B1c in BIP A combination of four primers comprising a second primer having a region or B2 region or as a B3 region (preferably as a B2 region in BIP);
3c ′) a base sequence of at least a part of the 1st to 1000th base sequences in the base sequence of SEQ ID NO: 4 or an equivalent base sequence thereof as an F1c region or F2 region in FIP, or as an F3 region (preferably A first primer having (as F2 region in FIP), and at least a part of the base sequence of positions 1001 to 1430 in the base sequence of SEQ ID NO: 4 or a complementary sequence equivalent thereto, B1c in BIP A combination of four primers comprising a second primer having a region or B2 region or as a B3 region (preferably as a B2 region in BIP);
3d ′) At least a part of the 1st to 1000th nucleotide sequences in the nucleotide sequence of SEQ ID NO: 4 or a complementary sequence thereof equivalent to the F1c region or F2 region in FIP or as the F3 region ( Preferably, the first primer having (as the F2 region in FIP), and at least a part of the base sequence of positions 1001 to 1430 in the base sequence of SEQ ID NO: 4 or an equivalent base sequence thereof is represented by B1c in BIP A combination of four primers comprising a second primer having a region or B2 region or as a B3 region (preferably as a B2 region in BIP);
3e ′) At least a part of the base sequence of the 1001-1430th base sequence in the base sequence of SEQ ID NO: 4 or an equivalent base sequence thereof as the F1c region or F2 region in FIP, or as the F3 region (preferably A first primer having (as an F2 region in FIP), and at least a part of the base sequence of nucleotides 1431 to 2430 in the base sequence of SEQ ID NO: 4 or a complementary sequence of the equivalent base sequence, B1c in BIP A combination of four primers comprising a second primer having a region or B2 region or as a B3 region (preferably as a B2 region in BIP);
3f ′) At least a part of the base sequence of positions 1001 to 1430 in the base sequence of SEQ ID NO: 4 or a complementary sequence equivalent thereto is used as the F1c region or F2 region in FIP, or as the F3 region ( Preferably, the first primer having (as the F2 region in FIP) and at least a part of the base sequence of the 1431st to 2430th base sequences in the base sequence of SEQ ID NO: 4 or an equivalent base sequence thereof are represented by B1c in BIP A combination of four primers comprising a second primer having a region or B2 region or as a B3 region (preferably as a B2 region in BIP).
In addition to the four primer combinations 3a ′) to 3f ′), two loop primers (LF and LB) may be used in combination.

More preferably, the LAMP primer set for the third target mPing comprises a primer consisting of the base sequence of SEQ ID NO: 17, a primer consisting of the base sequence of SEQ ID NO: 18, a primer consisting of the base sequence of SEQ ID NO: 19, and Includes primers consisting of a base sequence.
Most preferably, the LAMP primer set for the third target mPing further comprises a primer consisting of the base sequence of SEQ ID NO: 21 corresponding to the loop primer, and a base sequence of SEQ ID NO: 22. Therefore, in the present invention, the NK3 primer set described in the examples is preferably used.

(4) Primer set composed of two or more primers capable of detecting varieties other than Koshihikari As a primer set composed of two or more primers capable of detecting varieties other than Koshihikari, specific primer varieties other than Koshihikari Any primer set for the marker region can be used. Varieties other than Koshihikari may have a blast resistance gene Pi5-1 (Accession: EU869185). Here, regarding the first exon region of Pi5-1 and the region after the second exon, a region having high homology is found in the genome of Koshihikari, but the first intron region of Pi5-1 (in EU869185) Regarding the 5593 to 6567th base sequence), there is no region having high homology in the Koshihikari genome. Therefore, in the present invention, a primer set for the first intron region of Pi5-1 can be used as a primer set composed of two or more primers that can detect varieties other than Koshihikari (eg, Journal of the Japan Food Chemical Engineering Society). 58, No. 12, 591-596 (2011)).
In addition, there is no region having high homology in the Koshihikari genome with respect to the region before the first exon of Pi5-1 (the 1st to 5277th base sequence of EU869185). Therefore, in the present invention, as a primer set composed of two or more primers capable of detecting varieties other than Koshihikari, a primer set for a region before the first exon of Pi5-1 (eg, disclosed in the examples of the present application). Primer set) can be used.
Furthermore, many methods for determining varieties other than Koshihikari and Koshihikari using a plurality of markers have been reported (eg, Patent Publication 2012-55253, Patent Publication 2009-219498, Patent Publication 2005-73655, Patent Publication 2003). -135082, patent publication 2001-95589). Therefore, in the present invention, a plurality of primer sets composed of two or more primers that can detect varieties other than Koshihikari by using such a plurality of markers may be used.

Preferably, the primer set for varieties other than Koshihikari is a LAMP primer set.
More preferably, the LAMP primer set for varieties other than Koshihikari includes a primer consisting of the base sequence of SEQ ID NO: 23, a primer consisting of the base sequence of SEQ ID NO: 24, a primer consisting of the base sequence of SEQ ID NO: 25, and Includes primers consisting of a base sequence.
Most preferably, the LAMP primer set for varieties other than Koshihikari further includes a primer consisting of the base sequence of SEQ ID NO: 27 and the base sequence of SEQ ID NO: 28 corresponding to the loop primer. Therefore, in the present invention, the NK4 primer set described in the examples is preferably used.

(5) Primer set composed of two or more primers capable of specifically detecting Koshihikari As a primer set composed of two or more primers capable of specifically detecting Koshihikari, a marker region specific for Koshihikari Any primer set can be used. Koshihikari has a region having high base sequence homology with the first exon region of Pi5-1 and the region after the second exon. However, for the first intron region of Pi5-1 (base sequence 5593 to 6567 in EU869185), there is no region having high base sequence homology in the Koshihikari genome, and only a non-homologous region. Therefore, in the present invention, as a primer set composed of two or more primers capable of detecting Koshihikari, the non-homologous region (for example, the 9673280th to 9673302th of chromosome 9 of Nipponbare (IRGSP-1.0) The primer set for the base sequence can be used (see, for example, Journal of Japanese Society of Food Chemical Engineering, Vol. 58, No. 12, pages 591-596 (2011)).
In addition, there is no region having high homology in the Koshihikari genome with respect to the region before the first exon of Pi5-1 (the 1st to 5277th base sequence of EU869185). Therefore, in the present invention, as a primer set composed of two or more primers capable of detecting Koshihikari, such a region (e.g., Nipponbare (IRGSP-1.0)) is located behind the 9746618th base of chromosome 9. A primer set for the base sequence) can be used.
Furthermore, many methods for determining Koshihikari and varieties other than Koshihikari using a plurality of markers have been reported (eg, see the above-mentioned document). Therefore, in this invention, you may use the some primer set comprised from two or more primers which can detect Koshihikari by utilizing such a some marker.

Preferably, the primer set for Koshihikari is a LAMP primer set.
More preferably, the LAMP primer set for Koshihikari comprises a primer consisting of the base sequence of SEQ ID NO: 29, a primer consisting of the base sequence of SEQ ID NO: 30, a primer consisting of the base sequence of SEQ ID NO: 31, and a base sequence of SEQ ID NO: 32 A primer comprising
Most preferably, the LAMP primer set for Koshihikari further includes a primer consisting of the base sequence of SEQ ID NO: 33 corresponding to the loop primer, and the base sequence of SEQ ID NO: 34. Therefore, in the present invention, the NK5 primer set described in the examples is preferably used.

  The term “equivalent base sequence” in the various primers constituting the primer set mentioned above means that the base sequence corresponds to the variation of the base sequence between rice varieties, especially Koshihikari from Niigata Prefecture and Nipponbare. . Therefore, several base sequences equivalent to a predetermined base sequence (eg, at least a part of the base sequence of Y to Z in the base sequence of SEQ ID NO. X) are several (for example, 1 to The nucleotide sequence may include 10 (preferably 1 to 5, more preferably 1, 2 or 3) base modifications (eg, substitution, deletion, insertion, addition). Such an equivalent base sequence is obvious to those skilled in the art, and by referring to a database in which gene information of rice varieties is registered, or around the insertion region of the target mPing in rice varieties. This can be determined by decoding the base sequence.

  In the method of the present invention, by an amplification reaction using at least a primer set composed of two or more primers capable of detecting the presence or absence of insertion of the first target mPing found in the first chromosome of a constituent variety of Koshihikari produced in Niigata Prefecture, The presence or absence of insertion of the first target mPing is confirmed, and thereby the rice production area or variety is determined. For example, when it is confirmed that the first target mPing has been inserted, it can be determined that the sample is highly likely to be rice produced in Niigata Prefecture, or that the sample is likely to contain rice produced in Niigata Prefecture. On the other hand, when it is confirmed that the first target mPing has not been inserted, it can be determined that the sample is not likely to be rice produced in Niigata Prefecture, or is likely not to contain rice produced in Niigata Prefecture.

In one embodiment, the method of the present invention relates to a method for more accurately determining Koshihikari from Niigata Prefecture.
Such a method is characterized by confirming the following results (I), (II) and (III) in an amplification reaction using the primer sets (1), (2) and (3) below:
(1) A first primer set composed of two or more primers capable of detecting the presence or absence of insertion of the first target mPing found in the first chromosome of a constituent variety of Koshihikari produced in Niigata Prefecture;
(2) a second primer set comprising two or more primers capable of detecting the presence or absence of insertion of a second target mPing not found in chromosome 8 of a constituent variety of Koshihikari from Niigata Prefecture; and (3) A third primer set consisting of two or more primers capable of detecting the presence or absence of insertion of a third target mPing not found in chromosome 8 of a constituent variety of Koshihikari from Niigata Prefecture;
(I) Confirming that the first target mPing has been inserted by an amplification reaction using the first primer set;
(II) Confirm that the second target mPing is not inserted by an amplification reaction using the second primer set; and (III) Insert the third target mPing by an amplification reaction using the third primer set. Make sure that it is not.

In another embodiment, the method of the present invention relates to a method for more accurately determining that Koshihikari from Niigata Prefecture contains Koshihikari from other prefectures or rice of other varieties.
Such a method is characterized in that the following results (IV) and (V) are confirmed in an amplification reaction using the primer sets (1), (2) and (3):
(1) A first primer set composed of two or more primers capable of detecting the presence or absence of insertion of the first target mPing found in the first chromosome of a constituent variety of Koshihikari produced in Niigata Prefecture;
(2) a second primer set comprising two or more primers capable of detecting the presence or absence of insertion of a second target mPing not found in chromosome 8 of a constituent variety of Koshihikari from Niigata Prefecture; and (3) A third primer set consisting of two or more primers capable of detecting the presence or absence of insertion of a third target mPing not found in chromosome 8 of a constituent variety of Koshihikari from Niigata Prefecture;
(IV) confirming that the first target mPing is inserted by an amplification reaction using the first primer set; and (V) using one or both of the second primer set and the third primer set. Confirm that the second target mPing or the third target mPing is inserted by an amplification reaction.

In the above embodiment, as Koshihikari from other prefectures, for example, Tochigi Prefecture, Fukushima Prefecture, Ibaraki Prefecture, Toyama Prefecture, Chiba Prefecture, Nagano Prefecture, Ishikawa Prefecture, Mie Prefecture, Fukui Prefecture , Koshihikari from Shiga Prefecture, Shimane Prefecture, Hiroshima Prefecture, Hyogo Prefecture, and Yamagata Prefecture.
Other rice varieties include, for example, Hitomebore, Akitakomachi, Hinohikari, Nanatsuboshi, Haenuki, Kirara 397, Shinigura, Demon Buki, Tsuru Roman, Kinuhikari, Asahi no Yume, Yumepirika, Fusakogane, Hoshino Yume , And Hanaechizen.

In yet another embodiment, the method of the present invention relates to a method for more accurately determining rice of varieties other than Koshihikari.
Such a method is characterized by confirming the following results (VI), (VII) and (VIII) in an amplification reaction using the primer sets (1), (4) and (5) below:
(1) A first primer set composed of two or more primers capable of detecting the presence or absence of insertion of the first target mPing found in the first chromosome of a constituent variety of Koshihikari produced in Niigata Prefecture;
(4) a fourth primer set composed of two or more primers capable of detecting rice of varieties other than Koshihikari; and (5) a second primer composed of two or more primers capable of specifically detecting Koshihikari. 5 primer sets;
(VI) confirming that the first target mPing is not inserted by an amplification reaction using the first primer set;
(VII) Confirm that the rice is of a variety other than Koshihikari by an amplification reaction using the fourth primer set; and (VIII) Confirm that it is not Koshihikari by an amplification reaction using the fifth primer set. .

In another embodiment, the method of the present invention relates to a method for more accurately determining Koshihikari produced in prefectures other than Niigata prefecture.
Such a method is characterized by confirming the following results (IX), (X) and (XI) in an amplification reaction using the primer sets (1), (4) and (5) below:
(1) A first primer set composed of two or more primers capable of detecting the presence or absence of insertion of the first target mPing found in the first chromosome of a constituent variety of Koshihikari produced in Niigata Prefecture;
(4) a fourth primer set composed of two or more primers capable of detecting rice of varieties other than Koshihikari; and (5) a second primer composed of two or more primers capable of specifically detecting Koshihikari. 5 primer sets;
(IX) Confirming that the first target mPing is not inserted by an amplification reaction using the first primer set;
(X) Confirm that it is not a rice of a variety other than Koshihikari by an amplification reaction using the fourth primer set; and (XI) Confirm that it is Koshihikari by an amplification reaction using the fifth primer set. .

In still another embodiment, the method of the present invention relates to a method for more accurately determining whether the mixture is rice of a variety other than Koshihikari and Koshihikari from prefectures other than Niigata.
Such a method is characterized by confirming the following results (XII), (XIII) and (XIV) in an amplification reaction using the primer sets (1), (4) and (5) below:
(1) A first primer set composed of two or more primers capable of detecting the presence or absence of insertion of the first target mPing found in the first chromosome of a constituent variety of Koshihikari produced in Niigata Prefecture;
(4) a fourth primer set composed of two or more primers capable of detecting rice of varieties other than Koshihikari; and (5) a second primer composed of two or more primers capable of specifically detecting Koshihikari. 5 primer sets;
(XII) confirming that the first target mPing is not inserted by an amplification reaction using the first primer set;
(XIII) Confirm that the rice is of a variety other than Koshihikari by an amplification reaction using the fourth primer set; and (XIV) Confirm that it is Koshihikari by an amplification reaction using the fifth primer set. about.

  The present invention also relates to the above-described various primer sets used in the method of the present invention and a kit containing such various primer sets.

  The kit of the present invention may further contain a protein used in the amplification reaction. Examples of such proteins include DNA polymerase (eg, strand displacement DNA polymerase in LAMP), restriction enzyme, ribonuclease H, ligase, helicase, and recombinase. The kit of the present invention may also contain a fluorescent reagent, a substrate such as dNTPs, and a coenzyme such as ATP. The kit of the present invention further comprises a control DNA encoding a housekeeping gene (eg, GAPDH, β-actin, β2-microglobulin, HPRT1) that can be used as a control, and two or more primers for such control DNA. May be included. The kit of the present invention may also contain a positive control (eg, Koshihikari genomic DNA from Niigata Prefecture) and / or a negative control (eg, Koshihikari genomic DNA from other prefectures, multiple types of genomic DNA). The kit of the present invention may contain each component in a form isolated in individual containers (eg, tubes), but two or more kinds of components may be mixed in advance in the same container.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to these Examples.

Example 1: Determination of Koshihikari from Niigata Prefecture, Koshihikari from Tochigi Prefecture and Hitomebori from Miyagi Prefecture, and their mixed feed 1) Koshihikari from Niigata Prefecture (using rice produced in 2011), Koshihikari from Tochigi Prefecture and Hitomeboru from Miyagi Prefecture Brown rice was pulverized with a food mill, and DNA was extracted from 0.5 g of the pulverized sample using a GM Quicker 2 extraction kit.
2) An equivalent amount of extracted DNA from Koshihikari from Tochigi Prefecture or Hitomebo from Miyagi Prefecture was mixed with the extracted DNA from Koshihikari from Niigata Prefecture to prepare a pseudo mixed sample into Koshihikari from Niigata Prefecture.
3) A LAMP reaction solution was prepared for each of the five primer sets (NK1, NK2, NK3, NK4, NK5) shown in Table 1, and 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 8 mM MgSO _{4, respectively.} 0.1% Tween 20, 0.8M Betain, 1.4 mM dNTPs, 0.2 μM F3 and B3 primers, 1.6 μM FIP and BIP primers, 0.8 μM LF and LB primers, 8 units Bst DNA polymerase (New England Biolabs) A total volume of 25 μL containing 1 μL of Loopamp fluorescence / visual detection reagent (Eiken Chemical) and 10 ng of extracted DNA was used. NK1 is a LAMP primer set composed of six primers that can detect the presence or absence of the insertion of the first target mPing. When the first target mPing is inserted into the genome, an amplification product may be generated. However, it is designed so that an amplification product cannot be produced if it is not inserted. NK2 is a LAMP primer set composed of six primers that can detect the presence or absence of the insertion of the second target mPing. When the second target mPing is inserted into the genome, an amplification product may be generated. However, it is designed so that an amplification product cannot be produced if it is not inserted. NK3 is a LAMP primer set composed of six primers that can detect the presence or absence of the insertion of the third target mPing. When the third target mPing is inserted into the genome, an amplification product may be generated. However, it is designed so that an amplification product cannot be produced if it is not inserted. NK4 is a LAMP primer set composed of 6 primers that can detect rice of varieties other than Koshihikari. No amplification product can be produced in Koshihikari, but no amplification product can be produced in varieties other than Koshihikari. Designed to. NK5 is a LAMP primer set composed of six primers capable of detecting Koshihikari, and is designed so that an amplification product can be produced in Koshihikari, but an amplification product cannot be produced in varieties other than Koshihikari. In addition, the target site | part of NK4 and NK5 is the same as what is disclosed by the Japan Food Chemical Engineering Society Vol.58 No.12 page 591-596 (2011).
4) LAMP amplification was allowed to react at 63 ° C. for 30 minutes, and then heated at 80 ° C. for 2 minutes to stop the reaction.
5) The reaction solution was irradiated with ultraviolet rays of 365 nm, and a sample that developed fluorescence was judged as having amplification, and a sample that did not develop color was judged as having no amplification.
6) As a result, as shown in FIG. 7, in (A), it was determined that Koshihikari produced in Niigata Prefecture was free from contamination by amplification of NK1 and non-amplification of NK2 and NK3. On the other hand, in (D) and (E), amplification of NK1 was observed, but amplification was also detected in NK2 and NK3, and it was determined that Koshihikari from Niigata Prefecture was mixed with Koshihikari from other prefectures or other varieties. In (B), (C) and (F), it can be determined that the non-amplification of NK1 is other than Koshihikari produced in Niigata Prefecture, and from the amplification results of NK4 and NK5, (B) ) Was determined to be another variety, and (F) was determined to be a mixture of Koshihikari from other prefectures and other varieties. It was confirmed that the same results were obtained not only for Koshihikari produced in Niigata Prefecture produced in 2011 but also for Koshihikari produced in Niigata Prefecture in 2005-2010 and 2012.

Example 2: Determination of top 16 varieties of Koshihikari shipments excluding top 15 prefectures and Koshihikari shipments In the same manner as in Example 1, the top 15 varieties of Koshihikari shipments and top 16 varieties of shipments excluding Koshihikari were determined. In the LAMP reaction, five primer sets (NK1, NK2, NK3, NK4, NK5) shown in Table 1 were used. The results were as shown in Table 2. It was confirmed that the same results were obtained not only for Koshihikari produced in Niigata Prefecture produced in 2011 but also for Koshihikari produced in Niigata Prefecture in 2005-2010 and 2012.

Example 3: Decoding the base sequence around the target site of the NK1 primer set in Koshihikari from Niigata Prefecture The base sequence around the target site of the NK1 primer set in Koshihikari from Niigata Prefecture was decoded. As a result, the peripheral base sequence was as shown in the base sequence of SEQ ID NO: 2 shown in FIG. In addition, in the base sequence of SEQ ID NO: 2, the mPing insertion site (the site consisting of the 1001 to 1430th nucleotide residues underlined in FIG. 2) was confirmed.

  According to the present invention, it is possible to detect Koshihikari produced in Niigata Prefecture and the presence or absence of contamination from other prefectures and other varieties.

Claims (11)

Including a primer set composed of two or more primers capable of detecting the presence or absence of insertion of the first target mPing found in the first chromosome of Niigata Prefecture Koshihikari, determination of whether or not a Niigata Prefecture Koshihikari A kit for
The discriminating kit, wherein the insertion site of the first target mPing corresponds to the site consisting of the 1001 to 1430th nucleotide residues in the nucleotide sequence of SEQ ID NO: 2.   The discrimination kit according to claim 1, wherein the primer set is a LAMP primer set.   The primer set is a primer consisting of the base sequence of SEQ ID NO: 5, a primer consisting of the base sequence of SEQ ID NO: 6, a primer consisting of the base sequence of SEQ ID NO: 7, a primer consisting of the base sequence of SEQ ID NO: 8, the base sequence of SEQ ID NO: 9 The discriminating kit according to claim 2, comprising a primer consisting of: and a primer consisting of the base sequence of SEQ ID NO: 10. The following (1) and the primer set of the following (2), (3), (4) and (5) a least one primer set selected from the group consisting of, whether Niigata Prefecture Koshihikari Identification kit:
(1) composed of two or more primers capable of detecting the presence or absence of insertion of the first target mPing found in the first chromosome of Niigata Prefecture Koshihikari, a first primer set, the first target mPing the insertion site, the nucleotide sequence of SEQ ID NO: 2, corresponds to a site consisting of 1001 to 1430 th nucleotide residues, the first primer set; see the eighth chromosome of and (2) Niigata Prefecture Koshihikari A second primer set composed of two or more primers capable of detecting the presence or absence of insertion of a second target mPing that is not released;
(3) composed of two or more primers which existence capable of detecting the insertion of the third target mPing not found in the eighth chromosome of Niigata Prefecture Koshihikari, the third primer set;
(4) a fourth primer set comprising two or more primers capable of detecting varieties other than Koshihikari; and (5) a fifth primer comprising two or more primers capable of specifically detecting Koshihikari. Primer set. (1) The first primer set is a primer consisting of the base sequence of SEQ ID NO: 5, a primer consisting of the base sequence of SEQ ID NO: 6, a primer consisting of the base sequence of SEQ ID NO: 7, a primer consisting of the base sequence of SEQ ID NO: 8, Composed of a primer consisting of the base sequence of SEQ ID NO: 9 and a primer consisting of the base sequence of SEQ ID NO: 10,
(2) The second primer set is a primer consisting of the base sequence of SEQ ID NO: 11, a primer consisting of the base sequence of SEQ ID NO: 12, a primer consisting of the base sequence of SEQ ID NO: 13, a primer consisting of the base sequence of SEQ ID NO: 14, Composed of a primer consisting of the base sequence of SEQ ID NO: 15 and a primer consisting of the base sequence of SEQ ID NO: 16,
(3) The third primer set is a primer consisting of the base sequence of SEQ ID NO: 17, a primer consisting of the base sequence of SEQ ID NO: 18, a primer consisting of the base sequence of SEQ ID NO: 19, a primer consisting of the base sequence of SEQ ID NO: 20, Composed of a primer consisting of the base sequence of SEQ ID NO: 21 and a primer consisting of the base sequence of SEQ ID NO: 22,
(4) The fourth primer set is a primer consisting of the base sequence of SEQ ID NO: 23, a primer consisting of the base sequence of SEQ ID NO: 24, a primer consisting of the base sequence of SEQ ID NO: 25, a primer consisting of the base sequence of SEQ ID NO: 26, Composed of a primer consisting of the base sequence of SEQ ID NO: 27 and a primer consisting of the base sequence of SEQ ID NO: 28,
(5) the fifth primer set is a primer consisting of the base sequence of SEQ ID NO: 29, a primer consisting of the base sequence of SEQ ID NO: 30, a primer consisting of the base sequence of SEQ ID NO: 31, a primer consisting of the base sequence of SEQ ID NO: 32, The kit according to claim 4, comprising a primer consisting of the base sequence of SEQ ID NO: 33 and a primer consisting of the base sequence of SEQ ID NO: 34. At least using an amplification reaction primer set composed of two or more primers capable of detecting the presence or absence of insertion of the first target mPing found in the first chromosome of Niigata Prefecture Koshihikari, whether insertion of the first target mPing including that you check, a Niigata Prefecture whether or not the judgment method whether it is Koshihikari,
The discriminating method, wherein the insertion site of the first target mPing corresponds to a site consisting of the 1001 to 1430th nucleotide residues in the base sequence of SEQ ID NO: 2. In the amplification reaction using the primer sets of (1), (2) and (3) below, Koshihikari produced in Niigata Prefecture is characterized by confirming the results of (I), (II) and (III) below The discriminating method according to claim 6, wherein the discriminating method is:
(1) composed of two or more primers which existence capable of detecting the insertion of the first target mPing found in the first chromosome of Niigata Prefecture Koshihikari, the first primer set;
(2) Niigata Prefecture not found eighth chromosome of Koshihikari composed of two or more primers capable of detecting the presence or absence of insertion of the second target mPing, second primer set; and (3) Niigata A third primer set composed of two or more primers capable of detecting the presence or absence of insertion of a third target mPing not found in chromosome 8 of Koshihikari from the prefecture;
(I) Confirming that the first target mPing has been inserted by an amplification reaction using the first primer set;
(II) Confirm that the second target mPing is not inserted by an amplification reaction using the second primer set; and (III) Insert the third target mPing by an amplification reaction using the third primer set. Make sure that it is not. In the amplification reaction using the primer sets of (1), (2) and (3) below, the results of (IV) and (V) below are confirmed, and Koshihikari from other prefectures is confirmed. Or the discrimination method of Claim 6 which is a method of determining that there exists mixing of the rice of other varieties:
(1) composed of two or more primers which existence capable of detecting the insertion of the first target mPing found in the first chromosome of Niigata Prefecture Koshihikari, the first primer set;
(2) Niigata Prefecture not found eighth chromosome of Koshihikari composed of two or more primers capable of detecting the presence or absence of insertion of the second target mPing, second primer set; and (3) Niigata A third primer set composed of two or more primers capable of detecting the presence or absence of insertion of a third target mPing not found in chromosome 8 of Koshihikari from the prefecture;
(IV) confirming that the first target mPing is inserted by an amplification reaction using the first primer set; and (V) using one or both of the second primer set and the third primer set. Confirm that the second target mPing or the third target mPing is inserted by an amplification reaction. In the following amplification reaction using the primer set of (1), (4) and (5), the results of the following (VI), (VII) and (VIII) are confirmed. The discriminating method according to claim 6, wherein the discriminating method is:
(1) composed of two or more primers which existence capable of detecting the insertion of the first target mPing found in the first chromosome of Niigata Prefecture Koshihikari, the first primer set;
(4) a fourth primer set composed of two or more primers capable of detecting rice of varieties other than Koshihikari; and (5) a second primer composed of two or more primers capable of specifically detecting Koshihikari. 5 primer sets;
(VI) confirming that the first target mPing is not inserted by an amplification reaction using the first primer set;
(VII) Confirm that the rice is of a variety other than Koshihikari by an amplification reaction using the fourth primer set; and (VIII) Confirm that it is not Koshihikari by an amplification reaction using the fifth primer set. . Prefectures other than those produced in Niigata Prefecture, characterized by confirming the results of (IX), (X) and (XI) below in the amplification reaction using the primer sets of (1), (4) and (5) The discrimination method according to claim 6, which is a method for judging Koshihikari produced in Japan:
(1) composed of two or more primers which existence capable of detecting the insertion of the first target mPing found in the first chromosome of Niigata Prefecture Koshihikari, the first primer set;
(4) a fourth primer set composed of two or more primers capable of detecting rice of varieties other than Koshihikari; and (5) a second primer composed of two or more primers capable of specifically detecting Koshihikari. 5 primer sets;
(IX) Confirming that the first target mPing is not inserted by an amplification reaction using the first primer set;
(X) Confirm that it is not a rice of a variety other than Koshihikari by an amplification reaction using the fourth primer set; and (XI) Confirm that it is Koshihikari by an amplification reaction using the fifth primer set. . In the following amplification reaction using the primer set of (1), (4) and (5), the results of the following (XII), (XIII) and (XIV) are confirmed. The discrimination method according to claim 6, wherein the determination method is a mixture of Koshihikari from other prefectures than from Niigata prefecture.
(1) composed of two or more primers which existence capable of detecting the insertion of the first target mPing found in the first chromosome of Niigata Prefecture Koshihikari, the first primer set;
(4) a fourth primer set composed of two or more primers capable of detecting rice of varieties other than Koshihikari; and (5) a second primer composed of two or more primers capable of specifically detecting Koshihikari. 5 primer sets;
(XII) confirming that the first target mPing is not inserted by an amplification reaction using the first primer set;
(XIII) Confirm that the rice is of a variety other than Koshihikari by an amplification reaction using the fourth primer set; and (XIV) Confirm that it is Koshihikari by an amplification reaction using the fifth primer set. about.