JP2008259469A - Primer for discriminating rice variety, and method for discriminating rice variety using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a technique capable of discriminating rice variety and determining mixed rice presence/absence with a simple operation, in a short time, efficiently and inexpensively, in good visibility, and without the need of any special equipment and expertise. <P>SOLUTION: The technique for discriminating rice variety and determining mixed rice presence/absence is provided, comprising the following process: Using a primer set capable of amplifying a specific domain in rice's genomic sequence useful for the objective discrimination and determination, an amplified product obtained by rice's nucleic acid amplification treatment is detected by electrophoresis, and the formation of a hybrid of the above amplified product and a captured oligonucleotide group is detected. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a primer and a capture oligonucleotide for discriminating rice varieties and the presence or absence of mixed rice in Koshihikari, a method for discriminating rice varieties using these and the presence or absence of mixed rice in Koshihikari. The present invention relates to a method for determination and a kit for carrying out the method.

  In Japan, more than 200 rice varieties are currently cultivated, but the name of rice varieties is required to be displayed due to the enforcement of the revised JAS law. However, some fraudulent mixed rice is sold in which the display is misrepresented while rice other than the displayed variety is mixed. Considering the current situation in which good-tasting varieties such as Koshihikari are traded at high prices in response to consumers' preference for good-tasting taste, and the growing interest in information disclosure of food, the accuracy of the display of rice variety names is This is an important issue both administratively and industrially, and because of the necessity of preventing fraud due to mixed rice, etc., there is a need for the development of rice variety discrimination technology that is simple, highly accurate, and highly practical. However, most rice varieties cultivated in Japan are very similar because of the fact that they have been bred by cross-breeding between several closely related varieties based on several native varieties. It is very difficult to distinguish rice varieties. On the other hand, in recent years, analysis of rice genome sequences has progressed, and attempts have been made to distinguish rice varieties using genetic polymorphisms that show differences among rice varieties. As such a technique, an STS method, a detection method using a DNA array, and a SNPs method are used.

  The STS method (Sequence Tagged Site) extracts DNA from polished rice, performs a nucleic acid amplification reaction (hereinafter also simply referred to as PCR) using primers that show differences between varieties, It is a technology that distinguishes polymorphisms by size. When discriminating rice varieties using the STS method, it is necessary to select a primer pair to be used according to the rice varieties to be discriminated, and when the rice varieties are unknown, it is difficult to determine the primer pair to be used, which is not practical. There is a problem (Patent Document 1). In addition, it is possible to classify multiple varieties of rice into multiple distinction groups using multiple types of primer pairs, but it is necessary to perform multiple PCRs when rice of unknown varieties is used as a sample, which is complicated. Yes (Patent Document 2). In addition, in order to confirm the mixed rice of other varieties for a specific variety, it is necessary to search for a primer pair corresponding to the variety to be examined from a plurality of primer pairs, and a primer different from the mixed rice determination primer pair The pair is required as a primer pair for discriminating rice varieties, and the operation is complicated (Patent Document 2).

  Several kits used for the method of discriminating rice varieties by the STS method are commercially available. Examples of such kits include TaKaRa's "Kit discrimination PCR Kit I" and "Rice discrimination PCR Kit II (Koshihikari nega kit)", TaKaRa's "2005 Koshihikari BL discrimination PCR kit produced in 2005", There is “Kyoto Discrimination Kit“ Rice Appraisal Team ”” by Vision Bio. However, TaKaRa's “Kithi discriminating PCR Kit I” and “Rice discriminating PCR Kit II (Koshihikari negative kit)” are used to confirm whether rice of other varieties is mixed in Koshihikari. The operation is complicated because two PCRs, Kit I (positive detection) and Kit II (negative detection), are required for each sample. Also, the variety cannot be specified, and Koshihikari BL (Niigata Koshihikari) is determined to be mixed rice and cannot be identified as Koshihikari. The “Koshihikari BL PCR PCR Kit produced in 2005” manufactured by TaKaRa Co., Ltd. was not able to be discriminated by the above “Rice Discrimination PCR Kit I” and “Rice Discrimination PCR Kit II (Koshihikari negative kit)” It is used for re-inspection. However, since a plurality of rice grains cannot be inspected at a time and one rice grain must be inspected, it is very complicated. In addition, Vision Bio's “Kyoto Discrimination Kit“ Rice Appraisal Team ”” is a kit that uses six different primer sets to discriminate between Koshihikari and the top 20 varieties, but six times to discriminate one sample. PCR is required and is not convenient.

  In the detection method using a DNA array, a large number of capture oligonucleotides are immobilized on a support (hereinafter referred to as a DNA array or simply an array), and a labeled target nucleic acid is hybridized to the capture oligonucleotide and captured. This is a technique for analyzing the presence or absence of a target nucleic acid. However, when discriminating rice varieties using a DNA array, multiple types of multiplex primer sets are required to create a target (PCR product) for discriminating multiple varieties of rice, and PCR is performed multiple times per sample. There is a problem that it is necessary to carry out the inspection and the inspection takes time (Patent Document 3). In addition, an expensive apparatus is required to detect a fluorescently labeled PCR product and digitize the fluorescence intensity to determine the type (Patent Documents 3 and 4). Furthermore, although a limited number of rice varieties can be easily discriminated, there is a problem that there are few varieties that can be discriminated compared to other methods (Patent Document 4).

  The SNPs (Single Nucleotide Polymorphism) method uses a primer that is paired up to one base before the SNP site, performs a reaction that incorporates only one nucleotide of a fluorescently labeled nucleotide, and then measures the degree of polarization of each fluorescence and incorporates it. This is a technique for determining a polymorphism by identifying a base type. In order to discriminate rice varieties by the SNP method, it is necessary to select a primer pair to be used depending on the rice varieties to be discriminated. If the rice varieties are unknown, it is difficult to determine the primer pair to be used, which is not practical. There is a problem (Patent Document 5). In addition, based on Patent Document 5, there is a kit called “Komehikari Series“ Koshihikari Discrimination Kit ”” (manufactured by Plant Genome Center) that discriminates whether the rice displayed as 100% Koshihikari is really 100% Koshihikari. Although it is commercially available, it is effective only when other varieties (85 varieties) are mixed in Koshihikari and for the identification of pure Koshihikari rice, and the rice varieties cannot be identified.

As described above, various methods using genetic polymorphisms of genome sequences have been developed to discriminate rice varieties. However, the above-described method has the disadvantages that the operation is complicated, takes time, and is inefficient. Therefore, under the situation where the demand for rice variety discrimination inspection is increasing and the number of rice samples to be tested is rapidly increasing, there is a need for a technology that can distinguish many rice varieties in a simpler, more efficient and accurate manner. Yes.
Japanese Patent No. 3685478 JP 2005-73655 A JP 2005-245244 A JP 2006-158253 A JP 2006-174714 A

  An object of the present invention is to provide a technique that can easily and efficiently discriminate and determine the variety of rice samples and the presence or absence of mixed rice with a simple operation, at low cost and with excellent visibility.

  As a result of intensive studies by the present inventors in order to solve the above-mentioned problems, it has been found that a specific region in the rice genome sequence is useful for discriminating rice varieties and determining the presence or absence of mixed rice, and an oligo capable of detecting the region. By using nucleotides, it was found that the region can be efficiently detected using the difference in the electrophoresis pattern of the PCR product as an index and / or the difference in hybridization efficiency with the PCR product as an index, and the present invention was completed. .

That is, this invention consists of the following.
1. It consists of two kinds of oligonucleotides consisting of two kinds of base sequences selected from the following (1) to (5), and has 80 bases or more consisting of the base sequence represented by any one of SEQ ID NOs: 1 to 11 A primer pair capable of amplifying a nucleic acid.
(1) Two types of base sequences constituting any one combination selected from the following (a) to (k) (a) SEQ ID NO: 12 and SEQ ID NO: 23
(B) SEQ ID NO: 13 and SEQ ID NO: 24
(C) SEQ ID NO: 14 and SEQ ID NO: 25
(D) SEQ ID NO: 15 and SEQ ID NO: 26
(E) SEQ ID NO: 16 and SEQ ID NO: 27
(F) SEQ ID NO: 17 and SEQ ID NO: 28
(G) SEQ ID NO: 18 and SEQ ID NO: 29
(H) SEQ ID NO: 19 and SEQ ID NO: 30
(I) SEQ ID NO: 20 and SEQ ID NO: 31
(J) SEQ ID NO: 21 and SEQ ID NO: 32
(K) SEQ ID NO: 22 and SEQ ID NO: 33
(2) a base sequence complementary to the base sequence of (1) (3) a base sequence capable of hybridizing with the base sequence of (1) under stringent conditions (4) a base sequence of (1) A base sequence capable of hybridizing with a complementary base sequence under stringent conditions (5) one to several bases of the base sequences of (1) to (4) above are substituted, deleted, inserted, or 1. Added base sequence A primer set comprising at least two kinds of primer pairs according to item 1.
3. 3. The primer set according to item 2 above, comprising at least 11 kinds of primer pairs capable of amplifying a nucleic acid of 80 bases or more consisting of the base sequences represented by SEQ ID NOs: 1 to 11, respectively.
4). 4. The primer set according to item 3 above, wherein the primer pair capable of amplifying a nucleic acid of 80 bases or more consisting of the base sequences represented by SEQ ID NOs: 1 to 11 is the following primer pair (A) to (K):
(A) a nucleotide sequence represented by SEQ ID NO: 12 and SEQ ID NO: 23; a nucleotide sequence complementary to the nucleotide sequence; a nucleotide sequence capable of hybridizing with the nucleotide sequence under stringent conditions; and the nucleotide sequence A base sequence capable of hybridizing with a complementary base sequence under stringent conditions; one to several bases of these base sequences are selected from the base sequences substituted, deleted, inserted or added A primer pair (B) consisting of two kinds of oligonucleotides consisting of two kinds of base sequences and capable of amplifying a nucleic acid of 80 bases or more consisting of the base sequence represented by SEQ ID NO: 1 and SEQ ID NO: 24 A nucleotide sequence complementary to the nucleotide sequence; a nucleotide sequence capable of hybridizing with the nucleotide sequence under stringent conditions; a nucleotide sequence complementary to the nucleotide sequence and a string; A nucleotide sequence capable of hybridizing under a gentle condition; 1 to several of these nucleotide sequences are composed of two nucleotide sequences selected from a substituted, deleted, inserted, or added nucleotide sequence A primer pair (C) consisting of two kinds of oligonucleotides and capable of amplifying a nucleic acid of 80 bases or more consisting of a base sequence represented by SEQ ID NO: 2 and a base sequence represented by SEQ ID NO: 25; A base sequence complementary to the base sequence; a base sequence capable of hybridizing with the base sequence under stringent conditions; a base sequence capable of hybridizing with the base sequence complementary to the base sequence under stringent conditions; Two oligonucleotides consisting of two kinds of base sequences in which one to several bases are selected from base sequences substituted, deleted, inserted or added A primer pair (D) capable of amplifying a nucleic acid of 80 bases or more comprising the base sequence represented by SEQ ID NO: 3 and a base sequence represented by SEQ ID NO: 26; complementary to the base sequence A base sequence that can hybridize with the base sequence under stringent conditions; a base sequence that can hybridize with the base sequence complementary to the base sequence under stringent conditions; 1 to several bases are composed of two kinds of oligonucleotides composed of two kinds of base sequences selected from substituted, deleted, inserted or added base sequences, and from the base sequence represented by SEQ ID NO: 4 A primer pair (E) that can amplify a nucleic acid of 80 bases or more; a base sequence represented by SEQ ID NO: 16 and SEQ ID NO: 27; a base sequence complementary to the base sequence; A base sequence capable of hybridizing under lingent conditions; a base sequence complementary to the base sequence and a base sequence capable of hybridizing under stringent conditions; one to several bases of these base sequences are It consists of two types of oligonucleotides consisting of two types of base sequences selected from substitutions, deletions, insertions, or additions, and amplifies a nucleic acid of 80 bases or more consisting of the base sequence represented by SEQ ID NO: 5. A base sequence represented by SEQ ID NO: 17 and SEQ ID NO: 28; a base sequence complementary to the base sequence; a base sequence capable of hybridizing with the base sequence under stringent conditions; A base sequence that can hybridize with a base sequence complementary to the base sequence under stringent conditions; 1 to several of these base sequences are substituted or deleted Primer pair that consists of two kinds of oligonucleotides consisting of two kinds of base sequences selected from the inserted or added base sequences and can amplify a nucleic acid of 80 bases or more consisting of the base sequence represented by SEQ ID NO: 6 ( G) base sequences represented by SEQ ID NO: 18 and SEQ ID NO: 29; base sequences complementary to the base sequences; base sequences capable of hybridizing with the base sequences under stringent conditions; complementary to the base sequences A nucleotide sequence capable of hybridizing with a stringent nucleotide sequence under stringent conditions; 1 to several of these nucleotide sequences are selected from a substituted, deleted, inserted, or added nucleotide sequence 2 Primer pair (H) SEQ ID NO: 2 consisting of two types of oligonucleotides consisting of a base sequence and capable of amplifying a nucleic acid of 80 bases or more consisting of the base sequence represented by SEQ ID NO: 9 and a base sequence represented by SEQ ID NO: 30; a base sequence complementary to the base sequence; a base sequence capable of hybridizing with the base sequence under stringent conditions; a base sequence complementary to the base sequence Nucleotide sequences that can hybridize with each other under stringent conditions; two nucleotide sequences in which one to several of these nucleotide sequences are selected from the substituted, deleted, inserted, or added nucleotide sequences A primer pair (I) that can amplify a nucleic acid of 80 bases or more consisting of a base sequence represented by SEQ ID NO: 8, and a base sequence represented by SEQ ID NO: 31 A base sequence complementary to the base sequence; a base sequence capable of hybridizing under stringent conditions with the base sequence; a base sequence complementary to the base sequence and stringent conditions Base sequences that can hybridize underneath; two types of base sequences in which one to several bases are composed of two base sequences selected from base sequences with substitution, deletion, insertion, or addition A primer pair (J) that can amplify a nucleic acid of 80 bases or more consisting of an oligonucleotide and consisting of a base sequence represented by SEQ ID NO: 9, and a base sequence represented by SEQ ID NO: 32; Complementary base sequence; base sequence capable of hybridizing with the base sequence under stringent conditions; base sequence capable of hybridizing with the base sequence complementary to the base sequence under stringent conditions; these base sequences 1 to several bases of which consist of two oligonucleotides consisting of two base sequences selected from the base sequences substituted, deleted, inserted or added, and the sequence A primer pair (K) that can amplify a nucleic acid of 80 bases or more consisting of the base sequence represented by No. 10 and the base sequence represented by SEQ ID NO: 33; a base sequence complementary to the base sequence; A base sequence capable of hybridizing with the base sequence under stringent conditions; a base sequence complementary to the base sequence with a base sequence capable of hybridizing under stringent conditions; one to several of these base sequences Consisting of two oligonucleotides consisting of two types of base sequences selected from the nucleotide sequences of substitution, deletion, insertion or addition, and 80 bases or more consisting of the base sequence represented by SEQ ID NO: 11 4. Primer pair capable of amplifying nucleic acids of Capture oligonucleotide (1) sequence capable of detecting a nucleic acid consisting of a base sequence represented by any one of SEQ ID NOS: 1 to 11 selected from oligonucleotides consisting of the following base sequences (1) to (5) Base sequence represented by any one of Nos. 34 to 46 (2) Base sequence complementary to the base sequence represented by any one of SEQ ID Nos. 34 to 46 (3) Any of SEQ ID Nos. 34 to 46 A base sequence capable of hybridizing under stringent conditions with a base sequence represented by any one of the above (4) a base sequence complementary to the base sequence represented by any one of SEQ ID NOs: 34 to 46 and a stringent (5) a base sequence in which 1 to several bases of the base sequences (1) to (4) are substituted, deleted, inserted or added; 6. A capture oligonucleotide group comprising at least two capture oligonucleotides according to item 5 above.
7). 7. The group of capture oligonucleotides according to item 6 above, comprising at least 13 types of capture oligonucleotides each capable of detecting a nucleic acid comprising the base sequence described in SEQ ID NOs: 1 to 11.
8). The capture oligonucleotide group (I) according to item 7 above, wherein the capture oligonucleotides each capable of detecting the nucleic acid having the base sequence described in SEQ ID NOs: 1 to 11 are capture oligonucleotides of (I) to (XIII) below: The nucleotide sequence represented by SEQ ID NO: 34; a nucleotide sequence complementary to the nucleotide sequence; a nucleotide sequence capable of hybridizing with the nucleotide sequence under stringent conditions; a nucleotide sequence complementary to the nucleotide sequence and a stringent nucleotide sequence A nucleotide sequence that can hybridize under conditions; one to several of these nucleotide sequences is composed of an oligonucleotide selected from a substituted, deleted, inserted, or added nucleotide sequence; A capture oligonucleotide (II) capable of detecting a nucleic acid comprising the nucleotide sequence represented by the nucleotide sequence represented by SEQ ID NO: 35; a nucleotide sequence complementary to the nucleotide sequence; A base sequence that can hybridize with a base sequence under stringent conditions; a base sequence that can hybridize with a base sequence complementary to the base sequence under stringent conditions; one to several of these base sequences Capture oligonucleotide (III) SEQ ID NO: 36 which can detect a nucleic acid consisting of a base sequence represented by SEQ ID NO: 2 consisting of an oligonucleotide selected from base sequences substituted, deleted, inserted or added A base sequence complementary to the base sequence; a base sequence capable of hybridizing under stringent conditions; and a base sequence complementary to the base sequence under stringent conditions Hybridizable base sequence; selected from base sequences in which one to several bases of these base sequences are substituted, deleted, inserted or added A capture oligonucleotide (IV) that can detect a nucleic acid consisting of the base sequence represented by SEQ ID NO: 3; a base sequence represented by SEQ ID NO: 37; a base sequence complementary to the base sequence; A base sequence that can hybridize with a base sequence under stringent conditions; a base sequence that can hybridize with a base sequence complementary to the base sequence under stringent conditions; one to several of these base sequences Capture oligonucleotide (V) SEQ ID NO: 38, which comprises a nucleotide whose base is selected from substitution, deletion, insertion, or added base sequence, and which can detect a nucleic acid consisting of the base sequence represented by SEQ ID NO: 4 A base sequence complementary to the base sequence; a base sequence capable of hybridizing with the base sequence under stringent conditions; A base sequence that can hybridize with a base sequence complementary to the base sequence under stringent conditions; from a base sequence in which one to several bases of these base sequences are substituted, deleted, inserted, or added A capture oligonucleotide (VI) comprising the selected oligonucleotide and capable of detecting a nucleic acid comprising the base sequence represented by SEQ ID NO: 5; a base sequence represented by SEQ ID NO: 39; a base sequence complementary to the base sequence; A base sequence capable of hybridizing with the base sequence under stringent conditions; a base sequence complementary to the base sequence with a base sequence capable of hybridizing under stringent conditions; one to several of these base sequences Consisting of an oligonucleotide selected from the base sequences substituted, deleted, inserted or added, and consisting of the base sequence represented by SEQ ID NO: 6 Capture oligonucleotide (VII) capable of detecting nucleic acid: base sequence represented by SEQ ID NO: 40; base sequence complementary to the base sequence; base sequence capable of hybridizing with the base sequence under stringent conditions; the base A nucleotide sequence that can hybridize under stringent conditions with a complementary nucleotide sequence; selected from nucleotide sequences in which one to several of these nucleotide sequences are substituted, deleted, inserted, or added A capture oligonucleotide (VIII) that can detect a nucleic acid consisting of a base sequence represented by SEQ ID NO: 7; a base sequence represented by SEQ ID NO: 41; a base sequence complementary to the base sequence; A base sequence that can hybridize with a base sequence under stringent conditions; a hybrid with a base sequence complementary to the base sequence under stringent conditions Possible nucleotide sequence; a nucleotide sequence represented by SEQ ID NO: 8 consisting of an oligonucleotide wherein one or several of these nucleotide sequences are selected from a substituted, deleted, inserted or added nucleotide sequence A base sequence represented by SEQ ID NO: 42, which can detect a nucleic acid consisting of: a base sequence complementary to the base sequence; a base sequence capable of hybridizing with the base sequence under stringent conditions; A base sequence that can hybridize with a base sequence complementary to the base sequence under stringent conditions; a base sequence in which one to several bases of these base sequences are substituted, deleted, inserted, or added; A capture oligonucleotide (X) that is capable of detecting a nucleic acid consisting of the nucleotide sequence represented by SEQ ID NO: 9 and that is represented by SEQ ID NO: 43 A base sequence complementary to the base sequence; a base sequence capable of hybridizing with the base sequence under stringent conditions; a base capable of hybridizing with the base sequence complementary to the base sequence under stringent conditions A nucleic acid comprising a nucleotide sequence represented by SEQ ID NO: 9, comprising one or more nucleotides selected from substitution, deletion, insertion, or addition of the nucleotide sequence. A nucleotide sequence represented by SEQ ID NO: 44; a nucleotide sequence complementary to the nucleotide sequence; a nucleotide sequence capable of hybridizing with the nucleotide sequence under stringent conditions; the nucleotide sequence A nucleotide sequence that can hybridize under stringent conditions with a complementary nucleotide sequence; one to several of these nucleotide sequences are substituted or deleted A nucleotide sequence represented by SEQ ID NO: 45, which is a capture oligonucleotide (XII) consisting of an oligonucleotide selected from the inserted or added nucleotide sequences and capable of detecting a nucleic acid consisting of the nucleotide sequence represented by SEQ ID NO: 10; A base sequence complementary to the base sequence; a base sequence capable of hybridizing with the base sequence under stringent conditions; a base sequence capable of hybridizing with the base sequence complementary to the base sequence under stringent conditions; Among these base sequences, one to several bases are composed of oligonucleotides selected from the base sequences substituted, deleted, inserted or added, and a nucleic acid comprising the base sequence represented by SEQ ID NO: 10 is detected. A capture oligonucleotide (XIII) represented by SEQ ID NO: 46; a base sequence complementary to the base sequence; the base sequence and stringency A base sequence that can hybridize under conditions that are complementary to the base sequence; a base sequence that can hybridize under stringent conditions with a base sequence that is complementary to the base sequence; one to several of these base sequences are substituted 8. A capture oligonucleotide that can detect a nucleic acid consisting of a nucleotide sequence represented by SEQ ID NO: 11, consisting of an oligonucleotide selected from nucleotide sequences that are deleted, inserted, or added. A method for discriminating rice varieties, comprising performing the following steps (i) and (ii):
(I) Nucleic acid preparation step for preparing rice nucleic acid in test rice sample (ii) Using the nucleic acid prepared in step (i) as a template, using the primer set according to any one of the preceding items 2 to 4 9. Nucleic acid amplification step of obtaining nucleic acid amplification product by nucleic acid amplification treatment Furthermore, the following (iii) and / or the process of (iv) is performed, The discrimination | determination method of the rice varieties of the preceding clause 9 characterized by the above-mentioned.
(Iii) Detection step for detecting the nucleic acid amplification product obtained in step (ii) by electrophoresis (iv) The nucleic acid amplification product obtained in step (ii) and the capture described in any one of 6 to 8 above 10. Detection step for detecting hybridization with oligonucleotide group A method for determining the presence or absence of mixed rice in Koshihikari, comprising performing the following steps (i) and (ii):
(I) Nucleic acid preparation step for preparing rice nucleic acid in test rice sample (ii) Using the nucleic acid prepared in step (i) as a template, using the primer set according to any one of the preceding items 2 to 4 Nucleic acid amplification step of obtaining nucleic acid amplification product by nucleic acid amplification treatment12. Furthermore, the method of determining the presence or absence of the mixed rice to Koshihikari of the preceding clause 11 characterized by performing the following processes (iii) and / or (iv).
(Iii) Detection step for detecting the nucleic acid amplification product obtained in step (ii) by electrophoresis (iv) The nucleic acid amplification product obtained in step (ii) and the capture described in any one of 6 to 8 above 12. Detection step for detecting a hybrid with the oligonucleotide group The rice variety discrimination kit containing the primer set of any one of the preceding items 2-4, or the mixed rice determination kit to Koshihikari.
14 14. The kit according to item 13, further comprising the capture oligonucleotide group according to any one of items 6 to 8.

  When the primer set of the present invention is used, electrophoresis of amplification products by one PCR, discrimination of 15 or more rice varieties, determination of the presence or absence of other 118 varieties of mixed rice in Koshihikari pure rice, and It becomes possible to distinguish between Koshihikari and Koshihikari BL. Furthermore, according to the capture oligonucleotide of the present invention, by hybridizing the nucleic acid amplification product by the primer of the present invention and the oligonucleotide, discrimination of 119 rice varieties, and 118 other varieties of pure Koshihikari rice The presence / absence of mixed rice and the discrimination between Koshihikari and Koshihikari BL can be performed.

  Therefore, the method of determining the rice varieties of the present invention and the method of determining the presence or absence of mixed rice using the primer of the present invention and the capture oligonucleotide of the present invention is excellent in visibility, accurately and easily distinguishing rice varieties, Can determine mixed rice to Koshihikari pure rice. In addition, the mixed rice determination method of the present invention can determine mixed rice even if the amount of genomic DNA of the sample subjected to PCR is 0.025 ng / 20 μl. Further, if the mixed rice ratio is 1%, the mixed rice can be reliably determined. In addition, even if the test rice sample is cooked rice, there is an advantage that it is possible to determine the presence or absence of mixed rice.

The method for discriminating rice varieties and the method for determining the presence or absence of mixed rice in Koshihikari according to the present invention are characterized by performing the following steps (i) and (ii).
(I) Nucleic acid preparation step of preparing rice nucleic acid in test rice sample (ii) Nucleic acid amplification process using nucleic acid prepared in step (i) as a template to obtain nucleic acid amplification product Nucleic Acid Amplification Step Hereinafter, each step will be described.

(I) Nucleic acid preparation step The nucleic acid preparation step is a step of extracting and preparing rice nucleic acid from a test rice sample. As a method for preparing a nucleic acid from a test rice sample, a known nucleic acid preparation method can be appropriately selected and used. For example, it can be prepared according to the CTAB method (Murray MG, Thompson WF: Nucleic Acids Res., 8, p. 4321-4325), enzymatic method, alkaline SDS method and the like. These methods are standard preparation methods for extracting plant genomic DNA, but there are many alternative methods, any of which may be used. Moreover, it can prepare using commercially available DNA extraction kits, such as Wizard (R) Magnetic DNA Purification System for Food (Promega), for example.

  The test rice sample includes seedlings as well as edible seeds (rice grains). The seeds include not only polished rice and brown rice distributed in the market for food, but also polished rice flour, brown rice flour, and cooked rice and rice bran. Here, the cooked rice refers to a product obtained by cooking polished rice or brown rice with a normal electric rice cooker, gas cooker, steam cooking system or the like, and is also simply referred to as cooked rice. The test rice sample is not particularly limited as long as it is a rice-derived sample, but preferably the morphological variety is more difficult to discriminate, more preferably the morphological or biochemical variety is discriminated. The effect of the present invention is more significantly exhibited when the milled rice, brown rice, or a processed product thereof becomes more difficult.

(Ii) Nucleic acid amplification step The nucleic acid amplification step refers to a primer pair that can amplify a nucleic acid consisting of any one of the base sequences represented by SEQ ID NOs: 1 to 11, using the nucleic acid prepared in step (i) as a template. This is a step of subjecting the nucleic acid amplification treatment to the primer set.

(Primer)
The primer pair of the present invention is preferably a primer pair capable of amplifying a nucleic acid of 80 bases or more consisting of any one of the base sequences represented by SEQ ID NOs: 1 to 11, specifically, the following (1) to ( It consists of two kinds of oligonucleotides consisting of two kinds of base sequences selected from 5).
(1) Nucleotide sequence comprising any one of the following combinations (a) to (k) (a) Forward primer for nucleic acid amplification consisting of the nucleotide sequence represented by SEQ ID NO: 1: 5′-TCTATACATGGGCCGTACACATCATT -3 ′ (SEQ ID NO: 12)
Reverse primer: 5′-CACATACTCCCTCTCCCTCTGTCCT-3 ′ (SEQ ID NO: 23)
(B) Forward primer for nucleic acid amplification consisting of the base sequence represented by SEQ ID NO: 2: 5′-CCCGCAGTTAGATGCACCCATTAGA-3 ′ (SEQ ID NO: 13)
Reverse primer: 5′-GTGCTCTGGAGATGTGGGCATGTAAG-3 ′ (SEQ ID NO: 24)
(C) Forward primer for nucleic acid amplification consisting of the base sequence represented by SEQ ID NO: 3: 5'-AGGCCATCGTAGCAATTTCGAAAAG-3 '(SEQ ID NO: 14)
Reverse primer: 5′-CGTTCGCTCCCATGCAATCTGCAAA-3 ′ (SEQ ID NO: 25)
(D) Forward primer for nucleic acid amplification consisting of the base sequence represented by SEQ ID NO: 4: 5′-AGTTCCCTCTGTATCCCTGTCCCCCT-3 ′ (SEQ ID NO: 15)
Reverse primer: 5'-CACCCTCACATACAATGTGTCCTTGT-3 '(SEQ ID NO: 26)
(E) Nucleic acid amplification forward primer consisting of the base sequence represented by SEQ ID NO: 5: 5′-TGTAGCCCTTGCATCATCAATTTTA-3 ′ (SEQ ID NO: 16)
Reverse primer: 5'-TTCAGACATATACCTCCAAGCCAAT-3 '(SEQ ID NO: 27)
(F) Forward primer for nucleic acid amplification consisting of the base sequence represented by SEQ ID NO: 6: 5′-CTCAACTTACATTTCTACTAACACA-3 ′ (SEQ ID NO: 17)
Reverse primer: 5'-CATGCCAAAATATACACTGCATGTTT-3 '(SEQ ID NO: 28)
(G) Nucleic acid amplification forward primer consisting of the base sequence represented by SEQ ID NO: 7: 5′-CAGGCTTACAGCAAATGGATCTTGA-3 ′ (SEQ ID NO: 18)
Reverse primer: 5'-CTTGTCATGTACTTCTCTGTGCAA-3 '(SEQ ID NO: 29)
(H) Forward primer for nucleic acid amplification consisting of the base sequence represented by SEQ ID NO: 8: 5′-CGACTCCATATACCACCAAGGCGTTT-3 ′ (SEQ ID NO: 19)
Reverse primer: 5′-AGCTTTCTTAGGGTATTTATAGCGT-3 ′ (SEQ ID NO: 30)
(I) Nucleic acid amplification forward primer consisting of the base sequence represented by SEQ ID NO: 9: 5′-TCGGTCTCTGCTAGCTAGTTTCAG-3 ′ (SEQ ID NO: 20)
Reverse primer: 5'-ATTATTGAAAATGATGGTTTCGAGCG-3 '(SEQ ID NO: 31)
(J) Nucleic acid amplification forward primer consisting of the base sequence represented by SEQ ID NO: 10: 5′-GCTAAGTTTCCCTCAAAATCTTAT-3 ′ (SEQ ID NO: 21)
Reverse primer: 5'-CTACGTGGTTTATTGATTTAAAGGA-3 '(SEQ ID NO: 32)
(K) Nucleic acid amplification forward primer consisting of the base sequence represented by SEQ ID NO: 11: 5′-GCTGCTTGGATTTGCTTCTTTAGC-3 ′ (SEQ ID NO: 22)
Reverse primer: 5'-CATCCGATGGTATAGGGGACTTAAA-3 '(SEQ ID NO: 33)
(2) A base sequence complementary to the base sequence selected in (1) (3) A base sequence capable of hybridizing under stringent conditions with the base sequence selected in (1) (4) A base sequence that can hybridize under stringent conditions with a base sequence that is complementary to the base sequence selected in 1), and (5) one to several bases of the base sequences of (1) to (4) above , Substitutions, deletions, insertions, or additions

  The primer pair of the present invention is preferably used as a primer set comprising at least two or more kinds of the primer pairs, and each amplifies a nucleic acid of 80 bases or more comprising the base sequences represented by SEQ ID NOs: 1 to 11. More preferably, it is used as a primer set comprising at least 11 kinds of primer pairs.

The following primer pairs (A) to (K) are preferably exemplified as primer pairs capable of amplifying nucleic acids of 80 bases or more comprising the base sequences represented by SEQ ID NOs: 1 to 11, respectively.
(A) a nucleotide sequence represented by SEQ ID NO: 12 and SEQ ID NO: 23; a nucleotide sequence complementary to the nucleotide sequence; a nucleotide sequence capable of hybridizing with the nucleotide sequence under stringent conditions; and the nucleotide sequence A base sequence capable of hybridizing with a complementary base sequence under stringent conditions; one to several bases of these base sequences are selected from the base sequences substituted, deleted, inserted or added A primer pair (B) consisting of two kinds of oligonucleotides consisting of two kinds of base sequences and capable of amplifying a nucleic acid of 80 bases or more consisting of the base sequence represented by SEQ ID NO: 1 and SEQ ID NO: 24 A nucleotide sequence complementary to the nucleotide sequence; a nucleotide sequence capable of hybridizing with the nucleotide sequence under stringent conditions; a nucleotide sequence complementary to the nucleotide sequence and a string; A nucleotide sequence capable of hybridizing under a gentle condition; 1 to several of these nucleotide sequences are composed of two nucleotide sequences selected from a substituted, deleted, inserted, or added nucleotide sequence A primer pair (C) consisting of two kinds of oligonucleotides and capable of amplifying a nucleic acid of 80 bases or more consisting of a base sequence represented by SEQ ID NO: 2 and a base sequence represented by SEQ ID NO: 25; A base sequence complementary to the base sequence; a base sequence capable of hybridizing with the base sequence under stringent conditions; a base sequence capable of hybridizing with the base sequence complementary to the base sequence under stringent conditions; Two oligonucleotides consisting of two kinds of base sequences in which one to several bases are selected from base sequences substituted, deleted, inserted or added A primer pair (D) capable of amplifying a nucleic acid of 80 bases or more comprising the base sequence represented by SEQ ID NO: 3 and a base sequence represented by SEQ ID NO: 26; complementary to the base sequence A base sequence that can hybridize with the base sequence under stringent conditions; a base sequence that can hybridize with the base sequence complementary to the base sequence under stringent conditions; 1 to several bases are composed of two kinds of oligonucleotides composed of two kinds of base sequences selected from substituted, deleted, inserted or added base sequences, and from the base sequence represented by SEQ ID NO: 4 A primer pair (E) that can amplify a nucleic acid of 80 bases or more; a base sequence represented by SEQ ID NO: 16 and SEQ ID NO: 27; a base sequence complementary to the base sequence; A base sequence capable of hybridizing under lingent conditions; a base sequence complementary to the base sequence and a base sequence capable of hybridizing under stringent conditions; one to several bases of these base sequences are It consists of two types of oligonucleotides consisting of two types of base sequences selected from substitutions, deletions, insertions, or additions, and amplifies a nucleic acid of 80 bases or more consisting of the base sequence represented by SEQ ID NO: 5. A base sequence represented by SEQ ID NO: 17 and SEQ ID NO: 28; a base sequence complementary to the base sequence; a base sequence capable of hybridizing with the base sequence under stringent conditions; A base sequence that can hybridize with a base sequence complementary to the base sequence under stringent conditions; 1 to several of these base sequences are substituted or deleted Primer pair that consists of two kinds of oligonucleotides consisting of two kinds of base sequences selected from the inserted or added base sequences and can amplify a nucleic acid of 80 bases or more consisting of the base sequence represented by SEQ ID NO: 6 ( G) base sequences represented by SEQ ID NO: 18 and SEQ ID NO: 29; base sequences complementary to the base sequences; base sequences capable of hybridizing with the base sequences under stringent conditions; complementary to the base sequences A nucleotide sequence capable of hybridizing with a stringent nucleotide sequence under stringent conditions; 1 to several of these nucleotide sequences are selected from a substituted, deleted, inserted, or added nucleotide sequence 2 Primer pair (H) SEQ ID NO: 2 consisting of two types of oligonucleotides consisting of a base sequence and capable of amplifying a nucleic acid of 80 bases or more consisting of the base sequence represented by SEQ ID NO: 9 and a base sequence represented by SEQ ID NO: 30; a base sequence complementary to the base sequence; a base sequence capable of hybridizing with the base sequence under stringent conditions; a base sequence complementary to the base sequence Nucleotide sequences that can hybridize with each other under stringent conditions; two nucleotide sequences in which one to several of these nucleotide sequences are selected from the substituted, deleted, inserted, or added nucleotide sequences A primer pair (I) that can amplify a nucleic acid of 80 bases or more consisting of a base sequence represented by SEQ ID NO: 8, and a base sequence represented by SEQ ID NO: 31 A base sequence complementary to the base sequence; a base sequence capable of hybridizing under stringent conditions with the base sequence; a base sequence complementary to the base sequence and stringent conditions Base sequences that can hybridize underneath; two types of base sequences in which one to several bases are composed of two base sequences selected from base sequences with substitution, deletion, insertion, or addition A primer pair (J) that can amplify a nucleic acid of 80 bases or more consisting of an oligonucleotide and consisting of a base sequence represented by SEQ ID NO: 9, and a base sequence represented by SEQ ID NO: 32; Complementary base sequence; base sequence capable of hybridizing with the base sequence under stringent conditions; base sequence capable of hybridizing with the base sequence complementary to the base sequence under stringent conditions; these base sequences 1 to several bases of which consist of two oligonucleotides consisting of two base sequences selected from the base sequences substituted, deleted, inserted or added, and the sequence A primer pair (K) that can amplify a nucleic acid of 80 bases or more consisting of the base sequence represented by No. 10 and the base sequence represented by SEQ ID NO: 33; a base sequence complementary to the base sequence; A base sequence that can hybridize with the base sequence under stringent conditions; a base sequence that can hybridize with a base sequence complementary to the base sequence under stringent conditions; one to several of these base sequences Consisting of two oligonucleotides consisting of two types of base sequences selected from the base sequences substituted, deleted, inserted or added, and having 80 bases or more consisting of the base sequence represented by SEQ ID NO: 11 Primer pair that can amplify the nucleic acid of

  The rice varieties from which the nucleotide sequences represented by SEQ ID NOs: 1 to 11 of the present invention are derived are Nipponbare. In addition, information such as the presence or absence of polymorphisms in SEQ ID NOs: 1 to 11 is found based on rice genome sequences and RFLP marker information published in Rice Genome Research Program, sequences registered in GenBank, etc. It was originally discovered by Genetic ID Corporation and Japan Tobacco Inc.

  When a polymorphism exists in the nucleotide sequences represented by SEQ ID NOs: 1 to 11, it is preferable that a nucleic acid containing the polymorphic site can be amplified, and the polymorphic site is substituted by insertion, deletion, or the like. In such a case, it is more preferable to design a primer that makes it difficult to obtain a nucleic acid amplification product by the nucleic acid amplification treatment. Here, the polymorphism present in the nucleic acid derived from rice means a difference in base sequence between rices to be compared, and a concept including substitution, deletion, insertion and addition of 1 base to several hundred bases. It is.

  For example, polymorphisms exist at positions corresponding to positions 21 and 28 of the base sequence represented by SEQ ID NO: 6 and positions corresponding to position 145 of the base sequence represented by SEQ ID NO: 7. Therefore, the primer for nucleic acid amplification comprising the nucleotide sequences represented by SEQ ID NO: 6 and SEQ ID NO: 7 is preferably a primer comprising a nucleotide sequence containing the polymorphic site, a nucleotide sequence adjacent to the polymorphic site, and the like.

  Specifically, for example, the forward primer constituting the primer pair (F) for nucleic acid amplification consisting of the base sequence represented by SEQ ID NO: 6 is a polymorphism at position 21 of the base sequence represented by base sequence 6. A sequence consisting of a base sequence (SEQ ID NO: 17) containing a type site is preferred. Further, the reverse primer constituting the primer pair (G) for nucleic acid amplification consisting of the base sequence represented by SEQ ID NO: 7 is the base sequence of the site adjacent to the polymorphic site of SEQ ID NO: 7 (SEQ ID NO: 29) The sequence consisting of is preferred.

  The position of the polymorphism in the above sequence can be changed by insertion or deletion of nucleotides in the sequence upstream from the position. The above “corresponding position” indicates the corresponding position in the reference sequence represented by SEQ ID NOs: 1 to 11 when the absolute position in the gene sequence is changed as described above. That is, the polymorphism in the present invention is represented by SEQ ID NOs: 1 to 11 by comparing the base sequence represented by SEQ ID NOs: 1 to 11 with the base sequence of a nucleic acid derived from a test rice that can be identified. It includes polymorphisms that are understood to correspond to polymorphisms in the base sequence.

  The primer used in the method of the present invention may contain a labeling substance in consideration of a subsequent detection step. As a labeling substance, a radioisotope, a fluorescent substance, a chemiluminescent substance, biotin and the like well known in the art can be used.

  The oligonucleotide having a function as the primer of the present invention or the capture oligonucleotide of the present invention described later includes DNA, RNA, peptide nucleic acid, etc., but DNA or RNA is preferable, and DNA is more preferable. The oligonucleotide used in the present invention can be prepared by a genetic engineering technique or a synthetic method. Examples of the synthetic method include a normal solid phase synthesis method. Synthesis by this method can be performed by using a commercially available DNA synthesizer.

  The oligonucleotide having the function of the primer of the present invention or the capture oligonucleotide of the present invention described later can be produced by a method known per se, for example, chemically synthesized. Alternatively, a natural nucleic acid can be cleaved with a restriction enzyme or the like, modified so as to have the above base sequence, or ligated. Specifically, it can be synthesized using an oligonucleotide synthesizer (Expedite Model 8909 DNA synthesizer manufactured by Applied Biosystems) or the like. In addition, as a method for synthesizing a mutated oligonucleotide such as substitution, deletion, insertion or addition of one to several bases, a method known per se can be used. For example, site-directed mutagenesis method, gene homologous recombination method, primer extension method or polymerase chain amplification method (PCR) may be used alone or in combination as appropriate, for example, Molecular Cloning; A Laboratory Manual, 2nd edition, edited by Sambrook et al., Cold・ Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989; [Lab Manual Genetic Engineering], edited by Masami Muramatsu, Maruzen Co., 1988; [PCR Technology, Principles and Applications of DNA Amplification], Ehrlich, HE. Hen, Stockton Press, 1989, etc., or can be carried out by modifying those methods. For example, Ulmer's technology [Science (1983) 219: 666] can be used. .

  Here, generally known hybridization conditions can be selected, and examples thereof include 50% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate) solution, 50 mM sodium phosphate. , PH 7.6, 5 × Denhart's solution, 10% dextran sulfate, and 20 μg / ml DNA in a solution containing 2 μSSC / 0.1% SDS at room temperature after overnight hybridization at 42 ° C. The first washing is followed by the second washing with 0.1 × SSC / 0.1% SDS solution at about 65 ° C.

  There is no particular limitation on the method of performing nucleic acid amplification treatment in this step, and various nucleic acid amplification methods that can amplify the nucleic acid fragment to be detected can be used. Specifically, a PCR method, a LAMP method, or the like is used, and among them, the PCR method is preferably used. In this case, the 11 primer pairs of the primer pairs (A) to (K) of the present invention may be allowed to act individually in different reaction systems to obtain respective amplification products. In this case, they can then be mixed into one solution. Alternatively, a plurality of primer pairs selected from the primer pairs (A) to (K) may be mixed in advance in a predetermined amount in one reaction system, and an amplification reaction may be performed (hereinafter, this method is referred to as “multiplex method”). "). In this case, finally, the amplification products are mixed in such a combination that all the amplification products of the primer pairs (A) to (K) are present, so that one solution can be prepared. In particular, among the multiplex methods, it is most preferable to perform an amplification reaction at once in a reaction system in which all of the primer pairs (A) to (K) are mixed in a predetermined amount because of simplicity and efficiency. In such a multiplex method, a multiplex PCR method is preferably used.

  As described above, when there are 11 primer pairs, amplification by the multiplex method usually has a disadvantage that the balance of amplification efficiency for each primer is lost and a specific nucleic acid is extremely amplified. However, in the case of the primer pair of the present invention, amplification products corresponding to each primer pair can be obtained in a well-balanced manner by a single multiplex PCR by preparing them at a predetermined charge ratio.

  As a specific example of the mixing ratio of the primer pair, for example, the addition amount (molar ratio) of the primer pair (A) to (K) to the PCR reaction system is expressed as primer pair (A): primer pair (B): primer pair ( C): primer pair (D): primer pair (E): primer pair (F): primer pair (G): primer pair (H): primer pair (I): primer pair (J): primer pair (K) 2: 2: 2: 2: 2: 4: 4: 1: 2: 2: 2 makes it possible to amplify nucleic acids in a well-balanced manner by a single multiplex PCR. The presence / absence of mixed rice can be determined.

  The method for discriminating rice varieties and the method for determining the presence or absence of mixed rice in Koshihikari of the present invention includes the following (iii) step (ii) in addition to (i) nucleic acid preparation step and (ii) nucleic acid amplification step. A detection step for detecting the obtained nucleic acid amplification product by electrophoresis and / or (iv) a detection step for detecting hybridization between the nucleic acid amplification product obtained in step (ii) and the capture oligonucleotide of the present invention. You may go.

(Iii) Detection step for detecting the nucleic acid amplification product obtained in step (ii) by electrophoresis Conventional electrophoresis such as gel electrophoresis or capillary electrophoresis for the nucleic acid amplification product obtained in the nucleic acid amplification step (ii) Based on the length of the nucleic acid amplification product obtained by separation and detection by the method, it is possible to determine the rice variety contained in the test sample and the presence or absence of mixed rice in Koshihikari. In the case of gel electrophoresis, the amount of the nucleic acid amplification product to be subjected to electrophoresis is preferably 10 to 100 ng, more preferably 30 to 60 ng.

  In the case of discriminating rice varieties by detecting the nucleic acid amplification product by electrophoresis, the primer set is designed so that the length of the nucleic acid amplification product obtained differs depending on the rice variety to be detected. In addition, when the nucleic acid amplification product is detected by electrophoresis to determine the presence or absence of mixed rice in Koshihikari, it is represented by at least SEQ ID NOs: 2, 6, and 9 and / or 10 selected from the above primer pairs. A nucleic acid amplification treatment using a primer set including a primer pair [for example, the primer pair (B), (F), and further (I) and / or (J)] that can amplify a nucleic acid having a base sequence preferable. In addition, in the case of further discriminating by electrophoresis the discrimination of rice varieties and the presence or absence of mixed rice in Koshihikari, the above 11 primer pairs of the present invention (A) to (K) are included. It is preferable to perform a nucleic acid amplification treatment using a primer set.

  Furthermore, when the nucleic acid amplification product is detected by electrophoresis to discriminate Koshihikari BL (Niigata Koshihikari) from Koshihikari other than Koshihikari BL, at least SEQ ID NOs: 2, 7, 9, 10 selected from the above primer pairs. It is preferable to perform a nucleic acid amplification treatment using a primer set including a primer pair [for example, the primer pairs (B), (G), (I), (J)] that can amplify a nucleic acid having the base sequence represented by . Further, when discriminating between Koshihikari BL (Niigata Koshihikari) and Koshihikari other than Koshihikari BL, when further discriminating by electrophoresis, all 11 types of the above-mentioned primer pairs (A) to (K) of the present invention are selected. It is preferable to carry out nucleic acid amplification treatment using a primer set including a primer pair.

(Iv) Detection step for detecting hybridization between the nucleic acid amplification product obtained in step (ii) and the capture oligonucleotide of the present invention The nucleic acid amplification product obtained in the nucleic acid amplification step (ii) and immobilized on the support By hybridizing the captured oligonucleotide of the present invention and detecting the presence or absence of a hybridization signal, it is possible to determine the variety of rice and the presence or absence of mixed rice in Koshihikari.

  The amount of the nucleic acid amplification product provided to the DNA array is preferably in the range of 35 to 250 ng, more preferably 70 to 200 ng. In addition, the nucleic acid amplification product is preferably labeled in order to detect hybridization between the capture oligonucleotide and the nucleic acid amplification product.

  The nucleic acid labeling method is not particularly limited as long as it does not inhibit hybridization. Examples of such a method include a method of previously labeling a primer used for amplification of a target nucleic acid using a labeling substance, a target Examples include a method of labeling during production of a nucleic acid and a method of labeling after production of a target nucleic acid.

  As the labeling substance, a labeling substance used in a normal hybridization method can be used, and is not particularly limited. For example, examples of such a labeling substance include fluorescent substances and haptens. Specifically, fluorescein, rhodamine, phycoerythrin, Texas red, cyanine fluorescent dyes, and specific examples of haptens include biotin, digoxigenin, dinitrophenyl and the like.

  When discriminating rice varieties by detecting hybridization between the nucleic acid amplification product and the capture oligonucleotide of the present invention, a primer set is used so that a difference appears in the presence or absence of a hybridization signal depending on the rice cultivar to be detected. design. Further, when hybridization between the nucleic acid amplification product and the capture oligonucleotide of the present invention is detected by a DNA array to determine the presence or absence of mixed rice in Koshihikari, at least SEQ ID NOs: 2 and 6 selected from the above primer pairs. And a primer pair [for example, the primer pair (B), (F), and further (I) and / or (J)] capable of amplifying a nucleic acid having a base sequence represented by 9 and / or 10 It is preferable to perform a nucleic acid amplification treatment using a primer set. In addition, regarding the determination of rice varieties and the determination of the presence or absence of mixed rice in Koshihikari, in the case of detecting the hybrid formation of the nucleic acid amplification product and the capture oligonucleotide of the present invention, and further discriminating, It is preferable to perform nucleic acid amplification treatment using a primer set including all 11 types of primer pairs (A) to (K).

  Furthermore, when detecting hybridization between the nucleic acid amplification product and the capture oligonucleotide of the present invention to discriminate Koshihikari BL (Koshihikari from Niigata) from Koshihikari other than Koshihikari BL, at least a sequence selected from the primer pair described above Nucleic acid amplification using a primer set including a primer pair [for example, (B), (G), (I), (J)] that can amplify a nucleic acid having a base sequence represented by Nos. 2, 7, 9, 10 It is preferable to process. When discriminating between Koshihikari BL (Niigata Koshihikari) and Koshihikari other than Koshihikari BL, and when further discriminating by hybridization with the capture oligonucleotide of the present invention, the primer pair (A) of the present invention described above It is preferable to carry out nucleic acid amplification treatment using a primer set including all 11 types of primer pairs of (K).

(Capture oligonucleotide)
The capture oligonucleotide of the present invention is preferably an oligonucleotide having a length of 10 to 25 bases that can detect the base sequences represented by SEQ ID NOs: 1 to 11, and specifically, the following bases (I) to (XIII): Selected from oligonucleotides consisting of sequences.
(I) 5′-CTTGTGCAAGCAATG-3 ′ (SEQ ID NO: 34) for nucleic acid detection comprising the base sequence represented by SEQ ID NO: 1
(II) 5′-CTCTCCACAGCCAATG-3 ′ (SEQ ID NO: 35) for nucleic acid detection comprising the base sequence represented by SEQ ID NO: 2
(III) 5′-GACAACTACTCTACCA-3 ′ (SEQ ID NO: 36) for nucleic acid detection comprising the base sequence represented by SEQ ID NO: 3
(IV) 5′-TGGCAAACTGCCAGG-3 ′ (SEQ ID NO: 37) for nucleic acid detection comprising the base sequence represented by SEQ ID NO: 4
(V) 5′-GGTGTAGCACGCATG-3 ′ (SEQ ID NO: 38) for nucleic acid detection comprising the base sequence represented by SEQ ID NO: 5
(VI) 5′-ATCCTGCGGTCAGT-3 ′ (SEQ ID NO: 39) for nucleic acid detection comprising the base sequence represented by SEQ ID NO: 6
(VII) 5′-GCTCTCCCATTCCCA-3 ′ (SEQ ID NO: 40) for nucleic acid detection comprising the base sequence represented by SEQ ID NO: 7
(VIII) 5′-GATGCTCCAGTCACC-3 ′ (SEQ ID NO: 41) for nucleic acid detection comprising the base sequence represented by SEQ ID NO: 8
(IX) 5′-ACTCCTAGATGCGTA-3 ′ (SEQ ID NO: 42) for nucleic acid detection comprising the base sequence represented by SEQ ID NO: 9
(X) 5′-TATATACGCATCTAG-3 ′ (SEQ ID NO: 43) for nucleic acid detection comprising the base sequence represented by SEQ ID NO: 9
(XI) 5′-TATGAAATTTGCTCA-3 ′ (SEQ ID NO: 44) for nucleic acid detection comprising the base sequence represented by SEQ ID NO: 10
(XII) 5′-AGCCATTCTATAGGA-3 ′ (SEQ ID NO: 45) for nucleic acid detection comprising the base sequence represented by SEQ ID NO: 10
(XIII) 5'-TCAGCAGATAGGTTA-3 '(SEQ ID NO: 46) for nucleic acid detection comprising the base sequence represented by SEQ ID NO: 11

The capture oligonucleotide of the present invention is selected from (1) an oligonucleotide having the following base sequences (2) to (5) in addition to the base sequence represented by any one of SEQ ID NOs: 34 to 46 above. The oligonucleotide which can detect the nucleic acid which consists of any one of the base sequences represented by sequence number 1-11 is included.
(2) Base sequence complementary to the base sequence represented by any one of SEQ ID NOs: 34 to 46 (3) Base sequence represented by any one of SEQ ID NOs: 34 to 46 and stringent conditions (4) a base sequence that can hybridize under stringent conditions with a base sequence complementary to the base sequence represented by any one of SEQ ID NOs: 34 to 46 (5) A base sequence in which one to several bases in the base sequences of 1) to (4) are substituted, deleted, inserted or added

  The capture oligonucleotide of the present invention is preferably used as an oligonucleotide group containing at least two or more of the capture oligonucleotides, and 13 types of nucleic acids each having a base sequence set forth in SEQ ID NOs: 1 to 11 can be detected. It is preferable to use it as a group of capture oligonucleotides including at least a capture oligonucleotide.

Suitable capture oligonucleotides that can detect each of the nucleic acids comprising the nucleotide sequences of SEQ ID NOs: 1 to 11 include the following capture oligonucleotides (I) to (XIII).
(I) a base sequence represented by SEQ ID NO: 34; a base sequence complementary to the base sequence; a base sequence capable of hybridizing with the base sequence under stringent conditions; a base sequence complementary to the base sequence; A nucleotide sequence capable of hybridizing under stringent conditions; one or several of these nucleotide sequences is composed of an oligonucleotide selected from a substituted, deleted, inserted, or added nucleotide sequence; Capture oligonucleotide (II) capable of detecting a nucleic acid consisting of the base sequence represented by No. 1 (base sequence represented by SEQ ID NO: 35; base sequence complementary to the base sequence; stringent conditions with the base sequence) A base sequence that can hybridize under stringent conditions with a base sequence complementary to the base sequence; one of these base sequences A capture oligonucleotide (III) consisting of an oligonucleotide selected from a base sequence with substitution, deletion, insertion or addition of several bases and capable of detecting a nucleic acid consisting of the base sequence represented by SEQ ID NO: 2 ) A base sequence represented by SEQ ID NO: 36; a base sequence complementary to the base sequence; a base sequence capable of hybridizing with the base sequence under stringent conditions; a base sequence complementary to the base sequence and a stringent A nucleotide sequence capable of hybridizing under various conditions; one to several of these nucleotide sequences comprises an oligonucleotide selected from a substituted, deleted, inserted or added nucleotide sequence, SEQ ID NO: 3 A capture oligonucleotide (IV) capable of detecting a nucleic acid comprising the base sequence represented by SEQ ID NO: 37; a base sequence complementary to the base sequence; A base sequence that can hybridize with a base sequence under stringent conditions; a base sequence that can hybridize with a base sequence complementary to the base sequence under stringent conditions; one to several of these base sequences Capture oligonucleotide (V) SEQ ID NO: 38, which comprises a nucleotide whose base is selected from substitution, deletion, insertion, or added base sequence, and which can detect a nucleic acid consisting of the base sequence represented by SEQ ID NO: 4 A base sequence complementary to the base sequence; a base sequence capable of hybridizing under stringent conditions; and a base sequence complementary to the base sequence under stringent conditions Base sequences capable of hybridizing; one to several bases of these base sequences are selected from base sequences with substitutions, deletions, insertions or additions A capture oligonucleotide (VI) that can detect a nucleic acid consisting of a base sequence represented by SEQ ID NO: 5; a base sequence represented by SEQ ID NO: 39; a base sequence complementary to the base sequence; A base sequence capable of hybridizing under stringent conditions with a sequence; a base sequence complementary to the base sequence and capable of hybridizing under stringent conditions; one to several bases of these base sequences Is a capture oligonucleotide (VII) that can detect a nucleic acid consisting of a nucleotide sequence represented by SEQ ID NO: 6 consisting of an oligonucleotide selected from a base sequence that is substituted, deleted, inserted, or added. A nucleotide sequence complementary to the nucleotide sequence; a nucleotide sequence capable of hybridizing with the nucleotide sequence under stringent conditions; A base sequence that can hybridize with a base sequence complementary to the base sequence under stringent conditions; from a base sequence in which one to several bases of these base sequences are substituted, deleted, inserted, or added A capture oligonucleotide (VIII) comprising a selected oligonucleotide and capable of detecting a nucleic acid comprising a base sequence represented by SEQ ID NO: 7; a base sequence represented by SEQ ID NO: 41; a base sequence complementary to the base sequence; A base sequence capable of hybridizing with the base sequence under stringent conditions; a base sequence complementary to the base sequence with a base sequence capable of hybridizing under stringent conditions; one to several of these base sequences Consisting of an oligonucleotide selected from the nucleotide sequences substituted, deleted, inserted or added, and consisting of the nucleotide sequence represented by SEQ ID NO: 8. Capture oligonucleotide (IX) capable of detecting nucleic acid: base sequence represented by SEQ ID NO: 42; base sequence complementary to the base sequence; base sequence capable of hybridizing with the base sequence under stringent conditions; the base A nucleotide sequence that can hybridize under stringent conditions with a complementary nucleotide sequence; selected from nucleotide sequences in which one to several of these nucleotide sequences are substituted, deleted, inserted, or added A capture oligonucleotide (X) that can detect a nucleic acid consisting of the base sequence represented by SEQ ID NO: 9; a base sequence represented by SEQ ID NO: 43; a base sequence complementary to the base sequence; A base sequence capable of hybridizing under stringent conditions with a base sequence; hybridizing under stringent conditions with a base sequence complementary to the base sequence; Possible base sequence; one to several bases of these base sequences are composed of oligonucleotides selected from the base sequences substituted, deleted, inserted, or added, and from the base sequence represented by SEQ ID NO: 9 A capture oligonucleotide (XI) that can detect a nucleic acid, a base sequence represented by SEQ ID NO: 44; a base sequence complementary to the base sequence; a base sequence that can hybridize with the base sequence under stringent conditions; A base sequence that can hybridize with a base sequence complementary to the base sequence under stringent conditions; from a base sequence in which one to several bases of these base sequences are substituted, deleted, inserted, or added; Capture oligonucleotide (XII) consisting of a selected oligonucleotide and capable of detecting a nucleic acid consisting of a base sequence represented by SEQ ID NO: 10 represented by SEQ ID NO: 45 Base sequence; base sequence complementary to the base sequence; base sequence capable of hybridizing with the base sequence under stringent conditions; hybridizable with stringent base sequence complementary to the base sequence under stringent conditions Base sequence; 1 to several bases of these base sequences are composed of oligonucleotides selected from the base sequences substituted, deleted, inserted or added, and the base sequence represented by SEQ ID NO: 10 Capture oligonucleotide (XIII) capable of detecting nucleic acid: base sequence represented by SEQ ID NO: 46; base sequence complementary to the base sequence; base sequence capable of hybridizing with the base sequence under stringent conditions; the base A base sequence that can hybridize with a base sequence complementary to the sequence under stringent conditions; 1 to several of these base sequences are substituted Deletion, insertion, or addition consists oligonucleotide selected from the nucleotide sequence, the capture oligonucleotides capable of detecting a nucleic acid comprising the nucleotide sequence represented by SEQ ID NO: 11

  When a polymorphic site is present in the base sequences represented by SEQ ID NOs: 1 to 11, the capture oligonucleotide preferably comprises a base sequence containing the polymorphic site or a base sequence complementary thereto. In addition, when the base is substituted, deleted and / or added in the base sequences of SEQ ID NOs: 1 to 11, it is a capture oligonucleotide comprising a base sequence containing the polymorphic site or a base sequence complementary thereto. It is preferable.

  Specifically, a polymorphism exists at a position corresponding to position 55 of the base sequence represented by SEQ ID NO: 9. Therefore, the capture oligonucleotide for nucleic acid detection comprising the base sequence represented by SEQ ID NO: 9 preferably comprises a base sequence containing the polymorphic site. Specifically, for example, the capture oligonucleotide (IX) for nucleic acid detection comprising the base sequence represented by SEQ ID NO: 9 is a base comprising a polymorphic site at position 55 of the base sequence represented by SEQ ID NO: 9. It preferably consists of a sequence (SEQ ID NO: 42).

  In addition, in the base sequences represented by SEQ ID NOs: 1 to 11, when there is a site where differences such as deletion and insertion due to varieties occur frequently, from the base sequence containing the site and the base sequence not containing the site It is preferable to use two types of capture oligonucleotides in combination. For example, the nucleotide sequences of positions 38 to 45 of SEQ ID NO: 9 and positions 35 to 44 of SEQ ID NO: 10 are sites that are deleted depending on the rice variety. Therefore, the capture oligonucleotide for nucleic acid detection consisting of the base sequences represented by SEQ ID NOs: 9 and 10 preferably comprises a base sequence including the deletion site and a base sequence not including the deletion site.

  Specifically, for example, the capture oligonucleotide (IX) for nucleic acid detection consisting of the base sequence represented by SEQ ID NO: 9 is a base partially including the deletion site in the base sequence represented by SEQ ID NO: 9 It consists of a sequence (SEQ ID NO: 42), and the capture oligonucleotide (X) preferably consists of a base sequence (SEQ ID NO: 43) that does not contain the deletion site. Further, the capture oligonucleotide (XI) for nucleic acid detection comprising the base sequence represented by SEQ ID NO: 10 is a base sequence of the region containing the deletion site in the base sequence represented by SEQ ID NO: 10, It consists of a sequence (SEQ ID NO: 44) that does not include a deletion site, and the capture oligonucleotide (XII) is a base sequence of the region that includes the deletion site, and from a sequence (SEQ ID NO: 45) that includes the deletion site. It is preferable to become.

  When detecting the hybridization between the nucleic acid amplification product and the capture oligonucleotide of the present invention and determining the presence or absence of mixed rice in Koshihikari, the capture oligo capable of detecting the base sequence represented by SEQ ID NOs: 2, 6, 9, 10 It is preferable to use at least nucleotides [for example, the capture oligonucleotides (II), (VI), (IX), (X), (XI), (XII) above). In addition, when a nucleic acid amplification product is detected by a DNA array and Koshihikari BL and Koshihikari other than Koshihikari BL are discriminated, a capture oligonucleotide that can detect the nucleotide sequence represented by SEQ ID NO: 2, 7, 9, 10 [for example, the above capture Oligonucleotide (II), (VII), (IX), (X), (XI), (XII)] is preferably used. Further, when determining the presence or absence of mixed rice in Koshihikari and / or determining Koshihikari BL and Koshihikari other than Koshihikari BL, the hybridization between the nucleic acid amplification product and the capture oligonucleotide of the present invention is detected and further discriminated. In this case, it is preferable to use a capture oligonucleotide group including all 13 types of capture oligonucleotides of the capture oligonucleotides (I) to (XIII) of the present invention described above.

  When detecting hybridization between the nucleic acid amplification product and the capture oligonucleotide of the present invention and discriminating rice varieties, the capture oligonucleotide to be fixed to the support may be singular or plural, but the variety is more accurately identified. Therefore, it is preferable to use a capture oligonucleotide group including a plurality of capture oligonucleotides, and a capture oligonucleotide group including all 13 types of capture oligonucleotides of the capture oligonucleotides (I) to (XIII) of the present invention described above. It is more preferable to use Here, when selecting a plurality of capture oligonucleotides, the rice varieties that can be discriminated from the rice varieties to be discriminated, the rice varieties to be discriminated, the rice varieties to be discriminated, and the rice to be discriminated may be applicable. Design appropriately by considering information on rice varieties. Further, in order to reliably discriminate extremely closely related varieties, it is possible to comprehensively detect a plurality of polymorphisms, preferably 5 or more, more preferably 10 or more, and even more preferably 15 or more on the same support. From the viewpoints of simplicity of operation and accuracy and speed of identification.

(Support)
The support on which the capture oligonucleotide is immobilized is capable of stably immobilizing the capture oligonucleotide, and can withstand the reaction steps necessary for hybridization with the nucleic acid derived from the test rice sample and detection of the hybrid. If it exists, the material in particular is not restrict | limited, For example, a glass slide, an organic polymer, an inorganic polymer, a metal, a natural polymer, a ceramic etc. can be used.

  Examples of the organic polymer include polyethylene, polystyrene, polycarbonate, polypropylene, polyamide, phenol resin, epoxy resin, polycarbodiimide resin, polyvinyl chloride, polyvinylidene fluoride, polyfluorinated ethylene, polyimide, and acrylic resin. Examples of the inorganic polymer include glass, crystal, carbon, silica gel, and graphite. Examples of the metal include gold, platinum, silver, copper, iron, aluminum, a magnet, and a paramagnet. Examples of natural polymers include polyamino acids, cellulose, chitin, chitosan, alginic acid, and derivatives thereof. Examples of the ceramic include apatite, alumina, silica, silicon carbide, silicon nitride, and boron carbide.

  The form of the support is not particularly limited, and for example, a support having a plate shape, a film shape, a film shape, a particle shape or the like can be suitably used, but a plate shape is preferable.

  In the present invention, a support may be supported on a support, and the capture oligonucleotide may be immobilized on the support via the support. As the carrier, the carrier itself may have a binding property to the capture oligonucleotide, or may be one that can immobilize the capture oligonucleotide through a linker having a binding property to the capture oligonucleotide. Here, “support” refers to a water-soluble solvent or organic solvent used for the purpose of washing or the like when immobilizing the capture oligonucleotide on a support on a support and detecting a hybridization signal using the support. It means that the carrier is not substantially detached from the support by various solvents such as.

  As long as the carrier is supported on the support, it may be supported simply using physical adhesiveness, or may be chemically supported via a covalent bond or the like. Moreover, the said support | carrier may be carry | supported on the whole surface on a support body as needed, and may be carry | supported in the part.

  Examples of the carrier include small organic molecules, organic polymers, inorganic polymers, metals, natural polymers, and ceramics. Here, as an organic low molecule, a carbodiimide group containing compound, an isocyanate group containing compound, a nitrogen iperit group containing compound, an aldehyde group containing compound, an amino group containing compound etc. are mentioned, for example. Further, organic polymers, inorganic polymers, metals, and ceramics can be the same as those described above. A particularly preferred carrier in the present invention is a carbodiimide group-containing compound or an isocyanate group-containing compound.

(Immobilization of capture oligonucleotide to support)
The means for fixing the capture oligonucleotide to the support is not particularly limited as long as it can discriminate rice varieties or determine the presence or absence of mixed rice, and a known method may be appropriately selected and used. For example, it may be fixed simply using physical adhesiveness, or may be chemically fixed via a covalent bond or the like. Moreover, it can also fix by irradiating electromagnetic waves, such as an ultraviolet-ray. Further, in a state where a known compound such as a capture oligonucleotide and a carbodiimide resin, nitrogen ipellite, polyamino acid, nitrocellulose or the like is chemically bonded or physically bonded, the mixture and the carrier may be contacted and fixed. At this time, the fixing can be performed by appropriately irradiating an electromagnetic wave such as ultraviolet rays. Here, the place where the capture oligonucleotide is immobilized on the support may be the entire surface of the support or a part thereof.

  In addition, when the capture oligonucleotide is immobilized on the support, the shape of the place occupied by the immobilized oligonucleotide does not affect the hybridization with the nucleic acid derived from the test rice sample, and detection is not possible. The shape is not particularly limited as long as it is not difficult, but examples thereof include a circle and a rectangle.

  Examples of specific means used for immobilization include a method using a dispensing device, a method using a pin type spot device, a method using an ink jet type spot device, and the like. As a result, the capture oligonucleotide solution is usually almost circular. It can be fixed to a part of the support in the shape of

  The size of the site where each capture oligonucleotide is immobilized on the support is preferably immobilized at a diameter of 10 μm to 1,000 μm. If it is smaller than this, detection becomes difficult, and if it is larger than this, the area occupied by the entire arranged capture oligonucleotide becomes larger, which may cause a problem that operability is deteriorated.

  The arrangement of each capture oligonucleotide on the support is not particularly limited as long as the rice varieties can be discriminated or the presence of mixed rice can be determined. It is preferable to arrange them together or in a line.

When the carrier is supported on the support and the capture oligonucleotide is immobilized on the support via the support, the capture oligonucleotide is immobilized on the support in addition to the method of directly supporting the capture oligonucleotide. It can be carried out by a method of supporting on a carrier through a linker having a property. Here, examples of the linker include a carbodiimide group-containing compound, an isocyanate group-containing compound, a nitrogen iperit group-containing compound, an aldehyde group-containing compound, and an amino group-containing compound. More specifically, a linker having a nucleic acid homopolymer ( DNattach ^™ , manufactured by Nisshinbo Industries, Inc.).

(Hybrid formation)
Hybridization of the nucleic acid amplification product and the capture oligonucleotide can be carried out on, for example, a nucleic acid detection membrane to which the capture oligonucleotide is immobilized, but it is preferable to use a support on which the capture oligonucleotide is immobilized. By using the support, it is possible to quickly and easily and accurately identify rice varieties that are closely related to each other with excellent visibility. The method used for hybridization is not particularly limited, and a known nucleic acid hybrid formation method can be appropriately employed and used. One embodiment of a specific hybridization method is shown below.

  In a mixed solution containing a buffer salt solution such as Standard Saline Citrate (SSC), an inhibitor of nonspecific adsorption such as sodium dodecyl sulfate (SDS), bovine serum albumin (BSA), and an additive for promoting hybridization reaction Add a pre-labeled nucleic acid amplification product. When the nucleic acid amplification product forms a double strand, it is denatured by heating or the like. After adding a suitable amount, preferably 20 to 100 μl, of a mixed solution containing the above-described labeled nucleic acid amplification product on a support on which a capture oligonucleotide is fixed, heat at a suitable temperature, preferably 30 ° C. to 80 ° C., is applied. In addition to time, a hybrid specific to the base sequence of the capture oligonucleotide immobilized on the support and the nucleic acid amplification product is formed. Next, the above mixed solution is removed, and a solution such as 5 × SSC solution or 3M tetramethyl chloride solution is added onto the support and heated at an appropriate temperature, preferably 30 ° C. to 80 ° C., to form a non-specific hybrid. The nucleic acid amplification product is peeled off from the support, and only specific hybrids are selectively left on the support.

(Detection of hybridization)
Hybridization can be detected by a hybridization signal. Detection of a hybridization signal is usually performed continuously with the above hybridization. The method used for detecting the hybrid depends on the substance labeled with the nucleic acid amplification product. That is, for detecting the hybrid, a labeling substance such as a fluorescent substance or a hapten used for labeling the target nucleic acid is used. Therefore, a known method capable of detecting the labeling substance may be appropriately selected and used. For example, when a fluorescent substance is used, the fluorescence labeled on the hybridized nucleic acid amplification product is detected using a dedicated fluorescence scanner. On the other hand, when a hapten is used, a solution containing a protein that recognizes the hapten or another protein that binds to the hapten and a conjugate (referred to as an enzyme conjugate) such as alkaline phosphatase or horseradish peroxidase is added to the support. For example, the reaction is performed at room temperature for several tens of minutes.

  Hybrid visualization can be accomplished by adding a dye compound that forms an insoluble compound only when a conjugate of the hapten and the enzyme conjugate is present. The production of the insoluble compound is amplified by an enzymatic reaction, and the immobilization site of the capture oligonucleotide in which a hybrid is formed is visualized by a dye. Examples of the compound to be added include nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate-p-toluidine salt when the enzyme part of the enzyme conjugate is composed of alkaline phosphatase. In the case of being composed of horseradish peroxidase, 3,3 ′, 5,5′-tetramethylbenzidine and the like can be mentioned.

  Determine the presence or absence of amplification products obtained by nucleic acid amplification by determining the presence or absence of hybrid pigmentation or fluorescent coloration at the position where the capture oligonucleotide is immobilized, and distinguishing rice varieties contained in the test sample Alternatively, the presence or absence of mixed rice can be determined. That is, when a hybrid is detected, it means that the test sample contains a variety corresponding to the capture oligonucleotide immobilized at the position where the pigmentation or the like is present.

  The rice variety discrimination kit of the present invention or the mixed rice presence / absence determination kit to Koshihikari is a kit for carrying out the above-mentioned rice variety determination method or the mixed rice presence / absence determination method to Koshihikari, the primer set, the above It includes a group of capture oligonucleotides, reagents and instruments used for the detection of the above hybridization and hybrid.

  Examples of reagents used in hybridization include buffer salt solutions such as Standard Saline Citrate (SSC), inhibitors of nonspecific adsorption such as sodium dodecyl sulfate (SDS), bovine serum albumin (BSA), and hybridization reaction promotion. And the like.

  Examples of the reagent used for detecting the hybrid include a conjugate of a protein that recognizes a hapten and an enzyme (enzyme conjugate), nitro blue tetrazolium chloride, 5-bromo-4-chloro-3-indolyl phosphate-p-. Examples include reagents such as toluidine salts and 3,3 ′, 5,5′-tetramethylbenzidine.

  The reagents used for the exemplified hybridization and hybrid detection are not limited to these, and an appropriate reagent suitable for the purpose may be appropriately selected to form a kit.

  Furthermore, the kit preferably includes a reagent used for preparing the nucleic acid sample and / or a reagent used for preparing the nucleic acid amplification product.

  Examples of reagents used for the preparation of nucleic acid amplification products include PCR buffers, heat-resistant DNA polymerases, deoxynucleotide triphosphate mixed solutions, and the like, and reagents used in the nucleic acid preparation step include those for DNA extraction. Although a buffer solution etc. can be mentioned, it is not limited to these, What is necessary is just to select the suitable reagent suitable for the objective suitably and to make it the structure of a kit. A primer for producing the target nucleic acid can also be included in the kit.

  The method for determining rice varieties and the method for determining the presence or absence of mixed rice in Koshihikari, the rice variety determining kit and the kit for determining the presence or absence of mixed rice in Koshihikari include the determination of rice varieties and the presence or absence of mixed rice in Koshihikari. It can be used for all applications that require judgment. Specifically, for example, when discriminating rice seeds at the time of sowing, discriminating seedlings, discriminating rice at the time of harvest, discriminating varieties at the rice distribution stage or determining the presence or absence of mixed rice, It is preferably used when managing rice seed varieties, or when it is necessary to discriminate rice varieties after cooking or to determine the presence or absence of mixed rice.

  Hereinafter, the present invention will be described more specifically with reference to examples.

[Example 1]
(1) Primer design In carrying out the present invention, a forward primer and a reverse primer capable of amplifying a nucleic acid having a reference sequence including polymorphisms of SEQ ID NOs: 1 to 11 were designed. Table 1 shows the relationship between the reference sequence number including the polymorphism used in this example, the primer and the primer pair.

(2) Primer synthesis Using the oligonucleotide synthesizer (manufactured by Perkin-elmer Applied biosystems) according to a standard method, the primers shown in Table 1 (SEQ ID NOs: 12 to 33) were used as unmodified primers or at the 5 ′ end. A biotin-modified primer was synthesized, deprotected, and dried. Then, it melt | dissolved using Distilled Water (made by Invitrogen), and prepared the primer solution of 2 pmol / microliter, respectively.

(3) Preparation of primer set In order to carry out multiplex PCR, the primer solutions prepared in (2) above were mixed to prepare a primer set containing 11 types of primer pairs (A) to (K). Table 2 shows the formulation of the prepared primer set. A biotin-modified primer and an unmodified primer were mixed in the same manner to prepare a primer set.

(4) Extraction of template DNA DNA extraction from a test rice sample was performed using Wizard® Magnetic DNA Purification System for Food (manufactured by Promega) and used as template DNA for multiplex PCR. The sample rice samples are Koshihikari, Hitomebore, Hinohikari, Akitakomachi, Kirara 397, Kinuhikari, Haenuki, Hoshino Yume, Tsugaru Roman, Sasanishiki, Yume Akari, Nipponbare, Hanaechizen, Yumetsushi, Aichi Pure rice sample (Table 3, Sample Nos. 1-20) and 19 Koshihikari rice, each sample of Nokaori, Mutsu Homare, Asahi no Yume, Hashimomo, Nanatsuboshi, Brown Rice of Fusotome A total of 19 rice varieties of rice varieties from the above-mentioned squeeze to fusotome (Table 3, specimen numbers 2 to 20) were mixed to prepare 20 rice grains (Table 3, specimen number Kb2 to Table 2). Kb20).

(5) Multiplex PCR
A nucleic acid amplification product was prepared by multiplex PCR using the rice-derived genomic DNA extracted in (4) above as a template. Specific PCR reaction composition and reaction conditions are shown in Table 4.

(6) Electrophoresis The amplification product solution obtained by multiplex PCR is 2 μl in the case of pure rice, and 5 μl in the case of mixed rice, after separating each DNA by electrophoresis on 8% polyacrylamide gel, and then SYBR Green1 nucleic acid gel. After staining with stain (manufactured by Invitrogen), the electrophoresis pattern of the identification band was observed by ultraviolet irradiation. As a DNA molecular weight marker, 20 bp DNA Ladder (TaKaRa) was used.

(7) Results FIG. 1 shows genomic DNA extracted from 20 single-species test rice samples using a primer set containing 11 types of primer pairs (A) to (K) for pure rice detection. The electrophoresis pattern of a PCR product using as a template is shown. The lane numbers in FIG. 1 correspond to the sample numbers described in Table 3.

  In addition, since the band confirmed in the vicinity of 400 bp is a PCR product amplified by a forward primer and a reverse primer of different markers, it is not a PCR product for amplification purposes in the present invention. Therefore, a band of 301 bp or less was used as an identification band (hereinafter, the same was applied to Examples using a primer set including primer pairs (A) to (K)).

  In FIG. 2, in order to determine the presence or absence of mixed rice, a primer set including primer pairs (A) to (K) was used, and the varieties of sample number 2 to sample number 20 shown in Table 3 for 19 Koshihikari grains An electrophoresis pattern of a PCR product using genomic DNA extracted from a test sample mixed with one grain as a template is shown. In FIG. 2, lane 1 shows the result of Koshihikari pure rice, and lanes 2 to 20 show the electrophoresis pattern of 5% mixed rice corresponding to the specimen numbers (Kb2 to Kb20) shown in Table 3.

  Table 5 shows an identification band judgment table by electrophoresis of each pure rice variety in FIG. + And-in Table 5 indicate the presence or absence of an identification band, respectively. In primer pair number I and primer pair number J, there is a difference in the chain length of the PCR product amplified by the rice varieties used as a template during multiplex PCR. Differences in these chain lengths are indicated by a or b in Table 5. Taking Koshihikari as an example, it was found that a (100 bp) was identified in primer pair number I, a (87 bp) was identified in primer pair number J, and there was no other identification band.

  In Table 5, the identification bands a and b obtained by the primer pair number I and the identification bands a and b obtained by the primer pair number J are very close to each other in the molecular weight of the obtained PCR product. It was observed on the photograph in an overlapping manner. Therefore, the pure rice variety discrimination and the presence / absence of 5% mixed rice in FIGS. 1 and 2 were performed using Table 5.

(8) Consideration In the case of Koshihikari pure rice, two identification bands were obtained (lane 1 in FIGS. 1 and 2), but in the case of other pure rice (FIG. 1), and with mixed rice In the case (FIG. 2), three or more identification bands were detected. From this, it is possible to perform multiplex PCR using a primer set including 11 types of primer pairs (A) to (K), and examine the number of discriminating bands detected by electrophoresis of PCR products. It was found that it was possible to determine whether or not. When the test sample is pure rice, at least 15 rice varieties among the varieties of specimen numbers 1 to 20 shown in Table 3 due to the difference in the number of identification bands and the chain length of the identification bands. I knew that I could distinguish.

[Example 2]
(1) Design of Capture Oligonucleotide A capture oligonucleotide was designed to discriminate rice varieties and determine the presence of mixed rice in Koshihikari using a DNA array. Table 6 shows the relationship between the designed capture oligonucleotides (I) to (XIII), SEQ ID NOs (sequences 34 to 46), primers, and primer pairs (A) to (K). Capture oligonucleotides (IX) to (XII) comprising the nucleotide sequences represented by SEQ ID NOs: 42 to 45 contain characteristic nucleotide sequences that are specific to rice varieties or that frequently differ between rice varieties. Capture oligonucleotides (I) to (VIII) and (XIII), which are capture oligonucleotides and have the nucleotide sequences represented by SEQ ID NOs: 34 to 41 and SEQ ID NO: 46, are specific to rice varieties. Capture oligonucleotide that detects the presence or absence.

(2) Synthesis of Capture Oligonucleotide When the capture oligonucleotide is immobilized on a slide glass “Carbo Station (Trademark Registration No. 4574974, manufactured by Nisshinbo Co., Ltd.)” coated with a polycarbodiimide resin, the capture oligonucleotide 5 ′ When an amino linker is added to the end and the capture oligonucleotide is immobilized on a polycarbonate resin slide (manufactured by Teijin Ltd.), the linker having a nucleic acid homopolymer at the 5 ′ end of the capture oligonucleotide ( DNattach ^™ (manufactured by Nisshinbo Industries, Inc.) was added and synthesized according to a conventional method in the same manner as in Example 1.

(3) Immobilization of capture oligonucleotide on support: preparation of DNA array 10 μl of microspotting solution (manufactured by TeleChem International Inc.) was mixed with 10 μl of capture oligonucleotide solution and microtiter plate (manufactured by Greiner Laboratory Inc.). ) Dispensed above. Surface-untreated polycarbonate resin slide (made by Teijin Ltd.) or slide glass whose surface is treated with polycarbodiimide resin “Carbo Station (Trademark Registration No. 4574974, Nisshinbo Co., Ltd.)” The spotting machine was operated, and the capture oligonucleotide solution was spotted on the slide glass so that the spots shown in Table 7 were aligned.

  After the spotting, the sample irradiated with 600 mJ of ultraviolet light was soaked in 100 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1% Triton X-100 containing 3% BSA (bovine serum albumin) for 30 minutes for blocking. Then, it was washed with 10 mM Tris-HCl (pH 7.5) and 1 mM EDTA buffer. Finally, it was rinsed with sterilized water, the slide glass was dried, and stored in a cool and dark place in a dry state until use.

  Table 7 shows the arrangement of capture oligonucleotides immobilized on each spot on the support. The numbers in Table 7 indicate the sequence numbers of the capture oligonucleotides shown in Table 6, and biotin indicates a marker for knowing the position, and a biotinylated oligomer spotted to confirm that there is no problem in the color development operation.

(4) Multiplex PCR and hybridization step As in Example 1, extraction of genomic DNA from the test rice sample and multiplex PCR were performed, and the obtained PCR product was used as a target nucleic acid. However, the primer set used was prepared using a 5 ′ biotinylated primer. 14 μl of the target nucleic acid solution obtained by multiplex PCR was placed in a 0.5 ml tube, heat-treated at 95 ° C. for 3 minutes, and then immersed in ice for 3 minutes. This was returned to room temperature, and 36 μl of ArrayIt Unihyb Hybridization Solution (manufactured by TeleChem International Inc.) was added and mixed to obtain a probe nucleic acid solution. The entire amount of the probe nucleic acid solution was taken and placed on the DNA array prepared in (3) above, and a cover film was placed thereon. This was put in a moisturizing box, and further placed in a thermostat set at 50 ° C. and left still for 60 minutes. The array is removed and quickly soaked in 2 × SSC solution (2 × SSC: 0.033M NaCl, 0.033M sodium citrate) at room temperature to remove the cover glass and the array is placed in 2 × SSC solution at room temperature for 5 minutes. Soaked.

(5) Detection of hybridization signal Lightly remove the 2 × SSC solution on the DNA array, and then prepare an avidin-biotinylated peroxidase complex prepared on the array using VECTASTAIN Elite ABC kit (manufactured by VECTOR). 0.0 ml was dropped and allowed to stand at room temperature for 30 minutes. Thereafter, the avidin-biotinylated peroxidase complex solution is gently shaken off, and washed in TBST solution with [50 mM Tris-HCl (pH 7.6), 0.15 M NaCl, 0.05% Tween 20] for 5 minutes at room temperature. Repeated twice. 1.0 ml of a coloring solution prepared using TMB substrate kit for peroxide (manufactured by VECTOR) was dropped on the array and allowed to stand at room temperature for 30 minutes to develop color by an enzymatic reaction. The array was washed with distilled water to stop the enzyme reaction, and it was confirmed that the blue color was developed.

(6) Determination The color spot was visually confirmed to determine the result. The results were stored by scanning the DNA array at an image density of 600 dpi using an OA scanner (GT-8700F, manufactured by EPSON).

(7) Results Table 8 shows the presence or absence of a hybridization signal to the capture oligonucleotide for 20 Koshihikari grains (sample number 1) and the mixed rice sample (sample numbers Kb2 to Kb20) shown in Table 3 of Example 1. A color image of the array is shown in FIG. In Table 8, “+” indicates that a hybrid was formed and a hybridization signal was detected by a color development reaction, and “−” indicates that a hybrid was not formed and a hybridization signal was not detected.

(8) Discussion As can be seen from FIG. 3 and Table 8, by multiplex PCR using a primer set including primer pairs (A) to (K) and a DNA array using capture oligonucleotides (I) to (XIII), It turned out that the mixed rice to Koshihikari can be confirmed. In addition, since no color was observed in the negative control, it was found that there was no non-specific amplification.

[Example 3]
Using the DNA array prepared in Example 2, the top 20 cultivars shown in Table 3 of Example 1 and other varieties shown in Table 9 were extracted from 20 grains (pure rice), respectively. Variety discrimination was performed.

(1) Method DNA extraction from the test rice sample was performed in the same manner as in Example 1 and used as template DNA for multiplex PCR. Multiplex PCR, hybridization step using DNA array, detection of hybridization signal and determination thereof were performed in the same manner as in Example 2.

(2) Results FIGS. 4 to 8 show the results of detection using a DNA array when 20 rice varieties shown in Table 3 of Example 1 and Table 9 are used as test samples. Tables 10 to 12 show the presence or absence of hybridization to capture oligonucleotides for 119 varieties. In Tables 10 to 12, “+” indicates that a hybrid was formed and a hybridization signal was detected, and “−” indicates that a hybrid was not formed and a hybridization signal was not detected.

(3) Discussion As can be seen from Tables 10 to 12 and FIGS. As a result of detection by the DNA array used, different signal patterns were obtained for all 119 varieties shown in Tables 3 and 9. That is, according to electrophoresis of PCR products obtained using a primer set including primer pairs (A) to (K) and a DNA array using capture oligonucleotides (I) to (XIII), Table 3 and Table It was found that all 119 varieties shown in 9 could be distinguished by one PCR.

[Example 4]
When using commercially available rice varieties PCR kits “Tape Ra for Rice Discrimination PCR Kit I” and “Rice Discrimination PCR Kit II (Koshihikari nega kit)” (comparison) Example) was compared with the case where a primer set containing primer pairs (A) to (K) was used (Example).

(1) Multiplex PCR
Using the rice genomic DNA extracted in Example 1 as a template, multiplex PCR was performed using “Kit discrimination PCR Kit I” and “Rice discrimination PCR Kit II (Koshihikari negative kit)” manufactured by TaKaRa. . Since the PCR kit for distinguishing rice varieties manufactured by TaKaRa uses Kit I and Kit II as a set, PCR is required twice for one test. The specific PCR reaction composition and reaction conditions were as shown in the instructions. Details of the test rice sample and the genome concentration are shown in Table 13 and Table 15. The amount of DNA was measured by UV.

  On the other hand, in the primer set including the primer pairs (A) to (K), multiplex PCR was performed under the same conditions as in Example 1, using the rice genomic DNA extracted in Example 1 as a template. However, the primer set used was prepared using a 5 'biotinylated and / or unmodified primer. Details of the test rice sample and the genome concentration are shown in Table 14 and Table 16. The amount of DNA was measured by UV.

(2) Electrophoresis 5 μl of PCR product solution obtained using TaKaRa's “Kit discrimination PCR Kit I” and “Rice discrimination PCR Kit II (Koshihikarinega kit)” was electrophoresed on a 2% agarose gel. After separation of the amplification products by staining with ethidium bromide (hereinafter referred to as ethidium bromide or EtBr) and SYBR Green1 nucleic acid gel stain (manufactured by Invitrogen, hereinafter referred to as SYBR Green I), and then electrophoresis of the PCR products by ultraviolet irradiation The pattern was observed.

  On the other hand, in the primer set including the primer pairs (A) to (K), 5 μl of the PCR product solution obtained using the primer set is separated by electrophoresis on 8% polyacrylamide gel, and then ethidium bromide. , And stained with SYBR Green I, and then the electrophoresis pattern of the PCR product was observed by ultraviolet irradiation.

(3) Results of Electrophoresis FIG. 9 shows the results obtained by performing multiplex PCR using genomic DNA extracted from a single variety of rice Koshihikari using “Kit discrimination PCR Kit I” manufactured by TaKaRa. The result of electrophoresis of a PCR product is shown. The left figure of FIG. 9 shows the result of staining with ethidium bromide, and the right figure of FIG. 9 shows the result of staining with SYBR GreenI. Sample numbers shown in Table 13 are shown for lanes 1 to 10 in FIG. Table 13 shows the description of the test rice sample corresponding to each lane number in FIG. 9 and whether or not discrimination in electrophoresis is possible. In addition, Koshihikari 1 and Koshihikari 2 show that the sources of the samples are different.

  FIG. 10 shows the results of electrophoresis of a PCR product obtained by performing multiplex PCR using a genomic DNA extracted from a single variety of rice Koshihikari using a primer set including primer pairs (A) to (K). Results are shown. The left figure of FIG. 10 shows the result of staining with ethidium bromide, and the right figure shows the result of staining with SYBR GreenI. The sample numbers shown in Table 14 are shown for lanes 1 to 13 in FIG. Table 14 shows the description of the test rice sample corresponding to each lane number, whether or not discrimination is possible in electrophoresis, and whether or not discrimination is possible in hybridization. In addition, Koshihikari 1 and Koshihikari 2 show that the sources of the samples are different.

  Fig. 11 shows a sample of a rice cracker mixed with 19 rice grains of Koshihikari to determine the presence or absence of mixed rice using TaKaRa's "Kit discrimination PCR Kit II (Koshihikari negative kit)". The result of electrophoresis of the PCR product obtained by performing multiplex PCR using the extracted genomic DNA is shown. The left figure of FIG. 11 shows staining with ethidium bromide, and the right figure of FIG. 11 shows the results of staining with SYBR GreenI. The sample numbers shown in Table 15 are shown for lanes 1 to 12 in FIG. Table 15 shows the description of the test rice sample corresponding to each lane number, and whether or not discrimination in electrophoresis is possible. In addition, Koshihikari 1 and Koshihikari 2, and Aichi no Kaori 1 and Aichi no Kaori 2 indicate that the sources of the samples are different.

  In FIG. 12, a primer set including primer pairs (A) to (K) was used and extracted from a sample in which one rice cake was mixed with 19 rice grains of Koshihikari to determine the presence or absence of mixed rice. The result of electrophoresis of the PCR product obtained by performing multiplex PCR using genomic DNA is shown. The left figure of FIG. 12 shows staining with ethidium bromide, and the right figure of FIG. 12 shows the results of staining with SYBR GreenI. For lanes 1 to 13 in FIG. Table 16 shows the description of the test rice sample corresponding to each lane number, whether or not discrimination is possible in electrophoresis, and whether or not discrimination is possible in hybridization. In addition, Koshihikari 1 and Koshihikari 2 show that the sources of the samples are different.

(4) Detection of hybridization signal by DNA array using capture oligonucleotides (I) to (XIII) For the PCR product, the DNA array prepared in Example 2 was used, and the formation of a hybridization signal by the DNA array was confirmed in the same manner as in Example 3. Details of the test rice sample and the genome concentration are shown in Table 14 and Table 16.

(5) Hybridization Results FIG. 13 shows the detection results corresponding to Table 14 and FIG. 14 shows the results corresponding to Table 16 for the detection results of (4) above.

(6) Discussion ・ Sensitivity test in Koshihikari pure rice As can be seen from FIG. 9 and Table 13, in “Kithikari Genome PCR Kit I” manufactured by TaKaRa, 0.5 ng of Koshihikari genome in 20 μl of the PCR reaction solution was added, When the PCR product was detected by electrophoresis, it was found that the identification band that should be three may not be confirmed (FIG. 9, lane 7).

  On the other hand, when a primer set containing primer pairs (A) to (K) was used, as can be seen from FIG. 10 and Table 14, the amount of genomic DNA in 20 μl of the PCR reaction solution was detected by detecting the PCR product. It was found that an identification band could be confirmed even when the value was 0.5 ng or 0.1 ng (FIG. 10, lanes 6, 7, 11, 12).

  Further, as can be seen from FIG. 13 and Table 14, by detecting the PCR product with a DNA array using the capture oligonucleotides (I) to (XIII), the amount of Koshihikari genome in 20 μl of the PCR reaction solution was 0.1 ng. But it turned out that the identification band can be confirmed.

Sensitivity test with 5% mixed rice As can be seen from FIG. 11 and Table 15, the presence or absence of mixed rice was detected by electrophoresis of PCR products using TaKaRa's "Kithikari Nega Kit)" 5% of mixed rice could be confirmed up to 5 ng of genome in 20 μl of PCR reaction solution (1 ng for SYBR GreenI), but amplification product was confirmed when the amount of genome in 20 μl of PCR reaction solution was 0.5 ng could not.

  On the other hand, as can be seen from FIG. 12 and Table 16, if mixed rice is detected by electrophoresis of PCR products using a primer set including primer pairs (A) to (K), the genome amount in 20 μl of the PCR reaction solution is 0. It was found that the presence or absence of mixed rice could be determined even at 0.5 ng and 0.1 ng. Moreover, in SYBR GreenI, even a smaller amount of genome (0.025 ng) was confirmed to be mixed rice (FIG. 12, lane 7).

  In addition, by detecting the hybridization signal of the DNA array using the PCR product obtained using the primer set including the primer pairs (A) to (K) and the capture oligonucleotides (I) to (XIII), As shown in FIG. 16, it was found that even when the amount of genome in 20 μl of the PCR reaction solution was 0.025 ng, the mixed rice could be confirmed. That is, a sample with a small amount of genome is obtained by electrophoresis of a PCR product obtained using a primer set including primer pairs (A) to (K) and a DNA array using capture oligonucleotides (I) to (XIII). Even so, it was found that the presence or absence of mixed rice could be determined.

[Example 5]
In order to discriminate between Koshihikari from Chiba and Koshihikari from Niigata (BL), the DNA array prepared in Example 2 was used, and the genome extracted from each 20 grains was used for discrimination by electrophoresis and DNA array It was.

(1) Method In the same manner as in Example 1, genomic DNA was extracted from a test rice sample and used as template DNA for multiplex PCR. Further, multiplex PCR was performed in the same manner as in Example 2 to obtain a PCR product.
The discrimination by electrophoresis was performed in the same manner as in Example 1, and the discrimination by the preparation of the DNA array, the hybridization step by the DNA array, and the detection of the hybridization signal was performed in the same manner as in Example 2.

(2) Results FIG. 15 shows the result of multiplex PCR using genomic DNA derived from Koshihikari from Chiba Prefecture and Koshihikari from Niigata Prefecture (BL) as a template, and the resulting PCR product detected by electrophoresis (left figure in FIG. 15). , And the result (right figure of FIG. 15) detected by the DNA array. As a result of electrophoresis, two identification bands were detected in Koshihikari produced in Chiba Prefecture, and three identification bands were detected in Koshihikari produced in Niigata (FIG. 15 left figure). In addition, two spots of hybridization signals by DNA array were detected in Koshihikari from Chiba Prefecture, and three spots were detected in Koshihikari (BL) from Niigata Prefecture (the right figure in FIG. 15).

(3) Discussion Three discriminating bands (using a band of 301 bp or less as discriminating bands) detected by electrophoresis shown in the left diagram of FIG. 15 suggested Koshihikari BL. Therefore, it is possible to discriminate between Koshihikari (BL) from Niigata Prefecture and Koshihikari other than Koshihikari (BL) from Niigata Prefecture by electrophoresis of PCR products obtained using a primer set containing primer pairs (A) to (K). I understood.

  In addition, as shown in the right figure of FIG. 15, two spots of hybridization signals by DNA array were detected in Koshihikari from Chiba Prefecture and three spots in Koshihikari (BL) from Niigata Prefecture, and the pattern of hybridization signal was clearly different. It was. Furthermore, the pattern of hybridization signals by Koshihikari (BL) produced in Niigata Prefecture (right figure in FIG. 15) was different from any pattern of the 119 varieties shown in Example 3 (FIGS. 4 to 8). From these results, it was found that the DNA array using the capture oligonucleotides (I) to (XIII) can distinguish Koshihikari (BL) from Niigata Prefecture and Koshihikari other than Koshihikari (BL) from Niigata Prefecture.

[Example 6]
Regarding the percentage of mixed rice that is the detection limit, when using commercially available PCR kits for rice discrimination, “Kit discrimination PCR Kit I” and “Kit discrimination PCR Kit II (Koshihikari nega kit)” manufactured by TaKaRa (Comparative Example) was compared with the case (Example) in which a primer set including primer pairs (A) to (K) was used.

(1) Method Details of the test rice sample and the genome concentration were performed as shown in Table 17, and multiplex PCR, electrophoresis, and hybridization were performed in the same manner as in Example 4. However, lane 18 and lane 19 in Table 17 are used only in FIGS. 18 and 19.

(2) Results FIG. 16 shows the results obtained by performing multiplex PCR using TaKaRa's “Kit discrimination PCR Kit I” and using the genomic DNA extracted from the test rice sample shown in Table 17 as the template DNA. The PCR product was electrophoresed on a 2% agarose gel and the result of staining with SYBR Green I is shown. The left figure of FIG. 16 shows the result when 0.5 ng of genomic DNA amount is introduced into 20 μl of the PCR reaction solution, and the right figure of FIG. 16 shows the result when 10 ng genomic DNA amount is introduced into 20 μl of the PCR reaction solution. In addition, the sample numbers shown in Table 17 are shown for lanes 1 to 17 in FIG.

  FIG. 17 shows the results obtained by performing multiplex PCR using “Kit discrimination PCR Kit II (Koshihikari nega kit)” manufactured by TaKaRa and using genomic DNA extracted from the test rice sample shown in Table 17 as template DNA. The results obtained by subjecting the PCR product to 2% agarose gel electrophoresis and staining with SYBR Green I are shown. The left figure of FIG. 17 shows the result when 0.5 ng of genomic DNA is introduced into 20 μl of the PCR reaction solution, and the right figure of FIG. 17 shows the result when 10 ng of genomic DNA is introduced into 20 μl of the PCR reaction solution. For lanes 1 to 17 in FIG.

  16 and 17, Table 18 shows whether mixed rice can be determined when the genomic DNA amount is 0.5 ng or 10 ng.

  FIG. 18 shows a PCR product obtained by performing multiplex PCR using a primer set including primer pairs (A) to (K) and using genomic DNA extracted from the test rice sample shown in Table 17 as a template DNA. Shows the result of electrophoresis on 8% polyacrylamide gel and staining with SYBR Green I. For lanes 1 to 19 in FIG. In addition, multiplex PCR was performed by adding 0.5 ng of genomic DNA in 20 μl of PCR reaction solution. In addition, for lanes 1 to 19 in FIG.

  FIG. 19 shows a PCR product obtained by performing multiplex PCR using a primer set including primer pairs (A) to (K) and using genomic DNA extracted from the test rice sample shown in Table 17 as a template DNA. And results of hybridization of a DNA array using capture oligonucleotides (I) to (XIII). In addition, multiplex PCR was performed by adding 0.5 ng of genomic DNA in 20 μl of PCR reaction solution. The numbers in FIG. 19 correspond to the sample numbers described in Table 17.

  Based on the results of FIGS. 18 and 19, Table 19 shows the results of a summary of detection by electrophoresis and determination of mixed rice determination by DNA array.

(3) Discussion As can be seen from FIG. 16 and Table 18, when using “Kit discrimination PCR kit I” manufactured by TaKaRa, 95% or more exists when 0.5 ng of genomic DNA is introduced into 20 μl of the PCR reaction solution. It was difficult to detect the expected Koshihikari. The same was true when 10 ng of genomic DNA was added to 20 μl of the PCR reaction solution. In addition, as can be seen from FIG. 17 and Table 18, when “Rice discrimination PCR Kit II (Koshihikari negative kit)” manufactured by TaKaRa was used, 10 ng was added even when 0.5 ng of genomic DNA was introduced into 20 μl of the PCR reaction solution. Even when it was added, the identification band of the amplification product could not be detected except that a thin identification band could be detected only when 5% of Kirara 397 was mixed even though the rice was mixed.

  On the other hand, as can be seen from FIG. 18 and Table 19, when a primer set including primer pairs (A) to (K) is used, when 0.5 ng of genomic DNA is introduced into 20 μl of the PCR reaction solution, lane 17 of FIG. An identification band other than that detected by (Koshihikari Junmai) could be detected. From this result, 1% mixed rice can be reliably detected by detecting a PCR product obtained by using a primer set including primer pairs (A) to (K) by electrophoresis. It was found that even 1% mixed rice can be detected. In addition, as can be seen from FIG. 19 and Table 19, the results by the DNA array are also good, and in the case of 5% mixed rice, the mixed rice varieties can be distinguished, and in the case of 1% mixed rice, the mixed rice varieties are unknown. However, the presence or absence of mixed rice was detectable. From this result, it was found that the DNA array using the capture oligonucleotides (I) to (XIII) can be detected even if the mixed rice ratio is 1% using a small amount of genomic DNA as a template. It was.

[Example 7]
When using a commercially available rice discrimination PCR kit, “Kame discrimination PCR Kit I” and “Rice discrimination PCR Kit II (Koshihikari nega kit)”, which are commercially available rice discrimination PCR kits (Comparative example) and the case where the primer set of the present invention was used (Example).

(1) Extraction of genomic DNA Fast ID (manufactured by Genetic ID Co., Ltd.) from 20 rice grains of commercially available Akitakomachi cooked with 2 rice cookers in a household rice cooker (made by Tiger) and commercially available vacuum-packed Koshihikari rice cooked rice ) To extract genomic DNA.

(2) Multiplex PCR
Using the rice cooked rice genomic DNA extracted in (1) above as a template, TaKaRa's “Rice Discrimination PCR Kit I” and “Rice Discrimination PCR Kit II (Koshihikari nega kit)” were used for multiplex PCR. went. The specific PCR reaction composition and reaction conditions were as shown in the instructions. The amount of DNA was measured by UV, and 0.5 ng and 10 ng were used in the PCR reaction solution.

  Multiplex PCR using a primer set including primer pairs (A) to (K) was performed in the same manner as in Example 1. However, the primer set used was prepared using a 5 'biotinylated and / or unmodified primer. The amount of DNA was measured by UV, and 0.5 ng was used in the PCR reaction solution. Genomic DNA extracted from the polished rice was used as a control.

(3) Electrophoresis 5% of a PCR product solution obtained by multiplex PCR using “Kit discrimination PCR Kit I” and “Rice discrimination PCR Kit II (Koshihikarinega kit)” manufactured by TaKaRa, Inc. was added to 2% agarose gel. After electrophoresis with SYBR Green I, the electrophoresis pattern of the identification band was observed by ultraviolet irradiation.

  In addition, 5 μl of a PCR product solution obtained by multiplex PCR using a primer set including primer pairs (A) to (K) was electrophoresed on an 8% polyacrylamide gel, stained with SYBR Green I, and then identified by ultraviolet irradiation. The electrophoresis pattern of the band was observed.

(4) Results of electrophoresis In FIG. 20, PCR products obtained by multiplex PCR using “Kit discrimination PCR Kit I” and “Rice discrimination PCR Kit II (Koshihikari nega kit)” manufactured by TaKaRa The result of electrophoresis is shown. The left diagram of FIG. 17 shows the results when 0.5 ng of genomic DNA is introduced into 20 μl of the PCR reaction solution, and the right diagram of FIG. The sample numbers shown in Table 20 are shown for lanes 1 to 16 in FIG.

  FIG. 21 shows the results of electrophoresis of PCR products obtained by multiplex PCR using a primer set including primer pairs (A) to (K). The sample numbers shown in Table 21 are shown for lanes 1 to 9 in FIG.

(4) DNA array In the same manner as in Examples 2 and 3, using PCR products obtained by performing multiplex PCR using a primer set including primer pairs (A) to (K), DNA Hybridization was detected by the array.

(5) Results of hybridization In the description of the test rice sample, the results corresponding to Table 21 are shown in FIG.

(6) Consideration As can be seen from FIG. 20 and Table 20, as a result of electrophoresis of the PCR product obtained using “Kit discrimination PCR Kit I” manufactured by TaKaRa, the amount of genome extracted from cooked rice and polished rice was 0. When 5 ng was added, none of 100% Koshihikari, 100% Akitakomachi, and 5% Akitakomachi mixed rice could be detected (FIG. 20, lanes 2 to 7).

  In addition, as a result of electrophoresis of a PCR product obtained using TaKaRa's “Kit discrimination PCR Kit I”, 100% Koshihikari was difficult to detect when 10 ng of genome was extracted from cooked rice. (Fig. 20, right, lane 2), but 100% Akitakomachi and 5% Akitakomachi mixed rice detected electrophoretic patterns different from Koshihikari (Fig. 20, right lanes 3 and 4). The possibility of being a variety of In addition, when 10 ng of genome amount extracted from the polished rice was added, an electrophoresis pattern of 100% Koshihikari and other electrophoresis patterns were also detected (right side of FIG. 20, lanes 5 to 7).

  As can be seen from FIG. 20 and Table 20, as a result of electrophoresis of the PCR product obtained using “Kithikari nega kit for rice discrimination” manufactured by TaKaRa, the amount of genome extracted from cooked rice and polished rice was 0. When 5 ng and 10 ng were added, 100% Koshihikari had no problem because the identification band was not originally detected (left and right in FIG. 20, lane 8), but 100% Akitakomachi and 5% Akitakomachi mixed. Rice was not detected (Figure 20 left and right, lanes 9 and 10).

  In addition, as a result of electrophoresis of a PCR product obtained using “Kithikari Nega Kit” for discriminating rice produced by TaKaRa, 100% Akitakomachi was introduced when 0.5 ng of genome was extracted from polished rice. When the genome amount of 10 ng was added, both 100% Akitakomachi and 5% Akitakomachi mixed rice were detected (Fig. 20, right, lanes 12 and 13). From this, the possibility that it was a variety other than Koshihikari was shown.

  From the above results, it was found that the mixed rice determination test using cooked rice is not suitable for “Kit discrimination PCR Kit I” and “Rice discrimination PCR Kit II (Koshihikarinega kit)” manufactured by TaKaRa.

  On the other hand, as a result of electrophoresis of the PCR product obtained using the primer set including the primer pairs (A) to (K), 100% Koshihikari, 100% Akitakomachi, and 5% Akitakomachi for both cooked rice and polished rice It was shown that it was possible to determine that it was mixed rice.

  A comparison of cooked rice 100% Koshihikari (Fig. 21, lane 2) and polished rice 100% Koshihikari (Fig. 21, lane 5) shows that the cooked rice 100% Koshihikari (Fig. 21, lane 2) has a higher chain length side. Found that the band disappeared. The disappearance of the band on the high chain length side was thought to be due to the influence of additives such as acidulants contained in commercially available cooked rice and the fact that the genome during cooking was cleaved.

  In addition, as can be seen from FIG. 22, in the hybridization using the DNA array using the capture oligonucleotides (I) to (XIII), the results were good for both cooked rice and polished rice.

  That is, according to electrophoresis of a PCR product obtained using a primer set including primer pairs (A) to (K) and a DNA array using capture oligonucleotides (I) to (XIII), It was found that even if the rice sample was cooked rice, the variety could be distinguished.

The results of electrophoresis of PCR products after performing multiplex PCR using genomic DNA extracted from 20 single varieties of rice samples using the primer set of the present invention are shown. Example 1 In order to determine the presence or absence of mixed rice, multiplex PCR was performed with the primer set of the present invention, and the results of electrophoresis of PCR products are shown. Example 1 It is the figure which showed the example of the detection result of the mixed rice by a DNA array. (Example 2) It is the figure which showed the example of the detection result by a DNA array. (Example 3) It is the figure which showed the example of the detection result by a DNA array. (Example 3) It is the figure which showed the example of the detection result by a DNA array. (Example 3) It is the figure which showed the example of the detection result by a DNA array. (Example 3) It is the figure which showed the example of the detection result by a DNA array. (Example 3) The result of electrophoresis of PCR products obtained by performing multiplex PCR using genomic DNA extracted from rice cultivar Koshihikari using “Kithi discrimination PCR Kit I” manufactured by TaKaRa is shown. Example 4 The result of carrying out multiplex PCR using the primer set of the present invention and electrophoresis of the obtained PCR product is shown. Example 4 The multiplex PCR was performed using “Kit discrimination PCR Kit II (Koshihikari negative kit)” manufactured by TaKaRa, and the obtained PCR product was electrophoresed. Example 4 The result of carrying out multiplex PCR using the primer set of the present invention and electrophoresis of the obtained PCR product is shown. Example 4 It is the figure which showed the detection result by a DNA array. Example 4 It is the figure which showed the detection result by a DNA array. Example 4 The result of analyzing Koshihikari (BL) from Niigata and Koshihikari from Chiba Prefecture by DNA array and electrophoresis is shown. (Example 5) The multiplex PCR was performed using “Kit discrimination PCR Kit I” manufactured by TaKaRa, and the obtained PCR product was electrophoresed. (Example 6) The multiplex PCR was performed using “Kit discrimination PCR Kit II (Koshihikari negative kit)” manufactured by TaKaRa, and the obtained PCR product was electrophoresed. (Example 6) The result of carrying out multiplex PCR using the primer set of the present invention and electrophoresis of the obtained PCR product is shown. (Example 6) It is the figure which showed the detection result by a DNA array. (Example 6) The results of electrophoresis of multiplex PCR using the "Kit discrimination PCR Kit I" and "Rice discrimination PCR Kit II (Koshihikari nega kit)" manufactured by TaKaRa are shown. (Example 7) The result of carrying out multiplex PCR using the primer set of the present invention and electrophoresis of the obtained PCR product is shown. (Example 7) It is the figure which showed the detection result by a DNA array. (Example 7)

Claims (14)

It consists of two kinds of oligonucleotides consisting of two kinds of base sequences selected from the following (1) to (5), and has 80 bases or more consisting of the base sequence represented by any one of SEQ ID NOs: 1 to 11 A primer pair capable of amplifying a nucleic acid.
(1) Two types of base sequences constituting any one combination selected from the following (a) to (k) (a) SEQ ID NO: 12 and SEQ ID NO: 23
(B) SEQ ID NO: 13 and SEQ ID NO: 24
(C) SEQ ID NO: 14 and SEQ ID NO: 25
(D) SEQ ID NO: 15 and SEQ ID NO: 26
(E) SEQ ID NO: 16 and SEQ ID NO: 27
(F) SEQ ID NO: 17 and SEQ ID NO: 28
(G) SEQ ID NO: 18 and SEQ ID NO: 29
(H) SEQ ID NO: 19 and SEQ ID NO: 30
(I) SEQ ID NO: 20 and SEQ ID NO: 31
(J) SEQ ID NO: 21 and SEQ ID NO: 32
(K) SEQ ID NO: 22 and SEQ ID NO: 33
(2) a base sequence complementary to the base sequence of (1) (3) a base sequence capable of hybridizing with the base sequence of (1) under stringent conditions (4) a base sequence of (1) A base sequence capable of hybridizing with a complementary base sequence under stringent conditions (5) one to several bases of the base sequences of (1) to (4) above are substituted, deleted, inserted, or Added base sequence   A primer set comprising at least two kinds of primer pairs according to claim 1.   The primer set according to claim 2, comprising at least 11 kinds of primer pairs each capable of amplifying a nucleic acid of 80 bases or more consisting of the base sequences represented by SEQ ID NOs: 1 to 11. The primer set according to claim 3, wherein the primer pair capable of amplifying a nucleic acid of 80 bases or more consisting of the base sequences represented by SEQ ID NOs: 1 to 11 is a primer pair of the following (A) to (K).
(A) a nucleotide sequence represented by SEQ ID NO: 12 and SEQ ID NO: 23; a nucleotide sequence complementary to the nucleotide sequence; a nucleotide sequence capable of hybridizing with the nucleotide sequence under stringent conditions; and the nucleotide sequence A base sequence capable of hybridizing with a complementary base sequence under stringent conditions; one to several bases of these base sequences are selected from the base sequences substituted, deleted, inserted or added A primer pair (B) consisting of two kinds of oligonucleotides consisting of two kinds of base sequences and capable of amplifying a nucleic acid of 80 bases or more consisting of the base sequence represented by SEQ ID NO: 1 and SEQ ID NO: 24 A nucleotide sequence complementary to the nucleotide sequence; a nucleotide sequence capable of hybridizing with the nucleotide sequence under stringent conditions; a nucleotide sequence complementary to the nucleotide sequence and a string; A nucleotide sequence capable of hybridizing under a gentle condition; 1 to several of these nucleotide sequences are composed of two nucleotide sequences selected from a substituted, deleted, inserted, or added nucleotide sequence A primer pair (C) consisting of two kinds of oligonucleotides and capable of amplifying a nucleic acid of 80 bases or more consisting of a base sequence represented by SEQ ID NO: 2 and a base sequence represented by SEQ ID NO: 25; A base sequence complementary to the base sequence; a base sequence capable of hybridizing with the base sequence under stringent conditions; a base sequence capable of hybridizing with the base sequence complementary to the base sequence under stringent conditions; Two oligonucleotides consisting of two kinds of base sequences in which one to several bases are selected from base sequences substituted, deleted, inserted or added A primer pair (D) capable of amplifying a nucleic acid of 80 bases or more comprising the base sequence represented by SEQ ID NO: 3 and a base sequence represented by SEQ ID NO: 26; complementary to the base sequence A base sequence that can hybridize with the base sequence under stringent conditions; a base sequence that can hybridize with the base sequence complementary to the base sequence under stringent conditions; 1 to several bases are composed of two kinds of oligonucleotides composed of two kinds of base sequences selected from substituted, deleted, inserted or added base sequences, and from the base sequence represented by SEQ ID NO: 4 A primer pair (E) that can amplify a nucleic acid of 80 bases or more; a base sequence represented by SEQ ID NO: 16 and SEQ ID NO: 27; a base sequence complementary to the base sequence; A base sequence capable of hybridizing under lingent conditions; a base sequence complementary to the base sequence and a base sequence capable of hybridizing under stringent conditions; one to several bases of these base sequences are It consists of two types of oligonucleotides consisting of two types of base sequences selected from substitutions, deletions, insertions, or additions, and amplifies a nucleic acid of 80 bases or more consisting of the base sequence represented by SEQ ID NO: 5. A base sequence represented by SEQ ID NO: 17 and SEQ ID NO: 28; a base sequence complementary to the base sequence; a base sequence capable of hybridizing with the base sequence under stringent conditions; A base sequence that can hybridize with a base sequence complementary to the base sequence under stringent conditions; 1 to several of these base sequences are substituted or deleted Primer pair that consists of two kinds of oligonucleotides consisting of two kinds of base sequences selected from the inserted or added base sequences and can amplify a nucleic acid of 80 bases or more consisting of the base sequence represented by SEQ ID NO: 6 ( G) base sequences represented by SEQ ID NO: 18 and SEQ ID NO: 29; base sequences complementary to the base sequences; base sequences capable of hybridizing with the base sequences under stringent conditions; complementary to the base sequences A nucleotide sequence capable of hybridizing with a stringent nucleotide sequence under stringent conditions; 1 to several of these nucleotide sequences are selected from a substituted, deleted, inserted, or added nucleotide sequence 2 Primer pair (H) SEQ ID NO: 2 consisting of two types of oligonucleotides consisting of a base sequence and capable of amplifying a nucleic acid of 80 bases or more consisting of the base sequence represented by SEQ ID NO: 9 and a base sequence represented by SEQ ID NO: 30; a base sequence complementary to the base sequence; a base sequence capable of hybridizing with the base sequence under stringent conditions; a base sequence complementary to the base sequence Nucleotide sequences that can hybridize with each other under stringent conditions; two nucleotide sequences in which one to several of these nucleotide sequences are selected from the substituted, deleted, inserted, or added nucleotide sequences A primer pair (I) that can amplify a nucleic acid of 80 bases or more consisting of a base sequence represented by SEQ ID NO: 8, and a base sequence represented by SEQ ID NO: 31 A base sequence complementary to the base sequence; a base sequence capable of hybridizing under stringent conditions with the base sequence; a base sequence complementary to the base sequence and stringent conditions Base sequences that can hybridize underneath; two types of base sequences in which one to several bases are composed of two base sequences selected from base sequences with substitution, deletion, insertion, or addition A primer pair (J) that can amplify a nucleic acid of 80 bases or more consisting of an oligonucleotide and consisting of a base sequence represented by SEQ ID NO: 9, and a base sequence represented by SEQ ID NO: 32; Complementary base sequence; base sequence capable of hybridizing with the base sequence under stringent conditions; base sequence capable of hybridizing with the base sequence complementary to the base sequence under stringent conditions; these base sequences 1 to several bases of which consist of two oligonucleotides consisting of two base sequences selected from the base sequences substituted, deleted, inserted or added, and the sequence A primer pair (K) that can amplify a nucleic acid of 80 bases or more consisting of the base sequence represented by No. 10 and the base sequence represented by SEQ ID NO: 33; a base sequence complementary to the base sequence; A base sequence that can hybridize with the base sequence under stringent conditions; a base sequence that can hybridize with a base sequence complementary to the base sequence under stringent conditions; one to several of these base sequences Consisting of two oligonucleotides consisting of two types of base sequences selected from the base sequences substituted, deleted, inserted or added, and having 80 bases or more consisting of the base sequence represented by SEQ ID NO: 11 Primer pair that can amplify the nucleic acid of Capture oligonucleotide (1) sequence capable of detecting a nucleic acid comprising a base sequence represented by any one of SEQ ID NOS: 1 to 11 selected from oligonucleotides comprising the following base sequences (1) to (5) Base sequence represented by any one of Nos. 34 to 46 (2) Base sequence complementary to the base sequence represented by any one of SEQ ID Nos. 34 to 46 (3) Any of SEQ ID Nos. 34 to 46 A base sequence that can hybridize with the base sequence represented by any one of the above (4) a base sequence complementary to the base sequence represented by any one of SEQ ID NOs: 34 to 46 and a stringent Base sequence capable of hybridizing under various conditions (5) A base sequence in which one to several bases of the base sequences (1) to (4) are substituted, deleted, inserted or added   A group of capture oligonucleotides comprising at least two capture oligonucleotides according to claim 5.   The group of capture oligonucleotides according to claim 6, comprising at least 13 types of capture oligonucleotides each capable of detecting a nucleic acid having the base sequence of SEQ ID NOs: 1 to 11. The capture oligonucleotide group (I) according to claim 7, wherein the capture oligonucleotides capable of detecting each of the nucleic acids having the base sequences described in SEQ ID NOs: 1 to 11 are capture oligonucleotides (I) to (XIII) below: ) A base sequence represented by SEQ ID NO: 34; a base sequence complementary to the base sequence; a base sequence capable of hybridizing with the base sequence under stringent conditions; a base sequence complementary to the base sequence and a stringent A nucleotide sequence capable of hybridizing under various conditions; one to several of these nucleotide sequences comprises an oligonucleotide selected from a substituted, deleted, inserted, or added nucleotide sequence, SEQ ID NO: 1 A capture oligonucleotide (II) capable of detecting a nucleic acid comprising the base sequence represented by SEQ ID NO: 35; a base sequence complementary to the base sequence A base sequence that can hybridize with the base sequence under stringent conditions; a base sequence that can hybridize with a base sequence complementary to the base sequence under stringent conditions; one to several of these base sequences Capture oligonucleotide (III) SEQ ID NO: which can detect a nucleic acid consisting of a base sequence represented by SEQ ID NO: 2 consisting of an oligonucleotide selected from the base sequence of substitution, deletion, insertion or addition A nucleotide sequence complementary to the nucleotide sequence; a nucleotide sequence capable of hybridizing under stringent conditions; a nucleotide sequence complementary to the nucleotide sequence and stringent conditions A base sequence that can hybridize with one or more of these base sequences selected from the base sequences in which one to several bases are substituted, deleted, inserted, or added. A capture oligonucleotide (IV) that can detect a nucleic acid consisting of a base sequence represented by SEQ ID NO: 3 and a base sequence that is complementary to the base sequence; A base sequence that can hybridize with the base sequence under stringent conditions; a base sequence that can hybridize with a base sequence complementary to the base sequence under stringent conditions; one to several of these base sequences Capture oligonucleotide (V) SEQ ID NO: which can detect a nucleic acid consisting of a base sequence represented by SEQ ID NO: 4 consisting of an oligonucleotide selected from the base sequence of substitution, deletion, insertion, or addition A nucleotide sequence complementary to the nucleotide sequence; a nucleotide sequence capable of hybridizing with the nucleotide sequence under stringent conditions; A base sequence that can hybridize with a base sequence complementary to the base sequence under stringent conditions; from a base sequence in which one to several bases of these base sequences are substituted, deleted, inserted, or added; A capture oligonucleotide (VI) comprising the selected oligonucleotide and capable of detecting a nucleic acid comprising the base sequence represented by SEQ ID NO: 5; a base sequence represented by SEQ ID NO: 39; a base sequence complementary to the base sequence; A base sequence that can hybridize with the base sequence under stringent conditions; a base sequence that can hybridize with a base sequence complementary to the base sequence under stringent conditions; one to several of these base sequences Consisting of an oligonucleotide selected from a base sequence with substitution, deletion, insertion or addition, consisting of a base sequence represented by SEQ ID NO: 6. A capture oligonucleotide (VII) that can detect a nucleic acid, a base sequence represented by SEQ ID NO: 40; a base sequence that is complementary to the base sequence; a base sequence that can hybridize with the base sequence under stringent conditions; A base sequence that can hybridize with a base sequence complementary to the base sequence under stringent conditions; from a base sequence in which one to several bases of these base sequences are substituted, deleted, inserted, or added; A capture oligonucleotide (VIII) comprising a selected oligonucleotide and capable of detecting a nucleic acid comprising a base sequence represented by SEQ ID NO: 7; a base sequence represented by SEQ ID NO: 41; a base sequence complementary to the base sequence; A base sequence capable of hybridizing with the base sequence under stringent conditions; a hybrid sequence under stringent conditions with a base sequence complementary to the base sequence; A base sequence represented by SEQ ID NO: 8, wherein one or several bases of these base sequences comprise an oligonucleotide selected from a base sequence with substitution, deletion, insertion or addition A capture oligonucleotide (IX) that can detect a nucleic acid comprising the sequence; a base sequence represented by SEQ ID NO: 42; a base sequence complementary to the base sequence; a base sequence capable of hybridizing with the base sequence under stringent conditions A base sequence that can hybridize with a base sequence complementary to the base sequence under stringent conditions; a base in which one to several bases of these base sequences are substituted, deleted, inserted, or added; A capture oligonucleotide (X) comprising an oligonucleotide selected from the sequence and capable of detecting a nucleic acid comprising the base sequence represented by SEQ ID NO: 9 represented by SEQ ID NO: 43 Base sequence; base sequence complementary to the base sequence; base sequence capable of hybridizing with the base sequence under stringent conditions; hybridizable with stringent base sequence complementary to the base sequence under stringent conditions Base sequence; 1 to several bases of these base sequences are composed of oligonucleotides selected from the base sequences substituted, deleted, inserted or added, and the base sequence represented by SEQ ID NO: 9 Capture oligonucleotide (XI) capable of detecting nucleic acid: base sequence represented by SEQ ID NO: 44; base sequence complementary to the base sequence; base sequence capable of hybridizing with the base sequence under stringent conditions; the base A base sequence that can hybridize with a base sequence complementary to the sequence under stringent conditions; 1 to several bases of these base sequences are substituted or missing. A capture oligonucleotide (XII) consisting of an oligonucleotide selected from lost, inserted, or added base sequences and capable of detecting a nucleic acid consisting of the base sequence represented by SEQ ID NO: 10 Base sequence represented by SEQ ID NO: 45 A base sequence complementary to the base sequence; a base sequence capable of hybridizing with the base sequence under stringent conditions; a base sequence capable of hybridizing with the base sequence complementary to the base sequence under stringent conditions; A nucleic acid consisting of an oligonucleotide selected from a base sequence in which one to several bases of these base sequences are substituted, deleted, inserted, or added, and consisting of the base sequence represented by SEQ ID NO: 10; Detectable capture oligonucleotide (XIII) base sequence represented by SEQ ID NO: 46; base sequence complementary to the base sequence; base sequence and stringer A base sequence that can hybridize under a stringent condition; a base sequence that is complementary to the base sequence and a base sequence that can hybridize under a stringent condition; one to several bases of these base sequences are substituted A capture oligonucleotide that can detect a nucleic acid consisting of a nucleotide sequence represented by SEQ ID NO: 11, consisting of an oligonucleotide selected from nucleotide sequences that are deleted, inserted, or added A method for discriminating rice varieties, comprising performing the following steps (i) and (ii):
(I) Nucleic acid preparation step of preparing rice nucleic acid in test rice sample (ii) Using the nucleic acid prepared in step (i) as a template, using the primer set according to any one of claims 2 to 4 Nucleic acid amplification step to obtain nucleic acid amplification product by nucleic acid amplification treatment Furthermore, the following (iii) and / or (iv) processes are performed, The rice varieties discrimination method according to claim 9 characterized by things.
(Iii) Detection step for detecting the nucleic acid amplification product obtained in step (ii) by electrophoresis (iv) The nucleic acid amplification product obtained in step (ii) and the nucleic acid amplification product according to any one of claims 6 to 8 Detection process for detecting hybridization with capture oligonucleotides A method for determining the presence or absence of mixed rice in Koshihikari, comprising performing the following steps (i) and (ii):
(I) Nucleic acid preparation step of preparing rice nucleic acid in test rice sample (ii) Using the nucleic acid prepared in step (i) as a template, using the primer set according to any one of claims 2 to 4 Nucleic acid amplification step to obtain nucleic acid amplification product by nucleic acid amplification treatment Furthermore, the following (iii) and / or (iv) processes are performed, The method of determining the presence or absence of the mixed rice to Koshihikari of Claim 11 characterized by the above-mentioned.
(Iii) Detection step for detecting the nucleic acid amplification product obtained in step (ii) by electrophoresis (iv) The nucleic acid amplification product obtained in step (ii) and the nucleic acid amplification product according to any one of claims 6 to 8 Detection process for detecting hybridization with capture oligonucleotides   The rice variety discrimination kit containing the primer set of any one of Claims 2-4, or the mixed rice determination kit to Koshihikari. Furthermore, the kit of Claim 13 containing the capture oligonucleotide group of any one of Claims 6-8.