JP5749466B2 - Cultivar identification method for rice or tissues derived from it or processed products thereof - Google Patents

Description

  The present invention relates to rice or a tissue derived from it (eg, rice), or a method for identifying varieties of processed products thereof.

  Based on the JAS law, the labeling of “variety” is obligatory for brown rice and polished rice. However, in good-tasting varieties centered on Koshihikari, which are traded at high prices, camouflage labeling continues to follow, and as a method of analysis to clarify camouflage labeling and as a deterrent, model identification using breed-specific DNA Technology development has been underway. As a result, it is possible to identify major varieties and exert a certain deterrent effect. However, the conventional technology is the rice cultivar “identification” method, and it is an appraisal method that guarantees evidence ability. Therefore, it is necessary to improve the accuracy of human DNA testing.

  The rice variety identification methods developed so far include the RAPD-STS method (Patent Documents 1 and 2), the SNP method (Patent Documents 3 and 4), and the SSR (Simple Sequence Repeats) method (Patent Documents) depending on the technology background used. 5 to 7). The RAPD-STS method detects the presence or absence of DNA amplification by a technique developed based on analysis using random primers. The SNP method detects single base substitutions in the genome. The SSR method detects the number of repeats (sequence length) of repeat sequences of 2 to 4 base units in the genome.

JP 2001-095589 A JP 2005-073655 A JP 2004-248635 A JP2009-219498 JP 10-057073 A JP2004-065251 JP2007-037468

  However, in the RAPD-STS method and the SNP method, basically only two types of polymorphisms can be obtained by analysis with one marker (the amount of information is small), and in rice with many closely related species, analysis with many markers is performed. Need.

  On the other hand, in the SSR method, several types to a dozen types of polymorphisms are obtained by analyzing one marker (the amount of information is large), so that efficient identification is possible. However, since the SSR method developed in the past mainly uses SSR having two base repeats, the determination may be difficult due to the stutter product detected due to the characteristics of PCR. In human DNA analysis, only 4-base repeat SSRs with few stutter products are used.

  Accordingly, an object of the present invention is to provide a method for identifying varieties using a four-base repeat SSR region with high discrimination in rice.

  As a result of intensive studies, the present inventors succeeded in identifying an SSR region having a high discriminating 4-base repeat in rice. Moreover, by identifying such SSR region, the inventors succeeded in developing a rice DNA marker for detecting the SSR region and a variety identification method using the marker, and completed the present invention.

  That is, the present invention identifies rice or a tissue derived therefrom, or a variety of processed products thereof based on the genetic polymorphism of the SSR region of 4 base repeats, or a tissue derived therefrom or processed products thereof. And a primer pair comprising two or more primer pairs for amplifying a four-base repeat SSR region in rice and a combination of primers.

  Since the SSR method has a larger amount of information than the conventional RAPD-STS method and SNP method, it is possible to identify a wider variety of varieties more efficiently. In addition, by using SSR with 4 base repeats instead of the conventional 2 base repeats, the same high accuracy as human DNA analysis can be obtained (FIG. 1). Therefore, the appraisal method of the present invention is excellent in the appraisal ability of rice or tissues derived from it (eg, rice) or processed products thereof. In the appraisal method of the present invention, for efficient variety appraisal, only a 4-base repeat SSR region rich in genetic polymorphism among rice varieties may be used. Therefore, only a 4-base repeat SSR region exhibiting a (rare) genetic polymorphism specific to a particular rice variety (ie, highly discriminating for a particular variety) may be used.

FIG. 1 is a diagram showing an example of genotype analysis of SSR regions of 2-base repeats and 4-base repeats. In the two-base repetition, a plurality of peaks are detected before and after the original peak, and it may be difficult to distinguish the peaks when a plurality of types are mixed. A 4-base repeat has almost a single peak and thus has high discrimination. FIG. 2 is a diagram showing an example of genotype analysis of eight SSR regions (RM10000, RM11499, RM12927, RM14137, RM15043, RM15764, RM16604, RM17039) according to the present invention. The variety is identified by detecting the numerical value of each peak and comparing it with the standard variety. Nipponbare: (1) RM17039; (2) RM14137; (3) RM15043; (4) RM10000; (5) RM16604 ;, (6) RM15764; (7) RM12927; (8) RM11499. Koshihikari: (1 ′) RM17039; (2 ′) RM14137; (3 ′) RM15043; (4 ′) RM10000; (5 ′) RM16604; (6 ′) RM15764; (7 ′) RM12927; ') RM11499. Glance: (1 ″) RM17039; (2 ″) RM14137; (3 ″) RM15043; (4 ″) RM10000; (5 ″) RM15764 ;, (6 ″) RM16604; '') RM12927; (8 '') RM11499. In addition, Koshihikari, the peak of (1 ') and (2'), and the peaks of (1 ") and (2") overlap in appearance, but use labels with fluorescent substances that emit different fluorescent colors. By doing so, it is actually identifiable. FIG. 3 is a view showing the base sequence around the SSR region of RM10000 (first chromosome: 21845-22844). The box portion indicates the SSR region (Nippon Hare: “tatc” × 15). The underlined portion indicates the position of the primer used in the examples (the same applies to FIGS. 4 to 26). The position on the chromosome follows MSU Rice Genome Announce Release 6.1 (http: //rice.plantbiology.msu.index.html) (same for FIGS. 4 to 26). FIG. 4 is a diagram showing the base sequence around the SSR region of RM11499 (first chromosome: 27752831 to 27753830). The box part indicates the SSR region (Nippon Hare: “atag” × 13). FIG. 5 is a view showing the base sequence around the SSR region of RM12927 (second chromosome: 9609399 to 9610398). The box portion indicates the SSR region (Nippon Hare: “atac” × 23). FIG. 6 is a diagram showing the base sequence around the SSR region of RM14137 (second chromosome: 3429476 to 3294475). The box part indicates the SSR region (Nippon Hare: “atag” × 15). FIG. 7 is a diagram showing the base sequence around the SSR region of RM15043 (Chromosome 3: 14698350-1469349). The box part indicates the SSR region (Nippon Hare: “tcta” × 14). FIG. 8 is a view showing the base sequence around the SSR region of RM15764 (Chromosome 3: 28382145 to 28383144). The box part indicates the SSR region (Nippon Hare: “atag” × 12). FIG. 9 is a view showing the base sequence around the SSR region of RM16604 (Chromosome 4: 11461847 to 11462846). The box portion indicates the SSR region (Nippon Hare: “atag” × 14). FIG. 10 is a view showing the base sequence around the SSR region of RM17039 (Chromosome 4: 21798477 to 21179476). The box portion indicates the SSR region (Nippon Hare: “atac” × 12). FIG. 11 is a view showing a base sequence around the SSR region of RM18204 (chromosome 5: 9479123 to 9480122). The box portion indicates the SSR region (Nippon Hare: “atac” × 15). FIG. 12 is a view showing a base sequence around the SSR region of RM18950 (chromosome 5: 24953090 to 24954089). The box portion indicates the SSR region (Nippon Hare: “ttat” × 22). FIG. 13 is a view showing the base sequence around the SSR region of RM19232 (chromosome 6: 2158080-216879). The box portion indicates the SSR region (Nippon Hare: “atac” × 12). FIG. 14 is a diagram showing the base sequence around the SSR region of RM20092 (chromosome 6: 17296152 to 17297151). The box portion indicates the SSR region (Nippon Hare: “agat” × 14). FIG. 15 is a view showing a base sequence around the SSR region of RM21367 (chromosome 7: 10275871-10276870). The box part indicates the SSR region (Nippon Hare: “atgt” × 20). FIG. 16 is a view showing the base sequence around the SSR region of RM21859 (chromosome 7: 23228185 to 23229184). The box portion indicates the SSR region (Nippon Hare: “tatc” × 18). FIG. 17 is a diagram showing the base sequence around the SSR region of RM22194 (chromosome 8: 94372-95371). The box portion indicates the SSR region (Nippon Hare: “cat” × 13). FIG. 18 is a view showing the base sequence around the SSR region of RM23036 (chromosome 8: 18760442 to 18764411). The box portion indicates the SSR region (Nippon Hare: “agat” × 15). FIG. 19 is a view showing the base sequence around the SSR region of RM23778 (chromosome 9: 3965696 to 3966695). The box portion indicates the SSR region (Nippon Hare: “ataa” × 20). FIG. 20 is a diagram showing the base sequence around the SSR region of RM24481 (chromosome 9: 17263432-17264431). The box portion indicates the SSR region (Nippon Hare: “act” × 23). FIG. 21 is a diagram showing the base sequence around the SSR region of RM25036 (Chromosome 10: 3804619 to 3805618). The box part indicates the SSR region (Nippon Hare: “tatg” × 18). FIG. 22 is a diagram showing the base sequence around the SSR region of RM25416 (Chromosome 10: 14572963 to 14573962). The box portion indicates the SSR region (Nippon Hare: “act” × 9). FIG. 23 is a diagram showing the base sequence around the SSR region of RM26245 (Chromosome 11: 5590572-5599171). The box portion indicates the SSR region (Nippon Hare: “act” × 12). FIG. 24 is a diagram showing the base sequence around the SSR region of RM26957 (Chromosome 11: 22135614 to 21336613). The box portion indicates the SSR region (Nippon Hare: “atag” × 14). FIG. 25 is a view showing the nucleotide sequence around the SSR region of RM27695 (Chromosome 12: 4828388 to 4829387). The box part indicates the SSR region (Nippon Hare: “tgta” × 13). FIG. 26 is a diagram showing the base sequence around the SSR region of RM28174 (Chromosome 12: 17657607 to 17658606). The box portion indicates the SSR region (Nippon Hare: “tatg” × 15).

  The present invention provides a method for identifying a variety of rice or a tissue derived therefrom, or a processed product thereof (hereinafter abbreviated as “rice etc.” as necessary).

  In one embodiment, the present invention relates to a method for identifying rice varieties. In the present invention, “rice” means not only grown plants and immature plants (eg, seedlings) but also rice straw, which is a withered plant. Rice varieties include, for example, Koshihikari, Hitomebore, Akitakomachi, Hinohikari, Haenuki, Nanatsubo, Kirara 397, Tsugaru Roman, Shinigura, Kinuhikari, Kobuki, Asahi no Yume, Hoshino Yume, Yumetsushi, Hanaechizen, Chiba 28 No. (Fusakogane), Fusaotome, Sasanishiki, Menkoin, Aichi Kaori SBL (Nihonbare), Akebono, Yume Shizuku, Aya no Kagayaki, Yume Mizuho, Milky Queen, Tentaku, Orozuki, Fukukinko , Forest Bear, Mana Musume, Hatsushimo Gifu SL (Hatsushima), Kinusume, Yumepirika, Chiyoshiki, Autumn Poetry, Amakari, Nashirakari, Asahi, Hiyokumochi, Himenomochi, Kokanemochi, Hakuchomochi, Wataboshi, The children of the wind, Nishiki Yamada, 500 million stones, Nishiki MiyamaIn addition to these varieties, there can be many rice varieties that can be identified by using an SSR region having a 4-base repeat.

  In another embodiment, the present invention relates to a method for identifying a variety of tissue derived from rice. In the present invention, “tissue derived from rice” means a part of a plant body of rice, and examples thereof include stems, leaves, roots, and rice (that is, rice seeds). The rice may be either brown rice or polished rice. Examples of rice include glutinous rice, glutinous rice, and brewing rice. The varieties of glutinous rice, glutinous rice and brewing rice are clear from the description in Table 6.

  In yet another embodiment, the present invention relates to a method for identifying varieties of rice or processed tissue derived therefrom. In the present invention, the “processed product” of rice or a tissue derived therefrom is a substance containing rice or a tissue derived therefrom (eg, rice), which can extract rice DNA that can be used according to the present invention. There is no particular limitation. Examples of such processed products include processed rice products (eg, cooked rice, processed rice, miso, rice crackers, rice flour, packaging rice, rice noodles, brown rice tea, rice flour bread, rice flour noodles, sake, beer), Processed rice straw products (eg, feed, bedding, flooring, folk crafts).

  Rice has chromosomes 1-12. Therefore, in the identification of varieties such as rice, those present on these chromosomes are used as SSR regions having a 4-base repeat. In addition, when using SSR regions with 4 base repeats existing on the same chromosome for variety identification, the accuracy of variety identification is improved by avoiding the influence of genetic linkage while measuring a smaller number of SSR regions. To improve, SSR regions that are separated on the chromosome are used. Specifically, such a 4-base repeat SSR region can be identified by an RM (rice microsatellite) number. For example, such SSR regions of 4 base repeats include RM10000 and RM11499 present on chromosome 1, RM12927 and RM14137 present on chromosome 2, RM15043 and RM15764 present on chromosome 3, chromosome 4 RM16604 and RM17039 present on RM18204 and RM18950 present on chromosome 5, RM19232 and RM20092 present on chromosome 6, RM21367 and RM21859 present on chromosome 7, RM22194 present on chromosome 8 and RM23036, RM23778 and RM24481 present on chromosome 9, RM25036 and RM25416 present on chromosome 10, RM262 present on chromosome 11 5 and RM26957, RM27695 and RM28174 and the like present in the 12 chromosome. In Tables 1 to 3 below, genetic polymorphisms of the SSR region of 4 base repeats are shown for the genotypes of 48 major varieties. The data in Tables 1 to 3 were prepared based on Examples 1 and 2 and Tables 12 to 14, and the genetic polymorphism of the SSR region of 4-base repeats among varieties has been reported so far. It has not been.

  The number of SSR regions having 4 base repeats used for breeding is not particularly limited as long as it is 1 or more. For example, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, or 24 or more Good.

  Specifically, the identification method for rice and other varieties can be performed using a gene amplification method such as a PCR method. The present invention also provides a primer pair for amplifying a four base repeat SSR region in rice that can be used for such applications. Examples of the 4-base repeat SSR region include those specified by the RM number described above. Hereinafter, the 4-base repeat SSR region specified by each RM number and the primer pair for amplifying this region will be described in detail.

1) RM10000
The SSR region of RM10000 (eg, “tatc” × 15 in Nipponbare) is present in the region consisting of nucleotide residues 471 to 530 in the base sequence shown in SEQ ID NO: 1 (base sequence around the SSR region of RM10000) (FIG. 1). Therefore, as a primer pair for amplifying the SSR region of RM10000, the first primer corresponding to the upstream site (sense strand or antisense strand) of this region and the downstream site (antisense strand or sense strand) of this region A second primer combination corresponding to can be used. Examples of such a primer pair include: a) a first primer having at least a partial base sequence of the 1st to 470th base sequence in the base sequence represented by SEQ ID NO: 1 or an equivalent base sequence thereof, and a sequence A combination of second primers having at least a part of the 531 to 1000th base sequence in the base sequence shown in No. 1 or a complementary sequence of its equivalent base sequence, and b) a base shown in SEQ ID No. 1. A first primer having at least a partial base sequence of the 1st to 470th base sequences in the sequence or a complementary sequence thereof, and the 531st to 1000th base sequences in the base sequence shown in SEQ ID NO: 1 A combination of second primers having at least a part of the base sequence or an equivalent base sequence thereof. That (e.g., see Figure 1).

2) RM11499
The SSR region of RM11499 (eg, “atag” × 13 in Nipponbare) exists in the region consisting of nucleotide residues 475 to 526 in the base sequence shown in SEQ ID NO: 2 (base sequence around the SSR region of RM11499). (FIG. 2). Therefore, as a primer pair for amplifying the SSR region of RM11499, the first primer corresponding to the upstream site (sense strand or antisense strand) of this region and the downstream site (antisense strand or sense strand) of this region A second primer combination corresponding to can be used. Examples of such a primer pair include: a) a first primer having at least a partial base sequence of the 1st to 474th base sequences in the base sequence represented by SEQ ID NO: 2 or an equivalent base sequence thereof, and a sequence A combination of second primers having at least a part of the base sequence of the 527 to 1000th base sequence in the base sequence shown in No. 2 or a complementary sequence thereof, and b) a base shown in SEQ ID No. 2. A first primer having at least a partial base sequence of the 1st to 474th base sequences in the sequence or a complementary sequence equivalent thereto, and the 527th to 1000th base sequences of the base sequence shown in SEQ ID NO: 2 A combination of second primers having at least a part of the base sequence or an equivalent base sequence thereof. That (e.g., see Figure 2).

3) RM12927
The SSR region of RM12927 (eg, “atac” × 23 in Nipponbare) is present in the region consisting of nucleotide residues 455 to 546 in the base sequence shown in SEQ ID NO: 3 (base sequence around the SSR region of RM12927) (FIG. 3). Therefore, as a primer pair for amplifying the SSR region of RM12927, the first primer corresponding to the upstream site (sense strand or antisense strand) of this region and the downstream site (antisense strand or sense strand) of this region A second primer combination corresponding to can be used. Examples of such a primer pair include: a) a first primer having at least a partial base sequence of the 1st to 454th base sequences in the base sequence represented by SEQ ID NO: 3 or an equivalent base sequence thereof, and a sequence A combination of second primers having at least a part of the base sequence of the 547-1000th base sequence in the base sequence shown in No. 3 or a complementary sequence thereof, and b) the base shown in SEQ ID No. 3. A first primer having at least a partial base sequence of the 1st to 454th base sequences in the sequence or a complementary sequence thereof, and the 547th to 1000th base sequences of the base sequence shown in SEQ ID NO: 3 A combination of second primers having at least a part of the base sequence or an equivalent base sequence thereof. That (e.g., see Figure 3).

4) RM14137
The SSR region of RM14137 (eg, “atag” × 15 in Nipponbare) exists in the region consisting of nucleotide residues 471 to 530 in the base sequence shown in SEQ ID NO: 4 (base sequence around the SSR region of RM14137). (FIG. 4). Therefore, as a primer pair for amplifying the SSR region of RM14137, the first primer corresponding to the upstream site (sense strand or antisense strand) of this region and the downstream site (antisense strand or sense strand) of this region A second primer combination corresponding to can be used. Examples of such a primer pair include: a) a first primer having at least a partial base sequence of the 1st to 470th base sequences in the base sequence represented by SEQ ID NO: 4 or an equivalent base sequence thereof, and a sequence A combination of second primers having at least a part of the 531 to 1000th base sequence in the base sequence shown in No. 4 or a complementary sequence of its equivalent base sequence, and b) a base shown in SEQ ID No. 4 A first primer having at least a partial base sequence of the 1st to 470th base sequences in the sequence or a complementary sequence of the equivalent base sequence, and the 531st to 1000th base sequences of the base sequence shown in SEQ ID NO: 4 A combination of second primers having at least a part of the base sequence or an equivalent base sequence thereof. That (e.g., see Figure 4).

5) RM15043
The SSR region of RM15043 (eg, “tcta” × 14 in Nipponbare) exists in the region consisting of nucleotide residues 473 to 528 in the base sequence shown in SEQ ID NO: 5 (base sequence around the SSR region of RM15043) (FIG. 5). Therefore, as a primer pair for amplifying the SSR region of RM15043, the first primer corresponding to the upstream site (sense strand or antisense strand) of this region and the downstream site (antisense strand or sense strand) of this region A second primer combination corresponding to can be used. Examples of such a primer pair include: a) a first primer having at least a partial base sequence of the 1st to 472nd base sequences in the base sequence represented by SEQ ID NO: 5 or an equivalent base sequence thereof, and a sequence A combination of second primers having at least a part of the base sequence of the 529 to 1000th base sequence in the base sequence shown in No. 5 or a complementary sequence thereof, and b) a base shown in SEQ ID No. 5. A first primer having at least a partial base sequence of the 1st to 472th base sequences in the sequence or a complementary sequence thereof, and the 529th to 1000th base sequences in the base sequence shown in SEQ ID NO: 5; A combination of second primers having at least a part of the base sequence or an equivalent base sequence thereof. That (e.g., see Figure 5).

6) RM15764
The SSR region of RM15764 (eg, “atag” × 12 in Nipponbare) exists in the region consisting of nucleotide residues 477 to 524 in the base sequence shown in SEQ ID NO: 6 (base sequence around the SSR region of RM15764) (FIG. 6). Therefore, as a primer pair for amplifying the SSR region of RM15764, the first primer corresponding to the upstream site (sense strand or antisense strand) of this region and the downstream site (antisense strand or sense strand) of this region A second primer combination corresponding to can be used. Examples of such a primer pair include: a) a first primer having at least a partial base sequence of the 1st to 476th base sequence in the base sequence represented by SEQ ID NO: 6 or an equivalent base sequence thereof, and a sequence A combination of second primers having at least a part of the base sequence of the 525 to 1000th base sequence in the base sequence shown in No. 6 or a complementary sequence thereof, and b) the base shown in SEQ ID No. 6 A first primer having at least a partial base sequence of the 1st to 476th base sequences in the sequence or a complementary sequence thereof, and the 525th to 1000th base sequences of the base sequence shown in SEQ ID NO: 6 A combination of second primers having at least a part of the base sequence or an equivalent base sequence thereof. That (e.g., see FIG. 6).

7) RM16604
The SSR region of RM16604 (eg, “atag” × 14 in Nipponbare) exists in the region consisting of nucleotide residues 473 to 528 in the base sequence shown in SEQ ID NO: 7 (base sequence around the SSR region of RM16604) (FIG. 7). Therefore, as a primer pair for amplifying the SSR region of RM16604, the first primer corresponding to the upstream site (sense strand or antisense strand) of this region and the downstream site (antisense strand or sense strand) of this region A second primer combination corresponding to can be used. Examples of such a primer pair include: a) a first primer having at least a partial base sequence of the 1st to 472nd base sequences in the base sequence represented by SEQ ID NO: 7 or an equivalent base sequence thereof, and a sequence A combination of second primers having at least a portion of the base sequence of the 529 to 1000th base sequence in the base sequence shown in No. 7 or a complementary sequence thereof, and b) a base shown in SEQ ID No. 7 A first primer having at least a partial base sequence of the 1st to 472th base sequences in the sequence or a complementary sequence thereof, and the 529th to 1000th base sequences in the base sequence shown in SEQ ID NO: 7; A combination of second primers having at least a part of the base sequence or an equivalent base sequence thereof. That (e.g., see Figure 7).

8) RM17039
The SSR region of RM17039 (for example, “atac” × 12 in Nipponbare) is present in the region consisting of nucleotide residues 477 to 524 in the base sequence shown in SEQ ID NO: 8 (base sequence around the SSR region of RM17039). (FIG. 8). Therefore, as a primer pair for amplifying the SSR region of RM17039, the first primer corresponding to the upstream site (sense strand or antisense strand) of this region and the downstream site (antisense strand or sense strand) of this region A second primer combination corresponding to can be used. As such a primer pair, for example, a) a first primer having at least a partial base sequence of the 1st to 476th base sequence in the base sequence represented by SEQ ID NO: 8 or an equivalent base sequence thereof, and a sequence A combination of second primers having at least a part of the base sequence of the 525 to 1000th base sequence in the base sequence shown in No. 8 or a complementary sequence thereof, and b) the base shown in SEQ ID No. 8 A first primer having at least a partial base sequence of the 1st to 476th base sequence in the sequence or a complementary sequence thereof, and the 525th to 1000th base sequences of the base sequence shown in SEQ ID NO: 8 A combination of second primers having at least a part of the base sequence or an equivalent base sequence thereof. That (e.g., see Figure 8).

9) RM18204
The SSR region of RM18204 (eg, “atac” × 15 in Nipponbare) exists in the region consisting of nucleotide residues 471 to 530 in the base sequence shown in SEQ ID NO: 9 (base sequence around the SSR region of RM18204) (FIG. 9). Therefore, as a primer pair for amplifying the SSR region of RM18204, the first primer corresponding to the upstream site (sense strand or antisense strand) of this region and the downstream site (antisense strand or sense strand) of this region A second primer combination corresponding to can be used. Examples of such a primer pair include: a) a first primer having at least a partial base sequence of the 1st to 470th base sequences in the base sequence represented by SEQ ID NO: 9 or an equivalent base sequence thereof, and a sequence A combination of a second primer having at least a part of the 531 to 1000th base sequence in the base sequence shown in No. 9 or a complementary sequence of its equivalent base sequence, and b) a base shown in SEQ ID No. 9 A first primer having at least a partial base sequence of the 1st to 470th base sequences in the sequence or a complementary sequence thereof, and the 531st to 1000th base sequences of the base sequence shown in SEQ ID NO: 9; A combination of second primers having at least a part of the base sequence or an equivalent base sequence thereof. That (e.g., see Figure 9).

10) RM18950
The SSR region of RM18950 (eg, “ttat” × 22 in Nipponbare) is present in the region consisting of nucleotide residues 457 to 544 in the base sequence shown in SEQ ID NO: 10 (base sequence around the SSR region of RM18950) (FIG. 10). Therefore, as a primer pair for amplifying the SSR region of RM18950, the first primer corresponding to the upstream site (sense strand or antisense strand) of this region and the downstream site (antisense strand or sense strand) of this region A second primer combination corresponding to can be used. Examples of such a primer pair include: a) a first primer having at least a partial base sequence of the 1st to 455th base sequences in the base sequence represented by SEQ ID NO: 10 or an equivalent base sequence thereof, and a sequence A combination of second primers having at least part of the base sequence of the 545th to 1000th base sequences in the base sequence shown in No. 10 or a complementary sequence thereof, and b) the base shown in SEQ ID No. 10. A first primer having at least a partial base sequence of the 1st to 455th base sequences in the sequence or a complementary sequence thereof, and the 545th to 1000th base sequences in the base sequence shown in SEQ ID NO: 10; Combination of second primers having at least a part of the base sequence or an equivalent base sequence thereof Like (e.g., see Figure 10).

11) RM19232
The SSR region of RM19232 (eg, “atac” × 12 in Nipponbare) is present in the region consisting of the 477th to 524th nucleotide residues in the base sequence shown in SEQ ID NO: 11 (base sequence around the SSR region of RM19232). (FIG. 11). Therefore, as a primer pair for amplifying the SSR region of RM19232, the first primer corresponding to the upstream site (sense strand or antisense strand) of this region and the downstream site (antisense strand or sense strand) of this region A second primer combination corresponding to can be used. Examples of such a primer pair include: a) a first primer having at least a partial base sequence of the 1st to 476th base sequence in the base sequence represented by SEQ ID NO: 11 or an equivalent base sequence thereof, and a sequence A combination of second primers having at least a part of the base sequence of the 525 to 1000th base sequence in the base sequence shown in No. 11 or a complementary sequence thereof, and b) the base shown in SEQ ID No. 11 A first primer having at least a part of a base sequence of the 1st to 476th base sequence in the sequence or a complementary sequence of the equivalent base sequence, and a 525th to 1000th base sequence of the base sequence shown in SEQ ID NO: 11 Combination of second primers having at least a part of the base sequence or an equivalent base sequence thereof Like (e.g., see Figure 11).

12) RM20092
The SSR region of RM20092 (eg, “agat” × 14 in Nipponbare) exists in the region consisting of nucleotide residues 473 to 528 in the base sequence shown in SEQ ID NO: 12 (base sequence around the SSR region of RM20092). (FIG. 12). Therefore, as a primer pair for amplifying the SSR region of RM20092, the first primer corresponding to the upstream site (sense strand or antisense strand) of this region and the downstream site (antisense strand or sense strand) of this region A second primer combination corresponding to can be used. Examples of such a primer pair include: a) a first primer having at least a partial base sequence of the 1st to 472nd base sequences in the base sequence represented by SEQ ID NO: 12 or an equivalent base sequence thereof, and a sequence A combination of second primers having at least a partial base sequence of the 529 to 1000th base sequence in the base sequence shown in No. 12 or a complementary sequence thereof, and b) a base shown in SEQ ID No. 12. A first primer having at least a partial base sequence of the 1st to 472th base sequences in the sequence or a complementary sequence thereof, and the 529th to 1000th base sequences in the base sequence shown in SEQ ID NO: 12; Combination of second primers having at least a part of the base sequence or an equivalent base sequence thereof Like (e.g., see Figure 12).

13) RM21367
The SSR region of RM21367 (eg, “atgt” × 20 in Nipponbare) exists in the region consisting of the 461st to 540th nucleotide residues in the base sequence shown in SEQ ID NO: 13 (base sequence around the SSR region of RM21367). (FIG. 13). Therefore, as a primer pair for amplifying the SSR region of RM21367, the first primer corresponding to the upstream site (sense strand or antisense strand) of this region and the downstream site (antisense strand or sense strand) of this region A second primer combination corresponding to can be used. Examples of such a primer pair include: a) a first primer having at least a partial base sequence of the 1st to 460th base sequence in the base sequence represented by SEQ ID NO: 13 or an equivalent base sequence thereof, and a sequence A combination of second primers having at least a part of the base sequence of the 541-1000th base sequence in the base sequence shown in No. 13 or a complementary sequence of the equivalent base sequence, and b) the base shown in SEQ ID No. 13 A first primer having at least a partial base sequence of the 1st to 460th base sequences in the sequence or a complementary sequence of the equivalent base sequence, and the 541th to 1000th base sequences of the base sequence shown in SEQ ID NO: 13 Combination of second primers having at least a part of the base sequence or an equivalent base sequence thereof Like (e.g., see Figure 13).

14) RM21859
The SSR region of RM21859 (eg, “tatc” × 18 in Nipponbare) is present in the region consisting of the 465th to 536th nucleotide residues in the base sequence shown in SEQ ID NO: 14 (base sequence around the SSR region of RM21859) (FIG. 14). Therefore, as a primer pair for amplifying the SSR region of RM21859, the first primer corresponding to the upstream site (sense strand or antisense strand) of this region and the downstream site (antisense strand or sense strand) of this region A second primer combination corresponding to can be used. Examples of such a primer pair include: a) a first primer having at least a partial base sequence of the 1st to 464th base sequences in the base sequence represented by SEQ ID NO: 14 or an equivalent base sequence thereof, and a sequence A combination of second primers having at least a part of the base sequence of the 537-1000th base sequence in the base sequence shown in No. 14 or a complementary sequence thereof, and b) the base shown in SEQ ID No. 14 A first primer having at least a partial base sequence of the 1st to 464th base sequences in the sequence or a complementary sequence equivalent thereto, and the 537th to 1000th base sequences of the base sequence shown in SEQ ID NO: 14 Combination of second primers having at least a part of the base sequence or an equivalent base sequence thereof Like (e.g., see Figure 14).

15) RM22194
The SSR region of RM22194 (eg, “cat” × 13 in Nipponbare) exists in the region consisting of nucleotide residues 475 to 526 in the base sequence shown in SEQ ID NO: 15 (base sequence around the SSR region of RM22194). (FIG. 15). Therefore, as a primer pair for amplifying the SSR region of RM22194, the first primer corresponding to the upstream site (sense strand or antisense strand) of this region and the downstream site (antisense strand or sense strand) of this region A second primer combination corresponding to can be used. Examples of such a primer pair include: a) a first primer having at least a partial base sequence of the 1st to 474th base sequence in the base sequence represented by SEQ ID NO: 15 or an equivalent base sequence thereof, and a sequence A combination of second primers having at least a part of the base sequence of the 527th to 1000th base sequences in the base sequence shown in No. 15 or a complementary sequence thereof, and b) a base shown in SEQ ID No. 15 A first primer having at least a partial base sequence of the 1st to 474th base sequences in the sequence or a complementary sequence equivalent thereto, and the 527th to 1000th base sequences of the base sequence shown in SEQ ID NO: 15 Combination of second primers having at least a part of the base sequence or an equivalent base sequence thereof Like (e.g., see Figure 15).

16) RM23036
The SSR region of RM23036 (eg, “agat” × 15 in Nipponbare) exists in the region consisting of nucleotide residues 471 to 530 in the base sequence shown in SEQ ID NO: 16 (base sequence around the SSR region of RM23036) (FIG. 16). Therefore, as a primer pair for amplifying the SSR region of RM23036, the first primer corresponding to the upstream site (sense strand or antisense strand) of this region and the downstream site (antisense strand or sense strand) of this region A second primer combination corresponding to can be used. Examples of such a primer pair include: a) a first primer having at least a partial base sequence of the 1st to 470th base sequence in the base sequence represented by SEQ ID NO: 16 or an equivalent base sequence thereof, and a sequence A combination of second primers having at least a partial base sequence of the 531st to 1000th base sequences in the base sequence shown in No. 16 or a complementary sequence thereof, and b) a base shown in SEQ ID No. 16 A first primer having at least a partial base sequence of the 1st to 470th base sequences in the sequence or a complementary sequence thereof, and the 531st to 1000th base sequences of the base sequence shown in SEQ ID NO: 16; Combination of second primers having at least a part of the base sequence or an equivalent base sequence thereof Like (e.g., see Figure 16).

17) RM23778
The SSR region of RM23778 (eg, “ataa” × 20 in Nipponbare) exists in the region consisting of nucleotide residues 461 to 540 in the base sequence shown in SEQ ID NO: 17 (base sequence around the SSR region of RM23778) (FIG. 17). Therefore, as a primer pair for amplifying the SSR region of RM23778, the first primer corresponding to the upstream site (sense strand or antisense strand) of this region and the downstream site (antisense strand or sense strand) of this region A second primer combination corresponding to can be used. Examples of such a primer pair include: a) a first primer having at least a partial base sequence of the 1st to 460th base sequence in the base sequence represented by SEQ ID NO: 17 or an equivalent base sequence thereof, and a sequence A combination of second primers having at least a part of the base sequence of the 541-1000th base sequence in the base sequence shown in No. 17 or a complementary sequence thereof, and b) the base shown in SEQ ID No. 17 A first primer having at least a partial base sequence of the 1st to 460th base sequence in the sequence or a complementary sequence of the equivalent base sequence, and the 541th to 1000th base sequences of the base sequence shown in SEQ ID NO: 17 Combination of second primers having at least a part of the base sequence or an equivalent base sequence thereof Like (e.g., see Figure 17).

18) RM24481
The SSR region of RM24481 (eg, “act” × 23 in Nipponbare) is present in the region consisting of nucleotide residues 455 to 546 in the base sequence shown in SEQ ID NO: 18 (base sequence around the SSR region of RM24481). (FIG. 18). Therefore, as a primer pair for amplifying the SSR region of RM24481, the first primer corresponding to the upstream site (sense strand or antisense strand) of this region and the downstream site (antisense strand or sense strand) of this region A second primer combination corresponding to can be used. Examples of such a primer pair include: a) a first primer having at least a partial base sequence of the 1st to 454th base sequences in the base sequence represented by SEQ ID NO: 18 or an equivalent base sequence thereof, and a sequence A combination of second primers having at least a part of the base sequence of the 547 to 1000th base sequence in the base sequence shown in No. 18 or a complementary sequence thereof, and b) the base shown in SEQ ID No. 18. A first primer having at least a partial base sequence of the 1st to 454th base sequences in the sequence or a complementary sequence equivalent thereto, and the 547th to 1000th base sequences of the base sequence shown in SEQ ID NO: 18 Combination of second primers having at least a part of the base sequence or an equivalent base sequence thereof Like (e.g., see Figure 18).

19) RM25036
The SSR region of RM25036 (eg, “tatg” × 18 in Nipponbare) exists in the region consisting of the 465th to 536th nucleotide residues in the base sequence shown in SEQ ID NO: 19 (base sequence around the SSR region of RM25036) (FIG. 19). Therefore, as a primer pair for amplifying the SSR region of RM25036, the first primer corresponding to the upstream site (sense strand or antisense strand) of this region and the downstream site (antisense strand or sense strand) of this region A second primer combination corresponding to can be used. Examples of such a primer pair include: a) a first primer having at least a partial base sequence of the 1st to 464th base sequence in the base sequence represented by SEQ ID NO: 19 or an equivalent base sequence thereof, and a sequence A combination of second primers having at least a part of the base sequence of the 537-1000th base sequence in the base sequence shown in No. 19 or a complementary sequence thereof, and b) the base shown in SEQ ID No. 19 A first primer having at least a partial base sequence of the 1st to 464th base sequences in the sequence or a complementary sequence thereof, and the 537th to 1000th base sequences in the base sequence shown in SEQ ID NO: 19; Combination of second primers having at least a part of the base sequence or an equivalent base sequence thereof Like (e.g., see Figure 19).

20) RM25416
The SSR region of RM25416 (eg, “act” x 9 in Nipponbare) is present in the region consisting of nucleotide residues 483 to 518 in the base sequence shown in SEQ ID NO: 20 (base sequence around the SSR region of RM25416) (FIG. 20). Therefore, as a primer pair for amplifying the SSR region of RM25416, the first primer corresponding to the upstream site (sense strand or antisense strand) of this region and the downstream site (antisense strand or sense strand) of this region A second primer combination corresponding to can be used. Examples of such a primer pair include: a) a first primer having at least a partial base sequence of the 1st to 482th base sequences in the base sequence represented by SEQ ID NO: 20 or an equivalent base sequence thereof, and a sequence A combination of second primers having at least a partial base sequence of the 519th to 1000th base sequences in the base sequence shown in No. 20 or a complementary sequence thereof, and b) a base shown in SEQ ID No. 20 A first primer having at least a partial base sequence of the 1st to 482th base sequences in the sequence or a complementary sequence equivalent thereto, and the 519th to 1000th base sequences in the base sequence shown in SEQ ID NO: 20 Combination of second primers having at least a part of the base sequence or an equivalent base sequence thereof Like (e.g., see Figure 20).

21) RM26245
The SSR region of RM26245 (eg, “act” × 12 in Nipponbare) is present in the region consisting of nucleotide residues 477 to 524 in the base sequence shown in SEQ ID NO: 21 (base sequence around the SSR region of RM26245). (FIG. 21). Therefore, as a primer pair for amplifying the SSR region of RM26245, the first primer corresponding to the upstream site (sense strand or antisense strand) of this region and the downstream site (antisense strand or sense strand) of this region A second primer combination corresponding to can be used. As such a primer pair, for example, a) a first primer having at least a partial base sequence of the 1st to 476th base sequence in the base sequence shown in SEQ ID NO: 21 or an equivalent base sequence thereof, and a sequence A combination of second primers having at least a part of the base sequence of the 525 to 1000th base sequence in the base sequence shown in No. 21 or a complementary sequence thereof, and b) the base shown in SEQ ID No. 21 A first primer having at least a partial base sequence of the 1st to 476th base sequence in the sequence or a complementary sequence of the equivalent base sequence, and the 525th to 1000th base sequences of the base sequence shown in SEQ ID NO: 21 Combination of second primers having at least a part of the base sequence or an equivalent base sequence thereof Like (e.g., see Figure 21).

22) RM26957
The SSR region of RM26957 (eg, “atag” × 14 in Nipponbare) exists in the region consisting of nucleotide residues 473 to 528 in the base sequence shown in SEQ ID NO: 22 (base sequence around the SSR region of RM26957) (FIG. 22). Therefore, as a primer pair for amplifying the SSR region of RM26957, the first primer corresponding to the upstream site (sense strand or antisense strand) of this region and the downstream site (antisense strand or sense strand) of this region A second primer combination corresponding to can be used. Examples of such a primer pair include: a) a first primer having at least a partial base sequence of the 1st to 472nd base sequences in the base sequence represented by SEQ ID NO: 22 or an equivalent base sequence thereof, and a sequence A combination of second primers having at least a partial base sequence of the 529th to 1000th base sequences in the base sequence shown in No. 22 or a complementary sequence thereof, and b) a base shown in SEQ ID No. 22 A first primer having at least a partial base sequence of the 1st to 472th base sequences in the sequence or a complementary sequence thereof, and the 529th to 1000th base sequences in the base sequence represented by SEQ ID NO: 22 Combination of second primers having at least a part of the base sequence or an equivalent base sequence thereof Like (e.g., see Figure 22).

23) RM27695
The SSR region of RM27695 (eg, “tgta” × 13 in Nipponbare) exists in the region consisting of nucleotide residues 475 to 526 in the base sequence shown in SEQ ID NO: 23 (base sequence around the SSR region of RM27695) (FIG. 23). Therefore, as a primer pair for amplifying the SSR region of RM27695, the first primer corresponding to the upstream site (sense strand or antisense strand) of this region and the downstream site (antisense strand or sense strand) of this region A second primer combination corresponding to can be used. Examples of such a primer pair include: a) a first primer having at least a partial base sequence of the 1st to 474th base sequences in the base sequence represented by SEQ ID NO: 23 or an equivalent base sequence thereof, and a sequence A combination of second primers having at least a part of the base sequence of the 527 to 1000th base sequence in the base sequence shown in No. 23 or a complementary sequence thereof, and b) a base shown in SEQ ID No. 23 A first primer having at least a partial base sequence of the 1st to 474th base sequences in the sequence or a complementary sequence thereof, and the 527th to 1000th base sequences in the base sequence shown in SEQ ID NO: 23 Combination of second primers having at least a part of the base sequence or an equivalent base sequence thereof Like (e.g., see Figure 23).

24) RM28174
The SSR region of RM28174 (eg, “tatg” × 15 in Nipponbare) exists in the region consisting of nucleotide residues 471 to 530 in the base sequence shown in SEQ ID NO: 24 (base sequence around the SSR region of RM28174). (FIG. 24). Therefore, as a primer pair for amplifying the SSR region of RM28174, the first primer corresponding to the upstream site (sense strand or antisense strand) of this region and the downstream site (antisense strand or sense strand) of this region A second primer combination corresponding to can be used. Examples of such a primer pair include: a) a first primer having at least a partial base sequence of the 1st to 470th base sequences in the base sequence represented by SEQ ID NO: 24 or an equivalent base sequence thereof, and a sequence A combination of second primers having at least a part of the 531 to 1000th base sequence in the base sequence shown in No. 24 or a complementary sequence of the equivalent base sequence, and b) a base shown in SEQ ID No. 24 A first primer having at least a partial base sequence of the 1st to 470th base sequences in the sequence or a complementary sequence thereof, and the 531st to 1000th base sequences of the base sequence shown in SEQ ID NO: 24 Combination of second primers having at least a part of the base sequence or an equivalent base sequence thereof Like (e.g., see Figure 24).

The “equivalent base sequence” described above may be a base sequence corresponding to a variation in base sequence among rice varieties. Accordingly, a predetermined base sequence (e.g., Y～Z th least a portion of the nucleotide sequence of the nucleotide sequence in the nucleotide sequence shown in SEQ ID NO: X) in equivalent nucleotide sequence, Ri several (good in a given nucleotide sequence The base sequence may preferably include 1, 2 or 3) base modification (substitution, deletion, insertion or addition). Such an equivalent base sequence is obvious to those skilled in the art, and is identified by the RM number by referring to a database in which genetic information of rice varieties is registered or in rice varieties. This can be determined by decoding the base sequence around the SSR region.

  The number of nucleotide residues constituting the primer (the nucleotide residue of the complementary portion that anneals to the target site) is as long as the primer anneals to the target site and specifically amplifies the target product containing a 4-base repeat SSR region. It is not specifically limited, For example, 15 or more, 16 or more, 17 or more, 18 or more, or 20 or more may be sufficient. The number of nucleotide residues constituting the primer may be, for example, 50 or less, 40 or less, or 30 or less.

  The primer may be labeled with a fluorescent substance for detection of the amplification product. Examples of such fluorescent materials include 6-FAM, VIC, NED, and PET. The primer may also contain other parts in addition to the complementary part that anneals to the target site. Examples of such other part include a part for adding an A residue to the end of the amplification product (eg, gttcttt). If there is no such moiety for adding an A residue, a mixture of those with and without the addition of an A residue at the end of the amplification product may be obtained as the amplification product. In such a case, the peak detected by a detector such as a sequencer may be bifurcated, which is not preferable. Therefore, in order to detect one sharp peak, it is preferable to use such a portion.

  The size of the amplification product is not particularly limited as long as it can detect the difference in amplification length based on the genetic polymorphism of the SSR region of the 4-base repeat in the measuring instrument. For example, when a sequencer is used to determine the size of the amplification product, even a single base length difference can be detected, so that the amplification length difference based on the genetic polymorphism of the SSR region of the 4-base repeat is increased. Sensitivity can be detected. The lower limit size of the amplification product is not particularly limited as long as the size of the amplification product can be detected in the measuring instrument used, but may be, for example, 50 bp or more, 100 bp or more, 150 bp or more, 200 bp or more. The upper limit size of the amplification product is not particularly limited as long as the size of the amplification product can be detected in the measuring instrument used, but may be, for example, 500 bp or less, 450 bp or less, 400 bp or less, 350 bp or less, or 300 bp or less. Thus, primer pairs can be designed for two regions that are separated by such lengths so that amplification products of such length are amplified.

  As described above, the method for identifying rice and other varieties can be performed using a gene amplification method such as a PCR method, but the present invention also includes two or more primer pairs that can be used for such applications. Provide a set of primer pairs. There is no conventional motivation to combine two or more of the above primer pairs without the knowledge of the present invention. Two or more primer pairs included in the set of the present invention may be labeled with different fluorescent substances that emit different fluorescent colors. Using two or more primer pairs labeled with such separate fluorescent substances, the SSR region having a 4-base repeat is multiplexed and multiple amplification products emitting different fluorescent colors are obtained. By applying to one lane of the sequencer, the genetic polymorphism of the SSR region of a plurality of 4-base repeats can be efficiently analyzed (eg, see FIG. 2). Although the number of primer pairs included in the set of the present invention is not particularly limited as long as it is 2 or more, the upper limit number may be prepared according to the variation of the fluorescent color. Accordingly, the number of primer pairs included in the set of the present invention may be, for example, 2, 3, or 4.

The set of the present invention includes, for example, 2 or more primer combinations selected from the group consisting of the following (eg, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more) 12 or more) may include:
1) A combination of any one of the above-mentioned 1a) and 1b);
2) a combination of any one of the primers 2a) and 2b) above;
3) a combination of any one of the above-mentioned 3a) and 3b);
4) a combination of any one of the primers 4a) and 4b) above;
5) a combination of any one of the primers 5a) and 5b) above;
6) a combination of any one of the primers 6a) and 6b) above;
7) A combination of any one of the primers 7a) and 7b) above;
8) A combination of any one of the primers 8a) and 8b) above;
9) a combination of any one of the primers 9a) and 9b) above;
10) A combination of any one of the primers 10a) and 10b) above;
11) A combination of any one of the above-mentioned 11a) and 11b);
12) A combination of any one of the primers 12a) and 12b) above;
13) a combination of any one of the primers 13a) and 13b) above;
14) A combination of any one of the primers 14a) and 14b) above;
15) A combination of any one of the primers 15a) and 15b) above;
16) A combination of any one of the primers 16a) and 16b) above;
17) A combination of any one of the primers of 17a) and 17b) above;
18) A primer combination of any one of 18a) and 18b) above;
19) A primer combination of any one of 19a) and 19b) above;
20) A combination of any one of the primers 20a) and 20b) above;
21) a primer combination of any one of 21a) and 21b) above;
22) a combination of any one of the primers of 22a) and 22b) above;
23) a primer combination of any one of 23a) and 23b) above;
24) A combination of any one of the primers 24a) and 24b) above.

  More specifically, the identification method of the present invention is achieved by identifying a variety such as rice based on the genetic polymorphism of the SSR region of 4-base repeat in the test genomic DNA. The test genomic DNA can be prepared from rice or the like (eg, rice) by a method known per se. Specifically, rice or other varieties (eg, rice) are identified by: a) extracting genomic DNA from a tissue derived from rice (eg, rice) or a processed product of rice or rice-derived tissue; b) Using a gene amplification method such as the PCR method, a portion containing a 4-base repeat SSR region is amplified, and c) the amplification length of the 4-base repeat SSR region is determined (eg, a 4-base repeat SSR region (24 types)). Genotype was identified), and then the determined 4-base repeat SSR region amplification length information was matched with the 4-base repeat SSR region amplification length information for each variety (eg, identified) The genotype information is checked against the genotype information of each variety). Specifically, collation may be performed using data (for example, Tables 1 to 5 and Tables 12 to 14) that disclose information on the amplification length of the SSR region having a 4-base repeat. The collation is also performed by comparing the outline of the detection peak of the amplified product in which the portion containing the SSR region of 4 base repeats is amplified between the test sample and the sample by using the sample together. Also good. The specimen is an arbitrary rice variety and is not particularly limited, and examples thereof include Nipponbare, which is a standard variety, and specific varieties suspected to be the same variety as the test object. The appraisal method of the present invention is useful for, for example, determining rice varieties and determining the presence or absence of mixed rice.

  In the appraisal method of the present invention, the SSR region of the 4-base repeat specified by the RM number described above can be used, but depending on the purpose, the SSR region of the 4-base repeat specified by the specific RM number is used. Also good.

  For example, an SSR region rich in genetic polymorphism among rice varieties can be suitably used because it contributes to efficient variety identification. The genetic polymorphism of the SSR region is apparent from Table 4 below, for example. Therefore, in the present invention, for example, an SSR region (RM12927, RM14137, RM15764, RM16604, RM23036, RM24436, RM25036, RM26957, RM28174) in which at least 5 types of genetic polymorphisms are recognized, at least 6 types or more SSR regions (RM12927, RM25036, RM26957) in which types are recognized, and SSR regions (RM12927) in which at least 8 types of genetic polymorphisms are observed may be suitably used in breeding.

  In addition, the SSR region showing a genetic polymorphism that is highly specific (rare) for a specific rice variety (that is, having a high discriminability for a specific variety) contributes to the specific variety identification, and is therefore preferable. Can be used. The genetic polymorphism of the SSR region having high specificity for a specific variety is apparent from, for example, Table 5 below. Therefore, in the present invention, for example, when inspection of the following specific varieties is required (for example, when identification of the following specific varieties is required, and mixing of rice of the following specific varieties is suspected. In the case), the SSR region specified by the following specific RM number showing a genetic polymorphism specific to the following specific breed may be suitably used in the breed identification.

  The present invention will be described in detail below, but the present invention is not limited to the examples.

Example 1: Protocol for variety identification from cooked rice (granular) [general matters]
Based on the analysis result of the original species obtained from the breeding place or the largest production area (Table 6), the kind is discriminated by comparing the analysis result of the inspection object.

[Principle of measurement]
Using a primer that specifically amplifies a region containing a simple sequence repeat (SSR) that exists on genomic DNA, a DNA fragment is amplified by PCR, and cultivar identification is performed using the difference in length. Do.

[Summary of method]
DNA is extracted from one grain of cooked rice using an extraction kit (GM quicker 2). Using the DNA as a template, a DNA region containing SSR is PCR-amplified using a primer pair that is fluorescently labeled on one side. The resulting fluorescently labeled DNA fragment is electrophoresed using a Genetic Analyzer to detect the fragment length. This fragment analysis is performed on all DNA fragments obtained from the 24 primer sets, and the varieties are identified by comparing them with the reference varieties.

[Inspection sample]
One grain of rice is used as a sample.

[Purchased reagent]
-Isopropyl alcohol (99.5%, special grade)
-GM quicker 2 (Nippon Gene, Cat. No. 310-065091)
-Gotaq Colorless Master Mix (Promega, Cat. No. M7132 (5 mL), M7133 (25 mL))
HiDi formamide (Applied Biosystems, Cat. No. 431320)
-GeneScan 500LIZ Size Standard (Applied Biosystems, Cat. No. 4352759)
-Genetic Analyzer Buffer with EDTA (Applied Biosystems, Cat. No. 402824)
-POP-4 polymer (Applied Biosystems, depending on the Genetic Analyzer used)
-Primers (Table 7)

(Preparation reagent)
-Primer Mix
Equal amounts of fluorescently labeled primer (forward primer) and unlabeled primer (reverse primer) (both 100 μM stock solution) are mixed, and the mixture is diluted 50-fold with sterilized water (final concentration 1 μM each). Store frozen at -20 ° C.

[Equipment / Program]
-Microcentrifuge (a centrifugal force of 13,000 xg or more can be obtained and a 1.5 mL tube can be centrifuged)
-Vortex mixer-Constant temperature water bath or heat block (1.5 mL tube can be heated to 60 ° C)
-Thermal cycler (executable the reaction program shown in 4.6.6. In the developer, GeneAmp PCR System 9700 (Applied Biosystems), iCycler (Bio-Rad), Thermal Cycler Dice (Takara Bio) Confirmed)
-Genetic Analyzer (Applied Biosystems)
-GeneMapper software (Applied Biosystems)

[Experimental operation]
A) Operation preparation work The operator wears a lab coat, glasses and gloves.

B) Operation location From DNA extraction to PCR amplification reaction solution preparation, operate in a clean environment where DNA other than the test sample is not mixed.

C) Reagents and instruments-Sterile distilled water-Isopropyl alcohol (99.5%, special grade)
-GM quicker 2 (Nippon Gene, Cat. No. 310-065091)
-Gotaq Colorless Master Mix (Promega, Cat. No. M7132 (5 mL), M7133 (25 mL))
HiDi formamide (Applied Biosystems, Cat. No. 431320)
-GeneScan 500LIZ Size Standard (Applied Biosystems, Cat. No. 4352759)
-Genetic Analyzer Buffer with EDTA (Applied Biosystems, Cat. No. 402824)
-Primer Mix (see 4.4)
-1.5 mL microtube (used as autoclave)
96 well PCR plate and plate seal (or cap)
− Pipette tips (used as autoclave)
-Micropipette-Homogenization pestle (disposable)
-Microcentrifuge (a centrifugal force of 13,000 xg or more can be obtained and a 1.5 mL tube can be centrifuged)
-Vortex mixer-Constant temperature water bath or heat block (1.5mL tube can be heated to 60 ° C)
-Thermal cycler-Genetic Analyzer (Applied Biosystems)

D) Taking out test material One sample of cooked rice is used. Do not use damaged grains or other grains attached to them. If there is water droplets on the container wall surface, such as packaged cooked rice, collect it so that it does not touch the water droplets.

E) DNA extraction operation In this step, GM quicker 2 (Nippon Gene) is used. Reagents other than isopropanol and Spin Column use the kit contents.
(1) Take 1 grain of cooked rice in a 1.5 mL microtube and add 250 μL of GE1 Buffer.
(2) Using a homogenization pestle, the cooked rice is crushed until the solid substance disappears and becomes a paste.
(3) Add 10 μL of Proteinase K, 2 μL of α-Amylase, and 5 μL of RNase A to the disrupted solution. Stir for 30 seconds in a vortex mixer.
(4) Heat at 60 ° C. for 15 minutes in a constant temperature water bath or heat block. Stir with a vortex mixer every 5 minutes.
(5) Add 40 μL of GE2-K Buffer and mix well with a vortex mixer.
(6) Centrifuge at 13,000 × g or more for 5 minutes at room temperature.
(7) Collect 280 μL of the supernatant in a new 1.5 mL tube while avoiding as much of the precipitate or suspended matter as possible.
(8) After adding 105 μL GB3 Buffer and 105 μL isopropanol, invert the tube vigorously 10-12 times and mix well. If precipitates appear and become cloudy, mix well until the liquid is clear.
(9) Transfer the total amount of the mixture of (8) to Spin Column, and centrifuge at 13,000 xg or more for 30 seconds at room temperature. Discard the filtrate.
(10) Add 650 μL of GW Buffer to Spin Column, and centrifuge at 13,000 × g or more for 1 minute at room temperature. Discard the filtrate.
(11) Transfer Spin Column to a new 1.5 mL tube and add 50 μL of TE (pH 8.0) dropwise.
(12) After standing at room temperature for 3 minutes, centrifuge at 13,000 × g or more for 1 minute at room temperature. The obtained eluate is used as a DNA solution. The DNA solution is stored at 4 ° C for short-term and at -20 ° C for long-term.

F) PCR amplification operation This step uses Gotaq Colorless Master Mix (Promega) in which PCR enzymes, buffers, and dNTPs are mixed in advance. Preparation of the reaction solution is basically performed on ice or in an environment conforming thereto.

(1) Prepare a premix of 25 reactions (24 primers + 1) for each DNA solution.

(2) 24 types of primer mix (1 μM each, see 4.4) is added to 24 wells of the PCR plate by 2.5 μL each.
(3) Mix the premix of (1) well with a vortex mixer, spin down, and dispense 7.5 μL into 24 wells of (2).
(4) Close the plate with a seal or cap, spin down the solution, and then perform an amplification reaction according to the following program. The reaction conditions are common to all Primers.

G) Detection of DNA marker (1) In order to perform electrophoresis efficiently, the 24 PCR products ag are mixed (Pooling) (see Table 3. Each set consists of 4 fluorescent labels) One by one). Further, in order to adjust the concentration of the PCR product, the mixing ratio is also adjusted at the time of Pooling. In Table 3, the dilution factor (20 to 160 times) for each PCR product employed by the developer is described as a guide, but since it varies depending on the equipment used, the dilution factor is changed as appropriate. Table 3 also shows an example of preparing a diluted sample of 160 μL, but it is not always necessary to follow this, and dilution and mixing are performed in an efficient manner.

(2) Prepare an electrophoresis sample in a 96-well plate as follows. For Size Standard and HiDi formamide, a mixed solution of the number of samples plus one minute may be prepared in advance, and 9 μL of it may be added to 1 μL of the diluted PCR solution.

(3) Using a thermal cycler, heat at 95 ° C. for 5 minutes and immediately leave on ice for 5 minutes.
(4) Analysis is performed using a POP-4 polymer, Genetic Analyzer Buffer with EDTA, and capillary according to the fragment analysis protocol of the Genetic Analyzer model to be used.
(5) Analyze the results using GeneMapper software.

Example 2: Identification of genotypes of rice varieties According to the protocol described in Example 1, genotypes of 4-base repeat SSR regions (24 species) in which genetic polymorphism was observed for each variety Was identified. The results are shown in Tables 12-14. In Tables 12 to 14, the genotype is expressed by how many bp away from the value, with “Nippon Hare” as a reference [= A (bp)]. FIG. 2 shows an example of genotype analysis of 8 SSR regions according to the present invention. In addition, since all 24 types of markers were prepared using an SSR region having a 4-base repeat, a value separated by a multiple of 4 was basically detected except for some of the markers.

Example 3: Identification of rice varieties The assay is performed according to items A) to G) of [Experimental operations] in Example 1. Next, rice varieties are identified by collating the obtained genotype information with the information in Tables 1 to 3 and / or Tables 4 and 5 or Tables 12 to 14. Note that the blank part in the table is not used for determination because genetic polymorphism may be found in the breed.

Reference Example 1: Evaluation of genetic polymorphism of a specific SSR region among varieties of glutinous rice SSR regions of other four base repeats (18 species: RM11489, RM12281, RM12333, RM14272, RM16282, RM17331, RM17822, RM18102, RM20183, RM21172, RM21802, RM22195, RM24461, RM25283, RM25733, RM26012, RM27824, RM28504) were evaluated for genetic polymorphism among rice varieties. The rice varieties evaluated were 16 varieties of glutinous rice (Nipponbare, Koshihikari, Hitomebore, Akitakomachi, Hinohikari, Haenuki, Nanatsubo, Kirara 397, Tsugaru Romance, Shinigura, Kinuhikari, Koishibuki, Asahi no Yume, Hoshino Yume, Yume Tsukushi, Kaori Aichi).
As a result, the SSR region (18 species) showed no genetic polymorphism among the 16 varieties.

Claims (3)

The rice or tissue derived from it, or the processed product thereof is identified based on the genetic polymorphism of the SSR region of 4 base repeats in the test genomic DNA. A method for identifying varieties ,
Use RM12927 for appraisal of Asahi, Koganemochi or Nishiki Yamada,
Use RM14137 for cypress or five million stones,
Using RM16604 for appraisal of cypress or cotton buds,
Using RM17039 for Asahi appraisal,
Using RM18204 for the evaluation of Aya no Kagayaki,
Using RM22194 for appraisal of splashes;
Using RM23036 for appraisal of rice cake or Miyama Nishiki,
Using RM24481 for appraisal of cypress,
Using RM25036 for the identification of straight or hanaetizen,
Use RM26957 for autumn poetry or appraisal of Himenomochi, and / or
Use RM28174 for appraisal of natsubo or crispness;
An appraisal method characterized by The identification method according to claim 1, wherein a primer pair for amplifying an SSR region having 4 base repeats in rice, which is a combination of primers selected from the group consisting of :
3 a) a first primer having at least a part of the 1st to 454th base sequences in the base sequence shown in SEQ ID NO: 3 or an equivalent base sequence thereof, and 547 in the base sequence shown in SEQ ID NO: 3 A combination of second primers having at least a part of the base sequence of the 1000 th base sequence or a sequence complementary to the equivalent base sequence;
3b) a first primer having at least a partial base sequence of the 1st to 454th base sequences in the base sequence shown in SEQ ID NO: 3 or a complementary sequence thereof, and the base sequence shown in SEQ ID NO: 3 A second primer combination having at least a part of the base sequence of the 547th to 1000th base sequences or an equivalent base sequence thereof;
4a) a first primer having at least a partial base sequence of the 1st to 470th base sequences in the base sequence shown in SEQ ID NO: 4 or an equivalent base sequence thereof, and 531-1 in the base sequence shown in SEQ ID NO: 4 A combination of second primers having at least a part of the base sequence of the 1000th base sequence or a complementary sequence of the equivalent base sequence;
4b) a first primer having at least a partial base sequence of the 1st to 470th base sequence in the base sequence shown in SEQ ID NO: 4 or a complementary sequence thereof, and the base sequence shown in SEQ ID NO: 4 A combination of second primers having at least a part of the 531 to 1000th base sequences in SEQ ID NO .
7 a) a first primer having at least a part of the 1st to 472nd base sequences in the base sequence shown in SEQ ID NO: 7 or an equivalent base sequence thereof, and 529 in the base sequence shown in SEQ ID NO: 7 A combination of second primers having at least a part of the base sequence of the 1000 th base sequence or a sequence complementary to the equivalent base sequence;
7b) a first primer having at least a partial base sequence of the 1st to 472nd base sequences in the base sequence shown in SEQ ID NO: 7 or a complementary sequence thereof, and the base sequence shown in SEQ ID NO: 7 A combination of second primers having at least a part of the base sequences of the 529 to 1000th base sequences in FIG.
8a) a first primer having at least a partial base sequence of the 1st to 476th base sequences in the base sequence shown in SEQ ID NO: 8 or an equivalent base sequence thereof, and 525 in the base sequence shown in SEQ ID NO: 8 A combination of second primers having at least a part of the base sequence of the 1000th base sequence or a complementary sequence of the equivalent base sequence;
8b) a first primer having at least a partial base sequence of the 1st to 476th base sequence in the base sequence shown in SEQ ID NO: 8 or a complementary sequence thereof, and the base sequence shown in SEQ ID NO: 8 A combination of second primers having at least a part of the base sequence of the 525 to 1000th base sequence or an equivalent base sequence thereof;
9a) a first primer having at least a partial base sequence of the 1st to 470th base sequences in the base sequence shown in SEQ ID NO: 9 or an equivalent base sequence thereof, and 531-1 in the base sequence shown in SEQ ID NO: 9 A combination of second primers having at least a part of the base sequence of the 1000th base sequence or a complementary sequence of the equivalent base sequence;
9b) a first primer having at least a partial base sequence of the 1st to 470th base sequences in the base sequence shown in SEQ ID NO: 9 or a complementary sequence equivalent thereto, and the base sequence shown in SEQ ID NO: 9 A combination of second primers having at least a part of the 531 to 1000th base sequences in SEQ ID NO .
1 5a) arranged first primer 527 in the nucleotide sequence and shown in SEQ ID NO: 15, having at least a portion of the nucleotide sequence or its equivalent nucleotide sequence of 1 to 473 th nucleotide sequence in the nucleotide sequence shown in ID NO: 15 A combination of second primers having at least a part of the base sequence of the 1000 th base sequence or a sequence complementary to the equivalent base sequence;
15b) a first primer having at least a partial base sequence of the 1st to 473th base sequences in the base sequence shown in SEQ ID NO: 15 or a complementary sequence thereof, and the base sequence shown in SEQ ID NO: 15 A combination of second primers having at least a part of the base sequence of the 527 to 1000th base sequence or an equivalent base sequence thereof;
16a) a first primer having at least a partial base sequence of the 1st to 470th base sequences in the base sequence shown in SEQ ID NO: 16 or an equivalent base sequence thereof, and 531-1 in the base sequence shown in SEQ ID NO: 16 A combination of second primers having at least a part of the base sequence of the 1000th base sequence or a complementary sequence of the equivalent base sequence;
16b) a first primer having at least a partial base sequence of the 1st to 470th base sequences in the base sequence shown in SEQ ID NO: 16 or a complementary sequence thereof, and the base sequence shown in SEQ ID NO: 16 A combination of second primers having at least a part of the 531 to 1000th base sequences in SEQ ID NO .
1 8a) sequence the first primer 547 in the nucleotide sequence and shown in SEQ ID NO: 18, having at least a portion of the nucleotide sequence or its equivalent nucleotide sequence of 1 to 454 th nucleotide sequence in the nucleotide sequence shown in ID NO: 18 A combination of second primers having at least a part of the base sequence of the 1000 th base sequence or a sequence complementary to the equivalent base sequence;
18b) a first primer having at least a partial base sequence of the 1st to 454th base sequences in the base sequence shown in SEQ ID NO: 18 or a complementary sequence thereof, and the base sequence shown in SEQ ID NO: 18 A second primer combination having at least a part of the base sequence of the 547th to 1000th base sequences or an equivalent base sequence thereof;
19a) a first primer having at least a partial base sequence of the 1st to 464th base sequences in the base sequence shown in SEQ ID NO: 19 or an equivalent base sequence thereof, and 537 in the base sequence shown in SEQ ID NO: 19 A combination of second primers having at least a part of the base sequence of the 1000th base sequence or a complementary sequence of the equivalent base sequence;
19b) a first primer having at least a partial base sequence of the 1st to 464th base sequences in the base sequence shown in SEQ ID NO: 19 or a complementary sequence thereof, and the base sequence shown in SEQ ID NO: 19 A combination of second primers having at least a part of the base sequence of the 537-1000th base sequence or an equivalent base sequence thereof ;
2 2a) First primer having at least a part of the base sequence of positions 1 to 472 in the base sequence shown in SEQ ID NO: 22 or an equivalent base sequence thereof, and 529 in the base sequence shown in SEQ ID NO: 22 A combination of second primers having at least a part of the base sequence of the 1000 th base sequence or a sequence complementary to the equivalent base sequence;
22b) a first primer having at least a partial base sequence of the 1st to 472nd base sequences in the base sequence shown in SEQ ID NO: 22 or a complementary sequence thereof, and the base sequence shown in SEQ ID NO: 22 A combination of second primers having at least a part of the base sequences of the 529 to 1000th base sequences in FIG .
2 4a) The first primer having at least a part of the 1st to 470th nucleotide sequences in the nucleotide sequence shown in SEQ ID NO: 24 or an equivalent nucleotide sequence thereof, and 531 in the nucleotide sequence shown in SEQ ID NO: 24 A combination of second primers having at least a part of the base sequence of the 1000 th base sequence or a sequence complementary to the equivalent base sequence;
24b) a first primer having at least a partial base sequence of the 1st to 470th base sequences in the base sequence shown in SEQ ID NO: 24 or a complementary sequence thereof, and the base sequence shown in SEQ ID NO: 24 A combination of second primers having at least a part of the 531 to 1000th base sequence in SEQ .
And
Here, the at least part of the base sequence consists of 15 or more nucleotide residues,
The equivalent base sequence consists of 15 or more nucleotide residues, and corresponds to variations in the base sequence among rice varieties . Unrealized combination of primers selected from the group consisting of 2 or more, and at least one of the two or more combining uses a set of primer pairs comprising a combination of primers of claim 2, in claim 2, wherein Appraisal method:
1) The following 1a) and 1b either one primer combinations of);
1a) a first primer having at least a partial base sequence of the 1st to 470th base sequences in the base sequence shown in SEQ ID NO: 1 or an equivalent base sequence thereof, and 531-1 in the base sequence shown in SEQ ID NO: 1 A combination of second primers having at least a part of the base sequence of the 1000th base sequence or a complementary sequence of the equivalent base sequence;
1b) a first primer having at least a partial base sequence of the 1st to 470th base sequence in the base sequence shown in SEQ ID NO: 1 or a complementary sequence thereof, and the base sequence shown in SEQ ID NO: 1 A combination of second primers having at least a part of the 531 to 1000th base sequences in SEQ ID NO.
2) a primer combination of any one of 2a) and 2b) below ;
2a) a first primer having at least a partial base sequence of the 1st to 474th base sequences in the base sequence shown in SEQ ID NO: 2 or an equivalent base sequence thereof, and 527 to in the base sequence shown in SEQ ID NO: 2 A combination of second primers having at least a part of the base sequence of the 1000th base sequence or a complementary sequence of the equivalent base sequence;
2b) a first primer having at least a partial base sequence of the 1st to 474th base sequences in the base sequence shown in SEQ ID NO: 2 or a complementary sequence thereof, and the base sequence shown in SEQ ID NO: 2 A combination of second primers having at least a part of the base sequence of the 527 to 1000th base sequence or an equivalent base sequence thereof;
3) The combination of any one of the primers of 3a) and 3b) according to claim 2 ;
4) A combination of any one of the primers 4a) and 4b) according to claim 2 ;
5) a primer combination of any one of 5a) and 5b) below ;
5a) a first primer having at least a partial base sequence of the 1st to 472nd base sequences in the base sequence shown in SEQ ID NO: 5 or an equivalent base sequence thereof, and 529 to in the base sequence shown in SEQ ID NO: 5 A combination of second primers having at least a part of the base sequence of the 1000th base sequence or a complementary sequence of the equivalent base sequence;
5b) a first primer having at least a partial base sequence of the 1st to 472nd base sequences in the base sequence shown in SEQ ID NO: 5 or a complementary sequence thereof, and the base sequence shown in SEQ ID NO: 5 A combination of second primers having at least a part of the base sequences of the 529 to 1000th base sequences in FIG.
6) The following 6a) and 6b any one primer combinations of);
6a) a first primer having at least a partial base sequence of the 1st to 476th base sequences in the base sequence shown in SEQ ID NO: 6 or an equivalent base sequence thereof, and 525 in the base sequence shown in SEQ ID NO: 6 A combination of second primers having at least a part of the base sequence of the 1000th base sequence or a complementary sequence of the equivalent base sequence;
6b) a first primer having at least a partial base sequence of the 1st to 476th base sequence in the base sequence shown in SEQ ID NO: 6 or a complementary sequence thereof, and the base sequence shown in SEQ ID NO: 6 A combination of second primers having at least a part of the base sequence of the 525 to 1000th base sequence or an equivalent base sequence thereof;
7) The primer combination of any one of 7a) and 7b) according to claim 2 ;
8) The primer combination of any one of 8a) and 8b) according to claim 2 ;
9) The primer combination of any one of 9a) and 9b) according to claim 2 ;
10) The following 10a) and 10b one of primer combinations of);
10a) a first primer having at least a partial base sequence of the 1st to 455th base sequences in the base sequence represented by SEQ ID NO: 10 or an equivalent base sequence thereof, and 545 to 545 in the base sequence represented by SEQ ID NO: 10 A combination of second primers having at least a part of the base sequence of the 1000th base sequence or a complementary sequence of the equivalent base sequence;
10b) a first primer having at least a partial base sequence of the 1st to 455th base sequences in the base sequence shown in SEQ ID NO: 10 or a complementary sequence thereof, and the base sequence shown in SEQ ID NO: 10 A combination of second primers having at least a part of the base sequence from the 545th to the 1000th base sequence or an equivalent base sequence thereof;
11) A primer combination of any one of 11a) and 11b) below ;
11a) a first primer having at least a partial base sequence of the 1st to 476th base sequences in the base sequence shown in SEQ ID NO: 11 or an equivalent base sequence thereof, and 525 in the base sequence shown in SEQ ID NO: 11 A combination of second primers having at least a part of the base sequence of the 1000th base sequence or a complementary sequence of the equivalent base sequence;
11b) a first primer having at least a partial base sequence of the 1st to 476th base sequence in the base sequence shown in SEQ ID NO: 11 or a complementary sequence thereof, and the base sequence shown in SEQ ID NO: 11 A combination of second primers having at least a part of the base sequence of the 525 to 1000th base sequence or an equivalent base sequence thereof;
12) a primer combination of any one of 12a) and 12b) below ;
12a) a first primer having at least a partial base sequence of the 1st to 471st base sequences in the base sequence shown in SEQ ID NO: 12 or an equivalent base sequence thereof, and 529 to in the base sequence shown in SEQ ID NO: 12 A combination of second primers having at least a part of the base sequence of the 1000th base sequence or a complementary sequence of the equivalent base sequence;
12b) a first primer having at least a partial base sequence of the 1st to 471st base sequences in the base sequence shown in SEQ ID NO: 12 or a complementary sequence thereof, and the base sequence shown in SEQ ID NO: 12 A combination of second primers having at least a part of the base sequences of the 529 to 1000th base sequences in FIG.
13) The following 13a) and 13b one of primer combinations of);
13a) a first primer having at least a partial base sequence of the 1st to 459th base sequences in the base sequence shown in SEQ ID NO: 13 or an equivalent base sequence thereof, and 541 to 541 in the base sequence shown in SEQ ID NO: 13 A combination of second primers having at least a part of the base sequence of the 1000th base sequence or a complementary sequence of the equivalent base sequence;
13b) a first primer having at least a partial base sequence of the 1st to 459th base sequences in the base sequence shown in SEQ ID NO: 13 or a complementary sequence thereof, and the base sequence shown in SEQ ID NO: 13 A combination of second primers having at least a part of the base sequence from the 541st to the 1000th base sequence or an equivalent base sequence thereof;
14) a primer combination of any one of 14a) and 14b) below ;
14a) a first primer having at least a partial base sequence of the 1st to 464th base sequences in the base sequence shown in SEQ ID NO: 14 or an equivalent base sequence thereof, and 537 in the base sequence shown in SEQ ID NO: 14 A combination of second primers having at least a part of the base sequence of the 1000th base sequence or a complementary sequence of the equivalent base sequence;
14b) a first primer having at least a partial base sequence of the 1st to 464th base sequences in the base sequence shown in SEQ ID NO: 14 or a complementary sequence thereof, and the base sequence shown in SEQ ID NO: 14 A combination of second primers having at least a part of the base sequence of the 537-1000th base sequence or an equivalent base sequence thereof;
15) A combination of any one of the primers 15a) and 15b) according to claim 2 ;
16) The primer combination of any one of 16a) and 16b) according to claim 2 ;
17) The following 17a) and 17b one of primer combinations of);
17a) a first primer having at least a partial base sequence of the 1st to 460th base sequences in the base sequence shown in SEQ ID NO: 17 or an equivalent base sequence thereof, and 541 to 541 in the base sequence shown in SEQ ID NO: 17 A combination of second primers having at least a part of the base sequence of the 1000th base sequence or a complementary sequence of the equivalent base sequence;
17b) a first primer having at least a partial base sequence of the 1st to 460th base sequences in the base sequence shown in SEQ ID NO: 17 or a complementary sequence thereof, and the base sequence shown in SEQ ID NO: 17 A combination of second primers having at least a part of the base sequence from the 541st to the 1000th base sequence or an equivalent base sequence thereof;
18) The primer combination of any one of 18a) and 18b) according to claim 2 ;
19) A primer combination of any one of 19a) and 19b) according to claim 2 ;
20) a primer combination of any one of 20a) and 20b) below ;
20a) a first primer having at least a partial base sequence of the 1st to 482th base sequences in the base sequence shown in SEQ ID NO: 20 or an equivalent base sequence thereof, and 519 in the base sequence shown in SEQ ID NO: 20 A combination of second primers having at least a part of the base sequence of the 1000th base sequence or a complementary sequence of the equivalent base sequence;
20b) a first primer having at least a partial base sequence of the 1st to 482th base sequences in the base sequence shown in SEQ ID NO: 20 or a complementary sequence thereof, and the base sequence shown in SEQ ID NO: 20 A combination of second primers having at least a part of the base sequence from the 519th to the 1000th base sequence or an equivalent base sequence thereof;
21) a primer combination of any one of 21a) and 21b) below ;
21a) a first primer having at least a partial base sequence of the 1st to 476th base sequences in the base sequence shown in SEQ ID NO: 21 or an equivalent base sequence thereof, and 525 in the base sequence shown in SEQ ID NO: 21 A combination of second primers having at least a part of the base sequence of the 1000th base sequence or a complementary sequence of the equivalent base sequence;
21b) a first primer having at least a partial base sequence of the 1st to 476th base sequence in the base sequence shown in SEQ ID NO: 21 or a complementary sequence thereof, and the base sequence shown in SEQ ID NO: 21 A combination of second primers having at least a part of the base sequence of the 525 to 1000th base sequence or an equivalent base sequence thereof;
22) The primer combination of any one of 22a) and 22b) according to claim 2 ;
23) The following 23a) and 23b one of primer combinations of);
23a) a first primer having at least a partial base sequence of the 1st to 474th base sequences in the base sequence shown in SEQ ID NO: 23 or an equivalent base sequence thereof, and 527 to in the base sequence shown in SEQ ID NO: 23 A combination of second primers having at least a part of the base sequence of the 1000th base sequence or a complementary sequence of the equivalent base sequence;
23b) a first primer having at least a partial base sequence of the 1st to 474th base sequences in the base sequence shown in SEQ ID NO: 23 or a complementary sequence thereof, and the base sequence shown in SEQ ID NO: 23 A combination of second primers having at least a part of the base sequence of the 527 to 1000th base sequence or an equivalent base sequence thereof;
24) a combination of primers according to any one of 24a) and 24b) according to claim 2 ,
And
Here, the at least part of the base sequence consists of 15 or more nucleotide residues,
The equivalent base sequence consists of 15 or more nucleotide residues, and corresponds to variations in the base sequence among rice varieties .