JP5563206B2 - Rice variety identification method - Google Patents

Description

  The present invention relates to a method for designing a variety identification oligonucleotide for crops such as rice, a method for identifying a variety, an oligonucleotide for variety identification, a kit for performing the variety identification method, and a DNA fragment useful for variety identification. .

  In Japan, over 200 kinds of rice varieties are currently cultivated, but the name of the rice varieties is required to be displayed due to the enforcement of the revised JAS Law. However, in some cases, fraudulent mixed rice is sold in which the display is misrepresented while rice other than the displayed variety is mixed. Taking into account the current situation in which good-tasting varieties such as Koshihikari are traded at a high price in response to the consumer's good-tasting taste orientation, and the growing interest in the disclosure of food information, the accuracy of content display is This is an important issue both administratively and industrially, and because of the necessity of preventing fraud due to mixed rice, development of simple, highly accurate, and practical rice variety identification technology is required.

  However, most of the varieties cultivated in Japan are very similar due to the fact that they have been bred by crossing between closely related species based on several conventional varieties, especially for polished rice. Scientifically, it is very difficult to identify rice varieties.

  On the other hand, in recent years, analysis of rice genome sequences has progressed and attempts have been made to identify rice varieties using genetic polymorphisms that show differences among rice varieties.

  Specifically, PCR-RFLP method, microsatellite method, SNPs method, and RAPD method are performed as this method.

  Among the above methods, the RAPD (Random Amplified Polymorphic DNA) method is a method in which a single distinct primer is added to separate amplified DNA by electrophoresis, and a discriminating band is selected from a number of electrophoresis bands. Select and use for variety identification. It is necessary to perform amplification reactions as many times as the type of primer set to be used, which is complicated and takes time. In addition, since many common bands appear in addition to the bands used for identification, it is not possible to employ a multiplex method in which a plurality of primers are mixed and amplified by the PCR method. There are problems such as being easily affected by purity and concentration, difficult to set PCR conditions, and poor identification and reproducibility even when the conditions are set.

  As a means for solving the problems of the RAPD method, there is a RAPD-STS (Sequence Tagged Site) method. The base sequence of the identification band obtained by the RAPD method is analyzed, and the specificity is increased by extending the primer from the RAPD primer site to the inside of the amplification product, and the band is improved so that only the band necessary for identification appears. Therefore, the amplification reaction can be carried out by employing a multiplex method. However, since it depends on the identification band found by the RAPD method, when the identification band size is large, the number of DNA molecules that can serve as a template is reduced in processed foods in which the DNA contained in the sample is reduced in molecular weight by heating or the like. Depending on the degree of processing, identification may be difficult. In addition, in this technique, detection is often performed based on the presence or absence of an identification band. When a variety whose identification band is detected is a positive variety and a variety whose identification band is not detected is a negative variety, a so-called quantitative inspection is performed. However, in the case of so-called qualitative inspection in which a plurality of grains are inspected together, there is a disadvantage that negative varieties mixed in positive varieties cannot be detected.

  In addition, DNA variety identification technology (JP-A-2001-95589 (Patent Document 1) etc.) using a RAPD-STS primer pair developed by Otsubo et al., Or RAPD-STS primer prepared by Otsubo et al. In Japanese Patent Laid-Open No. 2005-73655 (Patent Document 2), which is a DNA variety identification technique in which the size of the identification band obtained by the pair is shortened, it is assumed that most of the major planted varieties grown in Japan can be identified. Yes. Considering these results, the rice variety identification band identified by the RAPD method may be identified by a genomic portion having a polymorphism with a considerably different base sequence. Furthermore, since most of the varieties grown in Japan are by artificial mating, the 13 identification regions disclosed in Japanese Patent Application Laid-Open No. 2005-73655 (Patent Document 2) lead to the production of new varieties. There is a possibility that the gene change accompanying the mating is detected. However, there was no knowledge about what kind of gene change occurred in these regions. Japanese Patent Laid-Open No. 2005-73655 (Patent Document 2) is a technique in which the identification band found by the RAPD method is shortened, but basically has a size of about 80% of the shortened original identification band. Yes, it cannot be said that it is sufficiently shortened, such as the presence of amplified products of about 1.6 kbp and 1.3 kbp. Further, the discrimination performance is not particularly different from the discrimination performance by the original RAPD-STS primer pair.

  As described above, although the identification area developed by the RAPD method is suggested to be important for identifying rice varieties grown by artificial mating, the identification band size is large, and the target to be identified in the case of qualitative inspection Depending on the combination of varieties, there are varieties that cannot be detected, and in some cases, it is necessary to develop another identification area.

JP 2001-95589 A JP 2005-73655 A JP 2005-245244 A

  An object of the present invention is to provide a rice variety identification technique that makes it easier to use an identification area based on the RAPD method, which is used for identifying varieties, particularly rice varieties grown in Japan.

  For example, “Kinuhikari” is a stable variety with a planted area ranking of about 5th in the country, but when identifying the variety, the type described in JP-A-2005-245244 (Patent Document 3) Among the primers useful for identification, the identification band can be obtained with the RAPD-STS-primed primer pair A6 having a small number of varieties that can obtain the identification band for the main varieties, so it is not very difficult to construct a so-called quantitative test reaction system. No. However, when constructing a reaction system for qualitative testing, it is necessary to select from among RAPD-STS-modified primer pairs that are negative for “Kinuhikari”. Even if six types of RAPD-STS primer pairs M2CG, B43A, G22, G49A, F6, and E30 satisfying this condition are used, “Yumetsukushi” or the like of a higher planted variety cannot be detected. Further, the amplified product by G49A is about 1.5 kbp, and the amplified products by M2CG and F6 are about 1.2 kbp and 1 kbp in size. On the other hand, “Yumetsukushi” and “Kinuhikari” can be identified by A6, and as described above, there are few varieties that can obtain an identification band by A6. Therefore, “Dream Tsukushi” can be detected if a primer pair whose discrimination is reversed from A6 (“Kinuhikari” is negative and “Yumetsukushi” is positive) can be paired. Furthermore, if the other A6 negative varieties become positive as well as “Yumetsukushi” by the primer pair, it is possible to reduce the number of primers necessary for detecting other varieties, and it is possible to construct a multiplex PCR system for qualitative testing. It becomes easy.

  “Kirara 397” is Hokkaido's main planting variety, and the planting is only in Hokkaido, but it is stable at around the sixth place in the national planting area ranking. When identifying the cultivar, among the primers useful for cultivar identification described in JP-A-2005-245244 (Patent Document 3), a pair of RAPD-STS-modified primer pairs with few varieties capable of obtaining an identification band in the main cultivar. Since an identification band is obtained by G4 and S13, it is not so difficult to construct a so-called quantitative test reaction system. However, when constructing a reaction system for qualitative testing, it is necessary to select from the primer pairs in which “Kirara 397” is negative, and four types of RAPD-STS-modified primer pairs of M11, B1, G49A, and F6 were used. However, “Ginfu” of Hokkaido variety cannot be detected. Since “Kirara 397” is G22 positive and “Ginfu” is G22 negative, the identification is possible. G4 positive varieties such as “Kirara 397” and “Nanatsuboshi” are limited to Hokkaido cultivars. Therefore, if there is an identification primer (“Kirara 397” is negative and “Ginfu” is positive) in which the appearance pattern of the identification band of the RAPD-STS primer pair G22 is reversed, “Ginfu” can be detected. In addition, if a primer pair in which the distinguishability of the RAPD-STS-modified primer pair G4 is reversed (the G4 positive varieties “Kirara 397” and “Nanatsuboshi” are negative and the other G4 negative varieties are positive) is almost the same. Can be detected, and the construction of a multiplex PCR system is facilitated. In addition, although RAPD-STS-ized primer pair G49 can be used for the detection of “Nanatsuboshi” mixed in “Kirara 397”, the size of the amplified product is about 1.5 kbp and a size exceeding 1 kbp.

  “Hoshino Yume” is Hokkaido's main cropping variety, and the planting is only in Hokkaido, but it is stable at around 8th place in the national planting area ranking. When identifying the cultivar, among the primers useful for cultivar identification described in JP-A-2005-245244 (Patent Document 3), a pair of RAPD-STS-modified primer pairs with few varieties capable of obtaining an identification band in the main cultivar. Since an identification band is obtained in S13, it is not so difficult to construct a so-called quantitative test reaction system. However, when constructing a reaction system for qualitative testing, it is necessary to select a primer pair that makes “Hoshinoyume” negative, and five types of RAPD-STS primer pairs of M11, B1, F6, B43, and G4 are used. Even so, the Hokkaido variety “Hotarou” cannot be detected. “Hoshino Yume” is S13 positive, and “Hotaro” is S13 negative, so identification is possible. Therefore, if there is an identification primer ("Hoshino Yume" is negative and "Hotaro" is positive) whose appearance pattern of the identification band of the RAPD-STS S13 primer pair is reversed, "Hotaro" can be detected. In addition, as with “Hotarou”, when other RAPD-STS-modified S13 primer pair negative varieties are also positive, a large number of varieties can be detected with the primer, and a multiplex PCR system for qualitative testing can be easily constructed. Become.

  “Nanatsuboshi” is expected to be Hokkaido's next major planted variety, and the planted area in 2005 has risen to 10th in the national ranking. As described in the above-mentioned section “Kirara 397”, among the main cultivated varieties nationwide, there are few varieties that are positive for the RAPD-STS primer pair G4 together with “Kirara 397”. Therefore, if a primer pair in which the distinguishability of the RAPD-STS-modified primer pair G4 is reversed (the G4 positive varieties “Kirara 397” and “Nanatsuboshi” are negative and the other G4 negative varieties are positive) is used. Most varieties can be detected, and a multiplex PCR system for qualitative inspection can be easily constructed. In addition, since “Kirara 397” is positive and “Nanatsuboshi” is a negative variety in the RAPD-STS-modified primer pair S13, “Kirara 397” can be detected by the primer pair. The identification band by the primer pair S13 has a size approaching about 1.8 kbp and 2 kbp. On the other hand, although the planting is gradually decreasing, “Akiho” and “Nanatsuboshi” which are Hokkaido varieties cannot be identified by the primer pairs described in JP-A-2005-245244 (Patent Document 3). .

  The present inventors have found that many of the identification bands obtained by the RAPD method have a region (hereinafter referred to as a recombination region) that appears to have undergone recombination within the identification band sequence. It was. As a result of further intensive studies, it was found that by considering the PCR primer design position using the base site indicating the recombination region as an index, the size of the amplified product can be adjusted appropriately while maintaining the variety identification performance of the original RAPD-STS-modified primer. I found it. In addition, in the negative cultivar genome sequence, the identification pattern of the identification band appears in the rice cultivar DNA that has been confirmed by designing a PCR primer pair in a DNA region that appears to be a locus corresponding to the identification region developed by the RAPD method. Found to reverse.

That is, the first invention in the present invention is an oligonucleotide design method for detecting a genomic DNA region useful for variety identification,
(1) Comparing a DNA region useful for variety identification present in the genomic DNA of a variety desired to be identified with a region corresponding to the region in the genomic DNA of another variety, and predicting a recombination region A step of finding a site, and (2) a step of designing an oligonucleotide based on the base sequence of the genomic DNA adjacent to the site or the site when the base region predicted to be a recombination region is found,
The present invention relates to a method for designing an oligonucleotide for breed identification, characterized by comprising

  In one aspect of the first invention, the varieties are rice varieties.

  Another aspect of the first invention is characterized by designing a pair of oligonucleotides for a nucleic acid amplification reaction capable of amplifying a DNA fragment containing a recombination region and a predicted base site.

  Another aspect of the first invention is a nucleic acid amplification reaction that can amplify a DNA fragment only in a region that is adjacent to a predicted recombination region and has a low sequence identity to the genome sequence of one of the varieties. A pair of oligonucleotides is designed.

The second invention of the present invention is a rice variety identification method, characterized in that the presence or absence of a specific base sequence selected from the following (1) to (15) in genomic DNA is used as an identification index: About rice variety identification method:
(1) a variety-identifying DNA region having base numbers 424 and 425 which are represented by SEQ ID NO: 1 and predicted to be a recombination region,
(2) a cultivar identification DNA region represented by SEQ ID NO: 2 and whose base site predicted to be a recombination region is base numbers 48 and 49;
(3) a variety-identifying DNA region having base numbers of 70 to 80, which is represented by SEQ ID NO: 3 and predicted to be a recombination region,
(4) a cultivar identification DNA region represented by SEQ ID NO: 4 and whose base site predicted to be a recombination region is base numbers 19 to 26;
(5) a DNA region for detecting a cultivar having base numbers 763 and 764 as the base sites predicted from the recombination region shown in SEQ ID NO: 5;
(6) a DNA region represented by SEQ ID NO: 6 and whose base site predicted to be a recombination region is base numbers 366 and 367,
(7) a DNA region represented by SEQ ID NO: 7 and whose base site predicted to be a recombination region is base numbers 353 to 361,
(8) a DNA region represented by SEQ ID NO: 8 and whose base site predicted to be a recombination region is base numbers 249 and 250;
(9) a DNA region represented by SEQ ID NO: 9 and whose base site predicted to be a recombination region is base numbers 550 and 551;
(10) a DNA region represented by SEQ ID NO: 10 and having a base site predicted to be a recombination region of base numbers 9 and 10;
(11) a DNA region represented by SEQ ID NO: 11 and whose base site predicted to be a recombination region is base numbers 15 and 16;
(12) a DNA region represented by SEQ ID NO: 12 and having a base site predicted to be a recombination region of base numbers 555 to 558,
(13) a DNA region represented by SEQ ID NO: 13 and whose base site predicted to be a recombination region is base numbers 474 and 475;
(14) a DNA region represented by SEQ ID NO: 57 and predicted to be a recombination region is a DNA region having base numbers 231 and 232, and (15) a base region predicted to be a recombination region represented by SEQ ID NO: 58 is a base. DNA regions having numbers 114 and 115.

In one aspect of the second invention, a cultivar having at least one specific base sequence region selected from the specific base sequence regions described in the following (2), (4), and (5) in genomic DNA is represented by “ It is characterized by identifying that it is not "Kinuhikari":
(2) a cultivar identification DNA region represented by SEQ ID NO: 2 and whose base site predicted to be a recombination region is base numbers 48 and 49;
(4) a variety-identifying DNA region whose base site is represented by SEQ ID NO: 4 and predicted to be a recombination region is base numbers 19 to 26; and (5) a base that is predicted to be a recombination region represented by SEQ ID NO: 5. A DNA region for detecting a variety whose sites are base numbers 763 and 764.

In another aspect of the second invention, a cultivar having at least one specific base sequence region selected from the specific base sequence regions described in the following (3), (11) and (13) is present in the genomic DNA: It is characterized by identifying that it is not "Kinuhikari":
(3) a variety-identifying DNA region having base numbers of 70 to 80, which is represented by SEQ ID NO: 3 and predicted to be a recombination region,
(11) a DNA region represented by SEQ ID NO: 11 and predicted to be a recombination region is a DNA region having base numbers 15 and 16, and (13) a base region predicted to be a recombination region represented by SEQ ID NO: 13 is a base. DNA region numbered 474,475.

According to another aspect of the second invention, a variety in which at least one specific base sequence region selected from the specific base sequence regions described in (11) and (12) below is present in genomic DNA Is characterized by not being:
(11) a DNA region represented by SEQ ID NO: 11 and predicted to be a recombination region is a DNA region having base numbers 15 and 16, and (12) a base region predicted to be a recombination region represented by SEQ ID NO: 12 is a base. A DNA region having the number 555-558.

In another aspect of the second invention, a cultivar having at least one specific base sequence region selected from the specific base sequence regions described in the following (3), (9), and (10) is present in genomic DNA: It is characterized by not being "Hoshino Yume":
(3) a variety-identifying DNA region having base numbers of 70 to 80, which is represented by SEQ ID NO: 3 and predicted to be a recombination region,
(9) a DNA region represented by SEQ ID NO: 9 and predicted to be a recombination region is a DNA region having base numbers 550 and 551; and (10) a base region predicted to be a recombination region represented by SEQ ID NO: 10 is a base. DNA regions numbered 9 and 10.

  Another aspect of the second invention is characterized in that the presence of a specific base sequence in genomic DNA is based on detection of a DNA fragment amplified by a nucleic acid amplification method.

  Another aspect of the second invention is characterized in that the primer used in the nucleic acid amplification method is an oligonucleotide designed according to the first invention.

  Another aspect of the second invention is characterized in that the nucleic acid amplification method is a PCR method.

Another aspect of the second invention is characterized in that, in the PCR method, a primer pair selected from DNAs described in the following (a) to (r) is used:
(A) DNA represented by SEQ ID NO: 14 and DNA represented by SEQ ID NO: 15,
(B) DNA represented by SEQ ID NO: 16 and DNA represented by SEQ ID NO: 17,
(C) DNA represented by SEQ ID NO: 18 and DNA represented by SEQ ID NO: 19,
(D) DNA represented by SEQ ID NO: 20 and DNA represented by SEQ ID NO: 21,
(E) DNA represented by SEQ ID NO: 22 and DNA represented by SEQ ID NO: 23,
(F) DNA represented by SEQ ID NO: 24 and DNA represented by SEQ ID NO: 25,
(G) DNA represented by SEQ ID NO: 26 and DNA represented by SEQ ID NO: 27,
(H) DNA represented by SEQ ID NO: 28 and DNA represented by SEQ ID NO: 29,
(I) DNA represented by SEQ ID NO: 30 and DNA represented by SEQ ID NO: 31,
(J) DNA represented by SEQ ID NO: 32 and DNA represented by SEQ ID NO: 33,
(K) DNA represented by SEQ ID NO: 34 and DNA represented by SEQ ID NO: 32,
(L) DNA represented by SEQ ID NO: 35 and DNA represented by SEQ ID NO: 36,
(M) DNA represented by SEQ ID NO: 37 and DNA represented by SEQ ID NO: 38,
(N) DNA represented by SEQ ID NO: 39 and DNA represented by SEQ ID NO: 40,
(O) DNA represented by SEQ ID NO: 41 and DNA represented by SEQ ID NO: 34,
(P) DNA represented by SEQ ID NO: 59 and DNA represented by SEQ ID NO: 60,
(Q) DNA represented by SEQ ID NO: 61 and DNA represented by SEQ ID NO: 60, and (r) DNA represented by SEQ ID NO: 62 and DNA represented by SEQ ID NO: 63.

Another aspect of the second invention includes the following steps (A) to (C):
(A) a nucleic acid preparation step of preparing rice nucleic acid in a rice sample;
(B) A step of performing a nucleic acid amplification method using the nucleic acid obtained in step (A) as a template, and (C) a step of analyzing the DNA fragment obtained in step (B).

The third invention of the present invention relates to an oligonucleotide selected from the following (i) to (xvi) for detecting the presence of a specific base sequence in genomic DNA:
(I) an oligo having a specific base sequence represented by SEQ ID NO: 1 and comprising a continuous base sequence of 10 to 100 bases in length and including the base sites of base numbers 424 and 425 of the base sequence represented by SEQ ID NO: 1 nucleotide,
(Ii) an oligo having a specific base sequence represented by SEQ ID NO: 2 and a length of 10 to 100 bases in which the base sequence is continuous, and including the base sites of base numbers 48 and 49 in the base sequence represented by SEQ ID NO: 2 nucleotide,
(Iii) An oligo having a specific base sequence represented by SEQ ID NO: 3 and a length of 15 to 100 bases in which the base sequence is continuous, and including base sites of base numbers 70 to 80 in the base sequence represented by SEQ ID NO: 3 nucleotide,
(Iv) An oligo having a specific base sequence represented by SEQ ID NO: 4 and a length of 20 to 100 bases in which the base sequence is continuous, and including the base sites of base numbers 14 to 31 in the base sequence represented by SEQ ID NO: 4 nucleotide,
(V) an oligo having a specific base sequence represented by SEQ ID NO: 5 and consisting of 10 to 100 bases in length of the base sequence and including the base sites of base numbers 763 and 764 in the base sequence represented by SEQ ID NO: 5 nucleotide,
(Vi) an oligo having a specific base sequence represented by SEQ ID NO: 6 and a length of 10 to 100 bases in which the base sequence is continuous, and including the base sites of base numbers 366 and 367 in the base sequence represented by SEQ ID NO: 6 nucleotide,
(Vii) an oligo having a specific base sequence represented by SEQ ID NO: 7, consisting of 15 to 100 bases in length of the base sequence, and comprising the base sites of base numbers 353 to 361 in the base sequence represented by SEQ ID NO: 7 nucleotide,
(Viii) an oligo having a specific base sequence represented by SEQ ID NO: 8, consisting of 10 to 100 bases in length of the base sequence, and comprising base sites of base numbers 249 and 250 in the base sequence represented by SEQ ID NO: 6 nucleotide,
(Ix) An oligo having a specific base sequence represented by SEQ ID NO: 9 and a length of 10 to 100 bases in which the base sequence is continuous, and including the base sites of base numbers 550 and 551 in the base sequence represented by SEQ ID NO: 6 nucleotide,
(X) an oligo having a specific base sequence represented by SEQ ID NO: 10 and a length of 10 to 100 bases in which the base sequence is continuous, and including the base sites of base numbers 9 and 10 in the base sequence represented by SEQ ID NO: 10 nucleotide,
(Xi) The specific base sequence is represented by SEQ ID NO: 11 and consists of 10 to 100 bases in length of the base sequence and includes the base sites of base numbers 15 and 16 in the base sequence represented by SEQ ID NO: 11. nucleotide,
(Xii) an oligo having a specific base sequence represented by SEQ ID NO: 12, comprising a continuous base sequence of 10 to 100 bases in length, and comprising a base site of base numbers 555 to 558 in the base sequence represented by SEQ ID NO: 12 nucleotide,
(Xiii) An oligo having a specific base sequence represented by SEQ ID NO: 13, consisting of 10 to 100 bases in length of the base sequence, and including the base sites of base numbers 474 and 475 in the base sequence represented by SEQ ID NO: 13 nucleotide,
(Xiv) an oligo having a specific base sequence represented by SEQ ID NO: 57, consisting of 10 to 100 bases in length of the base sequence and including the base sites of base numbers 231 and 232 in the base sequence represented by SEQ ID NO: 57 nucleotide,
(Xv) an oligo having a specific base sequence represented by SEQ ID NO: 58, consisting of 10 to 100 bases in length of the base sequence and including the base sites of base numbers 114 and 115 in the base sequence represented by SEQ ID NO: 57 A nucleotide, and (xvi) an oligonucleotide having a base sequence complementary to the oligonucleotides (i) to (xv).

  As one aspect of the third invention, the DNA is any one of DNAs represented by SEQ ID NOs: 21, 22, 24, 33, 34, 38, and 40.

  A fourth invention of the present invention is a kit for carrying out the second invention, and is used for the purpose of detecting the presence of a specific base sequence in genomic DNA as a DNA fragment amplified by a nucleic acid amplification method. The present invention relates to a kit containing a primer pair.

  The fifth aspect of the present invention relates to a DNA fragment represented by SEQ ID NO: 12, which can be used for variety identification.

  INDUSTRIAL APPLICABILITY According to the present invention, a rice variety identification method using a DNA region useful for variety identification developed by the RAPD method can be used to identify a variety using an identification band having a lower molecular weight than the identification band obtained by the RAPD method. This makes it easier to identify processed crop (eg, rice) products in which DNA is expected to have a low molecular weight. In addition, among cultivars that have been identified by the RAPD method, by using the locus in the genome sequence of the negative cultivar corresponding to the DNA region useful for cultivar identification found by the RAPD method as an identification region, it is completely novel. Qualitative inspection can be performed without developing an identification area.

An oligonucleotide designing method for detecting a genomic DNA region useful for breed identification provided in the present invention comprises:
(1) Comparing a DNA region useful for variety identification present in the genomic DNA of a variety desired to be identified with a region corresponding to the region in the genomic DNA of another variety, and predicting a recombination region A step of finding a site, and (2) a step of designing an oligonucleotide based on the base sequence of the genomic DNA adjacent to the site or the site when the base region predicted to be a recombination region is found,
The present invention relates to a method for designing an oligonucleotide for breed identification, characterized by comprising

  As used herein, the term “breed” refers to a population having genetic characteristics that produce different traits in the same species than other populations. In the following, mainly rice varieties will be described. In the present invention, any organism including plants and animals can be used as a crop.

  The “oligonucleotide” in the present specification may be a so-called chimeric oligonucleotide in which a part of DNA is substituted with RNA in addition to DNA and RNA. Oligonucleotides labeled with a fluorescent dye for detection and the like are also included in the present oligonucleotide.

  On the other hand, “variety identification oligonucleotide” in the present specification includes those that function as probes and primers that can be used for variety identification. A probe refers to an oligonucleotide having a sequence complementary to the sequence in order to detect a nucleic acid having a specific base sequence. A primer refers to an oligonucleotide having a sequence complementary to a template nucleic acid and serving as a starting point for a synthesis reaction by a nucleic acid synthase. In addition to Southern hybridization as a method for detecting a specific nucleotide sequence using a probe, it is detected whether there is a nucleic acid containing a DNA region for identifying a variety based on hybridization with a probe immobilized on an appropriate carrier. In addition, a technique such as a so-called DNA chip is also included.

  As used herein, the term “RAPD method” is a method in which a single primer is added to a reaction system containing nucleic acid (eg, genomic DNA) derived from a target variety, and amplified DNA is separated by electrophoresis. This refers to a method of selecting a band having distinctiveness from among a large number of migration bands, that is, a band having a different appearance mode between a specific variety and another variety and using it for variety identification. A primer whose identity is confirmed by this method is referred to as “RAPD primer”.

  The term “RAPD-STS method” used in the present specification analyzes the nucleotide sequence of the identification band obtained by the RAPD method, and extends the RAPD primer sequence into the amplification product (ie, 3 ′ side). A method improved by increasing the length of the primer to increase the specificity and so that only the band necessary for identification appears. Further, modifying the RAPD primer by this method is referred to as “RAPD-STS conversion”, and the primer obtained thereby is referred to as “RAPD-STS conversion primer”.

  The present invention can be suitably used for a variety identification region developed by the RAPD method, which is likely to include a recombination region and a predicted base site.

  As used herein, the term “recombination region” or “recombination point” is the same as a region with high sequence identity observed when comparing the genomic sequence of one cultivar with the genomic sequence of the other cultivar. A region or point that includes a boundary with a low-quality region. Such a recombination region is considered to result from, for example, homologous recombination of chromosomes during population mating. The “base site predicted to be a recombination region” refers to a base site at the boundary that is found by the above comparison and is predicted to have undergone recombination.

  Prediction of whether or not a recombination point is included in a DNA region useful for variety identification is, for example, comparing the nucleotide sequence of a locus that seems to correspond to the region between varieties identified by the DNA region. Can be implemented. In the present invention, amplified base sequences and whole genome sequences obtained by various RAPD-STS-modified primer pairs disclosed in Japanese Patent Application Laid-Open No. 2005-245244 (Patent Document 3), which is a DNA region useful for variety identification, are analyzed. The “Nippon Hare” genome sequence is classified into 6 groups of A-1, A-2, A-3, B-1, B-2, and C, which are useful for variety identification, An investigation was made as to whether the recombination region and the predicted base site were included. Here, the “Nippon Hare” genome sequence is targeted for GenBank, and information can be extracted by BLAST search using the sequence to be compared as Query. As the base sequence data, it is possible to use base sequence data described in a record such as a BAP library created for performing genome sequence analysis in addition to a record relating to the total length of a chromosome. Whether or not the sequence is derived from “Nihonbare” can be confirmed by the matter described in SOURCE in the FEATURES section of each record.

  In the amplified product sequence, the primer, particularly the region derived from the vicinity of the 5 ′ end of the RAPD primer used when amplifying the identification region, is bonded to the corresponding genomic DNA region by causing misannealing. Since there is a possibility, it is preferable to remove the RAPD primer sequence region from the comparison control.

  According to the present invention, it is possible to design a PCR primer that reverses the distinguishability of a conventional RAPD-STS primer. Here, “inversion” means that the result obtained for a specific variety is opposite to the result obtained using a conventional RAPD-STS-primed primer, ie, negative is converted to positive and positive is converted to negative. That means. For example, in order to construct a qualitative test reaction system to negate the contamination of other varieties, it was necessary to use a specific primer that yielded a negative result, and a conventional positive result was obtained It is particularly useful for this purpose if the discriminability of the RAPD-STS-modified primer can be reversed.

A) RAPD-STS-primed primer pairs in which a discriminating band is not detected in a PCR reaction using Nipponbare genomic DNA as a template. Among the RAPD-STS-primed primer pairs disclosed in JP 2005-245244 A (Patent Document 3), Some of the PCRs using the “Nippon Hare” genomic DNA as a template do not detect an identification band.

  When the sequence of the identification band amplification product obtained by these primer pairs was compared with the genome sequence of “Nippon Haru”, it could be further classified into the following A-1, A-2 and A-3.

A-1) RAPD-STS primer pair in which a region showing high sequence identity with the partial region of the amplified product is also present in the “Nippon Hare” genomic sequence No discrimination band is detected by PCR using “Nippon Hare” genomic DNA as a template However, compared to the “Nipponbare” genomic sequence, it does not have sequence identity across the entire identification band amplification product, but exists in the amplification product sequence where there is a site where the degree of sequence identity changes rapidly at some point did. Examples of the RAPD-STS-modified primer pair from which such an amplification product is obtained include WKA09, S13, G4, G49A, M11, B7, and A7.

  For example, in the case of an amplification product obtained by PCR using the RAPD-STS-primed primer pair WKA09 and “Hitomebore” genomic DNA as a template, including the base sequence represented by SEQ ID NO: 1, the base numbers 425 to 1608 are almost completely complete. There is a region that matches the sequence in the “Nipponbare” genome sequence, but the region of base numbers 1 to 424 has low sequence identity. In the case of an amplification product obtained by PCR using the RAPD-STS primer pair S13 and the “Kirara 397” genomic DNA as a template, including the base sequence represented by SEQ ID NO: 8, the region of base numbers 250 to 1721 is almost completely A region with a sequence match exists in the “Nipponbare” genome sequence, but the region of base numbers 1 to 249 has low sequence identity. In the case of an amplification product obtained by PCR using the RAPD-STS-primed primer pair G4 and “Kirara 397” genomic DNA as a template, including the base sequence represented by SEQ ID NO: 10, the region of base numbers 1 to 9, the base number A region that almost completely coincided with each of the regions 10 to 491 was present on the “Nipponbare” genomic sequence, but two regions were present at distant positions. In the case of an amplification product obtained by PCR using the RAPD-STS-modified primer pair G49 and the “Hoshino Yume” genomic DNA as a template, including the base sequence represented by SEQ ID NO: 13, the region of base numbers 475 to 1475 is almost completely A region with a sequence match exists in the “Nipponbare” genome sequence, but the region of base numbers 1 to 474 has low sequence identity.

  Currently, most rice varieties cultivated in Japan are cultivated by artificial mating technology. Considering that a variety having a new trait has been obtained by homologous recombination, the base site where the sequence identity changes rapidly is expected to be a site (= recombination region) where homologous recombination has occurred.

  On the other hand, the “Nippon Hare” genomic sequence region, which showed high sequence identity with the partial region of each RAPD-STS-modified primer pair amplification product in this analysis, is expected to be a locus corresponding to the variety identification region in the positive variety. By using the corresponding locus sequence information in this “Nipponbare” genomic sequence, an amplified product can be obtained by a PCR reaction using at least “Nipponbare” genomic DNA as a template, and the template DNA of the PCR amplified product used for the locus sequence analysis can be obtained. It should be possible to design a cultivar identification primer that does not yield an amplified product with the prepared positive cultivar.

  In the present invention, based on the nucleotide sequence information of each gene locus, gene regions that can be used for variety identification in the negative variety corresponding to the identification region of the positive variety are narrowed down.

  According to the present invention, the locus in the “Nippon Hare” genomic sequence corresponding to the region amplified by the RAPD-STS-primed primer pair WKA09 in the “Hitomebore” genomic sequence is presumed to be a region containing the base sequence represented by SEQ ID NO: 2, The 1-48 region is a sequence region almost completely identical to the WKA09 amplification product sequence, and the base numbers 49-416 are regions with low sequence identity. In addition, there are three loci candidate regions in the “Nippon Hare” genomic sequence corresponding to the amplified region by the RAPD-STS-modified primer pair S13 in the “Kirara 397” genomic sequence. Met. In the present invention, the region containing the base sequence represented by SEQ ID NO: 9 was used as a genomic DNA region useful for variety identification. In the base sequence shown in SEQ ID NO: 9, base numbers 1 to 550 are regions where the partial region of the S13 amplification product almost completely matches, and 551 to 844 regions are regions with low sequence identity. On the other hand, it was predicted that there were also three locus candidate regions in the “Nippon Hare” genomic sequence corresponding to the amplified region by the RAPD-STS-primed primer pair G4 in the “Kirara 397” genomic sequence. The nucleotide sequence of the amplification product obtained using “Kirara 397” as a template was 817 bp, but there was one region where the sequence almost completely matched 484 bp from the forward side primer, and the other two locations were 333 bp from the reverse side primer. Is a region where the sequences are almost identical. Two of these are irrelevant to the region showing variety discrimination, and narrow down the locus candidate regions in the “Nippon Hare” genomic sequence corresponding to the amplified region by the RAPD-STS-modified primer pair G4 in the “Kirara 397” genomic sequence. I was able to. This region is a region including the base sequence represented by SEQ ID NO: 11, the base number 16 to 482 region is a sequence region substantially matching the partial region of the G4 amplification product, and the base numbers 1 to 15 are identical in sequence. This is a low-quality area. Similarly, it was predicted that there were also two loci candidate regions in the “Nipponbare” genomic sequence corresponding to the amplified region by the RAPD-STS-modified primer pair G49A in the “Hoshino Yume” genomic sequence. That is, almost completely the same as the region of base numbers 475 to 1475 of the base sequence shown in SEQ ID NO: 13 included in the amplified base sequence obtained by the RAPD-STS primer pair G49A using “Hoshinoyume” as a template There was one matching region and one region that almost completely matched the nucleotide numbers 900 to 1475, but the present invention can narrow down the G49A allele candidate region in the “Nippon Hare” genomic sequence. It was.

A-2) RAPD-STS-modified primer pairs in which a region showing high sequence identity with the partial region of the amplified product does not exist in the “Nippon Hare” genomic sequence, , P5. Amplified sequences obtained with RAPD-STS-modified primer pairs belonging to this group have the ability to discriminate "Nipponbare" genomic DNA over the entire region, and are negative even if PCR primers are designed at any position within the amplified region. There is a possibility that the product identification performance for at least “Nipponbare” is maintained. This amplified region may be part of the region inserted due to an insertion mutation at the corresponding gene locus of the negative cultivar, and it is expected that the recombination region exists in the vicinity of the amplified region on the genome sequence. . The Inverse PCR technique is a technique for analyzing a base sequence in the vicinity of a specific base sequence in genomic DNA. If this technique is used, a base sequence in the vicinity of an amplification region in a positive variety genomic DNA can be analyzed. After comparing the analyzed base sequence with the negatively cultivated “Nippon Hare” genomic DNA sequence, if there is a region that is almost completely identical, a region with low sequence identity, and a region that can be divided into two, the boundary is the recombination region. Is predicted to be a base site.

A-3) RAPD-STS-primed primer pairs in which a region showing high sequence identity with the entire region of the amplified product is present in the “Nippon Hare” genomic sequence B18 ( Japanese Patent Laid-Open No. 2001-95589 (Patent Document 1). In the case of a RAPD-STS-modified primer pair belonging to this group, it is expected that a base site important for discrimination exists in the primer sequence, particularly in the RAPD primer sequence that has led to the development of this discrimination region.

  However, since the RAPD primer may be misannealed and the DNA fragment derived from the positive cultivar genomic DNA may be amplified, the accurate genomic sequence information of the primer binding region can be obtained by the Inverse PCR technique using the positive cultivar genomic DNA. It is preferable to obtain. In addition, when the variety discriminating property recognized by the RAPD method is lost by PCR using a primer pair designed with the internal sequence of the amplification product adjacent to the RAPD primer region, it is highly likely that the primer pair belongs to this type.

B) RAPD-STS-primed primer pair in which a discriminating band is detected by PCR reaction using “Nippon Hare” genomic DNA as a template Among the RAPD-STS-primed primer pair disclosed in Japanese Patent Laid-Open No. 2005-245244 (Patent Document 3) Then, M2CG, P3, B1, B43, and F6 are mentioned. Since there is a template region somewhere in the “Nipponbare” genome sequence, these cannot be further classified by comparison with the “Nipponbare” genome sequence. However, if it becomes comparable to a genomic DNA sequence that cannot be amplified by each primer pair (hereinafter referred to as a negative variety), it can be further classified into the following two groups, as in A.

B-1) RAPD-STS-primed primer pair in which a recombination point exists in the identification band amplification product sequence when compared with the negative cultivar genomic DNA sequence, B-2) high sequence identity with a partial region of the identification band amplification product RAPD-STS-primed primer pair in which the region showing no exists in the negative cultivar genome sequence

  In the case of the RAPD-STS primer pair belonging to B, since the base sequence around the amplification region in the “Nippon Hare” genomic sequence is known, the PCR primer pair is inserted at both ends of the amplification region so as to include the RAPD-STS primer binding region. It is possible to design. When PCR is performed with the two primer pairs designed in this way, if the variety distinguishability is lost with one primer pair, the recombination region is present in the amplified region on the genomic DNA by the RAPD-STS primer pair. Belongs to the expected B-1. As compared with the sequence disclosed in Japanese Patent Application Laid-Open No. 2005-245244 (Patent Document 3) and the “Nippon Hare” genome sequence, M2CG, P3, and B1 belonged to this group among the RAPD-STS-modified primer pairs. As for the recombination region, the negative PCR and the positive variety are expected to be conserved. The base sequence information from the “Nipponbare” genomic sequence in the vicinity of the recombination region and the genomic DNA prepared from the negative cultivar are used. By using this technique, it is possible to obtain base sequence information of the corresponding locus region containing the recombination point in the negative cultivar. By comparing this sequence information with the genome sequence information of the positive cultivar “Nipponbare”, the recombination region and the expected base site can be determined.

  For example, the region amplified in the RAPD-STS-primed primer pair M2CG in the “Nipponbare” genomic sequence includes the base sequence shown in SEQ ID NO: 5, but the inverse of the RAPD-STS-primed primer pair M2CG negative cultivar “Akitakomachi” When compared with the genomic DNA sequence analyzed by the PCR technique, the region of base numbers 1 to 763 of SEQ ID NO: 5 has a region almost completely identical in the sequence of “Akitakomachi” genomic DNA, and base numbers 764 to 1087. Was found to be a region with low sequence identity. In addition, the region amplified by the RAPD-STS primer pair P3 in the “Nippon Hare” genome sequence includes the base sequence represented by SEQ ID NO: 6, while the base number 1 to 366 region is the “Akitakomachi” genome which is a P3-negative variety. It was found that there was a region that almost completely matched DNA, and the region of base numbers 367 to 447 was a region with low sequence identity.

  On the other hand, the DNA region having the base sequence on the negative variety genome sequence used in the above analysis is also estimated to be a DNA region that can be used for variety identification.

  For example, the locus in the “Akitakomachi” genomic sequence corresponding to the RAPD-STS-primed primer pair M2CG amplification region in the “Nippon Hare” genomic sequence is presumed to be a region including the base sequence represented by SEQ ID NO: 44, The 821 region has an almost completely identical region on the “Nippon Hare” genome sequence, and the regions of SEQ ID NOs: 822 to 1578 are regions with low sequence identity. On the other hand, the locus in the “Akitakomachi” genomic sequence corresponding to the RAPD-STS-modified P3 primer pair amplification region in the “Nippon Hare” genomic sequence is presumed to be a region including the base sequence represented by SEQ ID NO: 7. The 1 to 360 region and the base number 354 to 521 region are present on the same chromosomal sequence of “Nippon Hare” with an approximately 3 kbp region in between. In the “Akitakomachi” locus region, compared to the “Nippon Hare” locus sequence that is expected to correspond to the region, the 7 base portion of base numbers 354 to 360 overlapped and a sequence in which about 3 kbp in the middle was deleted. It has become.

  On the other hand, in the RAPD-STS-modified primer pair classified as B, if both PCR primers are designed to be discriminated by the PCR primer pair designed at both ends of the amplification region, it is considered to belong to B-2. Among the RAPD-STS primer pairs disclosed in JP-A-2005-245244 (Patent Document 3), B43 and F6 are mentioned. The amplified region sequence is selective to negative cultivar genomic DNA, and even if a PCR primer pair is designed at an appropriate position in the amplified product region, the cultivar identification performance for the negative cultivar may be retained.

  On the other hand, this amplified product region may be a part of the region where insertion occurred at the corresponding gene locus of the negative cultivar. Therefore, in the genome sequence of positive varieties, it is considered that there is a recombination region in which insertion has occurred in the vicinity of the amplified region. Since the sequence near the amplification region in the “Nipponbare” genomic sequence is known, for example, if a primer pair is designed at an appropriate position based on the sequence information and an amplified product can be obtained even in a negative variety, It is expected that there is a site where recombination has occurred between the amplification region by the primer pair and the amplification region by the primer pair that has maintained the variety discrimination performance. After that, using the technology such as Inverse PCR, using the base sequence information from the “Nipponbare” genomic sequence near the recombination region that is expected to be preserved for both negative and positive varieties and genomic DNA prepared from the negative varieties Thus, the base sequence information of the corresponding locus region including the recombination region in the negative cultivar can be obtained. By comparing this sequence information with the genome sequence information of the positive cultivar “Nipponbare”, the recombination region and the expected base site can be determined.

C) RAPD-STS-modified primer pair in which identification bands of different sizes can be detected depending on the cultivar Hereinafter, amplification product L is defined as amplification product L, and amplification product S of short size identification band amplification product. The base sequence of the amplified product S and the base sequence of the amplified product S do not have sequence identity, and there are some cases where the base sequence of the amplified product L has base insertion regions that do not exist in the base sequence of the amplified product S in some places. It was. Regarding the RAPD-STS primer pair A6 disclosed in JP-A-2005-245244 (Patent Document 3), when the annealing temperature during the PCR reaction is lowered, amplification is performed with a negative variety in which an A6 identification band is not detected. The substance L was detected and classified into this group. Also, discrimination band amplification products obtained with RAPD primer G22 or G28 which are not RAPD-STS-modified primer pairs are also classified into this group. Of the identification band amplification products obtained in the RAPD-STS primer pair A6, when the longer one is A6L and the shorter one is A6S, the A6L amplification region includes the nucleotide sequence represented by SEQ ID NO: 4 from the Nipponbare genome sequence information. When compared with the A6S amplification product sequence obtained by PCR using “Haenuki” genomic DNA as a template, insertion of base numbers 19 to 26 of SEQ ID NO: 4 was observed. Moreover, when the longer one of the identification amplified products obtained in the RAPD primer G22 is G22L and the shorter one is G22S, the G22S amplified region is a region including the base sequence represented by SEQ ID NO: 3 from the Nipponbare genome sequence information. When compared with the G22L amplified product sequence obtained by PCR using “Koshihikari” genomic DNA as a template, G22L was a sequence in which insertion occurred in the base number 71 to 79 region of SEQ ID NO: 3.

  Next, based on the above confirmation results, the variety discrimination ability of the RAPD-STS-modified primer pair was considered.

  The reason why the RAPD-STS-modified primer pair including the base region predicted to be the recombination region in the amplified product sequence excluding the primer-derived region shows variety discrimination is not because the sequence of the RAPD-STS-modified primer pair is important. It was suggested that the region to which the primer binds is located on both sides of the recombination region in the cultivar genomic DNA sequence that becomes positive. Therefore, if the PCR primer is designed so that at least the PCR amplification product includes the recombination region, it is expected that the amplification product size can be appropriately adjusted regardless of the RAPD primer binding region.

  This is considered to be the same not only in the amplified region identified by the RAPD-STS-modified primer pair, but also in the genomic DNA locus region in the negative cultivar considered to correspond to the region.

  When there is no comparable variety genomic DNA sequence information, for example, the base sequence of the identification band DNA fragment detected by the RAPD method is analyzed. Next, as in the RAPD-STS-modified primer pairs classified as A-2, DNA base sequence information around the amplification region is collected by the Inverse PCR technique based on the DNA information. Thereafter, in the same manner as the RAPD-STS-modified primer pairs classified as B-1, a PCR primer pair is prepared in the peripheral region of the amplification region, and the amplified product is detected even by PCR using negative cultivar genomic DNA with one primer pair. In this case, it is considered that the recombination region is present in the detection DNA fragment sequence as an identification band by the RAPD primer. Then, if the DNA base sequence information around the DNA region in the negative cultivar genomic DNA is analyzed by the Inverse PCR technique based on the DNA sequence that is considered to be also held in the negative cultivar genomic DNA, the recombination region as in the above Can be guessed.

  Alternatively, the base sequence around the DNA region can be analyzed by a technique such as a primer walking method from the DNA regions held in the positive variety genomic DNA and the negative variety genomic DNA obtained by the above method.

  That is, as one aspect of the method for designing an oligonucleotide for identifying varieties in the present invention, a pair of oligonucleotides capable of amplifying a DNA fragment containing a base region predicted to be a recombination region by a nucleic acid amplification reaction using genomic DNA as a template is designed. And a method for designing an oligonucleotide for identifying varieties.

  In the case of oligonucleotides used as PCR primers, the base region predicted to be the recombination region is designed to exist within the amplification product excluding the primer-derived region, and the bases predicted to be the recombination region in the PCR primer sequence You may design so that a site | part may be included. However, if the primer used as a pair can be designed only in a region that almost matches the sequence that appears to be the corresponding locus due to the sequence characteristics of the genomic DNA region useful for variety identification, etc., the base site that is predicted to be the recombination region However, if present near the 5 'end of the primer, depending on the reaction conditions, the sequence identification as a primer may not be sufficiently exerted, and the variety identification will not be exhibited, that is, the variety genome which is originally considered not to possess the sequence. An amplified product may also be obtained in a PCR reaction using DNA as a template. Therefore, the target amplification product is obtained in the reaction using the cultivar genomic DNA that possesses the sequence as a template, showing the distinguishability under the set reaction conditions, and the cultivar genomic DNA that does not possess the sequence is used as the template. It is necessary to appropriately adjust the primer sequence and amplification conditions so that the desired amplification product cannot be obtained in the reaction. This adjustment varies depending on the characteristics of the DNA region sequence to be identified and the set PCR reaction conditions (such as the annealing temperature), and therefore needs to be appropriately dealt with.

  When an oligonucleotide is used as a probe, an oligonucleotide having a variety identification ability is designed by designing an oligonucleotide having a nucleotide sequence including a nucleotide region predicted to be a recombination region or a nucleotide sequence complementary to the nucleotide sequence. Can be designed.

  On the other hand, in a DNA region useful for discriminating a variety that is predicted to include a recombination region, if there is a region having low sequence identity with the genome sequence of a discriminable variety, a primer pair designed only within the region sequence , Or the probe should also have breed identification performance.

  That is, as one aspect of the method for designing an oligonucleotide for identifying varieties in the present invention, only a region adjacent to a recombination region and a predicted base site and having low sequence identity with the genome sequence of one cultivar is subjected to a nucleic acid amplification reaction. One example is a method for designing a variety identification oligonucleotide, which is characterized by designing a pair of oligonucleotides that can be amplified.

  The means for designing oligonucleotides as primers and probes is not particularly limited, but it is preferable to use commercially available design software based on the selected base sequence. Examples of such design software include OLIGO (registered trademark) Primer Software Software for Windows (Code NB100W) released by Takara Bio Inc.

A second invention of the present invention is a rice variety identification method, characterized in that the presence or absence of a specific base sequence selected from the following (1) to (15) in genomic DNA is used as an identification index: Regarding rice variety identification method:
(1) a variety-identifying DNA region having base numbers 424 and 425 which are represented by SEQ ID NO: 1 and predicted to be a recombination region,
(2) a cultivar identification DNA region represented by SEQ ID NO: 2 and whose base site predicted to be a recombination region is base numbers 48 and 49;
(3) a variety-identifying DNA region having base numbers of 70 to 80, which is represented by SEQ ID NO: 3 and predicted to be a recombination region,
(4) a cultivar identification DNA region represented by SEQ ID NO: 4 and whose base site predicted to be a recombination region is base numbers 19 to 26;
(5) a DNA region for detecting a cultivar having base numbers 763 and 764 as base sites shown in SEQ ID NO: 5 and predicted to be a recombination region;
(6) a DNA region represented by SEQ ID NO: 6 and whose base site predicted to be a recombination region is base numbers 366 and 367,
(7) a DNA region represented by SEQ ID NO: 7 and whose base site predicted to be a recombination region is base numbers 353 to 361,
(8) a DNA region represented by SEQ ID NO: 8 and whose base site predicted to be a recombination region is base numbers 249 and 250;
(9) a DNA region represented by SEQ ID NO: 9 and whose base site predicted to be a recombination region is base numbers 550 and 551;
(10) a DNA region represented by SEQ ID NO: 10 and having a base site predicted to be a recombination region of base numbers 9 and 10;
(11) a DNA region represented by SEQ ID NO: 11 and whose base site predicted to be a recombination region is base numbers 15 and 16;
(12) a DNA region represented by SEQ ID NO: 12 and having a base site predicted to be a recombination region of base numbers 555 to 558,
(13) a DNA region in which the base site shown in SEQ ID NO: 13 and predicted to be a recombination region is a region of base numbers 474 and 475;
(14) a DNA region in which the base site shown in SEQ ID NO: 57 is the region of base numbers 231 and 232, and (15) the base site shown in SEQ ID NO: 58 and predicted to be the recombination region Is a DNA region where the base numbers 114 and 115 are regions.

  The base site selected as the recombination region is the predicted recombination region and one base on each side of the recombination region in comparison with the base sequence considered to be the corresponding gene locus. (1), (2), (5), (6), (8), (9), (10), (11), (13), (14), (15) are used for identification The possible DNA regions were clearly divided into two regions with high and low sequence identity with respect to the compared base sequences, so 2 bases indicating the boundary of each region are described as base regions predicted as recombination regions did. On the other hand, with regard to (3), (7), and (12), the DNA region that can be used for identification cannot be clearly divided into two regions that have high and low sequence identity with respect to the compared base sequences. Therefore, the base site selected as the recombination region is one base each on the 5 ′ side and 3 ′ side adjacent to the overlapping region. Regarding (4), since insertion of 8 bases was observed, the insertion region was described as a recombination region.

  (12) is a variety identification DNA region newly developed for the purpose of detecting “Kirara 397” and “Akiho” which are RAPD-STS primer pair G4 positive varieties mixed in “Nana Tsuboshi”.

  The DNA region (1) is an amplified region by the RAPD-STS-ized primer pair WKA09 in the “Hitomebore” genomic DNA, and base numbers 424 and 425 are predicted to be recombination regions in comparison with the “Nippon Hare” genomic sequence. However, in the PCR primer pair designed to include the recombination base site in the amplification product sequence, an identification band is detected in the RAPD-STS-primed primer pair WKA09 positive variety, and no identification band is detected in the negative variety, but RAPD- The same discrimination performance as the STS-modified primer pair WKA09 was exhibited.

  DNA region (5) is a region amplified by RAPD-STS-primed primer pair M2CG in “Nippon Hare” genomic DNA, and base numbers 763 and 764 are predicted to be recombination regions in comparison with “Akitakomachi” genomic sequence. However, in the PCR primer pair designed to include the recombination base site in the amplification product sequence, an identification band is detected in the RAPD-STS-modified primer pair M2CG positive variety, and no identification band is detected in the negative variety. The same discrimination performance as that of the STS primer pair M2CG was shown.

  The DNA region (8) is a region amplified by the RAPD-STS primer pair S13 in the “Kirara 397” genomic DNA, and base numbers 249 and 250 are predicted to be recombination regions in comparison with the “Nippon Hare” genomic sequence. However, in the PCR primer pair designed to include the recombination base site in the amplification product sequence, the identification band is detected in the RAPD-STS-modified primer pair S13 positive variety, the identification band is not detected in the negative variety, and the RAPD -It showed the same discrimination performance as STS-modified primer pair S13.

  The DNA region (10) is an amplification region by the RAPD-STS-ized primer pair G4 in the “Kirara 397” genomic DNA. In comparison with the “Nippon Hare” genomic sequence, base numbers 9 and 10 are predicted to be recombination regions. However, in the PCR primer pair designed to include the recombination base site in the amplification product sequence, the identification band is detected in the RAPD-STS-modified primer pair G4 positive variety, the identification band is not detected in the negative variety, and the RAPD -It showed the same discrimination performance as the STS-modified primer pair G4.

  The DNA region (13) is an amplification region by the RAPD-STS primer pair G49 in the “Hoshinoyume” genomic DNA, and the region of base numbers 1 to 474 has a low sequence identity in the region compared with the “Nippon Hare” genomic sequence. In the PCR primer pair designed to amplify only the RAPD-STS primer pair G49 positive variety, the identification band was detected, and in the negative variety, the identification band was not detected, showing the same discrimination performance as the RAPD-STS primer pair G49. It was.

  On the other hand, the DNA region (2) is a locus sequence corresponding to the amplified region in the RAPD-STS-primed primer pair WKA09 positive varieties in the “Nipponbare” genomic DNA, and the base site expected to be a recombination region is base numbers 48 and 49. Expected. Surprisingly, in the PCR using various rice genomic DNAs described in Examples as a template by PCR primer pairs designed for the detection of the DNA region (2), an identification band was detected in the RAPD-STS-modified primer pair WKA09-positive cultivars. However, it was found that a discrimination band was detected in negative varieties including “Nipponbare”, and it was possible to design a PCR primer pair whose discrimination was reversed with the RAPD-STS-modified primer pair WKA09.

  The DNA region (3) is a gene locus sequence corresponding to the amplified region in the RAPD-STS-modified primer pair G22-positive variety in the “Nipponbare” genomic DNA, and the base region predicted to be the recombination region is predicted to have base numbers 70-80. It was done. Surprisingly, in PCR using various rice genomic DNAs described in the actual examples as a template with primer pairs designed for the detection of DNA region (3), PCR primers whose recognizability is reversed from RAPD-STS primer pair G22 It turns out that the pair can be designed.

  The DNA region (4) is a gene sequence corresponding to the amplified region in the RAPD-STS-modified primer pair A6 positive varieties in the “Nippon Hare” genomic DNA, and the base site expected to be the recombination region is predicted to be base numbers 19 to 26 It was. Surprisingly, PCR using primer pairs designed for the purpose of detecting the DNA region (4) using various rice genomic DNAs described in the examples as templates is a PCR primer whose discrimination is reversed from the RAPD-STS-modified primer pair A6. It turns out that the pair can be designed.

  The DNA region (7) is a locus sequence corresponding to the amplified region in the RAPD-STS-modified primer pair P3 positive cultivar in “Akitakomachi” genomic DNA. It was. Examples based on primer pairs designed to detect the DNA region (7) Surprisingly in PCR using various rice genomic DNA as a template, a PCR primer pair whose discrimination is reversed from the RAPD-STS primer pair P3 It turns out that it can be designed.

  The DNA region (9) was present in the “Nipponbare” genomic DNA, and the base sites expected to be recombination regions were predicted to be base numbers 550 and 551. Surprisingly, PCR using primer pairs designed for the purpose of detecting the DNA region (9) using various rice genomic DNAs described in the examples as a template is a PCR primer pair whose discrimination is reversed from the RAPD-STS primer pair S13. Was found to be designable.

  The DNA region (11) was predicted to be a recombination region in the gene sequence corresponding to the amplification region in the RAPD-STS-modified primer pair G4 positive cultivar in “Nippon Hare” genomic DNA with base numbers 15 and 16 . Surprisingly, PCR using primer pairs designed for the detection of DNA region (11) using various rice genomic DNAs described in the examples as templates was a PCR primer whose recognizability was reversed from that of RAPD-STS primer pair G4. It turns out that the pair can be designed.

  The DNA region (14) is a locus sequence corresponding to the amplified region in the RAPD-STS-primed primer pair M2CG positive cultivar in the “Akitakomachi” genomic DNA. It was. Surprisingly, PCR using various pairs of rice genomic DNA described in Examples as a template with primer pairs designed for the detection of the DNA region (14) is surprisingly a PCR primer whose reversibility is reversed from the RAPD-STS primer pair M2CG. It turns out that the pair can be designed.

  The DNA region (15) is a base sequence expected to be a recombination region in the locus sequence corresponding to the amplified region in the RAPD-STS-modified primer pair G49A positive cultivar in “Nipponbare” genomic DNA, and nucleotide numbers 114 and 115 were predicted. . Surprisingly, PCR using various pairs of rice genomic DNA described in Examples as a template with primer pairs designed for the purpose of detecting the DNA region (15) is a PCR primer whose discrimination is reversed from the RAPD-STS-modified primer pair G49A. It turns out that the pair can be designed.

  As described above, DNA regions (1) and (2) can be used for discrimination between varieties indicated by + and-in Table 1, rWKA09-1 column of the present specification. As a specific example, Koshihikari , Nipponbare, etc., and varieties consisting of Hitomebore, Hinohikari, Akitakomachi, etc.

  The DNA region (3) can be used for identification between varieties indicated by + and − in Table 2, column rG22-1 of the present specification. Specific examples include Koshihikari, Hitomebore, Hinohikari, Kirara 397, It can be used to distinguish between varieties consisting of Hoshino Yume, Nanatsuboshi, etc., and varieties consisting of Akitakomachi, Kinuhikari, Haenuki, Tsugaru Roman, Nipponbare, Mutsuhomare, Sasanishiki, Hashishima, and the like.

  The DNA region (4) can be used for identification between varieties indicated by + and − in Table 1, rA6-1 column of the present specification. Specific examples include Koshihikari, Hitomebore, Hinohikari, Akitakomachi, Kirara 397, Hoshino Yume, etc., and a variety group consisting of Kinuhikari, Haenuki, Hashishima, etc. can be used for identification.

  The DNA regions (5) and (14) can be used for discrimination between varieties indicated by + and − in the sM2CG-1 column of Table 1 of the present specification. Specific examples include Koshihikari, Akitakomachi, Kinuhikari, and Tsugari. It can be used to identify a variety group consisting of romance, sasanishiki, and the like, and a variety group consisting of Hitomebore, Hinohikari, Haenuki, Kirara 397, Hoshinoyume, Nanatsuboshi, Hatsushima, and the like.

  The DNA regions (6) and (7) can be used for discrimination between varieties indicated by + and-in Table 1, rP3-1 column of the present specification. Specific examples include Koshihikari, Hinohikari, Kinuhikari. , Kirara 397, Hoshino Yume, Nanatsuboshi, Hashishima, etc., and varieties consisting of Hitomebore, Akitakomachi, Tsugaru Roman, Haenuki, Sasanishiki, and the like.

  DNA regions (8) and (9) can be used for discrimination between varieties indicated by + and − in the rS13-1 column of Table 4 of the present specification. Specific examples include Koshihikari, Hitomebore, Hinohikari, It can be used to identify a variety group consisting of Akitakomachi, Kinuhikari, Tsugaru Romance, Nanatsuboshi, and the like, and a variety group consisting of Kirara 397, Hoshino Yume, and the like.

  The DNA regions (10) and (11) can be used for identification between varieties indicated by + and − in the rG4-1 column of Table 4 of the present specification. Specific examples include Koshihikari, Hitomebore, Hinohikari, It can be used to distinguish between a variety group consisting of Akitakomachi, Kinuhikari, Haenuki, Hoshino Yume, Tsugaru Romantic, and a variety group consisting of Kirara 397, Nanatsuboshi, and the like.

The DNA regions (12) and (15) can be used for discrimination between varieties indicated by + and − in the sG49A-1 column of Table 4 of the present specification. Specific examples include Koshihikari, Hitomebore, Kinuhikari, Kirara 397, Tsugaru Romance, etc., and a variety group consisting of Hinohikari, Akitakomachi, Hoshino Yume, Nanatsuboshi, and the like.

  The DNA region (13) can be used for discrimination between varieties indicated by + and-in the Nak-1 column of Table 5 of the present specification, and specifically comprises Nanatsubo, Nipponbare, etc. It can be used to identify a variety group consisting of a variety group and Kirara 397, Hoshino Yume, Akiho, and the like.

  The use of the present invention makes it possible to identify various varieties, but the number of specific base sequences to be used varies depending on the cultivar to be identified and its preconditions.

  For example, in the case of the condition “identification between two varieties of specimens”, the identification can be performed by using at least one specific base sequence. In the condition that “specimen is mutually distinguishable with three types”, at least two specific base sequences are required for identification. However, if “specimen is any one of the three varieties and whether it is a specific one or not?”, It is sufficient to be able to distinguish between two groups of one specific cultivar and the other two varieties. May be identified by using.

  At present, paddy rice straw is the staple food in Japan, and the top 20 varieties occupy 90% and the top 60 varieties account for about 97% of the nationwide area for paddy rice cultivation.

  Therefore, if rice paddy varieties can be distinguished from the top 60 cultivars, especially the top 20 varieties, it can be considered as a practical cultivar identification system.

  In the following, a variety identification system for the top ten cultivars is exemplified. Selection of a specific DNA sequence for identifying a variety is premised on a qualitative inspection system capable of detecting different varieties in the top 60 varieties as much as possible.

  For example, in the case of a variety identification system using the PCR method, the number of reactions can be reduced if a multiplex reaction system is used, but stabilization of the reaction system can be expected with a smaller number of primers. Therefore, it is preferable to use a combination that requires as few primers as possible.

  As a method of selection, for example, when “Kinuhikari” is selected as a test subject variety, the first condition is to select a specific base sequence that is not held in the “Kinuhikari” genomic DNA. Examples of this reaction system are described in Table 1. In this table, M11, rP3-1, rWKA09-1, rA6-1 and sM2CG-1 represent PCR primer pair names, and the genome of “Kinuhikari” When an identification band is detected in PCR using DNA as a template, it is represented by +, and when an identification band is not detected, it is represented by-. That is, in the case of this table, the specific base sequence including the sequence to be the amplification region of each of the primer pairs of rP3-1, rA6-1, sM2CG-1, and rWKA09-1 that indicates-in Kinuhikari meets the first condition. To do. The primer pair rWKA09-1 is a PCR primer pair capable of detecting the DNA region (2), the primer pair rA6-1 is the above (4), and the primer pair sM2CG-1 is a PCR primer pair capable of detecting the above (5).

  When the varieties listed in Table 1 are tested, among the primer pairs that satisfy the first condition, the varieties from which the amplification product can be obtained with the primer pair rA6-1 occupy 48 varieties out of 59 varieties except “Kinuhikari”. Therefore, when the primer pair rA6-1 is used, it is possible to distinguish 48 varieties with one primer. After that, it is sufficient to select a primer for which a product for which no amplified product is obtained with the primer pair rA6-1 is positive, but the primer pair sM2CG-1 is positive for 10 of the remaining 11 varieties. The remaining one variety can be identified by using the primer pair rWKA09-1, and all the other 59 varieties excluding Kinuhikari become + and can be distinguished from Kinuhikari with a total of 3 primer pairs. It should be noted that varieties in which any of the three primer pairs is + can be identified as not Kinuhikari, but just because all 3 primer pairs are-cannot be determined to be Kinuhikari.

  On the other hand, Table 1 shows the results in a multiplex PCR reaction system using RAPD-STS-modified primer pair M11 and primer pair rP3-1 in addition to the three primer pairs. In the RAPD-STS primer pair M11, an identification band is detected in Kinuhikari, which is added for the Positive Control reaction for confirming whether the PCR reaction is inhibited. When the primer pair rP3-1 is added to the four primer pairs, the combination of + and − patterns of each variety further expands, and more types can be distinguished from each other in one reaction. The reaction system consisting of the 5 primer pairs shown in Table 1 is designed for use in the qualitative inspection of “Kinuhikari”, but it can also be used for identifying the varieties in more detail in the quantitative inspection.

  An example of an identification method in which the specific base sequence represented by (1) to (13) in the present invention is appropriately selected based on the same selection criteria is at least one selected from (3), (11), and (13). A method of identifying a variety whose species specific base sequence is present in genomic DNA as not "Kirara 397", at least one specific base sequence selected from the above (11) and (12) is present in genomic DNA A method for identifying a variety as not “Nanatsuboshi”, “Hoshi no Yume” for a variety in which at least one specific base sequence selected from (3), (9) and (10) above is present in genomic DNA It is a method of identifying that it is not.

  The presence of the specific base sequences (1) to (15) in the genomic DNA can be carried out, for example, by detecting a DNA fragment amplified by a nucleic acid amplification method. The nucleic acid amplification method is preferably a PCR method, but the primer pair designed based on the first invention may be used as the primer pair.

Examples of such PCR primer pairs include the following (a) to (r):
(A) DNA represented by SEQ ID NO: 14 and DNA represented by SEQ ID NO: 15,
(B) DNA represented by SEQ ID NO: 16 and DNA represented by SEQ ID NO: 17,
(C) DNA represented by SEQ ID NO: 18 and DNA represented by SEQ ID NO: 19,
(D) DNA represented by SEQ ID NO: 20 and DNA represented by SEQ ID NO: 21,
(E) DNA represented by SEQ ID NO: 22 and DNA represented by SEQ ID NO: 23,
(F) DNA represented by SEQ ID NO: 24 and DNA represented by SEQ ID NO: 25,
(G) DNA represented by SEQ ID NO: 26 and DNA represented by SEQ ID NO: 27,
(H) DNA represented by SEQ ID NO: 28 and DNA represented by SEQ ID NO: 29,
(I) DNA represented by SEQ ID NO: 30 and DNA represented by SEQ ID NO: 31,
(J) DNA represented by SEQ ID NO: 32 and DNA represented by SEQ ID NO: 33,
(K) DNA represented by SEQ ID NO: 34 and DNA represented by SEQ ID NO: 32,
(L) DNA represented by SEQ ID NO: 35 and DNA represented by SEQ ID NO: 36,
(M) DNA represented by SEQ ID NO: 37 and DNA represented by SEQ ID NO: 38,
(N) DNA represented by SEQ ID NO: 39 and DNA represented by SEQ ID NO: 40,
(O) DNA represented by SEQ ID NO: 41 and DNA represented by SEQ ID NO: 34,
(P) DNA represented by SEQ ID NO: 59 and DNA represented by SEQ ID NO: 60,
(Q) DNA represented by SEQ ID NO: 61 and DNA represented by SEQ ID NO: 60, and (r) DNA represented by SEQ ID NO: 62 and DNA represented by SEQ ID NO: 63.

  When the identification of “Kinuhikari” is carried out by PCR, the primer pairs used are the primer pairs used when the identification of the above (b), (d), (e), and “Kirara 397” is carried out by PCR. As for the above (c), (k), (l) and the above-mentioned “Nanatsuboshi”, the primer pairs to be used are the above (m), (o) and “Hoshino Yume” (C), (i), and (j) are exemplified as the primer pairs to be used.

  Hereinafter, in this specification, a primer pair consisting of the DNA shown in (a) is referred to as sWKA09-1, a primer pair consisting of the DNA shown in (b) is referred to as rWKA09-1, and a primer pair consisting of the DNA shown in (c) is referred to as rG22-1, a primer pair consisting of the DNA shown in (d) is rA6-1, a primer pair consisting of the DNA shown in (e) is sM2CG-1, and a primer pair consisting of the DNA shown in (f) is sP3- 1. A primer pair consisting of DNA shown in (g) is rP3-1, a primer pair consisting of DNA shown in (h) is sS13-1, a primer pair consisting of DNA shown in (i) is rS13-1. The primer pair consisting of the DNA shown in (j) is sG4-1, the primer pair consisting of the DNA shown in (k) is rG4-1, (l) The primer pair consisting of the indicated DNA is indicated by sG49A-1, the primer pair consisting of the DNA indicated by (m) is indicated by Nak-1, the primer pair consisting of the DNA indicated by (n) is indicated by rG22-2 and (o). A primer pair consisting of DNA is rG4-2, a primer pair consisting of DNA indicated by (p) is rM2CG-1, a primer pair consisting of DNA indicated by (q) is rM2CG-2, and a DNA pair indicated by (r) This primer pair is called rG49A-1.

  The primer pair is designed to be suitably used as a PCR primer under the reaction conditions of PCR Kit I for rice variety discrimination (product code RR211A, manufactured by Takara Bio Inc.) sold by Takara Bio Inc. In the present invention, various primers can be designed to meet other conditions, and the present invention is not limited to the use of the above primer pairs.

  The PCR primer pair is designed on the premise of agarose gel electrophoresis as a PCR amplification product detection technique, but the technique for detecting a DNA fragment in the present specification is not particularly limited. For example, the DNA fragment may be detected by fluorescence by real-time PCR using a probe to which a fluorescent dye is added in addition to the PCR primer. Such a primer and probe set can be appropriately designed using commercially available design software based on the sequence information described in (1) to (13) above. In addition, it is detected by a technique such as a so-called DNA chip that detects whether or not there is a nucleic acid including a DNA region for variety identification based on hybridization with an oligonucleotide described in this specification fixed on an appropriate carrier. Also good.

When the nucleic acid amplification method is used as the rice cultivar identification method in the present invention, a method of specifically performing the following steps may be mentioned:
(A) a nucleic acid preparation step of preparing rice nucleic acid in a rice sample;
(B) A step of performing a nucleic acid amplification method using the nucleic acid obtained in step (A) as a template, and (C) a step of analyzing the DNA fragment obtained in step (B).

(A) Nucleic Acid Preparation Step for Preparing Rice Genomic DNA in Rice Sample In this step, rice genomic DNA is prepared from a rice-derived rice sample. As a method for preparing a nucleic acid from a test sample, a known genomic DNA preparation method can be appropriately selected and used. For example, it can be prepared according to the CTAB method, the enzymatic method (Otsubo et al., JP 2000-287691 A) and the like. In addition, it is not particularly limited as long as it is a preparation method capable of extracting plant genomic DNA. For example, a commercially available DNA extraction kit (rice DNA extraction kit manufactured by Takara Bio Inc. (Takara Bio Inc .: Code 9197, Code 9103) is available. ) Etc.).

  The test sample is not particularly limited as long as it is rice, and whole grass, leaves, stems, roots, seeds, and the like can be used in the present invention. The method of the present invention can be applied to seeds that are more difficult to identify morphological varieties, brown rice, or polished rice that is more difficult to identify morphological or biochemical varieties.

(B) A step of performing a nucleic acid amplification method using the nucleic acid obtained in step (A) as a template In this step, a nucleic acid amplification method is performed using the nucleic acid prepared in step (A) as a template DNA, and The presence of a specific base sequence in the present invention is detected.

  Containers, reagents, and compositions required for the reaction are not particularly limited, but commercially available PCR reagents and PCR kits may be used when the PCR method is used as a nucleic acid amplification method. The above steps may be carried out by adopting components and reaction conditions included in PCR Kit I for rice discrimination (Takara Bio Inc. product code RR211A) sold by Takara Bio Inc. In this case, it is also easy to identify the variety of rice by using the primer pair for variety identification contained in the product together with the primer pair provided by the present invention.

When the product is used, a PCR reaction solution can be prepared, for example, with the following composition.
2 μL: 10 × PCR Buffer (100 mM Tris-HCl (pH 8.3), 500 mM KCl)
2 μL: 25 mM MgCl ₂
2 μL: dNTP Mixture (2.5 mM each)
1.5 μL: PCR primer solution 2 μL: Template DNA solution 0.15 μL: TaKaRa Taq (5 u / μL)
10.35 μL: sterile water

  The conditions for the DNA amplification reaction in the above reaction solution are as follows. When Thermal Cycler DICE (product code TP650, manufactured by Takara Bio Inc.) is used as a PCR apparatus, heat denaturation (94 ° C. for 2 minutes) is performed. C. 1 minute), annealing (62.degree. C. 1 minute), and extension reaction (72.degree. C. 2 minutes) are exemplified by 35 cycles. In general, the reaction conditions may be set as appropriate in consideration of the DNA polymerase used, the size of the amplification product, and the capacity of the apparatus used.

  In the case of a multiplex PCR reaction in which a plurality of primer pairs are reacted at once, it is useful in that it suppresses DNA polymerase activity until high temperature and suppresses non-prime amplification due to preprime cycle mispriming and primer dimer. The use of an enzyme for Hot Start PCR, for example, TaKaRa Taq Hot Start Version (product code R007A manufactured by Takara Bio Inc.) is more preferable.

  Further, the template DNA solution is not particularly limited, but the use of a DNA solution corresponding to 10 ng in terms of absorbance is exemplified. In the case of the reaction solution composition exemplified above, a DNA solution prepared to 5 ng / μL in terms of absorbance may be used. The amount of primer used is not particularly limited, and may be appropriately adjusted so that, for example, an amount of DNA fragment suitable for detection is amplified under the above conditions.

(C) The step of analyzing the DNA fragment obtained in step (B) The amplified product obtained in step (B) is subjected to an appropriate DNA separation means to separate and detect the amplified DNA fragment. The presence of a DNA fragment that serves as an index for identifying rice varieties is tested. The DNA separation means used in this step is not particularly limited, and known separation means such as agarose gel electrophoresis and polyacrylamide gel electrophoresis can be used. Agarose gel electrophoresis is preferred from the viewpoint of simplicity of operation, and examples of the conditions include the following.
Agarose gel: 2.5% Agarose L03 (product code 5003, manufactured by Takara Bio Inc.), electrophoresis buffer: 1 × TAE, electrophoresis apparatus: Mupid-2 Plus (product code AD110, Takara Bio Inc.), sample migration amount : Step B: In case of the exemplified composition, injection at 10 μL / well, migration at 50 V for 60 minutes.

  The means for detecting the separated DNA fragment is not limited, but, for example, agarose gel is stained with ethidium bromide or SYBR (registered trademark) Green, and then irradiated with ultraviolet light, and a fluorescent band is detected at a predetermined position. It can be confirmed by whether or not.

  The technique for detecting a DNA fragment in the present specification is not particularly limited to the gel electrophoresis technique. For example, the DNA fragment may be detected by fluorescence by real-time PCR using a probe to which a fluorescent dye is added in addition to the PCR primer. Such a primer and probe set can be appropriately designed using commercially available design software based on the sequence information described in (1) to (13) above. In addition, it is detected by a technique such as a so-called DNA chip that detects whether or not there is a nucleic acid including a DNA region for variety identification based on hybridization with an oligonucleotide described in this specification fixed on an appropriate carrier. Also good.

The third aspect of the present invention is an oligonucleotide selected from the following (i) to (xvi), which is used for detecting the presence of a specific base in genomic DNA:
(I) an oligo having a specific base sequence represented by SEQ ID NO: 1 and consisting of 10 to 100 bases in length of the base sequence and including the base sites of base numbers 424 and 425 of the base sequence represented by SEQ ID NO: 1 nucleotide,
(Ii) an oligo having a specific base sequence represented by SEQ ID NO: 2 and a length of 10 to 100 bases in which the base sequence is continuous, and including the base sites of base numbers 48 and 49 in the base sequence represented by SEQ ID NO: 2 nucleotide,
(Iii) An oligo having a specific base sequence represented by SEQ ID NO: 3 and a length of 15 to 100 bases in which the base sequence is continuous, and including base sites of base numbers 70 to 80 in the base sequence represented by SEQ ID NO: 3 nucleotide,
(Iv) An oligo having a specific base sequence represented by SEQ ID NO: 4 and a length of 20 to 100 bases in which the base sequence is continuous, and including the base sites of base numbers 14 to 31 in the base sequence represented by SEQ ID NO: 4 nucleotide,
(V) an oligo having a specific base sequence represented by SEQ ID NO: 5 and consisting of 10 to 100 bases in length of the base sequence and including the base sites of base numbers 763 and 764 in the base sequence represented by SEQ ID NO: 5 nucleotide,
(Vi) an oligo having a specific base sequence represented by SEQ ID NO: 6 and a length of 10 to 100 bases in which the base sequence is continuous, and including the base sites of base numbers 366 and 367 in the base sequence represented by SEQ ID NO: 6 nucleotide,
(Vii) an oligo having a specific base sequence represented by SEQ ID NO: 7, consisting of 15 to 100 bases in length of the base sequence, and comprising the base sites of base numbers 353 to 361 in the base sequence represented by SEQ ID NO: 7 nucleotide,
(Viii) an oligo having a specific base sequence represented by SEQ ID NO: 8, consisting of 10 to 100 bases in length of the base sequence, and comprising base sites of base numbers 249 and 250 in the base sequence represented by SEQ ID NO: 6 nucleotide,
(Ix) An oligo having a specific base sequence represented by SEQ ID NO: 9 and a length of 10 to 100 bases in which the base sequence is continuous, and including the base sites of base numbers 550 and 551 in the base sequence represented by SEQ ID NO: 6 nucleotide,
(X) an oligo having a specific base sequence represented by SEQ ID NO: 10 and a length of 10 to 100 bases in which the base sequence is continuous, and including the base sites of base numbers 9 and 10 in the base sequence represented by SEQ ID NO: 10 nucleotide,
(Xi) The specific base sequence is represented by SEQ ID NO: 11 and consists of 10 to 100 bases in length of the base sequence and includes the base sites of base numbers 15 and 16 in the base sequence represented by SEQ ID NO: 11. nucleotide,
(Xii) an oligo having a specific base sequence represented by SEQ ID NO: 12, comprising a continuous base sequence of 10 to 100 bases in length, and comprising a base site of base numbers 555 to 558 in the base sequence represented by SEQ ID NO: 12 nucleotide,
(Xiii) An oligo having a specific base sequence represented by SEQ ID NO: 13, consisting of 10 to 100 bases in length of the base sequence, and including the base sites of base numbers 474 and 475 in the base sequence represented by SEQ ID NO: 13 nucleotide,
(Xiv) an oligo having a specific base sequence represented by SEQ ID NO: 57, consisting of 10 to 100 bases in length of the base sequence and including the base sites of base numbers 231 and 232 in the base sequence represented by SEQ ID NO: 57 nucleotide,
(Xv) an oligo having a specific base sequence represented by SEQ ID NO: 58, consisting of 10 to 100 bases in length of the base sequence, and including the base sites of base numbers 114 and 115 in the base sequence represented by SEQ ID NO: 58 A nucleotide, and (xvi) an oligonucleotide having a base sequence complementary to the oligonucleotides (i) to (xv).

  These oligonucleotides are oligonucleotides that include in their sequences a base site predicted to be a recombination region in a genomic DNA region useful for breed identification. Specifically, DNA shown by sequence number 21, 22, 24, 33, 34, 38, 40 is illustrated.

  A rice variety identification kit according to a fourth aspect of the present invention is a kit for carrying out the above-described rice variety identification method, and detects the presence of a specific nucleotide sequence in genomic DNA as a DNA fragment amplified by a nucleic acid amplification method. It contains the primer pair used for the purpose to do. In a preferred embodiment, the primer pair is suitable for PCR.

  This kit can further contain a heat-resistant DNA polymerase suitable for the PCR method and a reaction buffer for carrying out the reaction with the DNA polymerase. The thermostable polymerase is not particularly limited, but in the case of a kit employing a multiplex PCR reaction system that reacts a plurality of primer pairs at once, an enzyme for Hot Start PCR, for example, TaKaRa Taq Hot Start Version (Product code R007A manufactured by Takara Bio Inc.) is more preferable.

  The reaction buffer is not particularly limited, and a heat-resistant polymerase to be used may be used depending on the desired reaction conditions.

  The primer pair for PCR can be included in the kit of the present invention in the form of a solution. In this case, a plurality of solutions containing one kind of primer or primer pair may be prepared, or a plurality of primer pairs may be mixed in advance and combined into one.

  It relates to a DNA region that can be used for variety identification of the fifth invention, and is a DNA fragment represented by SEQ ID NO: 12 newly developed as a DNA region that can be used for variety identification.

  Hereinafter, the present invention will be described more specifically with reference to examples. In addition, this invention is not specifically limited to a present Example.

[Design of PCR Primer Pair for Variety Identification that Detects DNA Region Described in (1) and (2) of this Application]
The DNA region for discriminating varieties described in (1) and (2) is amplified sequence information by the RAPD-STS-modified primer pair WKA09 developed by Otsubo et al. (Japanese Patent Laid-Open No. 2005-245244 (Patent Document 3)): Sequence No. 7) was used. When the sequence information was compared with the genome sequence information of the rice cultivar “Nipponbare” disclosed in GenBank, the region was divided into a region having high sequence identity and a region having low sequence identity, and the border region was predicted to be a recombination region. Based on the DNA sequence information, the PCR primer design software Oligo6 (Takara Bio Inc.) was used to detect the variety identification DNA region (1) by the RAPD-STS-modified primer pair WKA09 in “Hitomebore” genomic DNA. Primer pair (a): sWKA09-1 and primer pair (b): rWKA09-1 for the purpose of detecting the DNA region for variety identification (2) were designed. The primer pairs sWKA09-1 and rWKA09-1 are PCR primer pairs designed to include a base site predicted to be a recombination region in each product identification DNA region in an amplification product excluding the primer-derived region.

[Design of PCR Primer Pair for Variety Identification that Detects DNA Region Described in (3) of this Application]
For the estimation of the DNA region for variety identification described in (3), amplification sequence information by RAPD-STS-modified primer pair G22 developed by Otsubo et al. (Japanese Patent Laid-Open No. 2005-245244 (Patent Document 3)): SEQ ID NO: 2). In addition, Unexamined-Japanese-Patent No. 2005-245244 (patent document 3): The 5 'terminal in sequence number 2 is not the sequence (agggccatgata) of RAPD primer G22. This is because when the identification band appearance pattern by the RAPD primer G22 is confirmed, an identification band of about 0.83 kbp is obtained for “Koshihikari” and “Hitomebore”, and an identification band of about 0.45 kbp is obtained for “Akitakomachi” and “Nihonbare”. This is because, as a result of the analysis of the base sequence of the amplified product obtained from “Koshihikari” and “Akitakomachi”, primers were designed that were positive for “Koshihikari” and negative for “Akitakomachi”. In the case of the base sequence of the “Koshihikari” amplified product, which is a G22 positive variety, it was estimated that insertion occurred between ttagccags of base numbers 71 to 79 of SEQ ID NO: 3. In addition, the above ttagtccag sequence was present at both ends of the insertion part. This time, based on the nucleotide sequence of the identification band obtained from “Akitakomachi”, it is represented by SEQ ID NOs: 42 and 43, which are extended 3 ′ from the RAPD primer G22 sequence part existing at both ends to the inside of the amplified product. A RAPD-STS primer pair G22-2 consisting of DNA was designed. This primer pair can amplify the entire DNA identification region (3).

  Furthermore, based on the DNA information, PCR primer design software Oligo6 was used to design a primer pair (c): rG22-1 and (r) rG22-2 for the purpose of detecting the type identification DNA region (3). In addition, the primer pair rG22-1 includes a base site predicted to be a recombination region in the variety identification region (3) (base numbers 70 to 80 in SEQ ID NO: 3 in the sequence listing) in the amplified product sequence excluding the primer-derived region. The primer pair rG22-2 is designed so that the base region predicted to be a recombination region is included in one primer sequence of the primer pair.

[Design of PCR Primer Pair for Variety Identification that Detects DNA Region Described in (4) of this Application]
For the estimation of the DNA region for identifying the variety described in (4), information on the sequence of the amplification product by the RAPD-STS primer pair A6 (see JP 2005-245244 (Patent Document 3): SEQ ID NO: 9) was used. . When the sequence information was compared with the genomic DNA sequence information of the A6 negative rice cultivar “Nipponbare” disclosed in GenBank, the region was divided into a region having a high sequence identity and a region having a low sequence identity, and the boundary region was a recombination region. Predicted. The main difference was the number of repeats of the TA repeat region present in the A6 amplification product and the number of repeats of tatc at base numbers 15 to 30 in the base sequence of SEQ ID NO: 4 in the sequence listing. Therefore, it was considered appropriate to design the RAPD-STS-modified primer pair A6 and a primer pair whose identification band pattern is inverted in a region where the number of tatc repeats is different. In addition, when a primer pair is designed between regions where the RAPD-STS-modified primer pair A6 is expected to bind from this region, the amplification product size is close when another multiplex PCR system is constructed with other cultivar identification primers. There was a possibility that separation by gel electrophoresis might be worsened. Therefore, from the “Nipponbare” genome sequence information, we decided to design a pair of primers outside the RAPD-STS-primed primer pair A6 amplification product region, and used the PCR primer design software Oligo6 to identify the DNA region (4 A primer pair (d): rA6-1 was designed. In SEQ ID NO: 4 in the sequence listing, the region of base numbers 380 to 459 corresponds to the outside of the RAPD-STS primer pair A6 amplification region in the “Nippon Hare” genomic sequence. In addition, this primer pair was designed to include a base site predicted to be a recombination region in the primer sequence shown in SEQ ID NO: 21 in the sequence listing.

[Design of PCR Primer Pair for Variety Identification that Detects DNA Region Described in (5) and (14) of the Application]
For the estimation of the DNA region for variety identification described in (5), information on the amplification product sequence by RAPD-STS primer pair M2CG (Japanese Patent Laid-Open No. 2005-245244 (Patent Document 3): see SEQ ID NO: 13) was used. . When comparing the sequence information with the genome sequence information of the rice cultivar “Nippon Hare” disclosed in GenBank, “Nippon Hare” is an M2CG-positive cultivar. Therefore, RAPD-STS conversion was performed except for a few bases derived from the RAPD primer M2. There was a region with high sequence identity over the entire amplification product sequence by the primer pair M2CG. In addition, when the primers were designed and PCR was performed at both ends of the M2CG amplification region in the “Nippon Hare” genome sequence based on the “Nippon Hare” genome sequence information, it was found that the variety identification performance was lost. Therefore, an attempt was made to determine the base sequence of the corresponding locus region in the genome sequence of the M2CG negative cultivar using the Inverse PCR method. First, EcoRI and PstI were selected as restriction enzymes that cleave this region from a partial sequence of an M2CG amplification product expected to be conserved in the genomic DNA of “Akitakomachi”, which is an M2CG negative variety. Subsequently, genomic DNA was prepared from “Akitakomachi” and digested with restriction enzymes EcoRI or PstI. The cleaved DNA was prepared by DNA Ligation Kit Ver. After the self-circularization reaction of the cleaved DNA piece at 1 (product code 6021), PCR was performed with IRM2-1F (ATATAGGGCAAACCCAACATATACACCAA) and IRM2-1R (ATATAAGGCCACAACCAACATATACACCAA) designed as primers for Inverse PCR. Among them, an amplified product A of about 1.5 kbp was obtained by inverse PCR using a PstI cut piece. As a precaution, PCR with the primer IRM2-2F (ACCAACATTAACCCAAAGATAAAGAAAAT) and the primer IRM2-2R (TGGCCATACTCACTACTACTACATGTGTGT) was performed using the above-mentioned about 1.5 kbp amplified product A as a template, and an amplified product B of about 1.4 kbp was obtained. Since the amplified product B was predicted to obtain an amplified product 75 bp shorter than the amplified product A based on the positional relationship of the primers, it was determined that about 1.4 kbp amplified product B was the target DNA fragment. The sequence of the DNA fragment is shown in SEQ ID NO: 44 in the sequence listing. When the base sequence of the amplified product B was compared with the base sequence of the M2CG amplified product, it was divided into a region having high sequence identity and a region having low sequence identity, and the border region was estimated to be a recombination region. Next, using the PCR primer design software Oligo6 based on the DNA fragment information, a primer pair (e): sM2CG-1 and SEQ ID NO: 45 and 46 in the sequence listing for the purpose of detecting the variety identification DNA region (5) The primer pair (p): sM2CG-2 consisting of the DNA shown in FIG. 5 and the primer pair (p): rM2CG-1 and (q) rM2CG-2 for the purpose of detecting the variety identification DNA region (14) were designed. The primer pairs sM2CG-1 and sM2CG-2, and rM2CG-1 and rM2CG-2 are both designed to include the base region predicted to be a recombination region in the amplification product sequence excluding the primer-derived region. It is a thing. In SEQ ID NO: 57, the region of base numbers 204 to 231 and the region of base numbers 294 to 321 have a palindromic structure. Since an oligonucleotide consisting of the base sequence shown in this region functions alone as a PCR primer pair, care must be taken when designing a PCR primer pair.

[Design of PCR Primer Pair for Variety Identification that Detects DNA Regions Described in (6) and (7) of this Application]
For the estimation of the DNA region for identifying the variety described in (6) and (7), the sequence information of the amplified product by the RAPD-STS primer pair P3 (Japanese Patent Laid-Open No. 2005-245244 (Patent Document 3): see SEQ ID NO: 16) ) Was used. When comparing the sequence information with the genome sequence information of the rice cultivar “Nippon Hare” disclosed in GenBank, “Nippon Hare” is a P3 positive cultivar. Therefore, the RAPD-STS primer pair except for the RAPD primer P3-derived region bases. There was a region with high sequence identity over the entire amplification product sequence by P3. In the “Nipponbare” genome sequence, there were about 0.8 kbp regions including a part 168 bp of the P3 amplified product sequence on the same chromosome genome sequence, and the two regions were located about 3 kbp apart. Assuming that the P3 amplification region in the P3 positive variety was generated by insertion of a DNA fragment of about 3 kbp into the corresponding locus region in the P3 negative variety, and included the about 3 kbp region based on the genomic sequence information of “Nippon Hare” A primer pair consisting of rP3-F (atcagaatatagaacaccaaaagt) and rP3-R (ttcgctagccctgtagaagaacaat) was designed and PCR was performed. As a result, an amplified product of about 0.2 kbp was obtained by a reaction using “Akitakomachi” genomic DNA and “Hitomebore” genomic DNA as P3 negative varieties as templates. Next, the base sequence of the amplification product C obtained using “Akitakomachi” genomic DNA as a template was determined and compared with the “Nippon Hare” genomic sequence. As a result, the “Nippon Hare” genomic sequence has the base sequence shown in SEQ ID NO: 7 in the sequence listing. Of these, insertion was predicted to occur between AACTGATs of base numbers 354 to 360. The AACTGAT sequence was present at both ends of the insertion region. Next, using the PCR primer design software Oligo6 based on the DNA fragment information, a primer pair (f): sP3-1 for detecting the type identification DNA region (6), type identification DNA region (7) Primer pair (g): rP3-1 was designed for detection of. The primer pair sP3-1 is designed to include a base site predicted to be a recombination region in the primer sequence shown in SEQ ID NO: 24 in the sequence listing, and the primer pair rP3-1 is a recombination in the DNA identification region (7). It is designed so that the base site predicted to be a region is included in the amplification product sequence excluding the primer-derived region.

[Design of PCR Primer Pair for Variety Identification that Detects DNA Region Described in (8) and (9) of the Application]
For the estimation of the DNA region for variety identification described in (8) and (9), amplification sequence information by RAPD-STS primer pair S13 developed by Otsubo et al. (Japanese Patent Laid-Open No. 2005-245244 (Patent Document 3)) ): See SEQ ID NO: 4). When the sequence information was compared with the genome sequence information of the rice cultivar “Nipponbare” disclosed in GenBank, it was divided into a region having high sequence identity and a region having low sequence identity, and the border region was a region where recombination occurred. Estimated. Next, a primer pair (h): sS13-1 capable of obtaining a DNA fragment (8) was designed based on the DNA fragment information using PCR primer design software Oligo6. On the other hand, in the “Nipponbare” genomic sequence, there were three regions that showed high sequence identity with a partial region of the S13 amplification product. Of these, two sites have approximately 3 kbp regions including a region showing high sequence identity with the partial region of S13, and a DNA region for identifying a variety (9) can be detected based on the sequences of these two sites. Primer pair (i): rS13-1 and primer pair (q): rS13-2 consisting of the DNAs shown in SEQ ID NOs: 47 and 48 were designed. In addition, a primer pair (r): rS13-3 consisting of the DNAs shown in SEQ ID NOs: 49 and 50 was designed at one remaining candidate variety identification DNA region. The primer pairs sS13-1, rS13-1, rS13-2, and rS13-3 are all included in the amplified product sequence excluding the primer-derived region in the variety identification region DNA sequence. Designed.

[Design of PCR Primer Pair for Variety Identification that Detects DNA Region Described in (10) and (11) of this Application]
For the estimation of the DNA region for identifying the varieties described in (10) and (11), the sequence information of the amplified product by the RAPD-STS primer pair G4 developed by Otsubo et al. (Japanese Patent Laid-Open No. 2005-245244 (Patent Document 3) ): See SEQ ID NO: 8). When the sequence information was compared with the genome sequence information of the rice cultivar “Nipponbare” disclosed in GenBank, it was divided into a region having high sequence identity and a region having low sequence identity, and the border region was a region where recombination occurred. Estimated. There were three regions on the Nipponbare genome sequence that showed high sequence identity with part of the base sequence of the G4 amplification product. When PCR primer pairs were designed in each region and reacted with various rice varieties genomic DNA, one site was estimated to be a locus corresponding to the amplified region in the RAPD-STS-modified primer pair G4 positive cultivar genomic DNA. Indicated. Next, using the PCR primer design software Oligo6 based on the DNA fragment information, a primer pair (j): sG4-1 for detecting the kind identification DNA region (10): kind identification DNA region (11 ) Primer pair (k): rG4-1 and primer pair (o): rG4-2 were designed for the purpose of detection. In the primer pair sG4-1, the primer represented by SEQ ID NO: 33 in the sequence listing, and the primer represented by SEQ ID NO: 34 in the sequence listing in the primer pairs rG4-1 and rG4-2, each have a DNA type identification region in each sequence. A primer including a base site predicted to be a recombination region in FIG.

[Design of PCR Primer Pair for Variety Identification that Detects DNA Region Described in (12) of this Application]
If a primer that can be discriminated from the RAPD-STS-modified primer pair G4 can be obtained, it will be easy to construct a qualitative test system for “Kirara 397” and “Nanatsuboshi” of the few Hokkaido varieties that are G4 positive. When “Nanatsuboshi” is targeted, detection means such as “Kirara 397” and “Akiho” are required. The phenotype of “Nanatsuboshi” has been reported as Pik−, but many other major Hokkaido varieties have been reported as Pik +. Although the nucleotide sequence information of the Pik gene was not available, the promising region was determined by comparing the genome sequence information of “Nippon Hare” disclosed in GenBank and other published data. When a primer pair consisting of the DNA represented by SEQ ID NO: 51 and the DNA represented by SEQ ID NO: 52 was designed in this region, each DNA of “Nana Tsuboshi” and “Nippon Hare” was about 0.45 kbp, “Hoshi An amplified product of about 0.65 kbp was obtained from each DNA of Taro, Kirara 397, and Akiho. Therefore, the base sequence of the amplified product obtained from “Hoshitaro” was analyzed and compared with the genome sequence of “Nippon Hare”. In “Hoshitaro”, the nucleotide region of SEQ ID NOs: 354 to 616 in the sequence listing was the “Nippon Hare” genome sequence. The result is estimated to be inserted. Next, using the PCR primer design software Oligo6 based on the DNA fragment information, a primer pair (m): Nak-1 was constructed for the purpose of detecting the DNA region for variety identification 13). In the primer pair Nak-1, the primer represented by SEQ ID NO: 38 in the sequence listing is a primer that includes a base site that is expected to be a recombination region in the variety-identifying DNA region.

[Design of PCR Primer Pair for Variety Identification that Detects DNA Regions Described in (13) and (15) of this Application]
For the determination of the DNA for discriminating the variety described in (13), the sequence information of the amplified product by the RAPD-STS-modified primer pair G49 developed by Otsubo et al. (Japanese Patent Laid-Open No. 2005-245244 (Patent Document 3)): SEQ ID NO: 12 Reference) was used. When the sequence information was compared with the genome sequence information of the rice cultivar “Nipponbare” disclosed in GenBank, it was divided into a region having high sequence identity and a region having low sequence identity, and the border region was a region where recombination occurred. Estimated. It was noted that the sequence identity with respect to the “Nippon Hare” genome sequence was confirmed again only in the region with low identity, but there was no region that matched the sequence. Therefore, it was expected that the entire region having low sequence identity with the “Nippon Hare” genomic sequence in the amplification region on the “Hoshinoyume” genomic DNA by the RAPD-STS-modified primer pair G49 shows selectivity for the “Nippon Hare” genomic sequence. . Therefore, using the primer design software Oligo6 based on the DNA fragment information, a primer pair (l): sG49A-1 for detection of the variety identification DNA region (13): of the variety identification DNA region (15) A primer pair (r): rG49A-1 for the purpose of detection was designed. In the primer pair sG49A-1, the region of base numbers 96 to 362 in SEQ ID NO: 13 in the sequence listing showing the product identification DNA region (13) is the amplification region. On the other hand, rG49A-1 is designed to include a base site predicted to be a recombination region within the amplified product sequence excluding the primer-derived region.

[Design of primer pair sB43-1]
Amplified sequence information by RAPD-STS primer pair B43 developed by Otsubo et al. (See JP 2005-245244 (Patent Document 3): SEQ ID NO: 3) and rice cultivar “Nippon Hare” disclosed in GenBank Compared with genome sequence information, “Nippon Hare” is a B43-positive variety, so there are regions with high sequence identity over the entire region of the amplified sequence by RAPD-STS primer pair B43, except for several bases derived from RAPD primer B43. did. Based on the “Nippon Hare” genome sequence information, primers were designed at both ends of the B43 amplification region in the “Nippon Hare” genome sequence, and PCR was performed. It was. Considering the results of the prior art, it was speculated that the entire B43 amplification region was selective for the negative breed genome sequence. Therefore, when the PCR primer design software Oligo6 is used and a multiplex PCR system is constructed with other primer pairs, the sequence table is presumed to be adjustable to the length of the amplified product that provides good band separation by agarose gel electrophoresis. The primer pair sB43-1 consisting of the DNAs shown in SEQ ID NOs: 53 and 54 was designed.

[Design of primer pair sF6-1]
Amplification sequence by RAPD-STS-modified primer pair F6 developed by Otsubo et al. The sequence information (see Japanese Patent Application Laid-Open No. 2005-245244 (Patent Document 3): SEQ ID NO: 5) and rice cultivar “Nippon Hare "Nippon Hare" is a F6 positive variety, and the region with high sequence identity over the entire region of the amplified product sequence by the RAPD-STS primer pair F6, excluding several bases derived from the RAPD Primer F6. Existed. In addition, based on the “Nipponbare” genome sequence information, primers were designed at both ends of the F6 amplification region in the “Nipponbare” genome sequence, and PCR was performed. I understood. The primer pair sF6-1 consisting of the DNAs shown in SEQ ID NOs: 55 and 56 in the sequence listing was designed with the expectation that the amplification performance was shortened with the same discrimination performance as the RPAD-STS-modified primer pair F6.

[Confirmation of product identification of designed primer pairs]
The reactivity of the various PCR primer pairs designed above was confirmed. The composition of the reaction solution is as follows.
2 μL: 10 × PCR Buffer (100 mM Tris-HCl (pH 8.3), 500 mM KCl)
2 μL: 25 mM MgCl ₂
2 μL: dNTP Mixture (2.5 mM each)
1.5 μL: PCR primer solution 2 μL: Template DNA solution 0.15 μL: TaKaRa TaqHS (5 u / μL)
10.35 μL: sterile water

The reagent attached to TaKaRa Taq (registered trademark) (product code R001AM) manufactured by Takara Bio Inc. can be used for 10 × PCR Buffer, 25 mM MgCl ₂ and dNTP Mixture (2.5 mM etch). TaKaRa TaqHS is TaKaRa Taq (registered trademark) Hot Start Version (product code R007A) manufactured by Takara Bio Inc.

  As the template DNA solution, a DNA solution having a concentration of 5 ng / μL prepared by a rice DNA extraction kit (product code 9197) sold by Takara Bio Inc. was used.

  In addition, about each prepared kind | species genomic DNA, before confirming the reactivity of the primer pair as described in this specification, RAPD-STS-ized primer pair WKA09 (Unexamined-Japanese-Patent No. 2005-245244 (patent document 3): Sequence | arrangement) No. 37 and 38), B43 (Japanese Patent Laid-Open No. 2005-245244 (Patent Document 3): see SEQ ID Nos. 25 and 26), M11 (Japanese Patent Laid-Open No. 2005-245244 (Patent Document 3): SEQ ID Nos. 19 and 20) G22 (Japanese Patent Laid-Open No. 2005-245244 (Patent Document 3): see SEQ ID Nos. 22 and 23), S13 (Japanese Patent Laid-Open No. 2005-245244 (Patent Document 3): see SEQ ID Nos. 28 and 29), F6 (Japanese Patent Laid-Open No. 2005-245244 (Patent Document 3): see SEQ ID Nos. 31 and 32), E30 (Japanese Patent Laid-Open No. 2005-245244 (Special Document 3): see SEQ ID NOs: 34 and 35), M2CG (Japanese Patent Laid-Open No. 2005-245244 (Patent Document 3): see SEQ ID NOs: 50 and 51), A6 (Japanese Patent Laid-Open No. 2005-245244 (Patent Document 3): SEQ ID NOs: 42 and 43), B1 (see JP 2005-245244 (Patent Document 3): SEQ ID NOS: 44 and 45), G28 (JP 2005-245244 (Patent Document 3): SEQ ID NO: 46, 47), P3 (Japanese Patent Laid-Open No. 2005-245244 (Patent Document 3): see SEQ ID Nos. 56 and 57), G4 (Japanese Patent Laid-Open No. 2005-245244 (Patent Document 3): see SEQ ID Nos. 40 and 41), For G49 (Japanese Patent Laid-Open No. 2005-245244 (Patent Document 3): see SEQ ID Nos. 48 and 49), an identification band appearance pattern was confirmed.

The DNA amplification reaction was performed as follows.
PCR apparatus: TaKaRa PCR Thermal Cycler DiceE (registered trademark) Standard (product code TP650, manufactured by Takara Bio Inc.)
Reaction conditions 96 ° C 2 minutes heat denaturation Heat denaturation (94 ° C 1 minute), annealing (62 ° C 1 minute), extension reaction (72 ° C minute) 35 steps, then cooled to 4 ° C

The analysis of the reaction results was performed by agarose gel electrophoresis under the following conditions.
Agarose gel: 2.5% Agarose L03 (product code 5003, manufactured by Takara Bio Inc.), electrophoresis buffer: 1 × TAE, electrophoresis apparatus: Mupid-2 Plus (product code AD110, Takara Bio Inc.), sample migration amount : 5 μL of electrophoresis buffer was added to the reaction-finished solution, injected into the gel at 10 μL / well, and electrophoresed at 50 V for 60 minutes.

  Whether or not the target DNA fragment was obtained was determined by staining the agarose gel after sample migration with a 1 μg / ml ethidium bromide aqueous solution for 30 to 60 minutes, and then using Image Master VDS (Pharmacia) at the target size. This was confirmed by the presence or absence of a fluorescent band.

[Confirmation of variety discrimination of primer pairs rWKA09-1, rA6-1, rP3-1, sM2CG-1]
Since the number of cultivar genomic DNAs to be confirmed may be large, the discrimination between each primer pair was confirmed in a multiplex PCR reaction system. In the actual confirmation, the RAPD-STS-modified primer pair M11 was further added and reacted. The result of electrophoresis is shown in FIG. In Table 1, the name of the cultivar genomic DNA subjected to the reaction and the presence or absence of an identification band are indicated by + and-. In Table 1, the number shown under the primer pair name represents the size (unit: kbp) of the identification band detected by the primer pair. In the confirmed DNA of 60 varieties, the primer pair rWKA09-1 did not detect the identification band in the PCR using the RAPD-STS-primed primer pair WKA09-positive cultivar genomic DNA as a template, and the RAPD-STS-modified primer containing “Nippon Haru” Discrimination bands were detected in all PCRs using WKA09 negative cultivar genomic DNA as a template. Similarly, in the primer pair rA6-1, the identification band was not detected by PCR using the RAPD-STS-modified primer pair A6 positive variety genomic DNA as a template, and the RAPD-STS-modified primer pair A6 negative variety genome containing “Nippon Hare” was included. In PCR using DNA as a template, an identification band was detected in all cases.

  Furthermore, the primer pair rP3-1 does not detect an identification band by PCR using the RAPD-STS-modified primer pair including “Nippon Hare” as a template, and the RAPD-STS-modified primer pair P3-negative variety genomic DNA Discrimination bands were detected in all the PCRs used as templates. That is, the identification band appearance patterns by the primer pairs rWKA09-1, rA6-1, and rP3-1 were reversed with the RAPD-STS-primed primer pairs WKA09, A6, and P3, respectively, and the appearance patterns of the identification bands.

  On the other hand, for the primer pair sM2CG-1, an identification band was detected in the PCR using the RAPD-STS-primed primer pair M2CG positive variety genomic DNA including “Nippon Hare” as a template, and in the PCR using the M2CG negative variety genomic DNA as a template. Was not detected. That is, the identification band by the primer pair sM2CG-1 is expected to be reduced in size from 1207 bp to 293 bp, but it has been clarified that it shows the same identification band appearance pattern as the RAPD-STS-modified primer pair M2CG.

  In this reaction system, only the M11 identification band appeared in “Kinuhikari”, but at least one of the other four identification bands appeared in other varieties, and this reaction system is useful for the qualitative test of “Kinuhikari”. It was a serious system.

[Speciation discrimination of primer pair rG22-1]
The primer pair rG22-1 was further confirmed in a multiplex PCR reaction system with a RAPD-STS-modified primer pair G28 and primer pairs sB43-1 and sF6-1. The electrophoresis results are shown in FIG. In Table 2, the name of the cultivar genomic DNA subjected to the reaction and the presence or absence of an identification band by each primer pair are indicated by + and-. In Table 2, the number shown under the primer pair name represents the size (unit: kbp) of the identification band obtained with the primer pair. In the confirmed 60 varieties of DNA, the 0.37 kbp discriminating band by the primer pair rG22-1 was not detected by PCR using the RAPD-STS-modified primer pair G22-positive cultivar genomic DNA as a template, and the RAPD-STS conversion containing “Nippon Hare” was made. It was detected by PCR using the primer pair G22 negative cultivar genomic DNA as a template. Therefore, it was revealed that the appearance pattern of the 0.37 kbp identification band by the primer pair rG22-1 was reversed with the identification band appearance pattern by the RAPD-STS-modified primer pair G22. In this reaction system, a band of about 0.7 kbp due to the primer pair rG22-1 was detected in the RAPD-STS primer pair G22 positive variety genomic DNA. On the other hand, the discrimination band by the primer pair sB43-1 is expected to be shortened in size from 827 bp to 267 bp with respect to the identification band by the RAPD-STS-ized primer pair B43, but is the same as the RAPD-STS-primed primer pair B43. It became clear that the identification band appearance pattern was shown. In addition, the identification band by the primer pair sF6-1 is also expected to be reduced in size from 1194 bp to 305 bp with respect to the identification band by the RAPD-STS primer pair F6, but the same identification as the RAPD-STS primer pair F6 It became clear to show the band appearance pattern. In “Hinohikari”, only the identification band of about 0.7 kbp appears and none of the four identification bands shown in Table 2 appear, but other varieties other than “Mori no Kumasan” are shown in Table 2. At least one of the identification bands of the book has appeared, and this reaction system is a system useful for the qualitative examination of “Hinohikari”.

[Confirmation of variety discrimination of primer pair rG22-2]
Although the size of the primer pair rG22-1 is different, an identification band is also detected by PCR using the RAPD-STS-modified primer pair G22 positive variety genomic DNA as a template. Thus, a primer pair designed so that the region of base numbers 71 to 79 shown in SEQ ID NO: 3 in which the insertion of the RAPD-STS-modified primer pair G22 positive cultivar genomic DNA sequence appears to have occurred in the PCR primer is rG22 -2. The discriminability of the primer pair rG22-2 was confirmed by a multiplex PCR reaction system using the RAPD-STS-modified primer pair M11 and G28 and the primer pair sB43-1, sF6-1. The result of electrophoresis is shown in FIG. Also, in Table 3, the name of the cultivar genomic DNA subjected to the reaction and the presence or absence of an identification band by each primer pair are indicated by + and-. In Table 3, the number shown under the primer pair name represents the size (unit: kbp) of the identification band obtained with the primer pair. In this reaction system, it was confirmed that a discrimination band by the primer pair rG22-2 appeared only by PCR using the RAPD-STS-modified primer pair G22 negative cultivar genomic DNA as a template.

  In the multiplex PCR reaction system of the four primer pairs (G28, rG22-1, sB43-1, sF6-1), “Kirara 397”, “Hoshino Yume”, “Koshibuki”, and “Aichi” Nokaori SBL and Asahi no Yume showed the same discernment band appearance pattern, but with the addition of primer pair M11, Kirara 397, Hoshino Yume, Koishibuki, and Aichi Kaori SBL and Asahi no Yume varieties obtained with different genomic DNAs, but the discriminating band appearance pattern showed different results, resulting in a reaction system with improved discrimination ability when using quantitative tests.

[Confirmation of variety discrimination of primer pairs rG4-1, rS13-1, sG49-1]
The confirmation of discrimination was performed in a multiplex PCR reaction system to which the primer pair rG22-1 was further added. FIG. 4 shows a photograph of the results of agarose gel electrophoresis, and Table 4 shows the names of the cultivar genomic DNA subjected to the reaction and the presence or absence of an identification band by + and −. In Table 4, the number shown under the primer pair name represents the size (unit: kbp) of the identification band obtained with the primer pair. In the confirmed 66 varieties of DNA, the appearance pattern of the 0.37 kbp identification band by the primer pair rG22-1 was reversed from the identification band appearance pattern of the RAPD-STS-modified primer pair G22, as in the “Hinohikari identification reaction system”. In addition, the identification band by the primer pair rG4-1 was not detected by PCR using the RAPD-STS primer pair G4 positive variety genomic DNA as a template, and PCR using a G4 negative variety genomic DNA including “Nippon Hare” as a template. The identification band was detected. Furthermore, the identification band by the primer pair rS13-1 is not detected by PCR using the RAPD-STS primer pair S13 positive breed genomic DNA as a template, but is detected by PCR using the negative breed genomic DNA including “Nippon Hare” as a template. It was. Therefore, the primer pair rG4-1 was a primer pair in which the identification band appearance pattern was inverted with respect to the RAPD-STS-modified primer pair G4, and the primer pair rS13-1 was inverted with respect to the RAPD-STS-modified primer pair S13. On the other hand, the identification band by the primer pair sG49A-1 was detected by PCR using the RAPD-STS-primed primer pair G49 positive variety genomic DNA as a template and not detected by PCR using a negative variety genomic DNA including “Nippon Hare” as a template. It was. Therefore, it was revealed that the discriminating band appearance pattern by the primer pair sG49-1 shows the same discriminating band appearance pattern as the RAPD-STS-modified primer pair G49 although the size is expected to be shortened from 1503 bp to 267 bp. In addition, the identification band of about 0.7 kbp shown in FIG. 4 is an identification band detected by PCR using a primer pair rG22-1 as a template with a RAPD-STS-modified primer pair G22 positive variety genomic DNA. In “Kirara 397”, only the identification band of about 0.7 kbp appears and the four identification bands shown in Table 4 are not detected, but at least one of the four identification bands shown in Table 4 appears in other varieties. Therefore, this reaction system is useful for the qualitative test of “Kirara 397”. In Table 4, for the varieties in which the identification band by the primer pair rS13-1 is +, at least one of the identification bands by the other three primer pairs becomes +, so even in the system excluding rS13-1, There is no impact on the number of varieties that can be detected.

[Confirmation of variety discrimination of primer pair Nak-1 and rG4-2]
In addition to Nak-1 and rG4-2, discrimination was confirmed by a multiplex PCR reaction system using a RAPD-STS-modified primer pair B43A (Forward: TggCCggCATgACTCACA, Reverse: ACTggCCggCATCAAgA). FIG. 5 shows a photograph of the electrophoresis result, and Table 5 shows the name of the cultivar genomic DNA subjected to the reaction and the presence or absence of an identification band by + and −. In Table 5, the number shown under the primer pair name represents the size (unit: kbp) of the identification band obtained with the primer pair. The identification band by the primer pair rG4-2 was not detected in the RAPD-STS-primed primer pair G4 positive variety genomic DNA by PCR using the confirmed 28 kinds of DNA as a template, but was detected in negative varieties including “Nippon Haru”. It was. Therefore, the primer pair rG4-2 can also be used as a primer pair whose discrimination is reversed from the RAPD-STS-modified primer pair G4. On the other hand, the identification band appearance pattern showed the same identification band appearance pattern as the primer pair rG4-1 described in the present specification, although the size was expected to be shortened from 482 bp to 268 bp. In “Nanatsuboshi”, only the identification band by B43A appears, but in other varieties, at least one of the identification bands of Nak-1 and rG4-2 appears. It is a system useful for qualitative inspection of

[Confirmation of variety discrimination of primer pair sG4-1]
In addition to the primer pair sG4-1, discrimination was confirmed in a multiplex PCR reaction system using rG22-1, rS13-1, and sB43-1. In Table 6, the name of the cultivar genomic DNA subjected to the reaction and the presence or absence of an identification band are indicated by + and-. In Table 6, the number shown under the primer pair name represents the size (unit: kbp) of the identification band obtained with the primer pair. The identified band by primer pair sG4-1 was detected by PCR using the RAPD-STS-modified primer pair G4 positive breed genomic DNA as a template in 66 confirmed breeds, and PCR using negative breed genomic DNA including “Nippon Haru” as a template. Was not detected. Therefore, it is clear that the primer pair sG4-1 is a primer pair that shows the same identification band appearance pattern as the RAPD-STS-modified primer pair G4 although the identification band size is expected to be shortened from 818 bp to 476 bp. It was. In addition, the RAPD-STS-primed primer pair G4 was slightly unstable under the reaction conditions at an annealing temperature of 62 ° C. even though it was STS based on the sequence characteristics of the region where the RAPD primer G04 was bound. Thus, the primer pair sG4-1 became a primer pair that produced a distinct discriminating band even in a reaction at an annealing temperature of 62 ° C. In “Hoshinoyume”, only the identification band of about 0.7 kbp by the primer pair rG22-1 detected in the RAPD-STS-modified primer pair G22-positive variety appeared, and all of the four identification bands shown in Table 5 were detected. However, at least one of the four identification bands shown in Table 5 has appeared in other varieties except “Haruru” and “Hanasatsuma”, and this reaction system is suitable for the “Hoshino Yume” test. It is a useful system. According to the 2005 cropping data, “Haruru” is a variety that is hardly cultivated outside Yamaguchi Prefecture and “Hanasatsuma” is not cultivated outside Kagoshima Prefecture.

[Confirmation of variety identification of RAPD-STS primer pair G22-2]
The reactivity of the RAPD-STS-modified primer pair G22-2 was confirmed, and in Table 7, the name of the cultivar genomic DNA subjected to the reaction and the presence or absence of an identification band are indicated by + and-. In Table 7, the number shown under the primer pair name represents the size (kbp) of the identification band obtained with the primer pair. About 0.78 kbp identification band was detected from the RAPD-STS-modified primer pair G22 positive variety, and about 0.43 kbp identification band was detected from the G22 negative variety.

[Confirmation of sM2CG-2 variety identification]
The reactivity of the primer pair sM2CG-2 was confirmed, and in Table 7, the name of the cultivar genomic DNA subjected to the reaction and the presence or absence of an identification band are indicated by + and-. The numbers shown under the primer pair names in Table 7 represent the size (unit: kbp) of the identification band obtained with the primer pair. Also in the primer pair sM2CG-2, an identification band was detected by PCR using the RAPD-STS-primed primer pair including “Nippon Hare” as a template and M2CG positive variety genomic DNA as a template, and no identification band was detected in the negative variety. In the primer pair sM2CG-2, the discriminating band size is expected to be shortened from 1207 bp to 1084 bp, but the primer pair M2CG-2 has the same discrimination as the RAPD-STS-modified primer pair M2CG and the primer pair sM2CG-1 in the present specification. It was revealed to be a primer pair showing a band appearance pattern.

[Confirmation of sP3-1 breed identification]
The reactivity of the primer pair sP3-1 was confirmed, and Table 7 shows the name of the cultivar genomic DNA subjected to the reaction and the presence or absence of an identification band by + and-. The numbers shown under the primer pair names in Table 7 represent the size (unit: kbp) of the amplification product obtained with the primer pair. In the primer pair sP3-1, an identification band is detected by PCR using the RAPD-STS-primed primer pair P3 positive cultivar genomic DNA including “Nippon Hare” as a template, and in the PCR using the negative cultivar genomic DNA as a template, an identification band is detected. There wasn't. Therefore, the primer pair sP3-1 is expected to have a discrimination band size shortened from 533 bp to 374 bp, but the primer pair sP3-1 and the RAPD-STS-modified primer pair P3 are primer pairs that show the same discrimination band appearance pattern. It became clear that there was.

[Confirmation of breed identification of primer pair sWKA09-1]
Regarding the primer pair sWKA09-1, the reactivity of each kind of genomic DNA of “Koshihikari”, “Hinohikari”, “Akitakomachi”, “Kinuhikari”, and “Nipponbare” was examined. In the primer pair sWKA09-1, the identification band was detected by PCR using the template DNAs of "Hinohikari", "Akitakomachi", and "Kinuhikari" which are RAPD-STS-primed primer pair WKA09-positive varieties, and the negative varieties "Nippon Hare" In addition, the identification band was not detected by PCR using the genomic DNA of each Koshihikari variety as a template. That is, the identification band by sWKA09-1 is expected to be reduced in size from 1663 bp to 260 bp, but the PCR using the confirmed 4 types of genomic DNA as a template shows the same identification band appearance pattern as the RAPD-STS-modified primer WKA09. It became clear. In JP-A-2005-73655 (Patent Document 2), the identification band size of the primer pair that seems to detect this variety identification DNA region is about 1.2 kbp, and the identification band can be further shortened. It was.

[Confirmation of variety discrimination of primer pair sS13-1]
Regarding the primer pair sS13-1, the reactivity of each kind of genomic DNA of “Kirara 397”, “Hoshino Yume”, “Hinohikari” and “Akitakomachi” was examined. A 0.3 kbp discriminating band was detected by PCR using each of the S13-positive varieties “Kirara 397” and “Hoshi no Yume” varieties genomic DNA as a template, and PCR using “Hinohikari” and “Akitakomachi” varieties genomic DNA as a template was about 0. A .3 kbp discriminating band was not detected, and the primer pair sS13-1 showed the same discriminability as the RAPD-STS-modified primer pair S13 in PCR using the confirmed four-variety genomic DNA as a template. In addition, in JP-A-2005-73655 (Patent Document 2), the size of the amplification product by the primer pair that is considered to detect the cultivar identification DNA region was about 1.6 kbp, and the identification band could be further shortened. .

[Confirmation of variety discrimination of primer pair rS13-2]
Regarding the primer pair rS13-2 consisting of the DNAs shown in SEQ ID NOs: 53 and 54 in the sequence listing, the reactivity of each kind of genomic DNA of “Hoshi no Yume”, “Hoshitaro” and “Nippon Haru” was examined. The PCR using the S13 negative varieties “Hotarou” and “Nippon Hare” varieties genomic DNA as a template detected an approximately 0.7 kbp identification band expected from the “Nippon Hare” genomic sequence, and the “Hoshino Yume” genomic DNA was used as a template. In the PCR, about 0.7 kbp identification band was not detected. Although the identification band size is larger than that of the primer pair rS13-1, there is a possibility that the same identification band appearance pattern is exhibited.

[Confirmation of variety discrimination of primer pair rS13-3]
As described in the present specification, the base sequence of the amplified product obtained from the RAPD-STS primer pair S13 using “Kirara 397” genomic DNA as a template was compared with the “Nippon Hare” genomic sequence which is an S13 negative cultivar. However, there were three regions that showed high sequence identity with the S13 amplified product partial region. Two of them are the DNA regions shown in the present specification (9), but the primer pair designed in the remaining one is the primer pair rS13-3 consisting of the DNAs shown in SEQ ID NOs: 55 and 56 in the Sequence Listing.

  Regarding the primer pair rS13-3, the reactivity of each genomic DNA of “Hoshi no Yume”, “Hotarou” and “Nippon Hare” was examined. In the PCR using RAPD-STS-primed primer pair S13-negative varieties “Hotarou” and “Nippon Hare” varieties genomic DNA as templates, an approximately 0.75 kbp discrimination band expected from the “Nippon Hare” genomic sequence was detected. About 1.3 kbp identification band was detected by PCR using the positive variety “Hoshi no Yume” genomic DNA as a template. The “Nippon Hare” genome sequence region where the primer pair rS13-3 was designed may also be a region where a PCR primer pair whose appearance pattern is inverted with respect to the identification band by the RAPD-STS-modified primer pair S13 can be designed.

[Confirmation of variety discrimination of primer pair rM2CG-1 and rM2CG-2]
Reactivity was confirmed for the primer pair rM2CG-1 consisting of the DNAs shown in SEQ ID NOs: 59 and 60 in the Sequence Listing and the primer pair rM2CG-2 consisting of the DNAs shown in SEQ ID NOS: 61 and 60 in the Sequence Listing. The name of the cultivar genomic DNA subjected to the reaction and the presence or absence of the identification band are indicated by + and-. The numbers shown under the primer pair names in Table 8 represent the size (unit: kbp) of the amplification product obtained with the primer pair. In the primer pair rM2CG-1 and rM2CG-2, the identification band was not detected by PCR using the RAPD-STS-primed primer pair including “Nippon Hare” as a template, and the primer pair M2CG negative variety genomic DNA as a template. In the PCR, an identification band was detected. Therefore, it was revealed that the primer pairs rM2CG-1 and rM2CG-2 are primer pairs in which the discrimination band appearance pattern is reversed with respect to the RAPD-STS-modified primer pair M2CG.

[Confirmation of variety discrimination of primer pair rG49A-1]
The reactivity of the primer pair rG49A-1 consisting of the DNAs shown in SEQ ID NOs: 62 and 63 in the Sequence Listing was confirmed, and Table 8 shows the name of the variety genomic DNA subjected to the reaction and the presence or absence of an identification band by + and-. It was. The numbers shown under the primer pair names in Table 8 represent the size (unit: kbp) of the amplification product obtained with the primer pair. In the primer pair rG49A-1, an identification band is detected by PCR using the RAPD-STS-primed primer pair G49A negative variety genomic DNA including “Nippon Hare” as a template, and in the PCR using the primer pair G49A positive variety genomic DNA as a template, the identification band is detected. Was not detected. Therefore, it was revealed that the primer pair rG49A-1 is a primer pair in which the identification band appearance pattern is reversed with respect to the RAPD-STS-modified primer pair G49A.

Production of PCR Kit for Variety Identification A PCR kit for rice variety identification (100 reactions / kit) comprising the following components was produced.
1. TaKaRa Taq HS (5u / μL) 20μL
2. 10 × PCR Buffer 250 μL
3.25 mM MgCl ₂ 250 μL
4). dNTP Mixture (2.5 mM eadh) 250 μL
5. Product Mixer Prime Mixure 150μL
6). Loading Buffer 500 μL for electrophoresis

  INDUSTRIAL APPLICABILITY According to the present invention, a rice variety identification method using a DNA region useful for variety identification developed by the RAPD method can be used to identify a variety using an identification band having a lower molecular weight than the identification band obtained by the RAPD method. This makes it easier to identify processed crop (eg, rice) products in which DNA is expected to have a low molecular weight. In addition, among cultivars that have been identified by the RAPD method, by using the locus in the genome sequence of the negative cultivar corresponding to the DNA region useful for cultivar identification found by the RAPD method as an identification region, it is completely novel. Qualitative inspection can be performed without developing an identification area.

  1 to 5 are agarose gel electrophoresis photographs when DNA prepared from 20 kinds of rice samples for various PCR reaction systems is used as a template. 1 is “Koshihikari”, 2 is “Hitomebori”, 3 is “Hinohikari”, 4 is “Akitakomachi”, 5 is “Kinuhikari”, 6 is “Kirara 397”, 7 is “Haenuki” , 8 for "Hoshino Yume", 9 for "Tsugaru Romance", 10 for "Nana Tsuboshi", 11 for "Yume Akari", 12 for "Sasanishiki", 13 for "Aichi no Kaori SBL" , 14 "Dream Tsukushi", 15 "Asahi no Yume", 16 "Nippon Hare", 17 "Hatsushimo", 18 "Hanaetizen", 19 "Koshibuki", 20 Shows the results of using “Festival Harvest” as the test rice sample. Further, N represents a negative control, and P represents a detection result in the case of using a plurality of varieties of rice DNA mixture so that all identification bands can be detected. M represents a lane in which ΦX174 HaeIII digest DNA was migrated as a molecular weight marker.

It is the figure which showed the multiplex PCR reaction result by primer pair M11, rA6-1, rP3-1, rWKA09-1, sM2CG-1. It is the figure which showed the multiplex PCR reaction result by primer pair G28, sF6-1, rG22-1 and sB43-1. It is the figure which showed the multiplex PCR reaction result by primer pair M11, G28, sF6-1, rG22-2, and sB43-1. It is the figure which showed the multiplex PCR reaction result by primer pair rG22-1, rG4-1, rS13-1, and sG49-1. It is the figure which showed the multiplex PCR reaction result by primer pair 43A, rG4-1, and Nak-1.

Claims (11)

A method for identifying rice varieties, wherein the presence or absence of a specific base sequence selected from the following (1) to (13) in genomic DNA is used as an identification index: Method:
(1) A variety-identifying DNA region represented by SEQ ID NO: 1 and having base numbers 424 and 425 as base sites predicted to be a recombination region,
(2) a cultivar identification DNA region represented by SEQ ID NO: 2 and whose base site predicted to be a recombination region is base numbers 48 and 49;
(3) a variety-identifying DNA region represented by SEQ ID NO: 3 and having a base number of 70 to 80 as a base site predicted to be a recombination region;
(4) a cultivar identification DNA region represented by SEQ ID NO: 4 and having a base site predicted to be a recombination region of base numbers 19 to 26,
(5) A variety detection DNA region represented by SEQ ID NO: 5 and having base numbers 763 and 764 as base sites predicted to be a recombination region,
(6) a DNA region represented by SEQ ID NO: 6 and whose base sites predicted to be a recombination region are base numbers 366 and 367,
(7) a DNA region represented by SEQ ID NO: 7 and whose base site predicted to be a recombination region is base numbers 353 to 361,
(8) a DNA region represented by SEQ ID NO: 8 and whose base site predicted to be a recombination region is base numbers 249 and 250;
(9) a DNA region represented by SEQ ID NO: 9 and whose base site predicted to be a recombination region is base numbers 550 and 551;
(10) a DNA region represented by SEQ ID NO: 10 and whose base sites predicted to be a recombination region are base numbers 9 and 10;
(11) a DNA region represented by SEQ ID NO: 11 and whose base site predicted to be a recombination region is base numbers 15 and 16;
(12) a DNA region represented by SEQ ID NO: 12 and predicted to be a recombination region is a DNA region having base numbers 555 to 558; and (13) a nucleotide region represented by SEQ ID NO: 13 and predicted to be a recombination region. Is a DNA region having base numbers 474 and 475.
(14) A DNA region represented by SEQ ID NO: 57 and having base numbers 231 and 232 as base sites predicted to be a recombination region.
(15) A DNA region represented by SEQ ID NO: 58 and having nucleotide numbers 114 and 115 as the base region predicted to be a recombination region. A cultivar wherein at least one specific base sequence selected from the specific base sequences described in (2), (4) and (5) below is present in genomic DNA is identified as not being "Kinuhikari". Item 1. Rice variety identification method according to item 1 :
(2) a cultivar identification DNA region represented by SEQ ID NO: 2 and whose base site predicted to be a recombination region is base numbers 48 and 49;
(4) A DNA region for discriminating varieties having the base number 19 to 26 shown in SEQ ID NO: 4 and predicted to be a recombination region; and (5) shown in SEQ ID NO: 5 and predicted to be a recombination region. A DNA region for detecting a variety whose base sites are base numbers 763 and 764. It is characterized in that a variety in which at least one specific base sequence selected from the specific base sequences described in the following (3), (11), and (13) is present in genomic DNA is identified as not “Kirara 397” The method for identifying rice varieties according to claim 1 :
(3) a variety-identifying DNA region represented by SEQ ID NO: 3 and having a base number of 70 to 80 as a base site predicted to be a recombination region;
(11) a DNA region represented by SEQ ID NO: 11 and predicted to be a recombination region, a DNA region having base numbers 15 and 16, and (13) a nucleotide region represented by SEQ ID NO: 13 and predicted to be a recombination region Is a DNA region having base numbers 474 and 475. The cultivar wherein at least one specific base sequence selected from the specific base sequences described in the following (11) and (12) is present in the genomic DNA is identified as not being "Nana Tsuboshi". Method for identifying rice varieties according to 1 :
(11) a DNA region represented by SEQ ID NO: 11 and predicted to be a recombination region, a DNA region having base numbers 15 and 16, and (12) a nucleotide region represented by SEQ ID NO: 12 and predicted to be a recombination region Is a DNA region having base numbers 555 to 558. It is characterized in that a variety in which at least one specific base sequence selected from the specific base sequences described in the following (3), (9), and (10) is present in genomic DNA is not identified as “Hoshinoyume” The method for identifying rice varieties according to claim 1 :
(3) a variety-identifying DNA region represented by SEQ ID NO: 3 and having a base number of 70 to 80 as a base site predicted to be a recombination region;
(9) a DNA region represented by SEQ ID NO: 9 and predicted to be a recombination region, a DNA region having base numbers 550 and 551, and (10) a nucleotide region represented by SEQ ID NO: 10 and predicted to be a recombination region Is a DNA region having base numbers 9 and 10. The presence of a particular nucleotide sequence of a genomic in the DNA, rice varieties identification method according to any one of claims 1 to 5 which comprises carrying out by the detection of DNA fragment amplified by the nucleic acid amplification method. The method for identifying rice varieties according to claim 6, wherein a primer pair selected from the DNAs described in (a) to (r) below is used in the PCR method:
(A) DNA represented by SEQ ID NO: 14 and DNA represented by SEQ ID NO: 15,
(B) DNA represented by SEQ ID NO: 16 and DNA represented by SEQ ID NO: 17,
(C) DNA represented by SEQ ID NO: 18 and DNA represented by SEQ ID NO: 19,
(D) DNA represented by SEQ ID NO: 20 and DNA represented by SEQ ID NO: 21,
(E) DNA represented by SEQ ID NO: 22 and DNA represented by SEQ ID NO: 23,
(F) DNA represented by SEQ ID NO: 24 and DNA represented by SEQ ID NO: 25,
(G) DNA represented by SEQ ID NO: 26 and DNA represented by SEQ ID NO: 27,
(H) DNA represented by SEQ ID NO: 28 and DNA represented by SEQ ID NO: 29,
(I) DNA represented by SEQ ID NO: 30 and DNA represented by SEQ ID NO: 31,
(J) DNA represented by SEQ ID NO: 32 and DNA represented by SEQ ID NO: 33,
(K) DNA represented by SEQ ID NO: 34 and DNA represented by SEQ ID NO: 32,
(L) DNA represented by SEQ ID NO: 35 and DNA represented by SEQ ID NO: 36,
(M) DNA represented by SEQ ID NO: 37 and DNA represented by SEQ ID NO: 38,
(N) DNA represented by SEQ ID NO: 39 and DNA represented by SEQ ID NO: 40,
(O) DNA represented by SEQ ID NO: 41 and DNA represented by SEQ ID NO: 34,
(P) DNA represented by SEQ ID NO: 59 and DNA represented by SEQ ID NO: 60,
(Q) DNA represented by SEQ ID NO: 61 and DNA represented by SEQ ID NO: 60, and (r) DNA represented by SEQ ID NO: 62 and DNA represented by SEQ ID NO: 63. The method for identifying rice varieties according to claim 7 , comprising the following steps (A) to (C):
(A) a nucleic acid preparation step for preparing rice genomic DNA in a rice sample;
(B) A step of performing a nucleic acid amplification method using the genomic DNA obtained in step (A) as a template, and (C) a step of analyzing the DNA fragment obtained in step (B). An oligonucleotide for detecting the presence of a specific base sequence in genomic DNA, which is any one of the DNAs represented by SEQ ID NOs: 21, 22, 24, 33, 34, 38, and 40. A kit for carrying out the method according to any one of claims 7 and 8 , comprising a primer pair used for the purpose of detecting the presence of a specific base sequence as a DNA fragment amplified by a nucleic acid amplification method A kit characterized in that the primer pair is the primer pair described in claim 7 .   A DNA fragment represented by SEQ ID NO: 12 that can be used for variety identification.

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