JP4359824B2 - Rice variety identification method - Google Patents

Description

  The present invention relates to a rice variety identification method, and more specifically, based on a polymorphism of DNA obtained by amplifying DNA (deoxyribonucleic acid) extracted from rice grains by a PCR (polymerase chain reaction) method, The present invention relates to a method for identifying a variety.

  Conventionally, as a method for identifying rice varieties as described above, there is known a method for identifying the varieties of a sample rice based on a DNA polymorphism obtained by amplifying DNA extracted from rice grains by PCR (Patent Document 1). And Non-Patent Document 2). In this method, rice to be measured is first pulverized to prepare a powder sample, and DNA is extracted from the powder sample by, for example, CTAB (cetyl-trimethyl-ammonium bromide) method to obtain a template DNA. Next, the template DNA and a pair of pairing primers or the like that selectively bind only to the base sequence site serving as an identification band useful for identifying the variety are placed in a container and applied to a PCR apparatus, thereby heating the container. The temperature is controlled, and denaturation, annealing, and elongation (replication) are repeated. Next, the amplified DNA obtained by this PCR step is applied to an electrophoresis apparatus to separate each DNA, and then ethidium bromide staining is performed, and a specific identification band is detected by ultraviolet irradiation. Based on this identification band, It identifies rice varieties.

JP 2001-95589 A Japanese Journal of Agricultural Chemistry Vol.76, No.4, pp.388-397, 2002

By the way, recently, there is an example of illegally displaying the rice varieties and mixed rice indications distributed in the market, so the technology for scientifically identifying rice varieties as described above is useful. It is possible to identify the top 50 cultivated varieties of the year. However, there are much more planted brands in Japan than 50 varieties, and there are more than 100 varieties. However, in the above technology, it is recognized that other than the above 50 varieties cannot be identified.
In view of the above problems, an object of the present invention is to provide a variety identification method that enables more variety identification of rice than before.

In order to solve the above-mentioned problems, the present invention uses a DNA extracted from rice as a template DNA, and generates a specific identification band by PCR in the presence of a pairing primer. a method for identifying a variety of rice based, the pairing primer, SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, and SEQ ID NO: 5 SEQ ID NO: 6, and SEQ ID NO: 7 SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20. Technical measures were taken to use all or a plurality of pairs of SEQ ID NO: 21 and SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, respectively.

  By using all or a plurality of selected 13 kinds of pairing primers of the present invention, it is possible to identify and identify more rice cultivars in Japan than in the past.

  The present invention will be described in detail below. In the rice variety identification method of the present invention, the sample rice for identification may be rice bran, brown rice, polished rice, cooked rice, or processed rice. In the following description, the sample rice is polished rice. In addition, when the sample rice is rice bran, brown rice from which rice husk has been removed is used.

  The basic rice variety identification method of the present invention will be described (see FIG. 1). First, 114 kinds of polished rice described below were prepared. “Koshihikari”, “Milky Queen”, “Hitomebore”, “Emi Mieno”, “Hinohikari”, “Kuma no Mori”, “Akitakomachi”, “Kirara 397”, “Kinuhikari”, “Tsukushi early life”, “Dream” `` Shizuku '', `` Hoshi no Yume '', `` Haenuki '', `` Mutsu Homare '', `` Nippon Hare '', `` Nishihomare '', `` Sasanishiki '', `` Tsugaru Romantic '', `` Hanaetizen '', `` Sagara '', `` Dream '' "Hatsushimo", "Morning light", "Moonlight", "Aichi no Kaori", "Mine Asahi", "Festival", "Seijiman", "Akiho", "Yukimaru", "Mukatsuori", "Manamusume", "Kakehashi", "Kiyonishiki", "Donnaka", "Dontokoi", "Koshiji Hayao", "Noto Hikari", "Chidori", "Yuki no Sei", "Hohohonoho", "Yume" "Akari", "Hatashirushi" `` Akebono '', `` Asahi '', `` Nakasei Shinsen '', `` Matsuyama Mitsui '', `` Kinan Kaze '', `` Yamaboshi '', `` Yamahikari '', `` Koganemasari '', `` Reihou '', `` Fusaotome '', `` Kari no "Mai", "Akinishi", "Fukuhikari", "Goropicari", "First Star", "Asahi no Yume", "Menkoina", "Norrin 22", "Yumehitachi", "Chionishiki", "Kokoromachi" , `` Hana no Mai '', `` Yume Minori '', `` Akane Sora '', `` Akitsuho '', `` Haruru '', `` Maihime '', `` Yume Adoration '', `` Ooseto '', `` Akiharu '', `` Golden Hare '', “Sunny”, “Todoroki Wase”, “Tokimeki 35”, “Koikoro”, “Akiman”, “Awaminori”, “Yumeumi”, “Autumn Poetry”, “Minonishiki”, “Aki Shiki” , “Aoi no Kaze”, “Yumeshinano”, “Yumekogane”, “Sato no Uta” , "Gin Umi", "Higonohana", "Shinrei", "Minami Hikari", "Diwa Hikari", "Bambanzai", "Fuji no Mai", "Ukon Nishiki", "Omachikane", "Miyako 95", “Ken no Hana”, “Horei”, “Koriyori”, “Omachi”, “Koi Omachi”, “Hachi 35”, “Hachiman Nishiki 1”, “Senbon Nishiki”, “Yamada Nishiki” “500,000 stones”, “Miyamanishiki”, “Yukihikari”, “Goldennishiki”, “Tosapika”, “Natsuhikari”, “Tosanishiki”.

  Next, a DNA extraction step is performed. DNA extraction is performed on all 114 varieties by the following method. In the DNA extraction step, one grain of rice of the above variety and 30 μL of sterilized water are placed in a predetermined container (tube) and left for 30 minutes. Next, the container is set in a heating device such as a heat block, and rice grains are cooked by applying a temperature of 90 ° C. for 30 minutes. Next, the cooked rice in the container is crushed using a pestle, and 0.43 mL of plant cell lysis reagent (main components: Tris, EDTA, NaCl, CTAB) is added and heated at 65 ° C. for 30 minutes, Dissolve rice grain plant cells. Thereafter, the container is set in an automatic DNA separation device (manufactured by Kurashiki Boseki Co., Ltd .: nucleic acid automatic separation device, model: NA-2000). Then, 0.57 mL of plant protein denaturing reagent (main component: SDS, other surfactant) was added and stirred to denature the rice grain protein, and then the plant protein denaturing reagent (main component: chloroform (90%)) Add and stir 0.20 mL to elute the lipids of rice grains. Next, 0.10 mL of a plant protein removal reagent (main component: potassium acetate) is added and stirred, and then a centrifugal action is applied to form a pellet containing chloroform, plant residues and the like, and the transfer of the supernatant is made smooth.

Next, the supernatant is transferred by decantation (gradient separation) to a collection container in which 0.75 mL of nucleic acid complex precipitation reagent (main components: Tris, EDTA, CTAB) has been put in advance, and stirred to obtain a CTAB-DNA complex. Form. After further centrifugal action, the supernatant is discarded by decantation. Next, 0.50 mL of a nucleic acid redissolving reagent (main component: NaCl) is added and stirred to dissociate the CTAB-DNA complex. Next, 0.60 mL of a precipitation reagent (main component: isopropanol (90% or more)) is added, stirred and centrifuged to precipitate DNA. Thereafter, the supernatant is again discarded by decantation. Further, 2.00 mL of a washing reagent (main components: ethanol (70% or less), isopropanol) is added, and the DNA is washed by stirring and centrifuging. Thereafter, the supernatant is again discarded by decantation and finally dried to obtain template DNA. The above is the DNA extraction step.

Next, a PCR (polymerase chain reaction) step is performed. The PCR process is also performed for all 114 varieties. First, 20 μL of a reaction solution is prepared in a predetermined container with the following composition.
Sterile water 5.05μL
10 x PCR buffer (100 mM Tris HCL (pH 8.3), 500 mM KCL)
2μL
MgCl2 2μL
dNTP mix (2.5 mM each) 2 μL
Primer (SEQ ID NO: 1) 0.4 μL (use 5 pmol / μL solution)
Primer (SEQ ID NO: 2) 0.4 μL (use 5 pmol / μL solution)
Template DNA solution 400ng (50ng / μL × 8μL)
Taq polymerase (5U / μL) 0.15μL

  Next, the container containing the reaction solution is set in a PCR device (ABI, Gene Amp PCR System 9700). The PCR apparatus is then heat-denatured by heating at 95 ° C. for 2 minutes, and heat denaturation (95 ° C. × 2 minutes), annealing (62 ° C. × 1 minute) and extension reaction (72 ° C. × 2 minutes) are performed in 30 to 30 minutes. 45 cycles are repeated and an extension reaction (72 ° C. × 4 minutes) is performed. After the extension reaction (72 ° C. × 4 minutes), the reaction solution is allowed to stand at 4 ° C. The above is the PCR process.

  Next, an electrophoresis step is performed. The electrophoresis process is also performed for all 114 varieties. Prepare a mixed solution consisting of 10 μL of the PCR solution of all 114 varieties obtained in the PCR step and 2 μL of Loading Buffer (bromophenol blue, etc.), and electrophorese 1.5% agarose gel (manufactured by Nacalai Tesque, 150-1500 bp) Set in an apparatus (Mupid-2, manufactured by Cosmo Bio Inc.), add the mixture, and perform electrophoresis at 100 volts for 40 minutes.

  Next, a densitograph image capturing step is performed. In the densitograph image capturing step, the gel obtained in the electrophoresis step is stained with ethidium bromide, and then the stained gel is set in a densitograph image device (AE-6920V-CX, manufactured by ATTO Corporation). Image. FIG. 2 shows an electrophoresis pattern photograph imaged using the first paired primer performed as described above (in the photograph, M is a molecular weight marker, and the values in lanes 1 to 114 are the results of amplification products of the above 114 varieties. The white bar is the identification band). From this photograph, the presence or absence of a specific identification band for each type can be confirmed.

In the above description, among the 13 paired primers found by the present inventor, the description was made using the first paired primer composed of the primer of SEQ ID NO: 1 (forward primer) and the primer of SEQ ID NO: 2 (reverse primer). It is. Thereafter, the densitograph image capturing process is performed from the DNA extracting process to the second to thirteenth pairing primers described later in the same manner as in the above method.
First pairing primer: SEQ ID NO: 1 (forward side), SEQ ID NO: 2 (reverse side)
Second pairing primer: SEQ ID NO: 3 (forward side), SEQ ID NO: 4 (reverse side)
Third pairing primer: SEQ ID NO: 5 (forward side), SEQ ID NO: 6 (reverse side)
Fourth pairing primer: SEQ ID NO: 7 (forward side), SEQ ID NO: 8 (reverse side)
Fifth pairing primer: SEQ ID NO: 9 (forward side), SEQ ID NO: 10 (reverse side)
Sixth pairing primer: SEQ ID NO: 11 (forward side), SEQ ID NO: 12 (reverse side)
Seventh pairing primer: SEQ ID NO: 13 (forward side), SEQ ID NO: 14 (reverse side)
Eighth pairing primer: SEQ ID NO: 15 (forward side), SEQ ID NO: 16 (reverse side)
Ninth pairing primer: SEQ ID NO: 17 (forward side), SEQ ID NO: 18 (reverse side)
Tenth pairing primer: SEQ ID NO: 19 (forward side), SEQ ID NO: 20 (reverse side)
Eleventh pairing primer: SEQ ID NO: 21 (forward side), SEQ ID NO: 22 (reverse side)
Twelfth pairing primer: SEQ ID NO: 23 (forward side), SEQ ID NO: 24 (reverse side)
13th pairing primer: SEQ ID NO: 25 (forward side), SEQ ID NO: 26 (reverse side)

  FIG. 3 shows a photograph of an electrophoretic pattern obtained by performing the densitograph image capturing process from the DNA extracting process using the second pairing primer. Similarly, FIG. 4 shows an electrophoresis pattern photograph using the third pairing primer, FIG. 5 shows an electrophoresis pattern photograph using the fourth pairing primer, and FIG. 6 shows an electrophoresis pattern photograph using the fifth pairing primer. FIG. 7 shows an electrophoresis pattern photograph using the sixth pairing primer, FIG. 8 shows an electrophoresis pattern photograph using the seventh pairing primer, FIG. 9 shows an electrophoresis pattern photograph using the eighth pairing primer, FIG. 10 shows an electrophoretic pattern photograph of the tenth pairing primer, FIG. 11 shows an electrophoretic pattern photograph of the tenth pairing primer, FIG. FIG. 13 shows an electrophoresis pattern photograph using the combined primer, and FIG. 14 shows an electrophoresis pattern photograph using the thirteenth paired primer. In each of these photographs, the presence or absence of a specific identification band for each type can be confirmed. In each electrophoresis pattern photograph, M represents a molecular weight marker (100 bp DNA Ladder).

  Based on electrophoresis pattern photographs using the first to thirteenth paired primers (FIGS. 2 to 14), the presence or absence of an identification band in the combination of the first to thirteenth paired primers and 114 varieties (PCR results) Are shown in Table 1, Table 2 and Table 3 below. Since there are many varieties, 114 varieties are listed in Tables 1 to 3. Table 1 shows 41 varieties from Koshihikari to Hohohonoho, Table 2 shows 39 varieties from Yume Akari to Awaminori, and Table 3 shows Yume Umi to Tosa Nishiki. 34 varieties are shown. In Tables 1 to 3, “+” indicates that an identification band has appeared, and a blank means that no identification band has appeared.

From Tables 1 to 3, it can be seen that 114 varieties can be identified by 91 identification groups (A to CM) by using the first to thirteenth paired primers.

Hereinafter, a method for discriminating rice varieties (sampled rice) based on the first to thirteenth paired primers and the PCR results shown in Tables 1 to 3 will be described (see FIG. 1). First, the sample rice is divided into 13 by a leveling machine, each is pulverized, and 13 pulverized rice samples are first prepared (instead of the above-mentioned sample obtained by cooking and crushing in a container). Next, the pulverized rice sample is used for each of the first to thirteenth paired primers, from the above-described DNA extraction step to the densitograph imaging step. And the PCR result by each paired primer and the said Table 1-Table 3 are compared, and a variety is identified. In addition, when it is desired to know only whether other than the above-mentioned specific varieties are mixed, not using all of the first to thirteenth pairing primers, referring to Tables 1 to 3 above, Discrimination is possible by selecting and using a pairing primer showing an identification band. As a result, if it is found that other than the specific varieties are included, all of the first to thirteenth paired primers can be used, and the included varieties can be specified in the 114 varieties.

  The method for discriminating 114 varieties of glutinous rice into 91 identification groups (A to CM) has been described above. A method for discriminating 28 varieties of glutinous rice will be described below. As for the method for identifying glutinous rice, a plurality of the above-mentioned paired primers are used.

The 28 varieties of glutinous rice are "Hyokumochi", "Hakuchomochi", "Himenomochi", "Mine no Mochimochi", "Miyakoganemochi", "Kaze no Komochi", "Wataboshi", "Kokonoemochi" ”,“ Mochiminori ”,“ Kagamochi ”,“ Shindai Shogo ”,“ Shigaha Nijutsu ”,“ Tatsukomochi ”,“ Shirayama Mochi ”,“ Kinu no Hada ”,“ Mochi Hikari ”,“ Tomichi ” "From", "Saiwaimochi", "Yashimomochi", "Tsukimimochi", "Kurenaimochi", "Surugamochi", "Anekomochi", "Kusutamochi", "Diwanomochi", "Ayumimochi", "Tanchomochi", It is “Megumi”.

  The first, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth and thirteenth pairing primers (10 types) are used to identify the 28 varieties of glutinous rice. . In the same manner as described above, the specific discriminating method includes the DNA extraction step, the PCR step, the electrophoresis step, and the densitograph imaging step, which are the first, fifth, sixth, seventh, eighth, ninth, tenth. The above 28 glutinous rice varieties are carried out for each paired primer of the eleventh, twelfth and thirteenth paired primers.

FIGS. 15 to 24 show electrophoresis pattern photographs by using the first, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth and thirteenth paired primers. Based on these FIG. 15 to FIG. 24, the presence or absence of the identification band (PCR result) in the combination of the paired primer and 28 varieties is summarized and shown in Table 4 below. In Table 4, “+” indicates that an identification band has appeared, and a blank means that no identification band has appeared. In each electrophoresis pattern photograph (FIGS. 15 to 24), M represents a molecular weight marker (100 bp DNA Ladder).

From Table 4, 28 varieties were obtained by using the first, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth and thirteenth pairing primers (10 types). It can be seen that 25 identification groups (A to Y) can be identified.

Hereinafter, for glutinous rice, the first, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth and thirteenth pairing primers (10 types) and the PCR of Table 4 are used. A method of discriminating rice variety (glutinous rice) based on the results will be described (see FIG. 1). First, the sample rice is divided into 10 by a leveling machine, each is pulverized, and 10 pulverized rice samples are prepared (in place of the above-mentioned sample obtained by cooking and crushing in a container). Next, using the pulverized rice sample, the above-described DNA extraction process to the densitograph image capturing process are performed for each of the 10 types of paired primers. Then, the PCR result by each paired primer is compared with Table 4 to identify the variety. When it is desired to know only whether or not other than the above-mentioned specific varieties are mixed, not all of the 10 kinds of pairing primers are used, and the specific varieties indicate an identification band with reference to Table 4 above. Identification is possible by selecting and using paired primers. As a result, if it is found that other than the specific varieties are included, it is also possible to use all of the 10 types of paired primers and specify the included varieties within the 28 varieties.

It is the flowchart which showed the basic process of the rice kind identification method.

It is the electrophoresis pattern photograph which showed the PCR result using the 1st matching primer.

It is the electrophoresis pattern photograph which showed the PCR result using the 2nd pairing primer.

It is the electrophoresis pattern photograph which showed the PCR result using the 3rd matching primer.

It is the electrophoresis pattern photograph which showed the PCR result using the 4th pairing primer.

It is the electrophoresis pattern photograph which showed the PCR result using the 5th paired primer.

It is the electrophoresis pattern photograph which showed the PCR result using the 6th pairing primer.

It is the electrophoresis pattern photograph which showed the PCR result using the 7th pairing primer.

It is the electrophoresis pattern photograph which showed the PCR result using the 8th paired primer.

It is the electrophoresis pattern photograph which showed the PCR result using the 9th paired primer.

It is the electrophoresis pattern photograph which showed the PCR result using the 10th pairing primer.

It is the electrophoresis pattern photograph which showed the PCR result using the 11th paired primer.

It is the electrophoresis pattern photograph which showed the PCR result using the 12th paired primer.

It is the electrophoresis pattern photograph which showed the PCR result using the 13th paired primer.

It is the electrophoresis pattern photograph which showed the PCR result using the 1st matching primer.

It is the electrophoresis pattern photograph which showed the PCR result using the 5th paired primer.

It is the electrophoresis pattern photograph which showed the PCR result using the 6th pairing primer.

It is the electrophoresis pattern photograph which showed the PCR result using the 7th pairing primer.

It is the electrophoresis pattern photograph which showed the PCR result using the 8th paired primer.

It is the electrophoresis pattern photograph which showed the PCR result using the 9th paired primer.

It is the electrophoresis pattern photograph which showed the PCR result using the 10th pairing primer.

It is the electrophoresis pattern photograph which showed the PCR result using the 11th paired primer.

It is the electrophoresis pattern photograph which showed the PCR result using the 12th paired primer.

It is the electrophoresis pattern photograph which showed the PCR result using the 13th paired primer.

Explanation of symbols

In FIG. 2 to FIG. 14 (114 varieties of glutinous rice), M represents a molecular weight marker, and lanes 1 to 114 each represent the following varieties (corresponding to the cultivar numbers in Tables 1, 2 and 3). 1: “Koshihikari”, 2: “Hitomebore”, 3: “Hinohikari”, 4: “Akitakomachi”, 5: “Kirara 397”, 6: “Kinuhikari”, 7: “Hoshino Yume”, 8: “Haenuki”, 9: “Mutsumere”, 10: “Nihonbare”, 11: “Sasanishiki”, 12: “Tsugaru Romantic”, 13: “Hanaetizen”, 14: “Yumetsukushi”, 15: “Hatsushima”, 16: “Morning” , 17: “Moonlight”, 18: “Kaori Aichi”, 19: “Festival”, 20: “Akiho”, 21: “Yukimaru”, 22: “Mukatsuori”, 23: “ Manamusume, 24: Kakehashi, 25: Kiyonishiki, 26: Domanaka, 27: Hayao Koshiji, 28: Yukinosei, 29: Hohohonoho, 30: Yume Akari 31: “Hikari Noto” 32: “Akebono” 33: Asahi, 34: “Yamahoshi”, 35: “Yamahikari”, 36: “Koganemasari”, 37: “Reihou”, 38: “Mine Asahi”, 39: “Fusaotome”, 40: “Kari no Mai”, 41: “Dontokoi”, 42: “Akinishi”, 43: “Fukuhikari”, 44: “Goropicari”, 45: “First Star”, 46: “Shinsen Nakasei”, 47: “Kuma no Mori”, 48: “Asahi no Yume”, 49: “Menkoina”, 50: “Milky Queen”, 51: “Noribayashi No.22”, 52: “Nishihomare”, 53: “Yumehitachi”, 54: “Chiyoshiki”, 55: “ Hana no Mai, 56: Yume Minori, 57: Akane Sora, 58: Akitsuho, 59: Haru, 60: Maihime, 61: Dreaming, 62: Ooseto ”, 63:“ Seijiman ”, 64:“ Golden Clear, 65: Fine, 66: Todoroki Wase, 67: Plover, 68: Matsuyama Mitsui, 69: Tokimeki 35, 70: Koikoro, 71: Akiman 72: “Emi Mieno”, 73: “Awaminori”, 74: “Yume Umi”, 75: “Autumn Poetry”, 76: “Minonishiki”, 77: “Hahajirushi”, 78: “Akageshiki”, 79: “Kaze no Kaze”, 80: “Yumeshinano”, 81: “Tsukushi Early Life”, 82: “Yumekogane”, 83: “Sato no Uta”, 84: “Gin Umi”, 85: “Shinrei”, 86: “Minamihikari”, 87: “Akiharu”, 88: “Diwahikari”, 89: “Yume Shizuku”, 90: “Bambanzai”, 91: “Kokoromachi”, 92: “Fuji no Mai”, 93: “Ukon Nishiki”, 94: “Omachikane”, 95: “Sagaura”, 96 “Miyako 95”, 97: “Ben no Hana”, 98: “Hourei”, 99: “Higo no Hana”, 100: “Kinnan Kaze”, 101: “Kuriyori”, 102: “Omachi”, 103: "Koi Omachi", 104: "Hachiman 35", 105: "Hachiman Nishiki 1", 106: "Senbon Nishiki", 107: "Million stone", 108: "Miyama Nishiki", 109: "Yuki Hikari, 110: “Yamada Nishiki”, 111: “Golden Nishiki”, 112: “Tosapika”, 113: “Natsuhikari”, 114: “Tosa Nishiki”.

In FIG. 15 to FIG. 24 (28 varieties of glutinous rice), M indicates a molecular weight marker, and lanes 1-114 indicate the following varieties (corresponding to the cultivar numbers in Tables 1, 2 and 3). 1: “Hiyokomochi”, 2: “Hakuchomochi”, 3: “Himenomochi”, 4: “Miyakoganemochi”, 5: “Kokonomochimochi”, 6: “Wataboshi”, 7: “ "Kokonoemochi", 8: "Mochiminori", 9: "Kagamochi", 10: "Shindai Masatsugu", 11: "Shigaha Double Duct", 12: "Tatsukomochi", 13: "Kinanohada", 14: “Mochihikari”, 15: “Saiwaimochi”, 16: “Mine no Mochimochi”, 17: “Yashiromochi”, 18: “Tsukimomochi”, 19: “Kurenaimochi”, 20: “Tomichikara”, 21: “Surugamochi”, 22: “Anekomochi”, 23: “Kustamamochi”, 24: “Dowanomochi”, 25: “Machi Hakusan”, 26: “Ayumimochi”, 27: “Tanchomochi”, 28: “Megumi”.

Claims (1)

The extracted DNA from rice as a template DNA, cause specific identification band by PCR in the presence of a pairing primers the template DNA, in the method for identifying the type of the rice on the basis of said specific identification bands There ,
The pairing primers are SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, SEQ ID NO: 23 And SEQ ID NO: 24, and SEQ ID NO: 25 and SEQ ID NO: 26 are used as a pair of rice varieties.