KR101286989B1 - Multiplex real time pcr method for discrimination of rice cultivar - Google Patents

Abstract

The present invention relates to a method for identifying rice varieties that can distinguish varieties of domestic and international rice varieties and a method for identifying rice varieties using real-time multiplex PCR, and can identify 514 species produced at home and abroad.

Description

MULTIPLEX REAL TIME PCR METHOD FOR DISCRIMINATION OF RICE CULTIVAR}

The present invention relates to multiple real-time PCR methods for identifying rice varieties, and more particularly, to an automated multiple real-time PCR method capable of quickly and accurately identifying 514 types of rice (or rice).

Rice is a plant (Gramineae) and genus Oryza, and there are about 60,000 species according to the Int'l Rice Research Institute (IRRI). According to the 2004 Ministry of Agriculture, there are 113 varieties and 195 breeding varieties supplied by the government. There are two species of cultivated rice: Asian rice (O. sativa) and African rice (O. glaberrima), among which Asian rice (O. sativa) is more widely cultivated worldwide.

Rice is being analyzed for genome sequencing of Nipponbare varieties through the International Rice Genome Sequencing Project (IRGSP), which has been participated by more than 10 countries (Japan, America, China, France, India, Taiwan, Korea, Brazil, Thailand and England), as of 2002, 92% of the approximately 400 million sequences of the Nippon Barre genome have been identified.

Meanwhile, in 2004, in order to encourage the production of high-quality rice in Korea, the government has implemented a government limited purchase system to encourage the production of high-quality rice before opening the rice market. In January 2004, the Ministry of Agriculture and Forestry promoted the establishment of a high quality rice and distribution order through the Brand Rice Evaluation System. Announcement of MAF: Law of rice management Subsection 1 of Article 20. This system aims to satisfy the right to know the quality of rice and aims to establish the production, consumption and transparent distribution process.

Therefore, attention has been focused on techniques for identifying rice varieties, and recently, many studies have been conducted to identify rice varieties using various DNA markers. Among them, random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP) and microsatellite are used for breed identification. For example, Wang, ZY, Tanksley, SD 1989 L. Genome. 32, 1113-1118), and Ryu's protein capillary electrophoresis method to identify varieties using restricted fragment length polymorphism (RFLP) markers. Method (Ryu, DJ 1998 J. food science and nutrition. 3, 43-347), identification method of 51 varieties of Japonica rice (Ji, HS et al., 1998 Korean J. Breed. 30, 350-360), reported 40 varieties of domestic varieties using RAPD, microsatellite, sequence tagged site (STS), and polymerase chain reaction (PCR) methods, and their advantages and disadvantages. It was also compared (Jeong OY et al., Korean 1998 J. Breed. 30, 136-137). In addition, the identification method of 31 rice varieties using RAPD and microsatellite (Kwon, S.J. et al., 1999 Korean J Crop Science 44, 112-116), the identification method of rice varieties using image analysis technology Kwon, S.J. et al., 1998 Korean J Crop Science 43, 33-34).

However, since these identification methods and imaging techniques cannot form a specific single band, it is difficult to identify each breed from mixed varieties and it is difficult to discuss the analysis results.

In addition, Korean Patent Application No. 2005-69858 discloses a rice variety identification method by the PCR method using a primer, which is currently published as a domestic standard test method. However, in the above method, when one rice sample was examined, the rice intrinsic gene was used as 9 SNP analysis primers with 24 DNAs extracted from randomly selected 24 rice (or rice) as templates. Including, a total of 240 or more PCR reactions should be performed. In addition, after completion of PCR, electrophoresis of 10 27-well agarose gels is required for 240 PCR products. In addition, this process is labor-intensive, which requires one inspector to perform for three days, there is a problem that takes a long time to obtain a result.

Therefore, there is a need for a more efficient method for simultaneously and accurately analyzing identification markers specific to each rice variety and calculating the result in an automated manner.

The present invention is to solve the above problems, an object of the present invention is to provide a quick, simple, automated identification method that can simultaneously analyze a variety of varieties of domestic and foreign rice with a plurality of markers.

In order to achieve the above object, the present invention provides a marker combination composition for identifying a rice variety, comprising two or more sets of markers for identifying a rice variety selected from the group consisting of (a) to (o):

(a) a primer comprising SEQ ID NO: 1, a primer comprising SEQ ID NO: 2, and a probe comprising SEQ ID NO: 3;

(b) a primer comprising SEQ ID NO: 4, a primer comprising SEQ ID NO: 5, and a probe comprising SEQ ID NO: 6;

(c) a primer comprising SEQ ID NO: 7, a primer comprising SEQ ID NO: 8, and a probe comprising SEQ ID NO: 9;

(d) a primer comprising SEQ ID NO: 10, a primer comprising SEQ ID NO: 11, and a probe comprising SEQ ID NO: 12;

(e) a primer comprising SEQ ID NO: 13, a primer comprising SEQ ID NO: 14, and a probe comprising SEQ ID NO: 15;

(f) a primer comprising SEQ ID NO: 16, a primer comprising SEQ ID NO: 17, and a probe comprising SEQ ID NO: 18;

(g) a primer comprising SEQ ID NO: 19, a primer comprising SEQ ID NO: 20, and a probe comprising SEQ ID NO: 21;

(h) a primer comprising SEQ ID NO: 22, a primer comprising SEQ ID NO: 23, and a probe comprising SEQ ID NO: 24;

(i) a primer comprising SEQ ID NO: 25, a primer comprising SEQ ID NO: 26, and a probe comprising SEQ ID NO: 27;

(j) a primer comprising SEQ ID NO: 28, a primer comprising SEQ ID NO: 29, and a probe comprising SEQ ID NO: 30;

(k) a primer comprising SEQ ID NO: 31, a primer comprising SEQ ID NO: 32, and a probe comprising SEQ ID NO: 33;

(l) a primer comprising SEQ ID NO: 34, a primer comprising SEQ ID NO: 35, and a probe comprising SEQ ID NO: 36;

(m) a primer comprising SEQ ID NO: 37, a primer comprising SEQ ID NO: 38, and a probe comprising SEQ ID NO: 39;

(n) a primer comprising SEQ ID NO: 40, a primer comprising SEQ ID NO: 41, and a probe comprising SEQ ID NO: 42; And

(o) a primer comprising SEQ ID NO: 43, a primer comprising SEQ ID NO: 44, and a probe comprising SEQ ID NO: 45.

In addition, the present invention comprises the steps of performing a real-time PCR with a DNA extracted from rice template using a marker combination composition for identifying the rice varieties; Detecting amplification of the fluorescence material implemented by the real-time PCR result and confirming PCR amplification by a set of markers for identifying each rice variety; And it provides a rice variety identification method using a plurality of rice varieties identification marker sets, comprising the step of identifying the rice varieties according to the PCR amplification.

As described above, the rice variety identification method of the present invention establishes a real-time PCR method using two, three, four, or five or more kinds of marker sets for identifying a rice variety, and to identify existing rice varieties. Not only can the PCR process that is performed for each marker significantly shorten, but all the test processes are operated by an automated system until the results are analyzed and the test is completed. It is very useful in that it is easy to collect and analyze data and minimizes inspection time and labor. Therefore, it can scientifically support a variety of policies, such as government varieties of purchase restrictions, packaging grain labeling, brand rice evaluation system, and can be usefully used to identify varieties for managing the origin of rice in circulation.

1 to 514 show the results of real-time PCR using a template extracted from 514 varieties of domestic and foreign rice.

Hereinafter, the present invention will be described in detail.

The present inventors recognize that the current domestic test method that can detect only one SNP is inefficient in terms of time, cost, and labor, and a combination of primer pairs and probes capable of detecting multiple SNPs at once, PCR conditions, and PCR amplification results While researching how to identify a combination of primer pairs and probes, the combination of primer pairs and probes that can efficiently perform their respective PCR amplification in one PCR reaction, even if different SNPs are targeted It was established a method that can simultaneously grasp the PCR conditions and PCR amplification results that can act at the same time, and completed the present invention based on this.

Accordingly, the present invention identifies a variety of rice tested by real-time PCR method by amplifying SNPs specific to each rice variety through PCR and simultaneously reacting labeled probes with hybridizable and detectable means to the amplification products. It is about a method. In particular, the present invention relates to a rice variety identification method using two or more sets of rice variety identification markers simultaneously using a primer set and a probe for amplifying SNPs specific to each rice variety. It is a highly economic method that has significantly shortened the process.

Table 1 shows a set of markers for identifying rice varieties of the present invention.

division Marker Name type denomination order SEQ ID NO: a DK17-1 SNP P1 (forward direction) atc ccc ata att ctt gac aa One P2 (Reverse) gga atc cgt ctt gat ttg tg 2 Probe 1 tgt cgc gga gaa cata 3 b DK34 In / Del P3 (forward direction) atg tca tgt ggc ttg tct gg 4 P4 (reverse direction) aac tca gga acc atg cca ctg 5 Probe 2 tga tca acg tca gac tc 6 c DK50 SNP P5 (forward direction) cgc gaa cta aac aag ccc ga 7 P6 (reverse direction) aac cct tcc gat atc ctg tc 8 Probe 3 ttg gtg tta cat cag atg 9 d DK63 SNP P7 (forward direction) aca cgc aat gag tgt ggc agt gtg 10 P8 (reverse direction) cgg cca tgt gtt tac cta ctg 11 Probe 4 tgt atg gct ttt gta cc 12 e DK560 SNP P9 (forward direction) gcc acg tct tct tta aag att 13 P10 (Reverse) att agg ctg cat aca tta gtc atc c 14 Probe 5 tag aat ccc gac ccc ct 15 f DK1412 SNP P11 (forward direction) tat tgg gcc gaa tct gat tcg t 16 P12 (Reverse) tct ccg aca gtt tag ggc tta ggg 17 Probe 6 tcg cag tac cgg ata tct 18 g DK2171 SNP P13 (forward direction) aca caa aat aat gtc cat att a 19 P14 (Reverse) aac tag caa att gcc tat gcg ttg 20 Probe 7 ctt tac aag tat gta gtg aag ga 21 h DK2394 SNP P15 (forward direction) tga tat ctt gga tta gaa gtt aac 22 P16 (Reverse) ctg ata cag aaa ctt tcc aac cat tgg 23 Probe 8 gaa cca caa cat ttt gcc gc 24 i DK2401 SNP P17 (forward direction) cac atc atc ttc aat ttc aac caa aat 25 P18 (reverse direction) tat aca cca ttg att tct cta aat gg 26 Probe 9 taa act ttg cgc tga ac 27 j DK1123 SNP P19 (forward direction) cga gct ctg gct ctg act atc a 28 P20 (reverse direction) cga tac ctc ctc aat aac cat aaa atc 29 Probe 10 cgt att tac aca ttc tta gtt 30 k DK1361 SNP P21 (forward direction) aca aac cag gga ttc caa acc 31 P22 (reverse direction) cgt aca aag tca ttg gag gtt ga 32 Probe 11 tat aat gcg aag tgg ttg aa 33 l DK600 In / Del P23 (forward direction) aac ggt gga gat ctg cgt aga 34 P24 (reverse direction) acg atc acg cag caa gat ctt 35 Probe 12 cta gga tgt tgg taa tgg tg 36 m DK2511
SNP P25 (forward direction) agc aat gct gat aat gaa agc tca a 37 P26 (reverse direction) tgc tcc ttt cat act tac aat tgg aag 38 Probe 13 ata ttg tgg aga cta ctt 39 n DK601 In / Del P27 (forward direction) gaa ccg ttt tca ccc cta g 40 P28 (reverse direction) gaa gga agc tac tac atc cta a 41 Probe14 caa gtc gag ggc tga ag 42 o
DK2708 SNP P29 (forward direction) gat aat gga ata gtc atc ccg gcc ttt gct 43 P30 (Reverse Direction) att ttt tct atc att tct gaa cta cac tcg 44 Probe 15 att aca att tag cac tct a 45

The markers of Table 1 describe the nucleotide sequences of SEQ ID NOs: 1 to 45. In Table 1, the marker name is arbitrarily set, and the types are SNP and In / Del marker types. SNP markers refer to markers in which the base portion of any allele is exactly matched, but is substituted with a base that is mismatched to a particular allele, while the In / Del marker is a base that exactly matches either allele. In other alleles, the marker refers to a marker to which a base is inserted or deleted and mismatched.

The primer according to the marker for identifying the rice variety according to the present invention is consistent with the nucleotide sequence of all rice varieties, the probe is consistent with the specific allele representing the SNP (the specific allele). Thus, the probe hybridizes specifically to the template DNA for complementary allelic types, but the probe does not hybridize to nonspecific alleles. In this case, since the probe is labeled with a detectable means, the SNP type of the product amplified by PCR can be confirmed. Rice varieties can be easily analyzed with more than two sets of primer pairs and probe sets specific to each rice variety.

The marker set for identifying a rice variety according to the present invention is designed for breed identification for japonica grown rice (rice). Japonica rice varieties Oryza sativa Domestic and foreign varieties of subspecies with the scientific name of L. The marker for identifying a rice variety according to the present invention uses a single base difference of DNA on the rice genome and is scattered within 12 chromosomes of rice. Each marker according to the present invention refers to the CAPS site for Japanese Nippon Barre varieties among 12 chromosomes of rice, and amplifies a certain region by PCR, and then analyzes the sequencing of the region. It was designed as an allele-specific probe that can identify the SNP or InDel position and identify the breed by using this position.

The characteristics of the marker set for identifying a rice variety according to the present invention are as follows.

DK17-1: present on chromosome 8 and site of site used for sequencing is clone AP003886

DK34: present on chromosome 1 and site of site used for sequencing is clone AP003209

DK50: present on chromosome 11 and site of site used for sequencing is clone of AC145367

DK63: located on chromosome 3 and site of site used for sequencing is AC135495 clone

DK560: located on chromosome 12 and site of site used for sequencing is BX000560 clone

DK1412: located on chromosome 7 and site of site used for sequencing is clone AP005198

DK2394: located on chromosome 7 and site of site used for sequencing is AP003866 clone

DK2401: located on chromosome 7 and site of site used for sequencing was cloned AP004010

DK1123: located on chromosome 2 and site of site used for sequencing is clone AC091238

DK1361: located on chromosome 10 and site of site used for sequencing is clone AC079888

DK600: located on chromosome 4 and site of site used for sequencing was cloned AL606660

DK2511: located on chromosome 2 and site of site used for sequencing is clone AP005111

DK601: located on chromosome 2 and site of site used for sequencing is clone AP005284

DK2708: located on chromosome 12 and site of site used for sequencing is AL731784 clone

In addition, as a positive control of the rice variety identification method of the present invention, genomic DNA present at the AB013614 clone position can be used as a CCS1 similar to the Cereal Centromeric Sequence in rice as an endogenous gene, such as an endogenous gene. have.

15 kinds of marker sets for identifying rice varieties of the present invention, DK17-1, DK34, DK50, DK63, DK560, DK1412, DK2171, DK2394, DK2401, DK1123, DK1361, DK600, DK600, DK2511, DK601 and DK2708 Real time PCR can be performed using more than one combination, preferably three or more combinations simultaneously. The combination of these 15 sets of rice varieties for identifying markers can be determined according to the type and number of detectable means available for probe labeling.

That is, the detectable means can be used simultaneously in one real time PCR reactant, and each detectable means used must be detectable respectively. For example, if there are two kinds of detectable means that can be used simultaneously for a single PCR reaction and can be specifically detected for each kind, the set of markers for identifying rice varieties of the present invention simultaneously performs a real-time PCR in combination of two kinds. can do. As another example, when three, four, five, six, or seven or more kinds of specific detectable means are used, the marker set for identifying the rice variety of the present invention is three, four, five, six, respectively. Or a combination of seven or more kinds. As used above, "specific" as used in specific detectable means means a property capable of using two or more kinds of a single PCR reaction at the same time as well as separately detecting each of those used under detectable conditions.

The detectable means means compounds, biomolecules or biomolecule mimetics, etc., which can be linked, bound, or attached to a probe to determine the density, concentration, amount, etc. in a conventional manner. Examples thereof include fluorescent materials, light emitting materials, bioluminescent materials, and isotopes which are commonly used, but are not limited thereto. As another example, fluorescent materials are most preferred. Since a large number of fluorescent materials are currently on the market, they are readily available. Examples of fluorescent materials include FAM ™ (6-carboxyfluorescein), SYBR  Green, VIC (2'-chloro-7'-phenyl-1,4-dichloro-6-carboxyfluorescein), JOE (carboxy-4 ', 5'-dichloro-2', 7'-dimethoxyfluoresce Phosphorus), NED ™ (2'-chloro-5'-fluoro-7 '8'-fused phenyl-1,4-dichloro-6-carboxyfluorescein), TAMRA ™ (carboxytetramethyldamine), Cy3 ™, ROX ™ (carboxy-X-rhodamine), HEX (2 ', 4', 5 ', 7'-tetrachloro-6-carboxy-4,7-dichlorofluorescein), TET (2', 7'-dichloro-6-carboxy-4,7-dichlorofluorescein), Texas Red, Cy5 ™ and the like, but is not limited thereto. If the fluorescent material has different excitation and emission wavelengths depending on the type, the method of use is also different. Therefore, the fluorescent material used together in one PCR reaction should be selected and used to determine whether the fluorescent material can be detected separately. Representative fluorescent material characteristics are briefly shown in Table 2 below.

Filter Excitation wavelength Radiation wavelength Fluorescent material One 490 520 FAM, SYBR Green 2 520 550 JOE, VIC, HEX, TET 3 550 580 NED, TAMRA, CY3 4 580 610 Taxas Red, ROX, RED610 5 640 670 CY5, RED670

In one embodiment of the present invention, the fluorescent material is labeled on a probe included in a marker set for identifying a rice variety according to the present invention in a conventional manner. Labeling methods include interchelating methods, TaqMan ™ probe methods, and molecular beacon methods. Intercalating method is a reagent that binds to double-stranded DNA and fluoresces (inter-chelator: SYBR).  Green I, EtBr, etc.) is added to the PCR reaction to detect fluorescence that develops with amplification.The intercalator binds to the double-stranded DNA synthesized by the PCR reaction and emits fluorescence. The amount of produced can be measured. TaqMan ™ probe method is a method in which an oligonucleotide (TaqManTM probe) modified with 5 ends of fluorescent material (FAM, etc.) and 3 ends of quencher material (TAMRA, etc.) is added to the PCR reaction solution. Fluorescence is inhibited by quencher on the probe, but only in the extension step, only the TaqMan ™ probe hybridized to the template by 5 → 3 exonuclease activity of the Taq DNA polymerase was decomposed to cause fluorescence. As it is released from the probe, the inhibition by the quencher is released and fluoresces. The molecular beacon method is a method of adding a oligonucleotide probe (Molecular Beacon probe) to the PCR reaction system to form a hairpin secondary structure in which both ends are modified with a fluorescent material (FAM, TAMRA, etc.) and a quencher material (DABCYL, etc.). Molecular beacon probes have a hairpin structure in the glass state and are close to the fluorescent material and quencher material to suppress fluorescence, but the distance between the fluorescent material and quencher material when specifically hybridized in the region complementary to the template DNA in the annealing step The quenching of fluorescence on the probes fluoresces because the inhibition by the quencher material is eliminated. However, unhybridized molecular beacon probes retain their hairpin structure and thus do not fluoresce.

This method can be applied to multiple real time PCR methods for identifying rice varieties of the present invention. Since the above methods are known in the art, specific methods may be selected in consideration of reaction efficiency, time, fluorescent material type, and the like. In the present invention, for example, a TaqMan ™ probe method may be used.

When three kinds of rice varieties for identifying marker sets according to the present invention are used for triplex real-time PCR, the FAM, VIC, and NED ^TM three species are used as fluorescents, respectively, to label the 5 'end of the probe, and the 3' end of the probe is MGBNFQ. (molecular grove binding non-fluorescence quencher) is attached. In one example, a combination of a set of markers for identifying rice variety that can be used for triplex real-time PCR can be as follows:

One marker set selected from the group consisting of DK2401, DK50, and DK2171;

One marker set selected from the group consisting of DK17-1, DK1412, and DK34;

One marker set selected from the group consisting of DK560, DK2394 and DK63;

One marker set selected from the group consisting of DK1123, DK1361, and DK600;

And

1 set of markers selected from the group consisting of DK2511, DK2708 and DK601.

The combination is based on the condition that a set of markers labeled with different fluorescent materials can be used together, and is only an example, and various combinations can be made while changing the fluorescent material in addition to the combination. This can be easily understood and implemented by those skilled in the art to which the present invention pertains. In another embodiment of the present invention, (1) DK2401 (FAM), DK50 (VIC), DK2171 (NED); (2) DK17-1 (FAM), DK1412 (VIC), DK34 (NED); (3) DK560 (FAM), DK2394 (VIC), DK63 (NED); (4) DK1123 (FAM), DK1361 (VIC), DK600 (NED); And (5) DK2511 (FAM), DK2708 (VIC), DK601 (NED); Combinations may be used.

In addition, the marker set for rice variety identification of this invention can be implemented using a conventional real-time PCR method and apparatus. The real-time PCR method is a method for detecting and quantitating fluorescence performed in real time every cycle of PCR based on the principle of DNA polymerase and Fluorescence Resonance Energy Transfer (FRET). This method distinguishes specific amplification products from non-specific amplification products and can easily obtain analysis results in an automated manner.

Real-time PCR devices that can be used in the present invention include, but are not limited to, AB Real-time PCR devices 7900, 7500, 7300, Stratagene Mx3000p, and BioRad Chromo 4 devices. At the end of PCR, the laser of the real-time PCR device senses the fluorescent material labeled on the probe of the amplified PCR product to implement the peak as shown in FIG. 1. Thus, the results can be automatically analyzed by executing a program embedded in the device without the electrophoresis process.

Through the marker set for identifying the rice variety of the present invention and the rice variety identification method using the same, it is possible to identify several kinds of rice varieties, and in particular, it is possible to identify a total of 514 domestic and foreign rice varieties, respectively, described in Table 3 below.

country kind Korea (286 species) Reclamation, Sura rice, Tam Jin rice, Alternative, Borami, Gemstone, Geuman, Cheongchungjinmi, Flavor No. 1, Blizzard, Hwasunchalb, Sinseonchalb, Goami 2, ARC11895, Cheongan, Groot, Geumo, Chogwang, CI5483-2, Samdeok , Geumnam, Hwamyeong, Hwajin, Gwangan, Goami, YJ06-01, Heukdang rice, Yeongdeok rice, Palgong rice, Ilmi, Karakchal, water ripple, Xi'an, Fine, Promotion, Flavor, Good rice, Gyunyoung-do, Hogwang, Sangok Rice, YJ06-14, Saegyehwa, Sampyeong, Hwayoung, Baekjinju No.1, Shinpung, Glory, Haiami, Sold, Reconstruction, Pung Jade, Dami rice, PAIKANTA + 8217, L + P ledves type, Eungu No.6, Patriotic, Consumption Rice , Cold, white snow, Sindongjin, black ore, Seorak Rice, YJ06-02, Ansan, YJ06-04, YJ06-15, Cheongah, Joami, Koshihikari, Golden Nuri, three hundred, Golden Node, Chilbo rice, Banggeum, Donghaemi, Horse Gummy, Junnam, Concentrate, Peace, Alanghyangbyeol, Josaeng Blackchal, Black Incense, Haipyeongchal, Palug, Saenuri, Haormi, Haepyeongbap, Dongjin No. 1, Koami 3, Baekjinju, Seolgang, One Piece Rice, Big Eyes, Gwanok, Fine , Rice paddy , Tadazo, oath, dwarf rice, bamboo, triangular road, morning dong, YJ06-07, Hong Jinju, Chang'an, crest rice, Hitomebore, Wonsanschal rice, Dobong rice, South wind rice, YJ06-10, YJ06-06, Eri239 No., Nokwonbil Rice, Unmi, Delivery, Handul, Namseon 102, Obong, PEMBE, Tillenag 8219, YJ06-03, YJ06-17, Old Mando, Unbongpyeon, Iljin, Samcheon, Saengdongbyeon, Sangsan, Odae, Unduk, Sinunbongbong Rice , Sinnong Black Sword, Gwanak Paddy, Geunjajo, Namseon No. 13, Red Pearl, Zhongshan Rice, Dunnae, Damarumum, White Sea Dal, Xi'an No. 1, Ungwang Rice, MAN + NG BALAY, Changzhou, Sinunbong No. 1, Samgwang Rice, Saesangju , Jinbu, Sangmi, Genuine, Delicacy rice, YJ06-16, Gem, Jinbong, Manna, Naengpung rice, Chinese food, Golden purple, Huadong, Sachuchu, while, Wonbwang rice, Jinmi, Chucheong, Anjungbap, Huaan, Nampyeong, Mangyeong , Manpung, Hwaseong, Nakdong rice, Jungmo 1006, Hwacheong rice, Edo, Manchu, Youngan rice, Dongjin, YJ06-11, Taebong, Seokjeong, Dongjinjak, Hanyangjo, Milseong, Eungu No.5, Sinmyeong blacksword, Beef beef 132, Huazhong , South Korea, Seojin, Erie 265, Manmi, Jado, Rec. No. 9, Scenery, Purple light, Jadae black, Jinbu-chalm rice, F1-312 8223, YJ06-13, Black snow, black pearl, Gyehwa rice, Namcheon rice, Beautiful rice, YJ06-05, YJ06-08, Nonglim Na Na 1, Sangnam field rice, Onnuri , YJ06-12, Ling Ta 150, Shin-Pa, YJ06-09, Miryang 30, Samgang rice, Yongmun rice, Wonpung rice, Hangang Chul No. 1, IR10179-23-1-3, Flavor Rice No. 1, IR667-98 -1-3-25, Dasan No. 1, Dasan Rice, Yeongnam Josaeng, Joseong Unification, Golden Rice, Namyoung Rice, Drachan, Baekyang Rice, Jangseong Rice, Jungwon Rice, Unification Temple, Pungsan Rice, Hanaummi Rice, Daejin, Brewing Rice, Bonsai, Unflavored rice, KASHIMIR BASMATI, Shandu-do, Taesung, Gems black flag, Joan, Odae 1, Nokyang, CR126-42-5, KAKAI 272/51, Danmi, Parsley, Crass-pd8244, Sobaek, YJ06 -19, cold water 13, water tank, rice paddy 3, BKNFR 76106-16-0-1-0, Pai Hsu, Yongju rice, Han river wax rice, BABUNIYA, flavor rice 2, IR9669-PP830-1, Lu Chin Tsi , Miryang 42, Chupung Rice, Taebaek Rice, Kaya Rice, Manseok Rice, Miryang 23, Soongbyeol Rice, Crystal Rice, Singwang Rice, Chungcheong Rice, Miryang 21, Miryang 22, Nongan, 2869 (16667), Plain, Residential Building, Hyangnam Rice, Daedae Rice No. 1, HP904B-1 8212 China (94 species) Use 3, Xi'an, Shan Gu, brown snow sweetness, salinity 11, heavy shaft 5, fascinator, SAN BANG QISHI LU +, shasani, shaft 7, butite ore, fasciitis 241, use 4, red blood, Yunnan collection 12, total 42, Bukji 18, Potpourri, Yeoksaeng 283, Chujung Soonjeong (Agida-machi), Yongnong 1, Use 2, Gil-tuk, Gando 10, Yonyeon 23, Yonggong 13, Tonggyeong 808, Yongnong 4, Tongwoo 163, China 9-23 , 100 points, cheonrakdo, Jangbaek No. 7, Gando 9, Dohwahyang No. 2, Gilneon No. 2, Ungang No. 20, Total 41, Heavy Shaft 6, Meat 201, Meat 211, Songgang 7, Total 35, Gilgang 61, Yonggang 12, salinity 11, Honggammi, black shell, meat 131, use 5, Dongnong 416, Gil Nongdae 19, Songgang 2, Heukdo, Bukji, Dongyeon 2, meat 308, Hyangmi, Yongsun 104, Yogong 151, Yoseong 2102, Songgyeong 3, Tongwoo 192, Tongyeon No. 5, Half Price 8, Segan Edo, Altitude No. 1, Dong Nong 415, 97-39-1, 98-49-1, BIR-H +, Hung-Fam-Goo, LIEN-CHAN -ZE-TH + U, NANTA 16, NAN-TA-H + W, Pl161010, SH + A-M + A-FAN, Altitude No. 2, Gilgang 88, Tunnel (Tianjin), Sensitivity 8, Gilgang 9, Tongyeon 6, tunnel (Yihong), road direction No. 1, Variety No.1, rice flavor, road nongdae 12, Daen-chon-Bir, Low-Shun-Dau, Shan-Tsan, scented, tunnels (resident), Mudanjiang 19 Japan (62 types) K + GY + KA, Yukdo Nabuji-style, Daneun Ark, Yukdo-wook, Power, Ungung, Hitome bare, Gyeongbuk Gokyangdo, Chungseungjae, Milky Queen, Gogane Mochi, D + IKU 154, Conviction, Gokyangdo, North Sea Red hair, rebirth of the sea, Sasanishi, Sun Ya Mona, Jana, Hoshiyudaka, SANTAM + MI, Kanto PL3, Nippon bare, Mauretsu, 5 million stone, Jado, Poonna, RIKUT + SAKUMAI, GINSUKEYAKAN, HATA K + GANE M + CHI, + NJ + HAKAM + D + SHI, TACHIMINAMI, Kuk Kwang Na, Daedo No. 3, Yeodo Agriculture and Forestry No. 16, Yeodo Agriculture and Forestry No. 19, Yeodo Agriculture and Forestry 21, Yeodo Agriculture and Forestry No. 13, Yeodo Agriculture and Forestry No. 1, and Yeodo Agriculture and Forestry No. 4 , Chungseungdo Island, HATA K + GANEM ++, + TSUKAM + D + SHI, Satominori
Goseong, Hong So Land 5, Daedo 4, B + ZUY AKAN, Improved No. 13, Yeodo No. 2, Yeokdo Edo No. 1, HIN + DE, Chubu Site, MIYAMAM + CHI, Daedo No. 1, Sagukna, Yeodo Agriculture and Forestry No. 9, Muskdo, Improvement 41, Yudo-do Agriculture 11, Mona, Yudo-do Agriculture 3 Guy An (2 species) NAR8F5-1-3-2, NAR163 F3-1-4 Nigeria (2 species) T + -3133-59-1-2-4-1, T + -3027-42-1-E-2-1-1-1 Nepal (2 species) Tadukan, P + KHARELIMASIN + Russia (3 species) Krasnodarskill, Novoselskill, H + 459 Madagassen (two kinds) ANG IN AF + TSY, WIR 7085 Mexico (1 species) + M987-1 United States (9 species) IRRI16302, Jasmine 85, California Blue Rose, 19b1014b-9n-1, NAHNGM + N S-4 SELECTI + N, anonymous 4, anonymous 1, anonymous 2, anonymous 3 Bangladesh (5 species) Bashtus, Boteswar-2, Mati Char, Dharga Sail, BR1543-9-2-1 Vietnam (2 species) JMANG PAI HAUTA +, Bui Tap49 Bhutan (2 types) Chumro, Bajo Kaapl North Korea (4 types) Yeongju 4, West Sea 13, Pyeongdo 1, Hamju Brazil (2 species) CAIAP +, MARAVILHA Sri Lanka (2 types) SUDAHAL + W, SEENATTY Spain (1 species) ARIETE Azerbaijan (1 species) kessa red Afghanistan (2 species) SPINMERE, LUN ++ PUN Uzbek (1 species) Debzere Iran (2 types) D + MS + F ++ D, HASSNAY Egypt (2 species) GIZA, GIZA 170 Italy (3 types) ZENA, ARIRTE, ARG + India (2 species) KAGI, BASMATI Indonesia (2 species) Mendawak, Mayang Chile (2 species) QUILLA, Diamaute Kazakh (1 species) Zarya Colombia (2 species) Blue bell, Lemont Thailand (1 species) + S248 Pakistan (2 species) BASAMATI, Muzzaffar-8 Peru (1 species) CAR + LINA Philippines (7 species) Giant GRAIN, KINAGAYKAY, IR1552, IR1750-F5B-22, IR1487-194--3-4, IRRI 10320, IR12780 Hungary (2 species) Cgi-Csing, DAMA

The determination of origin and variety is based on amplification of 15 markers, and each variety has a different combination of amplified markers. Real-time PCR results of 15 SNP markers for the above 514 varieties are shown in Table 4. If PCR amplification has occurred for the marker, it is marked with +, otherwise it is indicated with-.

In addition, in order to facilitate determination of the breed and the origin, scores were assigned to each marker to distinguish the sum of the scores for the markers amplified for each breed. The score for each marker is shown in Table 5 below.

SNP Marker Score DK2394 One DK50 2 DK2401 4 DK1412 8 DK17-1 16 DK34 32 DK63 64 DK560 128 DK2171 256 DK2708 512 DK1361 2048 DK2511 131072 DK601 524288 DK600 1048576 DK1123 2097152

By the real-time PCR method using two, three, four or five or more of the above-described marker set for identifying the rice variety of the present invention, the PCR process to be performed for each marker to distinguish the existing rice variety is remarkable. Not only can it be shortened, but the result can be quickly confirmed in real time. This method can be distinguished by laser sensitization of the presence of small DNA fragments and identification of PCR products of less than 100 bp that were difficult to distinguish by electrophoresis, and there is no concern about contamination by PCR products. All test procedures are operated as an automatic system until the end of the test period. Therefore, it is unlikely that a mistake of a tester or an error of a test will occur, and it is easy to collect and analyze data, and minimize test time and labor required. Very useful.

The present invention is described in detail with reference to the following examples. The following examples are merely to illustrate the invention, but not to limit the protection scope of the invention.

Example  1: for identifying rice varieties Plural Marker  Set Reclaimed rice  Breed Identification

1-1. Template DNA extraction

According to the method of patent application 2005-69858, DNA was extracted from the sample rice. Briefly, the CTAB method which is generally used when extracting DNA from a plant was modified and used in the present invention.

To prevent contamination of the sample, place it on oil paper and grind the rice sample into a very small piece with a hammer. The ground sample (about 30 mg) is put in a 1.5 ml tube, 500 µl of CTBA solution is added, mixed well, and soaked for 30 minutes in a 65 ° C constant temperature water bath. The composition of the CTAB solution is as follows: DNA extraction buffer (Sorbitol 63.75 g, Tris-base 12.1 g, EDTA-Na2 1.68 g / 1 L) and nuclear fusion buffer (CTAB 20 g, 1M Tris (pH 8.0) 200 ml, 0.25 200 ml of M EDTA, 400 ml / 1 L of 5M NaCl) is mixed 1: 1, and 0.2 volume 10% sarcosyl, 0.2 volume saline, 1% 2-mercaptoethanol is mixed in a 65 DEG C constant temperature water bath. After centrifugation at 14,240 xg (15,000 rpm) for 10 minutes, the supernatant is mixed with 1 volume of 3.8 g / L sodium bisulfite and 1 volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) at room temperature for 5 minutes. For 5 minutes at 14,240 xg. Transfer the supernatant to a 1.5 ml tube, mix 1 volume of chloroform: isoamyl alcohol (24: 1) and spin for 5 minutes at room temperature. Centrifuge at 14,240 × g for 5 minutes and transfer the supernatant to a 1.5 ml tube. Repeat the above procedure until the interface becomes transparent. Add the same amount of isopropanol and mix well for 5 minutes, put in 20 minutes at -20 ℃ and centrifuge for 15 minutes at 4 ℃. Discard the supernatant and centrifuge for 5 min at 4 ° C with 200 μl of 70% ethanol DNA pellet. Discard the supernatant and dry the remaining ethanol with air or vacuum dryer. Add 65 μl of sterile distilled water and 15 μl of RNase A (1 mg / ml) and place in a 37 ° C. water bath for 15 minutes.

1-2. Real time PCR reaction

Mix the reaction solution in a total volume of 25 μl, including 12.5 μl 2x Master mix, 10 μl template DNA, 4 μl of rice varietal marker set, and finally ddH 2 O. Proper concentrations of primers and probes are shown in Table 6 below. At this time, the marker set for rice product group identification is (1) DK2401 (FAM), DK50 (VIC), DK2171 (NED); (2) DK17-1 (FAM), DK1412 (VIC), DK34 (NED); (3) DK560 (FAM), DK2394 (VIC), DK63 (NED); (4) DK1123 (FAM), DK1361 (VIC), DK600 (NED); And (5) five triplex real-time PCRs were performed using a combination of DK2511 (FAM), DK2708 (VIC), and DK601 (NED).

Multiplex set SNP Marker Primer / probe Final concentration (pmol / rxn) Triplex 1 DK2401 Forward primer 7 Reverse primer 7 Probe 1.5 DK50 Forward primer 5 Reverse primer 5 Probe One DK2171 Forward primer 15 Reverse primer 15 Probe One Triplex 2 DK17-1 Forward primer 10 Reverse primer 10 Probe One DK1412 Forward primer 5 Reverse primer 5 Probe One DK34 Forward primer 10 Reverse primer 10 Probe One Triplex 3 DK560 Forward primer 5 Reverse primer 5 Probe 2 DK2394 Forward primer 10 Reverse primer 10 Probe 2 DK63 Forward primer 5 Reverse primer 5 Probe 2 Triplex 4 DK1123 Forward primer 10 Reverse primer 10 Probe 2 DK1361 Forward primer 10 Reverse primer 10 Probe 2 DK600 Forward primer 10 Reverse primer 10 Probe 2 Triplex 5 DK2511 Forward primer 10 Reverse primer 10 Probe 2 DK601 Forward primer 10 Reverse primer 10 Probe 2 DK2708 Forward primer 10 Reverse primer 10 Probe 2

The reaction solution was pre-denaturated for 2 minutes at 50 ° C. and 10 minutes at 95 ° C., followed by 40 minutes of reaction for 15 seconds at 95 ° C. and 1 minute at 60 ° C. to perform real-time PCR.

1-3. Analysis of PCR Products Using Real-Time PCR

The present invention can be applied to the Real-time PCR device 7500 of AB. When the PCR is terminated, the laser of the device detects the fluorescent material labeled on the probe of the amplified PCR product is implemented as a peak as shown in FIG. In other words, the results can be analyzed automatically by running the program embedded in the device without the electrophoresis process.

1-4. Final judgment

Figure 1 shows the results of real-time PCR with a template extracted from Nampyeong rice.

Referring to FIG. 1, it was confirmed that the sample rice was correctly judged as reclaimed rice by specifically amplifying the rice varieties marker sets DK2401 and DK560.

Example  2 to 50: for identifying rice varieties Plural Marker  Breed Identification Using Sets

According to the method of Example 1, real-time PCR was performed using the samples described in Table 7 below, and the results are shown in Table 7 and FIGS. 2 to 50.

Example sample Amplification marker 2 Sura rice DK2401, DK560 3 Rice paddy DK50, DK2401, DK1412, DK560 4 Alternatives DK50, DK2401, DK17-1, DK560 5 Borami DK1412, DK17-1, DK34, DK560 6 Jewelry DK50, DK2401, DK1412, DK17-1, DK34, DK560 7 Jinan DK63, DK560 8 Blue Delicacy DK63, DK560 9 Flavor 1 DK2401, DK560, DK2171 10 snowstorm DK2394, DK2401, DK560, DK2171 11 Rice line DK50, DK1412, DK560, DK2708 12 Fresh rice DK50, DK2401, DK1412, DK560, DK2708 13 Goami 2 DK63, DK560, DK2708 14 ARC11895 DK17-1, DK63, DK560, DK2171, DK2708 15 Cheongan DK50, DK2401, DK17-1, DK34, DK560, DK1361 16 Trees DK560, DK1361, DK2708 17 Geumoh DK34, DK560, DK1361, DK2708 18 Dimming DK560, DK2171, DK2511 19 CI5483-2 DK17-1, DK63, DK560, DK2171, DK2511, DK2708 20 Samdeok DK50, DK2401, DK17-1, DK560, DK601 21 Geumnam DK50, DK2401, DK1412, DK17-1, DK63, DK560, DK601 22 Hwamyeong DK50, DK2401, DK1412, DK17-1, DK63, DK560, DK601 23 Huajin DK50, DK2401, DK1412, DK17-1, DK63, DK560, DK601 24 Gwangan DK17-1, DK34, DK63, DK601, DK2708 25 Goami DK2394, DK1412, DK17-1, DK560, DK601, DK2708 26 YJ06-01 DK2394, DK50, DK2401, DK1412, DK63, DK560, DK601, DK2708 27 Black rice DK2394, DK50, DK2401, DK1412, DK63, DK560, DK601, DK2708 28 Yeongdeok Rice DK50, DK34, DK63, DK560, DK1361, DK601 29 Palgong Rice DK50, DK34, DK63, DK560, DK1361, DK601 30 Japan DK2401, DK1412, DK17-1, DK34, DK560, DK2171, DK1361, DK601 31 Rakrakchal DK560, DK1361, DK601, DK2708 32 Ripple DK50, DK560, DK600 33 West Coast DK2401, DK560, DK600 34 delicate DK2394, DK2401, DK560, DK600 35 Promotion DK2394, DK2401, DK560, DK600 36 zest DK50, DK17-1, DK560, DK600 37 Rice paddy DK50, DK17-1, DK560, DK600 38 Breaststroke DK2394, DK2401, DK17-1, DK560, DK600 39 Arc DK2394, DK2401, DK17-1, DK560, DK600 40 Jade rice DK50, DK2401, DK17-1, DK560, DK600 41 YJ06-14 DK2394, DK2401, DK1412, DK17-1, DK560, DK600 42 New world DK34, DK560, DK600 43 Sampyeong DK2394, DK2401, DK34, DK560, DK600 44 Hwa Young DK2394, DK50, DK2401, DK1412, DK17-1, DK34, DK560, DK600 45 Baek Jinju No. 1 DK63, DK560, DK600 46 New wind DK63, DK560, DK600 47 glory DK63, DK560, DK600 48 Haiami DK63, DK560, DK600 49 Sell DK2394, DK63, DK560, DK600 50 reconstruction DK1412, DK63, DK560, DK600

According to Table 7 and the results of FIGS. 2 to 50, the DNA extracted from the sample rice was specifically amplified in each rice variety marker set to confirm that the sample rice was correctly determined.

Example  51 to 100: for identifying rice varieties Plural Marker  Rice Variety Identification Using Sets

According to the method of Example 1, real-time PCR was performed using the samples described in Table 8 below, and the results are shown in Table 8 and FIGS. 51 to 100.

Example sample Amplification marker 51 Pung Jade DK1412, DK63, DK560, DK600 52 Rice DK50, DK17-1, DK63, DK560, DK600 53 PAIKANTA + 8217 DK2394, DK2401, DK17-1, DK63, DK560, DK600 54 L + P ledves type DK1412, DK17-1, DK63, DK560, DK600 55 Eun-gu 6 DK1412, DK17-1, DK63, DK560, DK600 56 Patriotic DK2394, DK1412, DK17-1, DK63, DK560, DK600 57 Consumption DK50, DK1412, DK17-1, DK63, DK560, DK600 58 Cold water DK2394, DK2401, DK1412, DK17-1, DK63, DK560, DK600 59 White snow DK17-1, DK34, DK63, DK560, DK600 60 Dongjin Shin DK2394, DK50, DK17-1, DK34, DK63, DK560, DK600 61 Black light DK50, DK17-1, DK34, DK63, DK560, DK600 62 Seorak Rice DK2394, DK17-1, DK63, DK560, DK2171, DK600 63 YJ06-02 DK2401, DK17-1, DK63, DK560, DK2171, DK600 64 Ansan DK2401, DK17-1, DK63, DK560, DK2171, DK600 65 YJ06-04 DK2401, DK1412, DK17-1, DK34, DK63, DK560, DK2171, DK600 66 YJ06-15 DK2401, DK1412, DK17-1, DK34, DK63, DK560, DK2171, DK600 67 Herring DK2394,, DK600, DK2708 68 Joami DK560, DK600, DK2708 69 Koshihikari DK2394, DK560, DK600, DK2708 70 Golden Nuri DK50, DK560, DK600, DK2708 71 three hundred DK2401, DK560, DK600, DK2708 72 Golden Node DK50, DK2401, DK560, DK600, DK2708 73 Cloisonne DK2394, DK17-1, DK560, DK600, DK2708 74 Eight gold DK2394, DK17-1, DK560, DK600, DK2708 75 Dong Hae-mi DK50, DK17-1, DK560, DK600, DK2708 76 Speech DK50, DK17-1, DK560, DK600, DK2708 77 Junnam DK2394, DK50, DK2401, DK17-1, DK560, DK600, DK2708 78 Concentrate DK2394, DK2401, DK17-1, DK560, DK600, DK2708 79 equability DK50, DK2401, DK17-1, DK560, DK600, DK2708 80 Alanghyang Sticky Rice DK1412, DK17-1, DK560, DK600, DK2708 81 Black and white DK2401, DK1412, DK17-1, DK560, DK600, DK2708 82 Black incense DK2394, DK2401, DK1412, DK17-1, DK560, DK600, DK2708 83 Haiyeong Police DK50, DK1412, DK34, DK560, DK600, DK2708 84 Eight DK2401, DK1412, DK34, DK560, DK600, DK2708 85 Saenuri DK50, DK1412, DK17-1, DK34, DK560, DK600, DK2708 86 Haormi DK50, DK17-1, DK34, DK560, DK600, DK2708 87 Haepyeong Rice DK50, DK17-1, DK34, DK560, DK600, DK2708 88 Dongjin No. 1 DK50, DK1412, DK17-1, DK34, DK560, DK600, DK2708 89 Goami 3 DK63, DK560, DK600, DK2708 90 White pearl DK63, DK560, DK600, DK2708 91 The DK63, DK560, DK600, DK2708 92 A la carte DK63, DK560, DK600, DK2708 93 Big eyes DK63, DK560, DK600, DK2708 94 Prison DK1412, DK63, DK560, DK600, DK2708 95 High quality DK17-1, DK63, DK560, DK600, DK2708 96 Upper wind rice DK17-1, DK63, DK560, DK600, DK2708 97 Snow ball DK17-1, DK63, DK560, DK600, DK2708 98 Jinbuol DK17-1, DK63, DK560, DK600, DK2708 99 Tea DK2394, DK17-1, DK63, DK560, DK600, DK2708 100 Oath DK1412, DK17-1, DK63, DK560, DK600, DK2708

According to Table 8 and the results of FIGS. 51 to 100, the DNA extracted from the sample rice was specifically amplified in each rice variety marker set to confirm that the sample rice was correctly determined.

Example  101 to 150: for identifying rice varieties Plural Marker  Breed Identification Using Sets

According to the method of Example 1, real-time PCR was performed using the samples described in Table 9 below, and the results are shown in Table 9 and FIGS. 101 to 150.

Example sample Amplification marker 101 Dwarf rice DK2394, DK1412, DK17-1, DK63, DK560, DK600, DK2708 102 God DK2394, DK1412, DK17-1, DK63, DK560, DK600, DK2708 103 Triangularity DK2394, DK1412, DK17-1, DK63, DK560, DK600, DK2708 104 A winter solstice DK2394, DK1412, DK17-1, DK63, DK560, DK600, DK2708 105 YJ06-07 DK2401, DK34, DK63, DK560, DK600, DK2708 106 Hong Jinju DK2401, DK17-1, DK34, DK63, DK560, DK600, DK2708 107 Chang'an DK17-1, DK34, DK2171, DK600, DK2708 108 Crest DK560, DK2171, DK600, DK2708 109 Hitomebore DK2394, DK2401, DK17-1, DK560, DK2171, DK600, DK2708 110 Wonsanzabap DK17-1, DK63, DK560, DK2171, DK600, DK2708 111 Dobong Rice DK2394, DK17-1, DK63, DK560, DK2171, DK600, DK2708 112 South wind DK2401, DK17-1, DK63, DK560, DK2171, DK600, DK2708 113 YJ06-10 DK34, DK63, DK560, DK2171, DK600, DK2708 114 YJ06-06 DK2401, DK1412, DK17-1, DK34, DK63, DK560, DK2171, DK600, DK2708 115 Erie239 DK1412, DK560, DK600, DK1361 116 Green rice DK2394, DK17-1, DK560, DK600, DK1361 117 Luck DK50, DK17-1, DK560, DK600, DK1361 118 delivery DK2394, DK1412, DK17-1, DK560, DK600, DK1361 119 Hand DK17-1, DK63, DK560, DK600, DK1361 120 Namsun 102 DK1412, DK17-1, DK63, DK560, DK600, DK1361 121 Obong DK17-1, DK2171, DK600, DK1361 122 PEMBE DK50, DK17-1, DK63, DK560, DK2171, DK600, DK1361 123 Tillenag 8219 DK2401, DK17-1, DK63, DK560, DK2171, DK600, DK1361 124 YJ06-03 DK2401, DK17-1, DK63, DK560, DK2171, DK600, DK1361 125 YJ06-17 DK2401, DK17-1, DK63, DK560, DK2171, DK600, DK1361 126 Old man DK2394, DK2401, DK17-1, DK34, DK63, DK560, DK2171, DK600, DK1361 127 Yunbong Rice DK2394, DK560, DK600, DK1361, DK2708 128 ILJIN DK1412, DK560, DK600, DK1361, DK2708 129 Three thousand DK17-1, DK560, DK600, DK1361, DK2708 130 Live insight DK2394, DK17-1, DK560, DK600, DK1361, DK2708 131 Sangsan DK34, DK560, DK600, DK1361, DK2708 132 Five DK2394, DK34, DK560, DK600, DK1361, DK2708 133 Mundo DK2394, DK17-1, DK34, DK560, DK600, DK1361, DK2708 134 Sinunbongpung DK2401, DK17-1, DK34, DK560, DK600, DK1361, DK2708 135 Sinnong Black Sword DK2394, DK50, DK2401, DK1412, DK63, DK560, DK600, DK1361, DK2708 136 Gwanak Rice DK2394, DK2401, DK17-1, DK63, DK560, DK600, DK1361, DK2708 137 Elderly group DK2394, DK34, DK63, DK560, DK600, DK1361, DK2708 138 Namsun 13 DK2394, DK17-1, DK34, DK63, DK560, DK600, DK1361, DK2708 139 Red pearl DK17-1, DK2171, DK600, DK1361, DK2708 140 Zhongshan rice DK2401, DK17-1, DK560, DK2171, DK600, DK1361, DK2708 141 Dungeon DK2394, DK63, DK560, DK2171, DK600, DK1361, DK2708 142 Damageum DK2394, DK17-1, DK34, DK63, DK560, DK2171, DK600, DK1361, DK2708 143 White Sea Otter , DK1412, DK17-1, DK34, DK63, DK560, DK2171, DK600, DK1361, DK2708 144 Xi'an 1 DK2401, DK34, DK600, DK2511 145 Ugwanghwa DK50, DK1412, DK17-1, DK560, DK600, DK2511 146 MAN + NG BALAY DK17-1, DK63, DK560, DK2171, DK600, DK2511 147 Resident DK17-1, DK600, DK2511, DK2708 148 Sinwoonbong No. 1 DK17-1, DK600, DK2511, DK2708 149 Samgwang Rice DK50, DK2401, DK17-1, DK34, DK600, DK2511, DK2708 150 New Residency DK2394, DK2401, DK560, DK600, DK2511, DK2708

According to Table 9 and the results of FIGS. 101 to 150, the DNA extracted from the sample rice was specifically amplified in each rice variety marker set to confirm that the sample rice was correctly determined.

Example  151 to 200: for identifying rice varieties Plural Marker  Rice Variety Identification Using Sets

According to the method of Example 1, real-time PCR was performed using the samples described in Table 10, and the results are shown in Table 10 and FIGS. 151 to 200.

Example sample Amplification Marker 151 banality DK2394, DK63, DK560, DK600, DK2511, DK2708 152 Ordinary DK17-1, DK63, DK560, DK600, DK2511, DK2708 153 curio DK50, DK2401, DK17-1, DK63, DK560, DK600, DK2511, DK2708 154 Delicacy DK17-1, DK560, DK2171, DK600, DK2511, DK2708 155 YJ06-16 DK2401, DK17-1, DK34, DK63, DK560, DK2171, DK600, DK2511, DK2708 156 jewelry DK2394,, DK600, DK1361, DK2511, DK2708 157 Jinbong DK560, DK600, DK1361, DK2511, DK2708 158 meet DK17-1, DK560, DK600, DK1361, DK2511, DK2708 159 Wind-resistant rice DK2394, DK2401, DK17-1, DK560, DK600, DK1361, DK2511, DK2708 160 Neutralization DK2394, DK2401, DK17-1, DK560, DK600, DK1361, DK2511, DK2708 161 Golden purple DK2171, DK600, DK1361, DK2511, DK2708 162 Huadong DK17-1, DK2171, DK600, DK1361, DK2511, DK2708 163 New Recommendation DK2401, DK17-1, DK600, DK601 164 During DK2401, DK1412, DK560, DK600, DK601 165 Yellow rice DK2401, DK1412, DK560, DK600, DK601 166 Jinmi DK50, DK17-1, DK560, DK600, DK601 167 Recommendation DK2401, DK17-1, DK560, DK600, DK601 168 Anjung Rice DK2394, DK34, DK560, DK600, DK601 169 Huaan DK2394, DK34, DK560, DK600, DK601 170 South pyeong DK2401, DK17-1, DK34, DK560, DK600, DK601 171 Mangyeong DK2394, DK2401, DK17-1, DK34, DK560, DK600, DK601 172 Full wind DK34, DK63, DK560, DK600, DK601 173 Mars DK34, DK63, DK560, DK600, DK601 174 Nakdong Rice DK50, DK34, DK63, DK560, DK600, DK601 175 Medium hair 1006 DK50, DK34, DK63, DK560, DK600, DK601 176 Flower garden DK50, DK34, DK63, DK560, DK600, DK601 177 ecliptic DK17-1, DK34, DK63, DK560, DK600, DK601 178 Late autumn DK560, DK2171, DK600, DK601, 179 Youngan Rice DK50, DK2401, DK1412, DK560, DK2171, DK600, DK601 180 easting DK50, DK2401, DK34, DK560, DK2171, DK600, DK601 181 YJ06-11 DK50, DK2401, DK1412, DK34, DK560, DK2171, DK600, DK601 182 Taebong DK2394, DK2401, DK17-1, DK600, DK601, DK2708 183 Stone DK2394, DK50, DK2401, DK34, DK600, DK601, DK2708 184 Medical examination DK2401, DK560, DK600, DK601, DK2708 185 Hanyang Brewery DK2401, DK560, DK600, DK601, DK2708 186 Denseness DK2394, DK2401, DK34, DK560, DK600, DK601, DK2708 187 Eun-gu No. 5 DK63, DK560, DK600, DK601, DK2708 188 Shinmyeong Black Flag DK2401, DK63, DK560, DK600, DK601, DK2708 189 Beef Cattle No.132 DK2394, DK1412, DK17-1, DK63, DK560, DK600, DK601, DK2708 190 Under fire DK50, DK2401, DK34, DK63, DK560, DK600, DK601, DK2708 191 South Japan DK50, DK34, DK2171, DK600, DK601, DK2708 192 letterweight DK2394, DK2401, DK560, DK600, DK1361, DK601 193 Erie 265 DK2394, DK2401, DK560, DK600, DK1361, DK601 194 Full rice DK2401, DK1412, DK560, DK600, DK1361, DK601 195 Sleeping DK17-1, DK63, DK560, DK600, DK1361, DK601 196 Reclamation 9 DK2401, DK1412, DK17-1, DK63, DK560, DK600, DK1361, DK601, 197 Scenery DK1412, DK34, DK63, DK560, DK600, DK1361, DK601, DK2708 198 Magnetic light DK17-1, DK560, DK2171, DK600, DK1361, DK601, DK2708 199 Black DK17-1, DK560, DK2171, DK600, DK1361, DK601, DK2708 200 Jinbyeol Rice DK17-1, DK560, DK2171, DK600, DK1361, DK601, DK2708

According to Table 10 and the results of FIGS. 151 to 200, the DNA extracted from the sample rice was specifically amplified in each rice variety marker set to confirm that the sample rice was correctly determined.

Example  201 to 250: for identifying rice varieties Plural Marker  Breed Identification Using Sets

According to the method of Example 1, real-time PCR was performed using the samples described in Table 11 below, and the results are shown in Table 11 and FIGS. 201 to 250.

Example sample Amplification marker 201 F1-312 8223 DK2394, DK1412, DK17-1, DK34, DK560, DK2171, DK600, DK1361, DK601, DK2708 202 YJ06-13 DK2401, DK17-1, DK34, DK63, DK560, DK2171, DK600, DK601, DK2511 203 Snow DK2401, DK17-1, DK34, DK63, DK560, DK600, DK601, DK2511, DK2708 204 Black pearl DK2401, DK17-1, DK63, DK560, DK2171, DK600, DK601, DK2511, DK2708 205 Gyehwa Rice DK2401, DK17-1, DK63, DK560, DK1123 206 Namcheon Rice DK560, DK2171, DK1123, DK2708 207 Beautiful DK560, DK2171, DK1123, DK2708 208 YJ06-05 DK17-1, DK63, DK560, DK2171, DK1123, DK2708 209 YJ06-08 DK17-1, DK63, DK560, DK2171, DK1123, DK2708 210 Agriculture and Forests No. 1 DK17-1, DK63, DK560, DK2171, DK1123, DK2708 211 Sangnam Paddy Rice DK17-1, DK63, DK560, DK2171, DK1123, DK2708 212 Onnuri DK50, DK2401, DK17-1, DK560, DK1123, DK1361, DK2708 213 YJ06-12 DK17-1, DK63, DK560, DK2171, DK1123, DK1361 214 Ling Ta 150 DK2171, DK1123, DK2511 215 Shin-pa DK2171, DK1123, DK2511 216 YJ06-09 DK2171, DK1123, DK2511 217 Miryang 30 DK2171, DK1123, DK2511 218 Samgang Rice DK2171, DK1123, DK2511 219 Yongmun Rice DK2171, DK1123, DK2511 220 Wonpung Rice DK2171, DK1123, DK2511 221 Han River Bridge No. 1 DK2171, DK1123, DK2511 222 IR10179-23-1-3 DK560, DK2171, DK1123, DK2511 223 Flavor Rice No. 1 DK1123, DK2511, DK2708 224 IR667-98-1-3-25 DK2171, DK1123, DK2511, DK2708 225 Dasan 1 DK2171, DK1123, DK2511, DK2708 226 Fertility rice DK2171, DK1123, DK2511, DK2708 227 Youngnam Early Students DK2171, DK1123, DK2511, DK2708 228 Reunification DK2171, DK1123, DK2511, DK2708 229 Golden rice DK2171, DK1123, DK2511, DK2708 230 Namyoung rice DK63, DK2171, DK1123, DK2511, DK2708 231 Drachan DK560, DK2171, DK1123, DK2511, DK2708 232 White rice DK560, DK2171, DK1123, DK2511, DK2708 233 Jangseong Rice DK560, DK2171, DK1123, DK2511, DK2708 234 Jungwon Rice DK560, DK2171, DK1123, DK2511, DK2708 235 Unification DK560, DK2171, DK1123, DK2511, DK2708 236 Pungsan Rice DK560, DK2171, DK1123, DK2511, DK2708 237 Hanam-ri rice DK560, DK2171, DK1123, DK2511, DK2708 238 Daejin DK2401, DK1412, DK17-1, DK2171, DK1123, DK1361, DK2511, DK2708 239 Brewed rice DK2401, DK1412, DK63, DK560, DK1123, DK601 240 Flower DK2401, DK1412, DK17-1, DK34, DK560, DK1123, DK1361, DK601, 241 Unflavored rice DK50, DK17-1, DK63, DK560, DK1123, DK1361, DK601, DK2708 242 KASHIMIR BASMATI DK34, DK560, DK2171, DK1123, DK601, DK2511, 243 Shantou Road DK2394, DK2401, DK17-1, DK63, DK560, DK1123, DK600, 244 Taesung DK17-1, DK63, DK1123, DK600, DK2708 245 Jewelry Inspector DK560, DK1123, DK600, DK2708 246 Joan DK2394, DK2401, DK34, DK560, DK1123, DK600, DK2708 247 Odae 1 DK63, DK560, DK1123, DK600, DK2708 248 Nokyang DK2171, DK1123, DK600, DK2708 249 CR126-42-5 DK50, DK34, DK63, DK560, DK2171, DK1123, DK600, DK2708 250 KAKAI 272/51 DK50, DK34, DK63, DK560, DK2171, DK1123, DK600, DK2708

According to Table 11 and the results of FIGS. 201 to 250, the DNA extracted from the sample rice was specifically amplified in each rice variety marker set to confirm that the sample rice was correctly determined.

Example  251 to 300: for identifying rice varieties Plural Marker  Rice Variety Identification Using Sets

According to the method of Example 1, real-time PCR was performed using the samples described in Table 12 below, and the results are shown in Table 12 and FIGS. 251 to 300.

Example sample Amplification marker 251 Sweet rice DK2394, DK2401, DK17-1, DK560, DK1123, DK600, DK1361 252 Parsley DK17-1, DK63, DK560, DK1123, DK600, DK1361 253 Crass-pd8244 DK2394, DK2401, DK17-1, DK63, DK560, DK1123, DK600, DK1361 254 Sobaek DK560, DK1123, DK600, DK1361, DK2708 255 YJ06-19 DK2394, DK2401, DK1412, DK17-1, DK560, DK2171, DK1123, DK600, DK1361, DK2708 256 Coldness13 DK2401, DK17-1, DK34, DK63, DK560, DK2171, DK1123, DK600, DK1361, DK2708 257 Water tank DK2401, DK17-1, DK34, DK63, DK560, DK2171, DK1123, DK600, DK1361, DK2708 258 Rice paddy 3 DK2401, DK1412, DK17-1, DK34, DK63, DK560, DK2171, DK1123, DK600, DK1361, DK2708 259 BKNFR 76106-16-0-1-0 DK2171, DK1123, DK600, DK2511 260 Pai hsu DK2171, DK1123, DK600, DK2511 261 Yongju Rice DK2171, DK1123, DK600, DK2511 262 Han River Rice DK2171, DK1123, DK600, DK2511 263 BABUNIYA DK34, DK2171, DK1123, DK600, DK2511 264 Flavor Rice 2 DK1123, DK600, DK2511, DK2708 265 IR9669-PP830-1 DK2171, DK1123, DK600, DK2511, DK2708 266 Lu chin tsi DK2171, DK1123, DK600, DK2511, DK2708 267 Miryang 42 DK2171, DK1123, DK600, DK2511, DK2708 268 Rice harvest DK2171, DK1123, DK600, DK2511, DK2708 269 Taebaek Rice DK2171, DK1123, DK600, DK2511, DK2708 270 Rice field DK560, DK2171, DK1123, DK600, DK2511, DK2708 271 Full rice DK560, DK2171, DK1123, DK600, DK2511, DK2708 272 Miryang 23 DK560, DK2171, DK1123, DK600, DK2511, DK2708 273 샛 Star Rice DK560, DK2171, DK1123, DK600, DK2511, DK2708 274 Crystal rice DK560, DK2171, DK1123, DK600, DK2511, DK2708 275 Singwang Rice DK560, DK2171, DK1123, DK600, DK2511, DK2708 276 Cheongchungbap DK560, DK2171, DK1123, DK600, DK2511, DK2708 277 Miryang 21 DK2394, DK560, DK2171, DK1123, DK600, DK2511, DK2708 278 Miryang 22 DK2394, DK560, DK2171, DK1123, DK600, DK2511, DK2708 279 Nong'an DK34, DK560, DK2171, DK1123, DK600, DK2511, DK2708 280 2869 (16667) DK63, DK560, DK2171, DK1123, DK600, DK2511, DK2708 281 plain DK2401, DK34, DK63, DK560, DK1123, DK600, DK601 282 Permanent residence DK2401, DK17-1, DK63, DK560, DK1123, DK600, DK601, DK2708 283 Hyangnam Rice DK50, DK17-1, DK560, DK2171, DK1123, DK600, DK601, DK2708 284 Opposition Rice No. 1 DK34, DK63, DK560, DK1123, DK600, DK1361, DK601 285 HP904B-1 8212 DK2401, DK17-1, DK560, DK1123, DK600, DK1361, DK601, DK2708 286 Car DK2401, DK1412, DK17-1, DK34, DK63, DK560, DK2171, DK1123, DK600, DK1361, DK601, DK2708 287 Use 3 DK2394, DK2401, DK17-1, DK34 288 West Coast DK560, 289 Shan gu DK17-1, DK63, DK560, DK2171 290 Brown snow sweetness DK17-1, DK34, DK63, DK560, DK2171 291 Salinity No. 11 DK17-1, DK63, DK560, DK2171, DK2708 292 Heavy shaft 5 DK17-1, DK34, DK560, DK2171, DK1361, DK2708 293 Fascinating DK2394, DK1412, DK63, DK560, DK600 294 SAN BANG QISHI LU + DK2401, DK17-1, DK63, DK560, DK2171, DK600 295 Shasani DK2394, DK2401, DK34, DK600, DK2708 296 Shaft 7 DK2394, DK2401, DK17-1, DK34, DK600, DK2708 297 Bute DK2394, DK2401, DK34, DK560, DK600, DK2708 298 Luster241 DK2394, DK2401, DK17-1, DK34, DK560, DK600, DK2708 299 Application 4 DK2394, DK2401, DK17-1, DK34, DK560, DK600, DK2708 300 Red blood , K17-1, DK63, DK560, DK600, DK2708

According to Table 12 and the results of FIGS. 251 to 300, the DNA extracted from the sample rice was specifically amplified in each rice variety marker set to confirm that the sample rice was correctly determined.

Example  301 to 350: for identifying rice varieties Plural Marker  Breed Identification Using Sets

According to the method of Example 1, real-time PCR was performed using the samples described in Table 13 below, and the results are shown in Table 13 and FIGS. 301 to 350.

Example sample Amplification marker 301 Yunnan Collection 12 DK2394, DK17-1, DK63, DK560, DK600, DK2708 302 Total 42 DK2401, DK17-1, DK63, DK560, DK600, DK2708 303 Northland18 DK2394, DK2401, DK17-1, DK34, DK63, DK560, DK600, DK2708 304 Homesickness DK17-1, DK2171, DK600, DK2708 305 Glamor 283 DK2401, DK34, DK2171, DK600, DK2708 306 Mid-Autumn Soup DK2394, DK560, DK2171, DK600, DK2708 307 Yongnong 1 DK17-1, DK560, DK2171, DK600, DK2708 308 Use 2 DK2394, DK17-1, DK560, DK2171, DK600, DK2708 309 Special way DK2401, DK34, DK560, DK2171, DK600, DK2708 310 Gando 10 DK2394, DK2401, DK17-1, DK34, DK560, DK2171, DK600, DK2708 311 Yin-yeon 23 DK2394, DK2401, DK63, DK560, DK2171, DK600, DK2708 312 Yonggang 13 DK2394, DK17-1, DK63, DK560, DK2171, DK600, DK2708 313 808 DK2394, DK17-1, DK63, DK560, DK600, DK1361, 314 Yongnong 4 DK17-1, DK560, DK2171, DK600, DK1361, 315 Tong163 DK2401, DK17-1, DK63, DK560, DK600, DK1361, DK2708 316 Chinese 9-23 DK17-1, DK560, DK2171, DK600, DK1361, DK2708 317 one hundred points DK2394, DK17-1, DK560, DK2171, DK600, DK1361, DK2708 318 Paradise DK63, DK560, DK2171, DK600, DK1361, DK2708 319 Changbai 7 DK2401, DK17-1, DK63, DK560, DK2171, DK600, DK1361, DK2708 320 Gando 9 DK1412, DK17-1, DK63, DK560, DK2171, DK600, DK1361, DK2708 321 Dohwahyang 2 DK2394, DK17-1, DK34, DK63, DK560, DK600, DK2511, 322 Gilnin 2 DK17-1, DK34, DK560, DK2171, DK600, DK2511, 323 Ungang 20 DK2394, DK2401, DK17-1, DK63, DK560, DK600, DK2511, DK2708 324 Total 41 DK2394, DK50, DK2401, DK17-1, DK63, DK560, DK600, DK2511, DK2708 325 Heavy shaft 6 DK2394, DK34, DK2171, DK600, DK1361, DK2511, DK2708 326 Meat201 DK2394, DK34, DK2171, DK600, DK1361, DK2511, DK2708 327 Meat211 DK2394, DK34, DK2171, DK600, DK1361, DK2511, DK2708 328 Songgang 7 DK2394, DK17-1, DK34, DK560, DK2171, DK600, DK1361, DK2511, DK2708 329 Total 35 DK2401, DK17-1, DK560, DK600, DK601, 330 Gilgang 61 DK2394, DK17-1, DK34, DK560, DK2171, DK600, DK601, 331 Yonggang 12 DK2394, DK560, DK2171, DK600, DK601, DK2708 332 Salinity No. 11 DK2394, DK2401, DK560, DK2171, DK600, DK601, DK2708 333 Red sweetness DK17-1, DK63, DK560, DK2171, DK600, DK601, DK2708 334 Black corner DK17-1, DK63, DK560, DK2171, DK600, DK601, DK2708 335 Education 131 DK2394, DK17-1, DK63, DK560, DK2171, DK600, DK601, DK2708 336 Use 5 DK2394, DK17-1, DK63, DK560, DK2171, DK600, DK601, DK2708 337 416 DK2394, DK17-1, DK34, DK63, DK560, DK2171, DK600, DK601, DK2708 338 19 years old DK17-1, DK560, DK2171, DK600, DK1361, DK601, 339 Songgang 2 DK17-1, DK34, DK560, DK2171, DK600, DK1361, DK601, 340 Blackdo DK34, DK63, DK560, DK2171, DK600, DK1361, DK601, 341 North DK2401, DK1412, DK17-1, DK34, DK63, DK560, DK2171, DK600, DK1361, DK601, 342 Dong Yeon 2 DK2394, DK2401, DK17-1, DK63, DK560, DK600, DK1361, DK601, DK2708 343 Breeding 308 DK17-1, DK560, DK2171, DK600, DK1361, DK601, DK2708 344 Flavor DK17-1, DK560, DK2171, DK600, DK1361, DK601, DK2708 345 Yongsun 104 DK2394, DK17-1, DK560, DK2171, DK600, DK1361, DK601, DK2708 346 Gangbang151 DK2401, DK17-1, DK560, DK2171, DK600, DK1361, DK601, DK2708 347 Yosung 2102 DK2401, DK17-1, DK560, DK2171, DK600, DK1361, DK601, DK2708 348 Song Kyung 3 DK2394, DK2401, DK17-1, DK63, DK560, DK2171, DK600, DK1361, DK601, DK2708 349 Tong192 DK17-1, DK560, DK2171, DK600, DK601, DK2511, DK2708 350 Tongyeon No.5 DK2394,-, DK34, DK560, DK2171, DK600, DK601, DK2511, DK2708

According to Table 13 and the results of FIGS. 301 to 350, the DNA extracted from the sample rice was specifically amplified in each rice variety marker set to confirm that the sample rice was correctly determined.

Example  351 to 400: for identifying rice varieties Plural Marker  Rice Variety Identification Using Sets

According to the method of Example 1, real-time PCR was performed using the samples described in Table 12 below, and the results are shown in Table 14 and FIGS. 351 to 400.

Example sample Amplification marker 351 Half price 8 DK2401, DK17-1, DK34, DK560, DK2171, DK600, DK1361, DK601, DK2511, DK2708 352 Zodiacal DK50, DK17-1, DK63, DK560, DK2171, DK1123 353 Altitude 1 DK560, DK1123, DK2708 354 Dong Nong 415 DK2394, DK17-1, DK63, DK560, DK2171, DK1123, DK2708 355 97-39-1 DK2171, DK1123, DK2511 356 98-49-1 DK2171, DK1123, DK2511 357 BIR-H + DK2171, DK1123, DK2511 358 Hung-Fam-Goo DK2171, DK1123, DK2511 359 LIEN-CHAN-ZE-TH + U DK2171, DK1123, DK2511 360 NANTA 16 DK2171, DK1123, DK2511 361 NAN-TA-H + W DK2171, DK1123, DK2511 362 Pl161010 DK2171, DK1123, DK2511 363 SH + A-M + A-FAN DK2171, DK1123, DK2511 364 Altitude 2 DK560, DK1123, DK601 365 Gilgang 88 DK560, DK2171, DK1123, DK601, DK2708 366 Tunnel (Tianjin) DK17-1, DK560, DK1123, DK600, DK2708 367 Sensitivity 8 DK2394, DK34, DK560, DK2171, DK1123, DK600, DK2708 368 Gil Gang 9 DK2401, DK34, DK560, DK2171, DK1123, DK600, DK2708 369 Tongue 6 DK17-1, DK63, DK560, DK2171, DK1123, DK600, DK2708 370 Tunnel DK1412, DK17-1, DK63, DK560, DK2171, DK1123, DK600, DK2708 371 Gilhyang 1 DK2394, DK17-1, DK63, DK560, DK1123, DK600, DK1361 372 Dohwahyang 1 DK2394, DK17-1, DK63, DK560, DK1123, DK600, DK1361 373 Flavor rice DK2394, DK2401, DK1412, DK17-1, DK560, DK2171, DK1123, DK600, DK1361, DK2708 374 1 year ago DK17-1, DK63, DK560, DK2171, DK1123, DK600, DK1361, DK2708 375 Daen-chon-bir DK2171, DK1123, DK600, DK2511 376 Low-shun-dau DK63, DK2171, DK1123, DK600, DK2511 377 Shan-tsan DK63, DK2171, DK1123, DK600, DK2511 378 guide DK2171, DK1123, DK600, DK2511, DK2708 379 Underground (resident) DK17-1, DK560, DK2171, DK1123, DK600, DK601, DK2708 380 Mudan River 19 DK2401, DK17-1, DK560, DK1123, DK600, DK1361, DK601, DK2708 381 K + GY + KA DK2394, DK2401, DK17-1, DK63, DK560, DK2171 382 Six Islands DK1412, DK17-1, DK34, DK63, DK560, DK601, DK2708 383 Daneun Ark DK17-1, DK560, DK600, 384 Dok Douk DK50, DK2401, DK17-1, DK560, DK600, 385 divine power DK2394, DK2401, DK17-1, DK63, DK560, DK600, 386 Ungung DK2394, DK2401, DK17-1, DK63, DK560, DK600 387 Hitome bare DK2394,, DK600, DK2708 388 Gyeongbuk Valley DK1412, DK63, DK560, DK600, DK2708 389 Chungseung-lae DK2394, DK2401, DK17-1, DK63, DK560, DK600, DK2708 390 Milky queen DK2394, DK560, DK2171, DK600, DK2708 391 Kogane Mochi DK17-1, DK63, DK560, DK2171, DK600, DK2708 392 D + IKU 154 DK2401, DK17-1, DK63, DK560, DK2171, DK600, DK2708 393 Strength DK2394, DK2401, DK17-1, DK34, DK560, DK600, DK1361 394 Curvature diagram DK2394, DK17-1, DK560, DK2171, DK600, DK1361 395 North Pirate DK17-1, DK63, DK560, DK2171, DK600, DK1361 396 Rebirth DK2394, DK1412, DK17-1, DK63, DK560, DK2171, DK600, DK1361, 397 Sasani DK2401, DK17-1, DK560, DK2171, DK600, DK1361, DK2708 398 Sunday DK17-1, DK63, DK560, DK2171, DK600, DK1361, DK2708 399 Jana DK17-1, DK63, DK560, DK2171, DK600, DK1361, DK2708 400 Hoshiyudaka DK2401, DK17-1, DK560, DK600, DK2511,

According to Table 14 and the results of FIGS. 351 to 400, the DNA extracted from the sample rice was specifically amplified in each rice variety marker set to confirm that the sample rice was correctly determined.

Example  401 to 450: for identifying rice varieties Plural Marker  Breed Identification Using Sets

According to the method of Example 1, real time PCR was performed using the samples described in Table 15 below, and the results are shown in Table 15 and FIGS. 401 to 450.

Example sample Amplification marker 401 SANTAM + MI DK2401, DK34, DK560, DK600, DK601 402 Kanto PL3 DK1412, DK17-1, DK34, DK600, DK601, DK2708 403 Nippon bare DK2401, DK1412, DK17-1, DK34, DK560, DK600, DK601, DK2708 404 Mauretsu DK2401, DK1412, DK17-1, DK560, DK2171, DK600, DK601, DK2708 405 Five million seats DK2394, DK17-1, DK34, DK560, DK2171, DK600, DK601, DK2708 406 Sleeping DK17-1, DK63, DK560, DK2171, DK600, DK601, DK2708 407 A good harvest DK17-1, DK63, DK560, DK2171, DK600, DK601, DK2708 408 RIKUT + SAKUMAI DK17-1, DK63, DK560, DK1123, DK2708 409 GINSUKEYAKAN DK17-1, DK63, DK560, DK2171, DK1123, DK2708 410 HATA K + GANE M + CHI DK17-1, DK63, DK560, DK2171, DK1123, DK2708 411 + NJ + HAKAM + D + SHI DK17-1, DK63, DK560, DK2171, DK1123, DK2708 412 TACHIMINAMI DK17-1, DK63, DK560, DK2171, DK1123, DK2708 413 Kwang Kwang Na DK17-1, DK63, DK560, DK2171, DK1123, DK2708 414 Daedo 3 DK17-1, DK63, DK560, DK2171, DK1123, DK2708 415 Yukdo Agriculture and Forestry # 16 DK17-1, DK63, DK560, DK2171, DK1123, DK2708 416 Yukdo Agriculture and Forestry # 19 DK17-1, DK63, DK560, DK2171, DK1123, DK2708 417 Yukdo Agriculture and Forest 21 DK17-1, DK63, DK560, DK2171, DK1123, DK2708 418 Ikdo Agriculture and Forests No. 13 DK17-1, DK63, DK560, DK2171, DK1123, DK2708 419 Yukdo Agriculture and Forests No. 1 DK17-1, DK63, DK560, DK2171, DK1123, DK2708 420 Yukdo Agriculture and Forests No. 4 DK17-1, DK63, DK560, DK2171, DK1123, DK2708 421 Chungseung Land DK17-1, DK63, DK560, DK2171, DK1123, DK2708 422 HATA K + GANEM ++ DK2394, DK17-1, DK63, DK560, DK2171, DK1123, DK2708 423 + TSUKAM + D + SHI DK2401, DK17-1, DK63, DK560, DK2171, DK1123, DK2708 424 Satominori DK2401, DK17-1, DK63, DK560, DK2171, DK1123, DK2708 425 Goseong DK2401, DK17-1, DK63, DK560, DK2171, DK1123, DK2708 426 Hong So Land 5 DK2401, DK17-1, DK63, DK560, DK2171, DK1123, DK2708 427 Daedo No. 4 DK1412, DK17-1, DK63, DK560, DK2171, DK1123, DK2708 428 B + ZUY AKAN DK2401, DK1412, DK17-1, DK63, DK560, DK2171, DK1123, DK2708 429 Improvement 13 DK2401, DK1412, DK17-1, DK63, DK560, DK2171, DK1123, DK2708 430 Sixth Road DK2401, DK1412, DK17-1, DK63, DK560, DK2171, DK1123, DK2708 431 Ikdo Strength 1 DK2401, DK1412, DK17-1, DK63, DK560, DK2171, DK1123, DK2708 432 HIN + DE DK2401, DK17-1, DK63, DK560, DK1123, DK1361, DK2708 433 Housewife DK2401, DK17-1, DK63, DK560, DK1123, DK1361, DK2708 434 MIYAMAM + CHI DK17-1, DK63, DK560, DK2171, DK1123, DK1361, DK2708 435 Daedo 1 DK17-1, DK63, DK560, DK2171, DK1123, DK1361, DK2708 436 Four countries DK17-1, DK63, DK560, DK2171, DK1123, DK1361, DK2708 437 Yukdo Agriculture and Forestry # 9 DK17-1, DK63, DK560, DK2171, DK1123, DK1361, DK2708 438 Musk DK2401, DK1412, DK17-1, DK63, DK560, DK2171, DK1123, DK1361, DK2708 439 Improvement 41 DK17-1, DK63, DK560, DK2171, DK1123, DK600, DK2708 440 Yukdo Agriculture and Forest No.11 DK17-1, DK63, DK560, DK2171, DK1123, DK600, DK2708 441 Mona DK17-1, DK63, DK560, DK2171, DK1123, DK600, DK1361, DK2708 442 Yukdo Agriculture and Forests No. 3 DK2171, DK1123, DK600, DK2511 443 NAR8F5-1-3-2 DK560, DK2171, DK600, DK2511 444 NAR163 F3-1-4 DK2171, DK1123, DK2511, DK2708 445 T + -3133-59-1-2-4-1 DK560, DK2171, DK1123, DK2511 446 T + -3027-42-1-E-2-1-1-1 DK2171, DK1123, DK2511, DK2708 447 Tadukan DK50, DK34, DK2171, DK1123, DK2511 448 P + KHARELIMASIN + DK63, DK2171, DK1123, DK2511 449 Krasnodarskill DK17-1, DK63, DK560, DK2171, DK600, DK1361 450 Novoselskill DK2394, DK17-1, DK63, DK560, DK2171, DK600, DK1361

According to Table 15 and the results of FIGS. 401 to 450, the DNA extracted from the sample rice was specifically amplified in each rice variety marker set to confirm that the sample rice was correctly determined.

Example  451 to 500: for identifying rice varieties Plural Marker  Rice Variety Identification Using Sets

According to the method of Example 1, real-time PCR was performed using the samples described in Table 16 below, and the results are shown in Table 16 and FIGS. 451 to 500.

Example sample Amplification marker 451 H + 459 DK50, DK17-1, DK34, DK63, DK560, DK2171, DK1123, DK600, DK2708 452 ANG IN AF + TSY DK17-1, DK560, DK2171, DK2511, DK2708 453 WIR 7085 DK2394, DK2401, DK17-1, DK63, DK560, DK1123, DK600, DK1361 454 #REF! DK560, DK2171, DK1123, DK2511 455 IRRI16302 DK50, DK17-1, DK63, DK560, DK2171, DK600, DK1361 456 Jasmine 85 DK34, DK560, DK2171, DK600, DK601, DK2511, DK2708 457 California blue rose DK2394, DK17-1, DK63, DK560, DK1123 458 19b1014b-9n-1 DK2171, DK1123, DK2511, DK2708 459 NAHNGM + N S-4 SELECTI + N DK2171, DK1123, DK600, DK2511 460 Cotton 4 DK560, DK2171, DK1123, DK600, DK2511, DK2708 461 Cotton 1 DK2401, DK17-1, DK63, DK560, DK2171, DK1123, DK600, DK2511, DK2708 462 Cotton 2 DK2401, DK17-1, DK63, DK560, DK2171, DK1123, DK600, DK2511, DK2708 463 Cotton 3 DK2401, DK17-1, DK63, DK560, DK2171, DK1123, DK600, DK2511, DK2708 464 Bashtus DK34, DK560, DK2171, DK1123 465 Boteswar-2 DK34, DK2171, DK1123, DK2511 466 Mati Char DK34, DK2171, DK1123, DK2511 467 Dharga sail DK34, DK560, DK2171, DK1123, DK2511 468 BR1543-9-2-1 DK34, DK560, DK2171, DK1123, DK600, DK2511, DK2708 469 JMANG PAI HAUTA + DK17-1, DK63, DK560, DK2171, DK600, DK2511 470 Bui Tap49 DK2394, DK2401, DK1412, DK560, DK2171, DK1123, DK2511 471 Chumro DK17-1, DK63, DK560, DK2171 472 Bajo kaapl DK2171, DK1123, DK2511 473 4 beads DK17-1, DK34, DK560, DK600 474 West Sea 13 DK2401, DK17-1, DK560, DK2171, DK600, DK1361, DK601, DK2708 475 Pyeongdo 1 DK2401, DK17-1, DK560, DK2171, DK1123, DK600, DK2511, DK2708 476 Hamzhou DK2401, DK17-1, DK34, DK1123, DK600, DK601, DK2511, DK2708 477 CAIAP + DK2401, DK17-1, DK63, DK560, DK2171, DK1123, DK2511, DK2708 478 MARAVILHA DK2401, DK17-1, DK63, DK560, DK2171, DK1123, DK2511, DK2708 479 SUDAHAL + W DK50, DK2171, DK1123 480 SEENATTY DK50, DK34, DK560, DK2171, DK1123, DK600, DK2511 481 ARIETE DK2401, DK17-1, DK63, DK560, DK2171, DK600 482 kessa red DK50, DK34, DK560, DK2171, DK1123, DK600, DK2708 483 SPINMERE DK34, DK560, DK2171, DK1123 484 LUN ++ PUN DK2394, DK2401, DK17-1, DK63, DK560, DK1123, DK600, DK1361 485 Debzere DK17-1, DK63, DK560, DK2171, DK1123, DK600, DK1361 486 D + MS + F ++ D DK34, DK560, DK2171, DK1123 487 HASSNAY DK50, DK34, DK63, DK560, DK2171, DK1123, DK600, DK2708 488 GIZA DK2171, DK1123, DK2511 489 GIZA 170 DK17-1, DK2171, DK1123, DK600, DK2511, DK2708 490 ZENA DK17-1, DK63, DK560, DK2171, DK600 491 ARIRTE DK2401, DK17-1, DK63, DK560, DK2171, DK600 492 ARG + DK17-1, DK63, DK560, DK2171, DK600, DK1361, DK2708 493 KAGI DK34, DK2171, DK1123 494 BASMATI DK34, DK2171, DK1123, DK600, DK2511 495 Mendawak DK34, DK560, DK2171, DK1123, DK2511 496 Mayang DK2171, DK1123, DK2511, DK2708 497 QUILLA DK2394, DK50, DK17-1, DK560, DK2171, DK600, DK1361 498 Diamaute DK50, DK17-1, DK63, DK560, DK2171, DK600, DK1361 499 Zarya DK2394, DK50, DK17-1, DK63, DK560, DK1123, DK600, DK1361 500 Blue bell DK17-1, DK63, DK560, DK2171, DK600, DK2708

According to the results of Table 16 and the results of FIGS. 451 to 500, the DNA extracted from the sample rice was specifically amplified in each rice variety marker set to confirm that the sample rice was correctly determined.

Example  501 to 514: for identifying rice varieties Plural Marker  Breed Identification Using Sets

According to the method of Example 1, real-time PCR was performed using the samples described in Table 17 below, and the results are shown in Table 17 and FIGS. 501 to 514.

Example sample Amplification marker 501 Lemont DK17-1, DK63, DK560, DK2171, DK600, DK2511, DK2708 502 #REF! DK34, DK560, DK2171, DK1123, DK2511 503 BASAMATI DK34, DK560, DK2171, DK1123, DK2511 504 Muzzaffar-8 DK34, DK560, DK2171, DK1123, DK601, DK2511 505 CAR + LINA DK17-1, DK560, DK2171, DK1123, DK2511 506 GIANT GRAIN DK17-1, DK63, DK560, DK2171 507 KINAGAYKAY DK17-1, DK63, DK560, DK2171, DK2708 508 IR1552 DK2171, DK1123, DK2511, DK2708 509 IR1750-F5B-22 DK560, DK2171, DK1123, DK2511, DK2708 510 IR1487-194--3-4 DK63, DK560, DK2171, DK1123, DK2511, DK2708 511 IRRI 10320 DK2171, DK1123, DK600, DK2511, DK2708 512 IR12780 DK2401, DK63, DK560, DK2171, DK1123, DK600, DK2511, DK2708 513 Cgi-csing DK2394, DK1412, DK17-1, DK63, DK560, DK600, DK2708 514 DAMA DK2394, DK1412, DK17-1, DK63, DK560, DK2171, DK600, DK1361

According to Table 17 and the results of FIGS. 501 to 514, DNA extracted from the sample rice was specifically amplified in each rice variety identification marker set to confirm that the sample rice was correctly determined.

Attach an electronic file to a sequence list

Claims (7)

The 5 'end of the probe is labeled with one fluorescent material selected from the group consisting of FAM, VIC, TET, JOE, HEX, TAMRA, CY3, CY5, ROX, RED610, TEXAS RED, RED670 and NED and the 3' end is MGBNFQ performing real-time PCR with a template extracted from rice using a marker combination composition consisting of (c), (g) and (i), labeled with a molecular grove binding non-fluorescencequencher;
The 5 'end of the probe is FAM, VIC, TET, JOE, HEX, TAMRA, CY3, CY5, ROX,
(A), (b) and (f) labeled with one fluorescent material selected from the group consisting of RED610, TEXAS RED, RED670 and NED and labeled with a molecular grove binding non-fluorescence quencher (MGBNFQ). Performing a real-time PCR with a template extracted from rice using a marker combination composition consisting of;
The 5 'end of the probe is labeled with one fluorescent material selected from the group consisting of FAM, VIC, TET, JOE, HEX, TAMRA, CY3, CY5, ROX, RED610, TEXAS RED, RED670 and NED and the 3' end is MGBNFQ performing real-time PCR with a template extracted from rice using a marker combination composition consisting of (d), (e) and (h), labeled with a molecular grove binding non-fluorescence quencher;
The 5 'end of the probe is labeled with one fluorescent material selected from the group consisting of FAM, VIC, TET, JOE, HEX, TAMRA, CY3, CY5, ROX, RED610, TEXAS RED, RED670 and NED and the 3' end is MGBNFQ performing real-time PCR with a template extracted from rice using a marker combination composition consisting of (j), (k) and (l), labeled with a molecular grove binding non-fluorescence quencher;
The 5 'end of the probe is labeled with one fluorescent material selected from the group consisting of FAM, VIC, TET, JOE, HEX, TAMRA, CY3, CY5, ROX, RED610, TEXAS RED, RED670 and NED and the 3' end is MGBNFQ performing real-time PCR with a template extracted from rice using a marker combination composition consisting of (m), (n) and (o), labeled with a molecular grove binding non-fluorescence quencher;
By detecting the peak of the fluorescent material implemented by the real-time PCR results, to identify each rice variety
Checking whether the PCR is amplified by a set of markers; And
Identifying a variety of rice according to whether the PCR amplification
Rice variety identification method using a set of identification markers for a plurality of rice varieties, including:
(a) a primer consisting of SEQ ID NO: 1, a primer consisting of SEQ ID NO: 2, and a probe consisting of SEQ ID NO: 3;
(b) a primer consisting of SEQ ID NO: 4, a primer consisting of SEQ ID NO: 5, and a probe consisting of SEQ ID NO: 6;
(c) a primer consisting of SEQ ID NO: 7, a primer consisting of SEQ ID NO: 8, and a probe consisting of SEQ ID NO: 9;
(d) a primer consisting of SEQ ID NO: 10, a primer consisting of SEQ ID NO: 11, and a probe consisting of SEQ ID NO: 12;
(e) a primer consisting of SEQ ID NO: 13, a primer consisting of SEQ ID NO: 14, and a probe consisting of SEQ ID NO: 15;
(f) a primer consisting of SEQ ID NO: 16, a primer consisting of SEQ ID NO: 17, and a probe consisting of SEQ ID NO: 18;
(g) a primer consisting of SEQ ID NO: 19, a primer consisting of SEQ ID NO: 20, and a probe consisting of SEQ ID NO: 21;
(h) a primer consisting of SEQ ID NO: 22, a primer consisting of SEQ ID NO: 23, and a probe consisting of SEQ ID NO: 24;
(i) a primer consisting of SEQ ID NO: 25, a primer consisting of SEQ ID NO: 26, and a probe consisting of SEQ ID NO: 27;
(j) a primer consisting of SEQ ID NO: 28, a primer consisting of SEQ ID NO: 29, and a probe consisting of SEQ ID NO: 30;
(k) a primer consisting of SEQ ID NO: 31, a primer consisting of SEQ ID NO: 32, and a probe consisting of SEQ ID NO: 33;
(l) a primer consisting of SEQ ID NO: 34, a primer consisting of SEQ ID NO: 35, and a probe consisting of SEQ ID NO: 36;
(m) a primer consisting of SEQ ID NO: 37, a primer consisting of SEQ ID NO: 38, and a probe consisting of SEQ ID NO: 39;
(n) a primer consisting of SEQ ID NO: 40, a primer consisting of SEQ ID NO: 41, and a probe consisting of SEQ ID NO: 42; And
(o) a primer consisting of SEQ ID NO: 43, a primer consisting of SEQ ID NO: 44, and a probe consisting of SEQ ID NO: 45. delete delete delete delete delete delete

Images