KR20120078193A - Multiplex real time pcr method for discrimination of rice cultivar - Google Patents

Abstract

PURPOSE: A method for identifying Oryza sativa species using real time multiple PCR is provided to shorten PCR process and to lower probability of error occurrence. CONSTITUTION: A marker complex composition for identifying Oryza sativa species contains two selected from: probes of sequence numbers 1, 2, and 3; probes of sequence numbers 4, 5, and 6; probes of sequence number 7, 8, and 9; probes of sequence numbers 10, 11, and 12; probes of sequence numbers 13, 14, and 15; probes of sequence numbers 16, 17, and 18; probes of sequence numbers 19, 20, and 21; probes of sequence numbers 22, 23, and 24; probes of sequence numbers 25, 26, and 27; probes of sequence numbers 28, 29, and 30; probes of sequence numbers 31, 32, and 33; and probes of sequence number 34, 35, and 36.

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 67 types of rice (or rice).

Rice is a plant of Gramineae and genus Oryza. According to the Int'l Rice Research Institute (IRRI), there are about 60,000 species. 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 has been recently analyzed for genome sequencing of Nipponbare varieties through the International Rice Genome Sequencing Project (IRGSP), which includes 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 rapid, simple, automated identification method that can simultaneously analyze a variety of varieties of domestic rice with a plurality of markers.

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

(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.

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 67 are Nampyeong rice, alternative rice, yongpyeong, Dongjin No. 1 rice, Sangmi rice, Saegyehwa rice, Saechu Cheong rice, Sura rice, Sindong Jin rice, Odae rice, Ilmi rice, a la carte rice, Junam rice, Chinese rice, Chu Cheong rice, Hwabong rice, Hwaseong rice, Hwayoung rice, Dongjin rice, Unduk rice, Taebong rice, crest rice, Koshihikari, Hitomebore, Daepyeong rice, Manju rice, Mihyang rice, Seoganb rice, Sobaek rice, Consumption rice, Youngan rice, Unbong rice, Red rice wine, Jinbu Rice, Hyangnam Rice, Flavor No. 1 Rice, Flavor No. 2 Rice, Hopyeong Rice, Black Incense Rice, Reclaimed Rice, Fine Rice, Geumo Rice, Blizzard Rice, Dami Rice, Samgwang Rice, Samdeok Rice, Sambaek Rice, Sangok Rice, Sangju Rice, Bird Sangju Paddy, Odae No. 1 Rice, Onnuri Rice, Ungwang Rice, Pyeongan Rice, Flavor No. 1 Rice, Flavored Rice, Unimodal Rice, Hoummi Rice, Hwarang Rice, Golden Nuri Rice, Cheongho Rice, Wonsul Rice, Horse Rice, Long Rice, Chilbo Rice And it shows the results of real-time PCR with a template extracted from Saenuri 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 DK2708 SNP P3 (forward direction) gat aat gga ata gtc atc ccg gcc ttt gct 4 P4 (reverse direction) att ttt tct atc att tct gaa cta cac tcg 5 Probe 2 att aca att tag cac tct a 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 DK1854 In / Del P19 (forward direction) agc atg gtc cca gac aaa tg 28 P20 (reverse direction) tgg atg att tct gtt agt ggt tca ag 29 Probe 10 ttc ttg atg ata aat tgc tct ca 30 k DK6 SNP P21 (forward direction) aac ttt agt gca ggg ttt tca tca c 31 P22 (reverse direction) gaa cta caa gct caa gtc aag ccc 32 Probe 11 agc ctc tag gta tta ttg 33 l DK721 SNP P23 (forward direction) acg ggt ttt ctt ttt cat ttc g 34 P27 (reverse direction) tag gga gca cct act ata gaa ggt atg 35 Probe 12


tt tgc gat ctt tc 36

The markers of Table 1 describe the nucleotide sequences of SEQ ID NOs: 1 to 36. 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

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

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

DK1854: located on chromosome 4 and site of site used for sequencing was cloned AL606616

DK6: present on chromosome 11 and site of site used for sequencing is AC119073 clone

DK721: located on chromosome 3 and site of site used for sequencing is AC119747 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.

12 kinds of marker sets for identifying rice varieties of the present invention, DK17-1, DK34, DK50, DK63, DK560, DK1412, DK2171, DK2394, DK2401, DK1854, DK6, and a combination of two or more thereof, preferably, Real-time PCR can be performed using three or more, most preferably four combinations simultaneously. The combination of these 12 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 Taq DNA polymerase is 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. For example, fluorescent labeling examples of the marker set for identifying the rice variety of the present invention for use in the TaqMan ™ probe method are shown in Table 3 below.

Marker set type denomination order DK17-1 SNP P1 (forward direction) atc ccc ata att ctt gac aa P2 (Reverse) gga atc cgt ctt gat ttg tg Probe 1 FAM-tgt cgc gga gaa cata-MGBNFQ DK2708 SNP P3 (forward direction) gat aat gga ata gtc atc ccg gcc ttt gct P4 (reverse direction) att ttt tct atc att tct gaa cta cac tcg Probe 2 Cy5-att aca att tag cac tct a-MGBNFQ DK50 SNP P5 (forward direction) cgc gaa cta aac aag ccc ga P6 (reverse direction) aac cct tcc gat atc ctg tc Probe 3 VIC-ttg gtg tta cat cag atg-MGBNFQ DK63 SNP P7 (forward direction) aca cgc aat gag tgt ggc agt gtg P8 (reverse direction) cgg cca tgt gtt tac cta ctg Probe 4 NED-tgt atg gct ttt gta cc-MGBNFQ DK560 SNP P9 (forward direction) gcc acg tct tct tta aag att P10 (Reverse) att agg ctg cat aca tta gtc atc c Probe 5 FAM-tag aat ccc gac ccc ct-MGBNFQ DK1412 SNP P11 (forward direction) tat tgg gcc gaa tct gat tcg t P12 (Reverse) tct ccg aca gtt tag ggc tta ggg Probe 6 VIC-tcg cag tac cgg ata tct-MGBNFQ DK2171 SNP P13 (forward direction) aca caa aat aat gtc cat att a P14 (Reverse) aac tag caa att gcc tat gcg ttg Probe 7 NED-ctt tac aag tat gta gtg aag ga-MGBNFQ DK2394 SNP P15 (forward direction) tga tat ctt gga tta gaa gtt aac P16 (Reverse) ctg ata cag aaa ctt tcc aac cat tgg Probe 8 VIC-gaa cca caa cat ttt gcc gc-MGBNFQ DK2401 SNP P17 (forward direction) cac atc atc ttc aat ttc aac caa aat P18 (reverse direction) tat aca cca ttg att tct cta aat gg Probe 9 FAM-taa act ttg cgc tga ac-MGBNFQ DK1854 In / Del P19 (forward direction) agc atg gtc cca gac aaa tg P20 (reverse direction) tgg atg att tct gtt agt ggt tca ag Probe 10 Cy5-ttc ttg atg ata aat tgc tct ca-MGBNFQ DK6 SNP P21 (forward direction) aac ttt agt gca ggg ttt tca tca c P22 (reverse direction) gaa cta caa gct caa gtc aag ccc Probe 11 Cy5-agc ctc tag gta tta ttg-MGBNFQ DK721 SNP P23 (forward direction) acg ggt ttt ctt ttt cat ttg g P27 (reverse direction) tag gga gca cct act ata gaa ggt atg Probe 12 NED-tt tgc gat ctt tc-MGBNFQ

Table 3 considers the case of using three sets of rice varieties for identifying markers according to the present invention for the Popplex real-time PCR, using four FAM, VIC, Cy5 and NED ^TM as the fluorescent material at the probe 5 'end, respectively. The probe 3 'end attaches a molecular grove binding non-fluorescence quencher (MBGNFQ). In one example, a combination of a set of markers for identifying rice variety that can be used for Popplex real-time PCR may be as follows:

One set of markers selected from the group consisting of DK2401, DK50, DK2171 and DK2708;

One marker set selected from the group consisting of DK17-1, DK1412, DK721 and DK1854; And

One marker set selected from the group consisting of DK560, DK2394, DK63, and DK6.

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), DK2708 (Cy5); (2) DK17-1 (FAM), DK1412 (VIC), DK721 (NED) and DK1854 (Cy5); And (3) combinations of DK560 (FAM), DK2394 (VIC), DK63 (NED), and DK6 (Cy5).

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 a rice variety of the present invention and a rice variety identification method using the same, it is possible to identify a variety of rice varieties. Saechu rice, Sura rice, Sindong Jin rice, Odae rice, Ilmi rice, a la carte rice, Junnam rice, Chinese rice, Chu cheung rice, Hwabong rice, Hwaseong rice, Hwayoung rice, Dongjin rice, Unduk rice, Taebong rice, crest rice, Koshihikari, Hitomebore, Daepyeong rice, Manju rice , Unflavored rice, Seogan rice, Sobaek rice, Consumption rice, Youngan rice, Unbong rice, Red rice wine, Jinbu rice, Hyangnam rice, Flavor No. 1 rice, Flavor No. 2 rice, Hopyeong rice, Black scent rice, Reclaimed rice , Gold rice, blizzard, dami rice, samgwang rice, samduk rice, sambaek rice, Sangok rice, Sangju rice, Sasang rice, Odae No. 1 rice, Onnuri rice, Ungwang rice, Pyeongan rice, Flavor No. 1 rice, flavor rice, 67 varieties of rice varieties, including one heart rice, good rice, hwarang rice, golden rice, blue rice, yellow rice, malmi rice, rice plant, chilbo rice, and saeuri rice Each can be identified.

Specific identification method is as follows.

 (1) Nampyeong: When the peaks appear at the location of the identification marker sets DK2401, DK17-1, DK560, DK721 and DK1854 for rice varieties, it is determined to be Nampyeong rice.

(2) Alternative: Identification Mark Set for Rice Varieties DK50, DK2401, DK17-1, DK560, DK721, DK1854 and DK6 will be judged as alternative rice if peaks appear at positions.

(3) During: When the peaks appear at the DK2401, DK1412, DK560, DK721, DK1854, and DK6 positions for the identification marker set for rice varieties, it is judged as rice.

(4) Dongjin 1: Identification Mark Set for Rice Varieties DK50, DK1412, DK17-1, DK560, DK2708, and DK721 indicate peaks at the locations of Dongjin 1 rice.

(5) Rice taste: If the peaks appear at the DK17-1, DK63, DK560, DK2708, DK1854, and DK6 positions for the rice varieties, it is determined as rice.

(6) Birdification: If peaks appear at the DK560 and DK721 positions for the identification markers set for rice varieties, it is determined as newage.

(7) Saecheongcheong: If the peaks appear at the DK2401, DK17-1, DK721, DK1854 and DK6 positions for the identification marker set for rice varieties, saecheongcheong rice shall be determined.

(8) Sura: If a peak appears at the DK2401, DK560, and DK1854 positions for the identification marker set for rice cultivar, it is judged as Sura rice.

(9) Sindongjin: If there is a peak at the position of DK50, DK17-1, DK63, DK560 and DK721 for the identification marker set for rice varieties, it is determined as Shindongjinbyeong.

(5) False: If a peak appears at the DK2394, DK560, DK2708, DK1854, and DK6 positions of the rice varieties, it is judged as a large rice.

(11) Japan-US: If the peaks appear at the positions DK2401, DK1412, DK17-1, DK560, DK2171, DK721, and DK1854 for the rice variety, it is determined as one rice.

(12) Dishes: When the peaks appear at the DK63, DK560, DK2708 and DK6 positions for the identification marker sets for rice varieties, they are judged as a single rice.

(13) Chunam: A distinctive marker set for rice varieties. If peaks appear at positions DK50, DK17-1, DK560, DK2708 and DK1854, they are judged to be rice.

(14) Neutralization: Identifying marker sets for rice varieties DK2394, DK2401, DK17-1, DK560, DK2708 and DK1854 indicate peaks at the positions of neutralized rice.

(15) Referral: If the peaks appear at the DK2401, DK17-1, DK560, DK721, DK1854, and DK6 positions of the rice varieties, it is judged as a chopped rice.

(16) Hwabong: Identification marker sets for rice varieties DK2401, DK1412, DK17-1, DK560, DK721, DK1854 and DK6 if peaks appear at the positions shall be determined.

(17) Mars: Identification marker sets for rice varieties DK63, DK560, DK721, DK1854 and DK6854 If peaks appear at the positions, it is determined as Hwaseong rice.

(18) Hwayoung: If a peak appears at the DK50, DK17-1, DK560 and DK721 positions for the rice varieties, it is determined as Hwayoung rice.

(19) Dongjin: If a peak appears at the DK50, DK2401, DK560, DK2171 and DK721 positions for the rice varieties, it is determined as the Dongjin rice.

(20) Mounds: Identification marker sets for rice varieties DK2394, DK17-1, DK560, DK2708, DK721 and if peaks appear at the positions DK6, it is determined as muddy rice.

(21) Taebong: If the peaks appear at the DK2394, DK2401, DK560, DK2708 and DK1854 positions for the rice varieties, it is determined as Taebong rice.

(22) Sentence: Identification Mark Set for Rice Varieties If peaks appear at positions DK2394, DK560, DK2171, DK2708, and DK6, they are judged to be rice.

(23) Koshihikari: When the peaks appear at the positions DK2394, DK560, DK2708 and DK1854 for the identification marker sets for rice varieties, it is determined as Koshihikari rice.

(24) Hitomebore: Identification marker sets for rice varieties If peaks appear at positions DK2394, DK17-1, DK560, DK2171, DK2708 and DK1854, it is determined as Hitomebore rice.

(25) Large flat: If the peaks appear at the positions DK50, DK2401, DK1412, DK17-1, DK560, DK721, and DK1854 for the rice variety, the flat verdict is determined.

(26) Autumn: When peaks appear at the positions DK560, DK2171, DK721 and DK1854 for the identification marker set for rice varieties, it is determined as the autumn rice.

(27) Flavour: A set of identification markers for rice varieties DK50, DK17-1, DK63, DK560, DK2708, DK721, and DK6, where peaks appear, are determined as unflavored rice.

(28) Letter: When the peaks appear at the DK50, DK2401, DK63, and DK560 positions of the identification marker sets for rice varieties, it is determined as the letter rice.

(29) Sobaek: If peaks appear at the DK560, DK2708, DK1854, and DK6 positions for the rice varieties, it is determined as bovine rice.

(30) Consumption: Identification marker sets for rice varieties. If peaks appear at the positions DK50, DK1412, DK17-1, DK63 and DK560, it is determined as consumption rice.

(31) Mortuary: Identification marker sets for rice varieties. If peaks appear at positions DK50, DK2401, DK1412, DK560, DK2171, DK721, and DK1854, they shall be judged as young rice.

(32) Clouds: Identification marker sets for rice varieties If peaks appear at the positions DK2394, DK560, DK2708, DK721, DK1854 and DK6, they are considered to be clouded rice.

(33) Red pearls: Set of identification markers for rice varieties DK17-1, DK2171, DK2708, DK721, DK1854 and DK6 shall be judged as red pearls when peaks appear.

(34) Truth: A set of identification markers for rice varieties. If peaks appear at positions DK2394, DK63, DK560, DK2708 and DK1854, they are judged as true rice.

(35) Hyangnam: Identification Mark Set for Rice Varieties DK50, DK17-1, DK560, DK2171, DK2708, DK721, DK1854, and DK6 indicate peaks at the positions of Hyangnam Rice.

(36) Flavor No. 1: If there is a peak at the DK2708 and DK721 positions for the identification marker set for rice varieties, it is determined as flavor No. 1 rice.

(37) Flavor No. 2: Identification Mark Set for Rice Varieties If a peak appears at position DK2708, flavor 2 is determined.

(38) Reputation: Set of identification markers for rice varieties If peaks appear at the positions DK1412, DK17-1, DK63, DK560 and DK1854, they are judged as favorable rice.

(39) Black scent: A set of identification markers for rice varieties DK2394, DK2401, DK1412, DK17-1, DK560, DK2708, DK1854 and DK6.

(40) Black pearl: A set of identification markers for rice varieties DK2401, DK17-1, DK63, DK560, DK2171, DK2708 and DK6. If peaks appear at the positions, it is determined as black pearl rice.

(41) Reclamation: Set of identification markers for rice varieties. DK50, DK2401, DK1412, DK560, DK721, DK1854 and DK6 If peaks appear at the positions, reclamation is judged.

(42) High quality: discriminating marker sets for rice varieties If peaks appear at the positions DK17-1, DK63, DK560, DK2708 and DK1854, they are judged as high quality rice.

(43) Geumo: If the peaks appear at the positions DK560, DK2708 and DK1854 of the marker set for rice varieties, they are judged as gold.

(44) Blizzard: Identification Mark Set for Rice Varieties If peaks appear at positions DK2394, DK2401, DK560, DK2171, DK2708, and DK1854, they are considered to be blizzards.

(45) Damiers: Identification marker sets for rice varieties If peaks appear at the positions DK50, DK17-1, DK63 and DK560, they are judged as dami-mi rice.

(46) Samkwang: Identification marker set for rice varieties. DK50, DK2401, DK17-1, DK2708, DK1854, and DK6 indicate peaks at the positions of three light rice.

(47) Samdeok: Identification marker set for rice varieties. If peaks appear at positions DK50, DK2401, DK17-1, DK560, DK721, and DK1854, they are judged as samdeok rice.

(48) Three hundred: Set of identification markers for rice varieties. DK2401, DK560, DK1708, DK1854 and DK6854 If peaks appear at positions D, it is determined as three hundred rice.

(49) Jade: A distinctive marker set for rice varieties. If peaks appear at positions DK50, DK2401, DK17-1, DK560, DK721 and DK6, they are judged as jade rice.

(50) Resident: A set of identification markers for rice varieties, DK17-1, DK2708, DK1854 and DK6, if peaks appear at the position is determined as permanent rice.

(51) New resident rice: If the peaks appear at DK2394, DK2401, DK560, DK2708, DK1854 and DK6 positions for rice varieties, the new resident rice is determined.

(5) Five No. 1: Identification Mark Set for Rice Varieties If peaks appear at the positions DK63, DK560, DK2708 and DK1854, it is determined as five No.1 rice.

(53) Onnuri: Identification marker sets for rice varieties. If peaks appear at positions DK50, DK2401, DK17-1, DK560 and DK1854, they are judged as paddy rice.

(54) Clouds: Identification marker sets for rice varieties. If peaks appear at positions DK50, DK1412, DK17-1, DK560, DK1854 and DK6, they are judged as paddy rice.

(55) Peace: A set of identification markers for rice varieties. If peaks appear at positions DK50, DK2401, DK17-1, and DK560, they are judged as peace rice.

(56) Flavor No. 1: Identification marker set for rice varieties If peaks appear at positions DK2401, DK560, DK2171, DK721 and DK1854, it is determined as flavor No. 1 rice.

(57) Flavor: Identification marker sets for rice varieties. If peaks appear at positions DK50, DK17-1, DK560, DK721 and DK1854, they are judged as flavored rice.

(58) One heart: When the peaks appear at the positions DK50, DK17-1, DK560 and DK2708 for the identification marker set for rice varieties, they are judged as one heart.

(59) Articles: When peaks appear at the positions DK50, DK17-1 and DK560 of the marker set for rice varieties, they are judged to be rice.

(60) Galleries: Identification marker sets for rice varieties DK2401, DK560, DK721, DK1854 and DK6854 If peaks appear at the positions, judge the galley rice.

(61) Golden nursing: When the peaks appear at the positions DK50, DK560, DK2708 and DK1854 for the identification marker set for rice varieties, the golden nursing is determined.

(62) Blue lakes: Set of identification markers for rice varieties. If peaks appear at positions DK1412, DK560 and DK6, they are determined as green lakes.

(63) Pale color: Identification marker sets for rice varieties DK2401, DK1412, DK560, DK721 and DK6 If peaks appear at positions D, it is determined as Pale rice.

(64) Malmium: A distinctive marker set for rice varieties shall be judged as malmium if peaks appear at positions DK50, DK17-1, DK560, DK2708 and DK721.

(65) Long: When the peaks appear at positions DK63, DK560 and DK2708 for the identification marker sets for rice varieties, the rice is judged to be old.

(66) Cloisonne: Identification marker sets for rice varieties. If peaks appear at positions DK2394, DK17-1, DK560 and DK2708, the result is judged as cloisonne rice.

(67) Saenuri: If a peak appears at the location of the identification marker set DK50, DK17-1, DK560, DK2708, DK721, and DK1854 for the rice variety, it is determined as saeuri.

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 Multiple Marker  Set Nampyeong 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 Nampyeong 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 900 nM and 300 nM, respectively. At this time, a set of markers for identifying the rice varieties (1) DK2401 (FAM), DK50 (VIC), DK2171 (NED), DK2708 (Cy5); (2) DK17-1 (FAM), DK1412 (VIC), DK721 (NED), DK1854 (Cy5); And (3) three 4-plex real-time PCRs were performed using a combination of DK560 (FAM), DK2394 (VIC), DK63 (NED), and DK6 (Cy5).

The reaction solution was denatured at 95 ° C. for 10 minutes, followed by 40 seconds of 15 seconds at 95 ° C. and 1 minute at 60 ° C. to perform real-time PCR. The real time PCR instrument uses ABI 7500 Real Time PCR.

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

The present invention can be applied to AB Real-time PCR instruments 7900, 7500, 7300, Stratagene Mx3000p, and BioRad Chromo 4 instruments. 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 determined to be Nampyeong rice by specifically amplifying the rice seed set markers DK2401, DK17-1, DK560, DK721, and DK1854.

Example  2: for identifying rice varieties Multiple Marker  Alternative Rice Variety Identification Using Sets

According to the method of Example 1, real-time PCR was performed using alternative rice as a sample, and the results are shown in FIG. 2.

According to the results of FIG. 2, DNA extracted from the sample rice was specifically amplified in the marker sets DK50, DK2401, DK17-1, DK560, DK721, DK1854 and DK6 for identifying the rice varieties, confirming that the sample rice was correctly determined as alternative rice. It was.

Example  3: for identifying rice varieties Multiple Marker  Set Rice  Breed Identification

In accordance with the method of Example 1, real-time PCR was performed using the rice paddy as a sample, and the results are shown in FIG. 3.

According to the results of FIG. 3, the DNA extracted from the sample rice was specifically amplified in the marker sets DK2401, DK1412, DK560, DK721, DK1854, and DK6 for identifying the rice varieties to confirm that the sample rice was correctly determined as rice.

Example  4: For identifying rice varieties Multiple Marker  Dongjin using sets No. 1 rice  Breed Identification

According to the method of Example 1, real-time PCR was performed using Dongjin No. 1 rice as a sample, and the results are shown in FIG. 4.

According to the results of FIG. 4, the DNA extracted from the sample rice was specifically amplified in the marker sets DK50, DK1412, DK17-1, DK560, DK2708, and DK721 for rice variety identification to confirm that the sample rice was correctly determined to be Dongjin No. 1 rice. It was.

Example  5: for identifying rice varieties Multiple Marker  Identification of Rice Varieties Using Rice Set

According to the method of Example 1, real-time PCR was performed using Sangmi rice as a sample, and the results are shown in FIG. 5.

According to the results of FIG. 5, the DNA extracted from the sample rice was specifically amplified in the marker sets DK17-1, DK63, DK560, DK2708, DK1854, and DK6 for identifying the rice varieties, confirming that the sample rice was correctly determined to be rice.

Example  6: For identifying rice varieties Multiple Marker  Identification of Saengyehwa Rice Varieties Using a Set

According to the method of Example 1, real-time PCR was performed using Saegyehwa as a sample, the results are shown in FIG.

According to the results of FIG. 6, the DNA extracted from the sample rice was specifically amplified in the marker sets DK560 and DK721 for identifying the rice variety, and it was confirmed that the sample rice was correctly determined to be saehwa-si.

Example  7: for identifying rice varieties Multiple Marker  Set Sachuchu rice  Breed Identification

According to the method of Example 1, real-time PCR was performed using Saechu Cheongbap as a sample, and the results are shown in FIG. 7.

According to the results of FIG. 7, the DNA extracted from the sample rice was specifically amplified in the marker sets DK2401, DK17-1, DK721, DK1854, and DK6 for identifying the rice varieties, confirming that the sample rice was correctly determined as Sachuchu rice.

Example  8: For identifying rice varieties Multiple Marker  Set Sura rice  Breed Identification

According to the method of Example 1, real-time PCR was performed using Sura rice as a sample, and the results are shown in FIG. 8.

According to the results of FIG. 8, the DNA extracted from the sample rice was specifically amplified in the marker sets DK2401, DK560, and DK1854 for identifying the rice variety, confirming that the sample rice was correctly determined to be rice rapeseed.

Example  9: For identifying rice varieties Multiple Marker  Set Shindongjin Rice  Breed Identification

According to the method of Example 1, real-time PCR was performed using Shindong Jinpyeon as a sample, and the results are shown in FIG. 9.

According to the results of FIG. 9, the DNA extracted from the sample rice was specifically amplified in the marker sets DK50, DK17-1, DK63, DK560, and DK721 for identifying the rice varieties, confirming that the sample rice was correctly determined to be Shindongjinp.

Example  10: for identifying rice varieties Multiple Marker  Identification of the Five Rice Varieties Using a Set

According to the method of Example 1, real-time PCR was performed using five rice as a sample, and the results are shown in FIG.

According to the results of FIG. 10, the DNA extracted from the sample rice was specifically amplified in the marker sets DK2394, DK560, DK2708, DK1854 and DK6 for identifying the rice varieties, confirming that the sample rice was correctly determined as five rice.

Example  11: For identifying rice varieties Multiple Marker  Set Rice  Breed Identification

According to the method of Example 1, real-time PCR was performed using ilmi rice as a sample, and the results are shown in FIG. 11.

According to the results of FIG. 11, the DNA extracted from the sample rice was specifically amplified in the marker sets DK2401, DK1412, DK17-1, DK560, DK2171, DK721, and DK1854 for identifying the rice varieties to confirm that the sample rice was correctly determined to be one rice. It was.

Example  12: for identifying rice varieties Multiple Marker  Identification of A La Carte Rice Varieties Using Sets

According to the method of Example 1, real-time PCR was performed using a la carte rice as a sample, the results are shown in FIG.

According to the results of FIG. 12, the DNA extracted from the sample rice was specifically amplified in the marker sets DK63, DK560, DK2708, and DK6 for identifying the rice varieties, and confirmed that the sample rice was correctly determined to be excellent rice.

Example  13: For identifying rice varieties Multiple Marker  Set Rice  Breed Identification

According to the method of Example 1, real-time PCR was performed using Junambap as a sample, and the results are shown in FIG. 13.

According to the results of FIG. 13, the DNA extracted from the sample rice was specifically amplified in the marker sets DK50, DK17-1, DK560, DK2708 and DK1854 for identifying the rice varieties, confirming that the sample rice was correctly judged as main rice.

Example  14: for identifying rice varieties Multiple Marker  Set Chinese rice  Breed Identification

According to the method of Example 1, real-time PCR was performed using a rice paddy as a sample, the results are shown in FIG.

According to the results of FIG. 14, the DNA extracted from the sample rice was specifically amplified in the marker sets DK2394, DK2401, DK17-1, DK560, DK2708, and DK1854 for rice variety identification, confirming that the sample rice was correctly determined as neutralized rice.

Example  15: For identifying rice varieties Multiple Marker  Identification of Cheongpyeong Rice Varieties Using a Set

According to the method of Example 1, real-time PCR was performed using Chuchubap as a sample, and the results are shown in FIG. 15.

According to the results of FIG. 15, the DNA extracted from the sample rice was specifically amplified in the marker sets DK2401, DK17-1, DK560, DK721, DK1854 and DK6 for identifying the rice varieties to confirm that the sample rice was correctly determined to be cold rice.

Example  16: For identifying rice varieties Multiple Marker  Set Flower bud  Breed Identification

According to the method of Example 1, real-time PCR was performed using Hwabongbap as a sample, and the results are shown in FIG. 16.

According to the results of FIG. 16, the DNA extracted from the sample rice was specifically amplified in the marker sets DK2401, DK1412, DK17-1, DK560, DK721, DK1854, and DK6 for identifying the rice varieties, confirming that the sample rice was correctly determined to be flower bud rice. It was.

Example  17: For identifying rice varieties Multiple Marker  Set Hwaseong Rice  Breed Identification

According to the method of Example 1, real-time PCR was performed using Hwaseong rice as a sample, and the results are shown in FIG. 17.

According to the results of FIG. 17, the DNA extracted from the sample rice was specifically amplified in the marker sets DK63, DK560, DK721, DK1854 and DK6 for identifying the rice varieties, confirming that the sample rice was correctly determined as hwaseong rice.

Example  18: For identifying rice varieties Multiple Marker  Set Hwayoung Rice  Breed Identification

According to the method of Example 1, real-time PCR was performed using Hwayoungbap as a sample, and the results are shown in FIG. 18.

According to the results of FIG. 18, the DNA extracted from the sample rice was specifically amplified in the marker sets DK50, DK17-1, DK560, and DK721 for identifying the rice variety, confirming that the sample rice was correctly determined as Hwayoung rice.

Example  19: For identifying rice varieties Multiple Marker  Identification of Dongjin Rice Varieties Using a Set

According to the method of Example 1, real-time PCR was performed using Dongjinbyeong as a sample, and the results are shown in FIG. 19.

According to the results of FIG. 19, the DNA extracted from the sample rice was specifically amplified in the marker sets DK50, DK2401, DK560, DK2171 and DK721 for identifying the rice varieties, confirming that the sample rice was correctly determined to be dongjinbyeong.

Example  20: for identifying rice varieties Multiple Marker  Set Muddy rice  Breed Identification

According to the method of Example 1, real-time PCR was performed using muddy rice as a sample, and the results are shown in FIG. 20.

According to the results of FIG. 20, the DNA extracted from the sample rice was specifically amplified in the marker sets DK2394, DK17-1, DK560, DK2708, DK721, and DK6 for rice variety identification, confirming that the sample rice was correctly determined to be mungbean rice.

Example  21: for identifying rice varieties Multiple Marker  Set Taebong Rice  Breed Identification

According to the method of Example 1, real-time PCR was performed using Taebong rice as a sample, and the results are shown in FIG. 21.

According to the results of FIG. 21, the DNA extracted from the sample rice was specifically amplified in the marker sets DK2394, DK2401, DK560, DK2708 and DK1854 for identifying the rice variety, and confirmed that the sample rice was correctly determined to be taebong rice.

Example  22: for identifying rice varieties Multiple Marker  Set Crest  Breed Identification

According to the method of Example 1, real-time PCR was performed using a rice plant as a sample, and the results are shown in FIG. 22.

According to the results of FIG. 22, the DNA extracted from the sample rice was specifically amplified in the marker sets DK2394, DK560, DK2171, DK2708 and DK6 for identifying the rice varieties to confirm that the sample rice was correctly determined to be rice.

Example  23: For identifying rice varieties Multiple Marker  Set Koshihikari rice  Breed Identification

According to the method of Example 1, real-time PCR was performed using Koshihikari rice as a sample, and the results are shown in FIG. 23.

According to the results of FIG. 23, the DNA extracted from the sample rice was specifically amplified in the marker sets DK2394, DK560, DK2708 and DK1854 for identifying the rice variety, and confirmed that the sample rice was correctly determined to be Koshihikari rice.

Example  24: For identifying rice varieties Multiple Marker  Identification of Hitomebore Rice Varieties Using a Set

According to the method of Example 1, real-time PCR was performed using Hitomebolebium as a sample, and the results are shown in FIG. 24.

According to the results of FIG. 24, the DNA extracted from the sample rice was specifically amplified in the marker sets DK2394, DK17-1, DK560, DK2171, DK2708 and DK1854 for identifying the rice varieties, confirming that the sample rice was correctly determined as Hitomebore rice. It was.

Example  25: For identifying rice varieties Multiple Marker  Set Flat rice  Breed Identification

According to the method of Example 1, real-time PCR was performed using large flat rice as a sample, and the results are shown in FIG. 25.

According to the results of FIG. 25, the DNA extracted from the sample rice was specifically amplified in the marker sets DK50, DK2401, DK1412, DK17-1, DK560, DK721, and DK1854 for identifying the rice variety, confirming that the sample rice was correctly determined to be flat rice. It was.

Example  26: for identifying rice varieties Multiple Marker  Set Late rice  Breed Identification

According to the method of Example 1, real-time PCR was performed using a rice paddy as a sample, and the results are shown in FIG. 26.

According to the results of FIG. 26, the DNA extracted from the sample rice was specifically amplified in the marker sets DK560, DK2171, DK721, and DK1854 for identifying the rice variety, and confirmed that the sample rice was correctly determined to be full rice.

Example  27: for identifying rice varieties Multiple Marker  Set Unflavored rice  Breed Identification

According to the method of Example 1, real-time PCR was performed using unflavored rice as a sample, and the results are shown in FIG. 27.

According to the results of FIG. 27, the DNA extracted from the sample rice was specifically amplified in the marker sets DK50, DK17-1, DK63, DK560, DK2708, DK721, and DK6 for rice variety identification, confirming that the sample rice was correctly determined as unflavored rice. It was.

Example  28: for identifying rice varieties Multiple Marker  Set Episodic  Breed Identification

In accordance with the method of Example 1, real-time PCR was performed using a rice paddy as a sample, and the results are shown in FIG. 28.

According to the results of FIG. 28, the DNA extracted from the sample rice was specifically amplified in the marker sets DK50, DK2401, DK63, and DK560 for identifying the rice varieties to confirm that the sample rice was correctly determined to be episodic rice.

Example  29: For identifying rice varieties Multiple Marker  Sobaek with the set rice plant  Breed Identification

In accordance with the method of Example 1, real-time PCR was performed using bovine rice as a sample, and the results are shown in FIG. 29.

According to the results of FIG. 29, the DNA extracted from the sample rice was specifically amplified in the marker sets DK560, DK2708, DK1854, and DK6 for identifying the rice variety, and confirmed that the sample rice was correctly determined to be small white rice.

Example  30: For identifying rice varieties Multiple Marker  Consumption with sets rice plant  Breed Identification

According to the method of Example 1, real-time PCR was performed using consumable rice as a sample, and the results are shown in FIG.

According to the results of FIG. 30, the DNA extracted from the sample rice was specifically amplified in the marker sets DK50, DK1412, DK17-1, DK63 and DK560 for identifying the rice varieties to confirm that the sample rice was correctly determined to be consumed rice.

Example  31: for identifying rice varieties Multiple Marker  Set Youngan Rice  Breed Identification

In accordance with the method of Example 1, real-time PCR was performed using a young rice as a sample, and the results are shown in FIG. 31.

According to the results of FIG. 31, the DNA extracted from the sample rice was specifically amplified in the marker sets DK50, DK2401, DK1412, DK560, DK2171, DK721, and DK1854 for the rice variety identification, and confirmed that the sample rice was correctly determined to be young rice.

Example  32: for identifying rice varieties Multiple Marker  Set Yunbong Rice  Breed Identification

According to the method of Example 1, real-time PCR was performed using Unbongpung as a sample, and the results are shown in FIG. 32.

According to the results of FIG. 32, the DNA extracted from the sample rice was specifically amplified in the marker sets DK2394, DK560, DK2708, DK721, DK1854, and DK6 for identifying the rice varieties, confirming that the sample rice was correctly determined to be Yunbong rice.

Example  33: for identifying rice varieties Multiple Marker  Set Red rice wine  Breed Identification

According to the method of Example 1, real-time PCR was performed using red rice wine as a sample, and the results are shown in FIG. 33.

According to the results of FIG. 33, the DNA extracted from the sample rice was specifically amplified in the marker sets DK17-1, DK2171, DK2708, DK721, DK1854 and DK6 for identifying the rice varieties, confirming that the sample rice was correctly determined as red rice wine. .

Example  34: For identifying rice varieties Multiple Marker  Cliché using set rice plant  Breed Identification

According to the method of Example 1, real-time PCR was performed using Jinbu rice as a sample, and the results are shown in FIG. 34.

According to the results of FIG. 34, the DNA extracted from the sample rice was specifically amplified in the marker sets DK2394, DK63, DK560, DK2708, and DK1854 for rice variety identification, confirming that the sample rice was correctly determined to be true rice.

Example  35: for identifying rice varieties Multiple Marker  Set Hyangnam Rice  Breed Identification

According to the method of Example 1, real-time PCR was performed using hyangnam rice as a sample, the results are shown in FIG.

According to the results of FIG. 35, DNA extracted from the sample rice was specifically amplified in the marker sets DK50, DK17-1, DK560, DK2171, DK2708, DK721, DK1854, and DK6 for the rice variety identification, and the sample rice was Dongjin No. 1 rice. It was confirmed that it was determined correctly.

Example  36: for identifying rice varieties Multiple Marker  Flavor with set No. 1 rice  Breed Identification

According to the method of Example 1, real-time PCR was performed using flavor No. 1 as a sample, and the results are shown in FIG. 36.

According to the results of FIG. 36, the DNA extracted from the sample rice was specifically amplified by the marker set DK2708 for rice variety identification to confirm that the sample rice was correctly determined to be No. 1 rice.

Example  37: for identifying rice varieties Multiple Marker  Flavors using sets 2 Hump  Breed Identification

According to the method of Example 1, real-time PCR was performed using flavor No. 2 as a sample, and the results are shown in FIG. 37.

According to the results of FIG. 37, the DNA extracted from the sample rice was specifically amplified in the marker sets DK1412, DK17-1, DK63, DK560, and DK1854 for rice variety identification, confirming that the sample rice was correctly determined to be Dongjin No. 1 rice.

Example  38: for identifying rice varieties Multiple Marker  Set Popular rice  Breed Identification

According to the method of Example 1, real-time PCR was performed using Hopyeong rice as a sample, and the results are shown in FIG. 38.

According to the results of FIG. 38, DNA extracted from the sample rice was specifically amplified in the marker sets DK1412, DK17-1, DK63, DK560 and DK1854 for identifying the rice variety, and confirmed that the sample rice was correctly determined to be well-reputed rice.

Example  39: for identifying rice varieties Multiple Marker  Set Black rice  Breed Identification

In accordance with the method of Example 1, real-time PCR was performed using black sauerkraut as a sample, and the results are shown in FIG. 39.

According to the results of FIG. 39, DNA extracted from the sample rice was specifically amplified in the marker sets DK2394, DK2401, DK1412, DK17-1, DK560, DK2708, DK1854, and DK6 for rice variety identification, and the sample rice was correctly judged as black vinegar rice. It was confirmed.

Example  40: For identifying rice varieties Multiple Marker  Black pearl using set rice plant  Breed Identification

According to the method of Example 1, real-time PCR was performed using black pearl rice as a sample, and the results are shown in FIG. 40.

According to the results of FIG. 40, the DNA extracted from the sample rice was specifically amplified in the marker sets DK2401, DK17-1, DK63, DK560, DK2171, DK2708 and DK6 for identifying the rice variety, confirming that the sample rice was correctly judged as black pearl rice. It was.

Example  41: for identifying rice varieties Multiple Marker  Reclamation with Sets rice plant  Breed Identification

According to the method of Example 1, real-time PCR was performed using reclaimed rice as a sample, and the results are shown in FIG. 41.

According to the results of FIG. 41, the DNA extracted from the sample rice was specifically amplified in the marker sets DK50, DK2401, DK1412, DK560, DK721, DK1854, and DK6 for rice variety identification, and confirmed that the sample rice was correctly determined as reclaimed rice.

Example  42: for identifying rice varieties Multiple Marker  Identification of High Quality Rice Varieties Using Sets

According to the method of Example 1, real-time PCR was performed using high quality rice as a sample, and the results are shown in FIG. 42.

According to the results of FIG. 42, the DNA extracted from the sample rice was specifically amplified in the marker sets DK17-1, DK63, DK560, DK2708 and DK1854 for identifying the rice variety, and confirmed that the sample rice was correctly judged as high quality rice.

Example  43: for identifying rice varieties Multiple Marker  Identification of Rice Varieties Using Rice Sets

According to the method of Example 1, real-time PCR was performed using gold rice as a sample, and the results are shown in FIG. 43.

According to the results of FIG. 43, the DNA extracted from the sample rice was specifically amplified in the marker sets DK560, DK2708 and DK1854 for identifying the rice variety, confirming that the sample rice was correctly determined to be gold rice.

Example  44: for identifying rice varieties Multiple Marker  Snowstorm with sets rice plant  Breed Identification

According to the method of Example 1, real-time PCR was performed using blizzard as a sample, and the results are shown in FIG. 44.

According to the results of FIG. 44, the DNA extracted from the sample rice was specifically amplified in the marker sets DK2394, DK2401, DK560, DK2171, DK2708, and DK1854 for identifying the rice variety, confirming that the sample rice was correctly determined to be blizzard.

Example  45: for identifying rice varieties Multiple Marker  Set Rice  Breed Identification

In accordance with the method of Example 1, real-time PCR was performed using dama rice as a sample, and the results are shown in FIG. 453.

According to the results of FIG. 45, the DNA extracted from the sample rice was specifically amplified in the marker sets DK50, DK17-1, DK63, and DK560 for identifying the rice varieties to confirm that the sample rice was correctly determined as dama rice.

Example  46: For identifying rice varieties Multiple Marker  Set Samgwang Rice  Breed Identification

According to the method of Example 1, real-time PCR was performed using Samgwang rice as a sample, and the results are shown in FIG. 46.

According to the results of FIG. 46, the DNA extracted from the sample rice was specifically amplified in the marker sets K50, DK2401, DK17-1, DK2708, DK1854, and DK6 for rice variety identification, confirming that the sample rice was correctly determined to be three-light rice.

Example  47: for identifying rice varieties Multiple Marker  Set Samdeok Rice  Breed Identification

In accordance with the method of Example 1, real-time PCR was performed using Samdeok rice as a sample, and the results are shown in FIG. 471.

According to the results of FIG. 47, the DNA extracted from the sample rice was specifically amplified in the marker sets DK50, DK2401, DK17-1, DK560, DK721, and DK1854 for rice variety identification, confirming that the sample rice was correctly judged as Samduk.

Example  48: For identifying rice varieties Multiple Marker  Set Three hundred rice  Breed Identification

According to the method of Example 1, real-time PCR was performed using three hundred rice as a sample, the results are shown in FIG.

According to the results of FIG. 48, the DNA extracted from the sample rice was specifically amplified in the marker sets DK2401, DK560, DK1708, DK1854 and DK6 for identifying the rice variety, and confirmed that the sample rice was correctly determined to be three hundred rice.

Example  49: for identifying rice varieties Multiple Marker  Set Jade rice  Breed Identification

According to the method of Example 1, real-time PCR was performed using sanggobap as a sample, the results are shown in FIG.

According to the results of FIG. 49, the DNA extracted from the sample rice was specifically amplified in the marker sets DK50, DK2401, DK17-1, DK560, DK721, and DK6 for identifying the rice variety, confirming that the sample rice was correctly determined to be jade rice.

Example  50: for identifying rice varieties Multiple Marker  Set Sangju Rice  Breed Identification

According to the method of Example 1, real-time PCR was performed using Sangjub as a sample, and the results are shown in FIG. 25.

According to the results of FIG. 25, the DNA extracted from the sample rice was specifically amplified in the marker sets DK17-1, DK2708, DK1854 and DK6 for identifying the rice varieties, confirming that the sample rice was correctly determined to be permanent rice.

Example  51: for identifying rice varieties Multiple Marker  New resident using set rice plant  Breed Identification

According to the method of Example 1, real-time PCR was performed using Sasangjub as a sample, and the results are shown in FIG. 25.

According to the results of FIG. 25, the DNA extracted from the sample rice was specifically amplified in the marker sets DK2394, DK2401, DK560, DK2708, DK1854, and DK6 for identifying the rice varieties, confirming that the sample rice was correctly judged as Sasangju.

Example  52: for identifying rice varieties Multiple Marker  Identification of Five No.1 Rice Varieties Using a Set

In accordance with the method of Example 1, real-time PCR was performed using five No. 1 rice as a sample, and the results are shown in FIG. 25.

According to the results of FIG. 25, the DNA extracted from the sample rice was specifically amplified in the marker sets DK63, DK560, DK2708 and DK1854 for identifying the rice variety, and confirmed that the sample rice was correctly determined to be 5 rices.

Example  53: for identifying rice varieties Multiple Marker  Set Onnuri rice  Breed Identification

According to the method of Example 1, real-time PCR was performed using Onnuribap as a sample, and the results are shown in FIG. 21.

According to the results of FIG. 21, the DNA extracted from the sample rice was specifically amplified in the marker sets DK50, DK2401, DK17-1, DK560, and DK1854 for identifying the rice variety, confirming that the sample rice was correctly determined to be onnuri rice.

Example  54: for identifying rice varieties Multiple Marker  Meteor with set rice plant  Breed Identification

According to the method of Example 1, real-time PCR was performed using a meteorite rice as a sample, and the results are shown in FIG. 22.

According to the results of FIG. 22, the DNA extracted from the sample rice was specifically amplified in the marker sets DK50, DK1412, DK17-1, DK560, DK1854, and DK60 for identifying the rice varieties, confirming that the sample rice was correctly determined to be meteorite rice.

Example  55: for identifying rice varieties Multiple Marker  Set Peace  Breed Identification

According to the method of Example 1, real-time PCR was performed using Pyeongan Rice as a sample, and the results are shown in FIG. 23.

According to the results of FIG. 23, the DNA extracted from the sample rice was specifically amplified in the marker sets DK50, DK2401, DK17-1, and DK560 for identifying the rice varieties, confirming that the sample rice was correctly determined to be flat rice.

Example  56: for identifying rice varieties Multiple Marker  Flavor using set No. 1 rice  Breed Identification

According to the method of Example 1, real-time PCR was performed using flavor 1 rice as a sample, and the results are shown in FIG. 24.

According to the results of FIG. 24, the DNA extracted from the sample rice was specifically amplified in the marker sets DK2401, DK560, DK2171, DK721, and DK1854 for rice variety identification, confirming that the sample rice was correctly determined to be flavor 1 rice.

Example  57: for identifying rice varieties Multiple Marker  Set Flavor  Breed Identification

According to the method of Example 1, real-time PCR was performed using flavor rice as a sample, and the results are shown in FIG. 21.

According to the results of FIG. 21, the DNA extracted from the sample rice was specifically amplified in the marker sets DK50, DK17-1, DK560, DK721, and DK1854 for identifying the rice varieties, confirming that the sample rice was correctly judged as a flavor rice.

Example  58: for identifying rice varieties Multiple Marker  Set Unison rice  Breed Identification

According to the method of Example 1, real-time PCR was performed using Hanmam rice as a sample, and the results are shown in FIG.

According to the results of FIG. 22, the DNA extracted from the sample rice was specifically amplified in the marker sets DK50, DK17-1, DK560, and DK2708 for rice variety identification, confirming that the sample rice was correctly determined to be a unified rice.

Example  59: for identifying rice varieties Multiple Marker  Good article using set rice plant  Breed Identification

According to the method of Example 1, real-time PCR was performed using a rice product as a sample, and the results are shown in FIG. 23.

According to the results of FIG. 23, the DNA extracted from the sample rice was specifically amplified in the marker sets DK50, DK17-1 and DK560 for identifying the rice variety, confirming that the sample rice was correctly determined to be good rice.

Example  60: for identifying rice varieties Multiple Marker  Set Hwarang Rice  Breed Identification

In accordance with the method of Example 1, real-time PCR was performed using Hwarang-Rip as a sample, and the results are shown in FIG. 24.

According to the results of FIG. 24, the DNA extracted from the sample rice was specifically amplified in the marker sets DK2401, DK560, DK721, DK1854, and DK6 for rice variety identification, and confirmed that the sample rice was correctly determined to be a rice plant.

Example  61: for identifying rice varieties Multiple Marker  Set Golden rice  Breed Identification

In accordance with the method of Example 1, real-time PCR was performed using golden rice as a sample, and the results are shown in FIG. 25.

According to the results of FIG. 25, the DNA extracted from the sample rice was specifically amplified in the marker sets DK50, DK560, DK2708 and DK1854 for identifying the rice variety, and confirmed that the sample rice was correctly determined to be golden rice.

Example  62: for identifying rice varieties Multiple Marker  Set Cheongho Rice  Breed Identification

According to the method of Example 1, real-time PCR was performed using Chungho rice as a sample, and the results are shown in FIG. 25.

According to the results of FIG. 25, the DNA extracted from the sample rice was specifically amplified in the marker sets DK1412, DK560 and DK6 for identifying the rice variety, and confirmed that the sample rice was correctly determined to be green rice.

Example  63: for identifying rice varieties Multiple Marker  Set Yellow rice  Breed Identification

In accordance with the method of Example 1, real-time PCR was performed using raw sulfur rice as a sample, and the results are shown in FIG. 25.

According to the results of FIG. 25, the DNA extracted from the sample rice was specifically amplified in the marker sets DK2401, DK1412, DK560, DK721, and DK6 for rice variety identification to confirm that the sample rice was correctly determined to be yellow rice.

Example  64: for identifying rice varieties Multiple Marker  Mummies using sets rice plant  Breed Identification

According to the method of Example 1, real-time PCR was performed using the rice cake as a sample, and the results are shown in FIG. 25.

According to the results of FIG. 25, the DNA extracted from the sample rice was specifically amplified in the marker sets DK50, DK17-1, DK560, DK2708 and DK721 for identifying the rice varieties to confirm that the sample rice was correctly determined to be rice.

Example  65: for identifying rice varieties Multiple Marker  Set Long  Breed Identification

According to the method of Example 1, real-time PCR was performed using a rice paddy as a sample, and the results are shown in FIG. 25.

According to the results of FIG. 25, the DNA extracted from the sample rice was specifically amplified in the marker sets DK63, DK560, and DK2708 for rice variety identification, confirming that the sample rice was correctly judged as long rice.

Example  66: for identifying rice varieties Multiple Marker  Set Cloisonne  Breed Identification

According to the method of Example 1, real-time PCR was performed using Chilbo rice as a sample, and the results are shown in FIG. 25.

According to the results of FIG. 25, the DNA extracted from the sample rice was specifically amplified in the marker sets DK2394, DK17-1, DK560, and DK2708 for rice variety identification, to confirm that the sample rice was correctly determined to be chilbo rice.

Example  67: for identifying rice varieties Multiple Marker  Set Saenuri rice  Breed Identification

In accordance with the method of Example 1, real-time PCR was performed using Saenuribap as a sample, and the results are shown in FIG. 2.

According to the results of FIG. 2, the DNA extracted from the sample rice was specifically amplified in the marker sets DK50, DK17-1, DK560, DK2708, DK721, and DK1854 for rice variety identification, confirming that the sample rice was correctly determined as saeuri rice.

<110> Kogene Biotech Co., LTD <120> MULTIPLEX REAL TIME PCR METHOD FOR DISCRIMINATION OF RICE          CULTIVAR <130> DP100322 <160> 36 <170> Kopatentin 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DK17-1 forward primer <400> 1 atccccataa ttcttgacaa 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DK17-1 reverse primer <400> 2 ggaatccgtc ttgatttgtg 20 <210> 3 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> DK17-1 probe <400> 3 tgtcgcggag aacata 16 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> DK2708 forward primer <400> 4 gataatggaa tagtcatccc ggcctttgct 30 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> DK2708 reverse primer <400> 5 attttttcta tcatttctga actacactcg 30 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> DK2708 probe <400> 6 attacaattt agcactcta 19 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DK50 forward primer <400> 7 cgcgaactaa acaagcccga 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DK50 reverse primer <400> 8 aacccttccg atatcctgtc 20 <210> 9 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> DK50 probe <400> 9 ttggtgttac atcagatg 18 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> DK63 forward primer <400> 10 acacgcaatg agtgtggcag tgtg 24 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> DK63 reverse primer <400> 11 cggccatgtg tttacctact g 21 <210> 12 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> DK63 probe <400> 12 tgtatggctt ttgtacc 17 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> DK560 forward primer <400> 13 gccacgtctt ctttaaagat t 21 <210> 14 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> DK560 reverse primer <400> 14 attaggctgc atacattagt catcc 25 <210> 15 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> DK560 probe <400> 15 tagaatcccg accccct 17 <210> 16 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> DK1412 forward primer <400> 16 tattgggccg aatctgattc gt 22 <210> 17 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> DK1412 reverse primer <400> 17 tctccgacag tttagggctt aggg 24 <210> 18 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> DK1412 probe <400> 18 tcgcagtacc ggatatct 18 <210> 19 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> DK2171 forward primer <400> 19 acacaaaata atgtccatat ta 22 <210> 20 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> DK2171 reverse primer <400> 20 aactagcaaa ttgcctatgc gttg 24 <210> 21 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> DK2171 probe <400> 21 ctttacaagt atgtagtgaa gga 23 <210> 22 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> DK2394 forward primer <400> 22 tgatatcttg gattagaagt taac 24 <210> 23 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> DK2394 reverse primer <400> 23 ctgatacaga aactttccaa ccattgg 27 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DK2394 probe <400> 24 gaaccacaac attttgccgc 20 <210> 25 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> DK2401 forward primer <400> 25 cacatcatct tcaatttcaa ccaaaat 27 <210> 26 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> DK2401 reverse primer <400> 26 tatacaccat tgatttctct aaatgg 26 <210> 27 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> DK2401 probe <400> 27 taaactttgc gctgaac 17 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DK1854 forward primer <400> 28 agcatggtcc cagacaaatg 20 <210> 29 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> DK1854 reverse primer <400> 29 tggatgattt ctgttagtgg ttcaag 26 <210> 30 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> DK1854 probe <400> 30 ttcttgatga taaattgctc tca 23 <210> 31 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> DK6 forward primer <400> 31 aactttagtg cagggttttc atcac 25 <210> 32 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> DK6 reverse primer <400> 32 gaactacaag ctcaagtcaa gccc 24 <210> 33 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> DK6 probe <400> 33 agcctctagg tattattg 18 <210> 34 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> DK721 forward primer <400> 34 acgggttttc tttttcattt cg 22 <210> 35 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> DK721 reverse primer <400> 35 tagggagcac ctactataga aggtatg 27 <210> 36 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> DK721 probe <400> 36 tttgcgatct ttc 13

Claims (8)

A marker combination composition for identifying a rice variety, comprising two or more marker sets for identifying a rice variety selected from the group consisting of (a) to (i):
(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. According to claim 1, Marker combination composition comprising three or more marker sets for identifying the rice variety. The combination of markers according to claim 1, wherein the marker combination composition comprises a probe labeled with a separate detectable means according to the type of probe, and the detectable means is detected when a specific PCR product is amplified. Composition. 4. A marker combination composition according to claim 3, wherein said detectable means is a fluorescent material. According to claim 3, The probe is labeled with a fluorescent material selected from the group consisting of FAM, VIC, TET, JOE, HEX, TAMRA, CY3, CY5, ROX, RED610, TEXAS RED, RED670 and NED at the 5 'end, Marker combination composition, characterized in that the probe 3 'end is labeled with a molecular grove binding non-fluorescence quencher (MGGBFF). The method of claim 1, wherein the marker combination composition for identifying rice varieties is
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 one marker set selected from the group consisting of (b), (c) (g) and (i), 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 one marker set selected from the group consisting of (a), (f) (j) and (l), labeled with a molecular grove binding non-fluorescence quencher; And
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 a marker set selected from the group consisting of (d), (e) (h) and (k), labeled with a molecular grove binding non-fluorescence quencher,
Probe included in the selected marker set is a marker combination composition, characterized in that each labeled with a separate fluorescent material. The method of claim 1, wherein the marker combination composition for identifying the rice varieties Nampyeong rice, alternative rice, rice plant, Dongjin No. 1 rice, Sangmi rice, Saegyehwa rice, Sachuchu rice, Sura rice, Sindongjin rice, Odae rice, Ilmi rice, one rice , Chunambyeon, Chinese rice, Chuchung rice, Huabong rice, Hwaseong rice, Hwayoung rice, Dongjin rice, Unduk rice, Taebong rice, crest rice, Koshihikari, Hitomebore, Great flat rice, Manju rice, Unscented rice, Seoganb rice, Sobaek rice, Consumption rice, Yeongan Rice, Unbong Rice, Red Jinju Rice, Jinbu Rice, Hyangnam Rice, Flavor No. 1 Rice, Flavor No. 2 Rice, Hopyeong Rice, Heukhyang Rice, Reclaimed Rice, Fine Rice, Geummyeon Rice, Blizzard Rice, Dami Rice, Samgwang Rice, Samdeok rice, three hundred rice, Sangok rice, Sangju rice, Sasangju rice, Odae No. 1 rice, Onnuri rice, Ungwang rice, Pyeongan rice, Flavor No. 1 rice, Flavor rice, Haneum rice, Houmb rice, Hwarang rice, Golden rice, Characterized in that it is used to identify rice varieties selected from the group consisting of Cheongho rice, yellow rice, horse rice, long rice, chilbo rice, and saeuri rice. Marker combination composition. Performing real-time PCR with a template extracted from rice using a marker combination composition for identifying rice varieties according to any one of claims 1 to 7;
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
Identifying a variety of rice according to whether the PCR is amplified;
Rice variety identification method using a plurality of rice varieties identification marker set, comprising a.

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