KR100974264B1 - Primer combination for discrimination of rice cultivar and method for discrimination of rice cultivar using same - Google Patents

Abstract

The present invention relates to a combination of 10 pairs of primers for identifying rice varieties and a method for identifying rice varieties using the same, wherein using 10 pairs of primers simultaneously in a single PCR reaction, rice varieties specific SNPs are determined by PCR amplification for each primer pair. By identifying, rice varieties can be identified specifically, quickly and accurately.

Rice, Breed Identification, Primer

Description

Primer combination for rice variety identification and identification method using same {PRIMER COMBINATION FOR DISCRIMINATION OF RICE CULTIVAR AND METHOD FOR DISCRIMINATION OF RICE CULTIVAR USING SAME}

The present invention relates to a primer combination for identifying a variety of rice varieties and an identification method using the same, and more specifically, 10 pairs of primer combinations capable of quickly and accurately identifying 30 kinds of rice (or rice) varieties and the primer combination The present invention relates to a method for identifying rice varieties according to SNP characteristics specific to each rice variety by analyzing the rice varieties specific PCR amplification characteristics using an automatic genetic analyzer.

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 (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, SJ et al., 1999 Korean J Crop Science 44, 112-116), the identification method of rice varieties using image analysis technology (Kwon, SJ 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, electrophoresis of 10 27 well agarose gels is required for 240 PCR products after the completion of PCR. 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 a plurality of identification markers specific to each rice variety in one PCR reaction, and calculating the result in an automated manner.

The present invention is to provide a primer combination for identifying the rice variety.

In addition, the present invention is to provide a method for quickly and simply identifying the varieties of rice.

The present invention is to solve the above problems, an object of the present invention is to identify the rice varieties designed to achieve the same results as when using each primer pair by using a plurality of primer pairs in one PCR reaction at the same time It is to provide a primer combination.

In addition, an object of the present invention is to amplify two or more SNPs specific to rice varieties in a single PCR reactant with a plurality of primer sets, and automated rice to quickly and simply determine whether rice amplification specific PCR amplification It is to provide a way to identify varieties.

Primer combination for identifying the rice varieties of the present invention and the identification method using the same can not only significantly shorten the PCR process to be carried out for each marker to identify the existing rice varieties, the results are analyzed until the test is completed Since all the test procedures are operated by automatic system, it is very useful in that the error of tester and error of test is low, and it is easy to collect and analyze data and minimize the test time and labor required. 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.

In order to achieve the above object, the present invention provides a primer combination for identifying rice varieties comprising a set of nine pairs of primers comprising the base sequence set forth in SEQ ID NO: 3 to SEQ ID NO: 20.

In addition, the present invention comprises the steps of PCR using a template extracted from rice using a primer combination for identifying the rice varieties; Detecting a fluorescent material labeled on the PCR amplification product by an automatic gene analyzer to identify the size of the PCR product in which the fluorescent material is detected; And identifying a variety of rice according to the size of the PCR product identified in the above step.

Identifying the varieties of rice is carried out according to the following identification criteria:

(1) Nampyeong: PCR amplification products of 284 bp, 473 bp, 484 bp, and 193 bp were detected;

(2) alternative: PCR amplification products of 205 bp, 284 bp, 473 bp, and 193 bp were detected;

During (3): PCR amplification products of 284 bp, 346 bp, and 193 bp were detected;

(4) Dongjin 1: PCR amplification products of 205 bp, 346 bp, 473 bp, 484 bp, and 193 bp were detected;

(5) phase: PCR amplification products of 473 bp, 368 bp, and 193 bp were detected;

(6) phageization: 484 bp, and 193 bp PCR amplification products detected;

(7) Saturn: 284 bp, 473 bp PCR amplification products detected;

(8) Sura: PCR amplification products of 284 bp and 193 bp were detected;

(9) Shin Dong Jin: PCR amplification products of 205 bp, 473 bp, 484 bp, 368 bp, and 193 bp were detected;

(10) False: PCR amplification products of 177 bp, 484 bp and 193 bp were detected;

(11) Japan: 284 bp, 346 bp, 473 bp, 484 bp, 193 bp, and 298 bp PCR amplification products were detected;

(12) gem: 368 bp, and 193 bp PCR amplification products were detected;

(13) Chunam: PCR amplification products of 205 bp, 473 bp, and 193 bp were detected;

(14) neutralization: PCR amplification products of 177 bp, 284 bp, 473 bp and 193 bp were detected;

(15) Recommendation: PCR amplification products of 284 bp, 473 bp and 193 bp were detected;

(16) flower: PCR amplification products of 284 bp, 346 bp, 473 bp, 484 bp, and 193 bp were detected;

(17) Mars: 484 bp, 368 bp, and 193 bp PCR amplification products detected;

(18) Hwayoung: PCR amplification products of 205 bp, 473 bp, 484 bp, and 193 bp were detected;

(19) Daejin: PCR amplification products of 284 bp, 346 bp, 473 bp and 298 bp were detected;

(20) Dongjin: PCR amplification products of 205 bp, 284 bp, 484 bp, 193 bp, and 298 bp were detected;

(21) three thousand: 473 bp, and 193 bp PCR amplification products detected;

(22) Mundo: PCR amplification products of 177 bp, 473 bp, 484 bp, and 193 bp were detected;

(23) saliva: PCR amplification products of 177 bp, 284 bp, and 473 bp were detected;

(24) Huadong: 473 bp, and 298 bp PCR amplification products detected;

(25) sentence: PCR amplification products of 193 bp and 298 bp were detected;

(26) Koshihikari: PCR amplification products of 177 bp, and 193 bp were detected;

(27) Hitomebore: PCR amplification products of 177 bp, 473 bp, 193 bp, and 298 bp were detected;

(28) three light: PCR amplification products of 205 bp, 284 bp, 473 bp, and 484 bp were detected;

(29) resident: PCR amplification products of 177 bp, 284 bp, and 193 bp were detected;

(30) Clouds: PCR amplification products of 205 bp, 346 bp, 473 bp, and 193 bp were detected.

Hereinafter, the present invention will be described in detail.

The present inventors recognize that the current domestic test method, which can detect only one SNP, is inefficient in terms of time, cost, and labor, and thus confirms a combination of primer pairs, PCR conditions, and PCR amplification results that can detect multiple SNPs at once. While studying the method, the PCR conditions that can be a combination of a pair of primers, the combination of the primer pairs that can be performed simultaneously in a single PCR reaction, even if they target different SNPs And to establish a method that can grasp the PCR amplification results at once, to complete the present invention based on this.

Therefore, the present invention performs multiple PCR using 10 pairs of primer combinations that can quickly and accurately identify 30 kinds of rice (or rice), and analyze the size of the PCR product with an automatic genetic analyzer. Thus, the present invention relates to a method for identifying rice varieties based on PCR amplification according to SNP characteristics specific to each rice variety.

Thus, the present invention provides 10 pairs of primer combinations shown in Table 1 below.

Table 1

number Primer Name order Fluorescent marker PCR product size (bp) SEQ ID NO: One CCS-F CAA CGC AAC GTC TTG TAT GG FAM 120 One CCS-R TGA ACC CTG AGT TCC TCT CG 2 2 DK2394-F ATA GAT CTT TGT AAC TCA GAA ACA CA FAM 177 3 DK2394-R AAA TGC TCA ACA AGT GCT TGG 4 3 DK560-F CAA TGG AAT CCC GCC C FAM 193 5 DK560-R ATT AGG CTG CAT ACA TTA GTC ATC C 6 4 DK50-F TTC AAT TCT TAC GAA CAC AAC G FAM 205 7 DK50-R AAC ACC TGT CAA ACA TCT GAT TTA 8 5 DK2401-F CGC TGA ACT AAA CAC AAC CTA TCT A FAM 284 9 DK2401-R CCA ATT CCT GCG TTT TTC C 10 6 DK2171-F AAA AGT GGA TGG CAC ATT GG FAM 298 11 DK2171-R ATA TTG ATT CCT TCA CTA CAT AC 12 7 DK1412-F ATC CGG GGG AGG GAC GAG G FAM 346 13 DK1412-R TAA TAG TTG GAG AGT CGA GAT ATA CG 14 8 DK63-F AAT GAG TGT GGC AGT GTG GAT T FAM 368 15 DK63-R ATG AGA CGA GAA GAC GAC CAG G 16 9 DK17-1-F AGG TAG TCC CTT ATT TTT CCC TAT CC FAM 473 17 DK17-1-R AGA AGT CTT GGT CCT AAG ATA TGT GCT 18 10 DK34-F ATG TCA TGT GGC TTG TCT GG FAM 484 19 DK34-R GCT GCC TAT CCA AGA GAA CG 20

The primers in Table 1 set forth the nucleotide sequences in SEQ ID NOs: 1-20. In Table 1, the marker name is arbitrarily set.

Each primer (or probe) according to the rice cultivar identification marker according to the present invention completely matches one allele, and the 3 'terminal base does not match the non-specific allele. Allele specific primers selectively amplify specific alleles because the 3 'ends that are not matched are stretched by the Tag polymerase at a much lower efficiency than the perfectly matched ends. The presence or absence of the product amplified by PCR using such a rice variety identification marker can be easily analyzed using a detectably labeled probe according to the rice variety identification marker.

The nine pairs of primer combinations according to the present invention are specifically designed to produce uniform PCR amplification results in the same manner as when PCR is performed in one PCR reaction, ie, each primer pair in a separate PCR reaction under the same PCR conditions. Each primer pair is designed to amplify the PCR product in different sizes. Therefore, by identifying the amplified PCR product size, it is easy to know the primer pairs that acted on the amplification. By identifying the rice varieties specific SNP portions targeted by the primers, rice varieties can be easily identified.

Features of the nine pairs of primer combinations according to the present invention are specifically as follows.

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

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

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

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

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

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

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

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. The primer pair specific for CCS1 is a forward primer comprising SEQ ID NO: 1 and a reverse primer comprising SEQ ID NO: 2.

Ten pairs of primer combinations according to the invention can be labeled with detectable means to analyze the size of the PCR amplification products with an automated automated genetic analyzer. The detectable means may be used in the same or different kinds according to each primer pair, and preferably, the same kind of detectable means may be used. In addition, the detectable means can be linked, coupled, or attached to the forward primer, reverse primer, or forward and reverse primer of each primer pair, and inserted into the PCR product at the 5 'end, 3' end of the primer or during PCR amplification. Can be

The detectable means is a compound, biomolecule or biomolecule mimetic that can be labeled, such as linking, binding, or attaching to a primer, or inserted into a PCR product to determine the density, concentration, amount, etc. in a conventional manner. Means. 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. Fluorescent materials are currently available on the market and include 6-carboxyfluorescein (FAM), SYBR Green ^® , VIC ^® , 2 ', 4', 5 ', 7'-tetrachloro-4,7-dichloro Fluorescein (2 ', 4', 5 ', 7'-tetrachloro-4,7-dichlorofluorescein, TET), 2', 7'-dimethoxy-4 ', 5'-dichloro-6-carboxyrodamine (2 ', 7'-dimethoxy-4', 5'-dichloro-6-carboxyrhodamine (JOE), HEX ™, 6-carboxytetramethyl rhodamine (TAMRA), CY3 ™, CY5 ™, ROX ™, RED610 ™, Texas Red ^® , RED670 ™ and NED ™ are available. Examples of the fluorescent material include, but are not limited to. If the fluorescent material has different excitation and emission wavelengths depending on the type, the method of use is also different.

Ten pairs of primers according to the present invention show specific PCR amplification results according to rice varieties, respectively. Therefore, after 10-plex PCR is performed using a combination of 10 pairs of primers, varieties are identified based on specific PCR results. The analysis method of the PCR amplification product used in the present invention is a method of confirming the size of the PCR product labeled with the detected fluorescent material by detecting a detectable means labeled with the PCR amplification product, such as a fluorescent material by laser photosensitization. An example is an automated genetic analyzer. The laser of the device detects the fluorescent material labeled on the amplified PCR product, and implements whether or not the PCR amplification and the product size to the 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. Specific methods and principles will be understood or appreciated by those skilled in the art.

Automatic Genetic Analyzers (Automatic Genetic Analyzer) usable in the present invention include, but are not limited to 377, 3100, 3130, etc. of AB.

Through the primer combination for identifying the rice variety of the present invention and the identification method using the same, it is possible to identify several kinds of rice varieties. 30 kinds of rice, including Japan, Japan, Drama, Chunan, Chinese, Chuchong, Hwabong, Hwaseong, Hwayoung, Daejin, Dongjin, Samcheon, Mundo, Taebong, Hwabong, Heraldry, Koshihikari, Hitomebore, Samgwang, Sasang, and Individual varieties can be identified.

Specific identification methods are as follows (see Table 2).

(1) Nampyeong: When PCR amplification products by primer pairs DK2401, DK17-1, DK34, and DK560 are detected, they are determined to be Nampyeong.

(2) Alternative: When PCR amplification products by primer pairs DK50, DK2401, DK17-1, and DK560 are detected, it is determined as alternative rice.

(3) During: When PCR amplification products by primer pairs DK2401, DK1412, and DK560 are detected, it is judged as rice.

(4) Dongjin 1: When PCR amplification products by primer pairs DK50, DK1412, DK17-1, DK34, and DK560 are detected, it is determined to be Dongjin 1 rice.

(5) Seasoning: When the PCR amplification products by the primer pairs DK17-1, DK63, and DK560 are detected, it is judged as rice.

(6) Birdification: When PCR amplification products by primer pairs DK34 and DK560 are detected, it is determined as bird seedling rice.

(7) Saecheongcheong: When PCR amplification products by primer pairs DK2401 and DK17-1 are detected, saecheongcheong rice is determined.

(8) Sura: When PCR amplification products by primer pair DK2401 and DK560 are detected, it is determined to be Sura rice.

(9) Shindongjin: When PCR amplification products by primer pairs DK50, DK17-1, DK34, DK63, and DK560 are detected, it is determined as shindongjin.

(10) False: When the PCR amplification products by primer pairs DK2394, DK34, and DK560 are detected, it is judged as a large rice.

(11) Japan-US: When PCR amplification products by primer pairs DK2401, DK1412, DK17-1, DK34, DK560, and DK2171 are detected, it is determined as one rice.

(12) One article: When the PCR amplification products by the primer pair DK63 and DK560 are detected, it is determined as one article rice.

(13) Chumnam: When PCR amplification products by primer pairs DK50, DK17-1, and DK560 are detected, it is judged as rice.

(14) Neutralization: When PCR amplification products by primer pairs DK2394, DK2401, DK17-1, and DK560 are detected, it is determined to be neutralized rice.

(15) Recommendation: When PCR amplification products by the primer pairs DK2401, DK17-1, and DK560 are detected, it is determined as a rice seed.

(16) Flower bud: When PCR amplification products by primer pair DK2401, DK1412, DK17-1, DK34, and DK560 are detected, it is determined as flower bud rice.

(17) Mars: When the PCR amplification products by the primer pairs DK34, DK63, and DK560 are detected, it is determined as martian rice.

(18) Hwayoung: When PCR amplified products by primer pairs DK50, DK17-1, DK34, and DK560 are detected, Hwayoung is determined.

(19) Daejin: When PCR amplification products by primer pairs DK2401, DK1412, DK17-1, and DK2171 are detected, it is determined as Daejin rice.

(20) Dongjin: When PCR amplification products by primer pairs DK50, DK2401, DK34, DK560, and DK2171 are detected, it is determined to be Dongjinbyeong.

(21) Three thousand: When PCR amplification products by primer pair DK17-1 and DK560 are detected, it is determined to be three thousand rice.

(22) Mung Bean: When PCR amplification products by primer pairs DK2394, DK17-1, DK34, and DK560 are detected, it is determined as mungbean rice.

(23) Taebong: When PCR amplification products by primer pairs DK2394, DK2401, and DK17-1 are detected, it is determined as Taebong rice.

(24) Flower copper: When PCR amplification products by primer pair DK17-1 and DK2171 are detected, it is determined as flower bud rice.

(25) Sentence: When the PCR amplification product by primer pair DK560 and DK2171 is detected, it is judged by sentence.

(26) Koshihikari: When PCR amplification products by primer pair DK2394 and DK560 are detected, it is judged as Koshihikari.

(27) Hitomebore: When PCR amplification products by primer pairs DK2394, DK17-1, DK560, and DK2171 are detected, it is determined as Hitomebore rice.

(28) Three light: When the PCR amplification products by the primer pairs DK50, DK2401, DK17-1, and DK34 are detected, it is determined as three light rice.

(29) Sasangju: If PCR amplification products by primer pairs DK2394, DK2401, and DK560 are detected, it is determined as sasangjub.

(30) Clouding: When PCR amplification products by primer pairs DK50, DK1412, DK17-1, and DK560 are detected, it is determined as clouding rice.

(Table 2)

By simultaneously using the 10 pairs of primer combinations for the identification of rice varieties of the present invention, not only can significantly shorten the PCR process to be performed for each marker to identify the existing rice varieties, but also the results are automated. Can be quickly verified. 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  Real time with sets PCR

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 avoid contamination of the sample, place it on oil paper and grind the rice sample into tiny pieces 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) and nuclear fusion buffer (CTAB 20 g, 1M Tris pH 8.0) 200 ml, 0.25 200 mL M EDTA, 400 mL 5M NaCl) are mixed 1: 1 and 0.2 volume 10% sarcosyl, 0.2 volume saline, 1% 2-mercaptoethanol is mixed in a 65 ° C. constant temperature water bath. After centrifugation at 14,240 × g (15,000 rpm) for 10 minutes, mix 1 volume of 3.8 g / L sodium bisulfite and 1 volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) in the supernatant Leave for minutes, then centrifuge for 5 minutes at 14,240 × g. 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, mix well for 5 minutes, keep at -20 ° C for 20 minutes and centrifuge for 4 to 15 minutes. Discard the supernatant and centrifuge for 5 min at 4 ° C by adding 200 μl of 70% ethanol to the DNA pellet. Discard the supernatant and dry the remaining ethanol with air or vacuum dryer. 65 μl of sterile distilled water and 15 μl of RNase A (1 mg / ml) were added thereto and placed in a 37 ° C. water bath for 15 minutes.

1-2. 10-plex PCR reaction

50 ng template DNA, 2 μl of a mixture of 10 to 5 pmole pairs of primers, 4 μl of 5X PCR reaction buffer (50 mM Tris-HCl, 250 mM KCl, 10 mM MgCl 2, 10 mM dNTP), 0.8 μl of Taq polymerase (1.25 U) / ul), and mix the reaction solution in a 20 μl volume containing ddH2O. The PCR reaction was denatured at 94 ° C. for 10 minutes, repeated 32 times in a series of reactions (20 sec at 94 ° C., 20 sec at 58 ° C., 20 sec at 72 ° C.), extended at 45 ° C. at 60 ° C. and terminated at 8 ° C. It was. Use PCR devices such as Biometra T-3000, MJ Research PTC-200, AB GeneAmp PCR system 9600, 9700, 2720

1-3. Analysis of PCR Products Using the Automatic Genetic Analyzer

Genotyping devices such as AB's Automatic Genetic Analyzer 377, 3100, 3130 can be used. In order to analyze the PCR result, 1 μl of PCR product was applied with Hi-Di Formamide ( Applied). Biosystems ) 9.3 μl and GenScan-500LIZ Size standard (Applied Biosystems) are mixed and run on AB Automatic Genetic Analyzer 3130. The laser of the instrument detects the fluorescent material labeled on the primer of the amplified PCR product and analyzes the result. Finally, the GeneMapper ID software built into the instrument is run to automatically identify rice varieties based on the size of the PCR product for each SNP marker.

Example  2: Rice Varieties Identification

After synthesizing the primers of SEQ ID NOS: 1-20, 6-carboxyfluorescein (FAM) was labeled on the forward primer. Each primer set is designed to amplify PCR products of different sizes.

With rice sample, Nampyeong, Alternative, During, Dongjin 1, Sangmi, Saehwahwa, Saecheongcheong, Sura, Sindongjin, Odae, Ilmi, One Piece, Junnam, China, Chucheong, Hwabong, Hwaseong, Hwayoung, Daejin, Dongjin, Samcheon, Undo Thirty varieties of rice varieties were used: Taebong, Hwadong, Heraldry, Koshihikari, Hitomebore, Samgwang, Sasangju, and Meteorite. Each rice sample was extracted according to the method of Example 1, 10-plex PCR was performed, and then analyzed by the AB Automatic Genetic Analyzer to determine whether the PCR amplification and the size of the amplification products, the programmed rice varieties specific Rice varieties were analyzed as compared to PCR amplification product size combinations (Table 2).

The results are shown in FIGS. 1A-1E. According to Figs. 1A to 1E, the PCR results of each sample rice were compared with the results described in the programmed rice varieties determination table, and the sample rice varieties used and the rice varieties identified by PCR were 100% identical.

1a to 1e is a 10-plex PCR using a combination of 10 pairs of primers for identifying rice varieties, rice varieties Nampyeong, alternative, while, Dongjin 1, Sangmi, Saegyehwa, Saechucheong, Sura, Sindongjin, Odae, Ilmi, Dish It shows the results of Joonam, Chinese, Chucheong, Hwabong, Hwaseong, Hwayoung, Daejin, Dongjin, Samcheon, Undo, Taebong, Hwadong, Heraldry, Koshihikari, Hitomebore, Samgwang, Sasangju and Meteorite. SNP amplification is seen.

<110> NATIONAL AGRICULTURAL PRODUCTS MANAGEMENT SERVICE <120> PRIMER COMBINATION FOR DISCRIMINATION OF RICE CULTIVAR AND METHOD          FOR DISCRIMINATION OF RICE CULTIVAR USING SAME <160> 20 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 1 caacgcaacg tcttgtatgg 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 2 tgaaccctga gttcctctcg 20 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 3 atagatcttt gtaactcaga aacaca 26 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 4 aaatgctcaa caagtgcttg g 21 <210> 5 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 5 caatggaatc ccgccc 16 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 6 attaggctgc atacattagt catcc 25 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 7 ttcaattctt acgaacacaa cg 22 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 8 aacacctgtc aaacatctga ttta 24 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 9 cgctgaacta aacacaacct atcta 25 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 10 ccaattcctg cgtttttcc 19 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 11 aaaagtggat ggcacattgg 20 <210> 12 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 12 atattgattc cttcactaca tac 23 <210> 13 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 13 atccggggga gggacgagg 19 <210> 14 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 14 taatagttgg agagtcgaga tatacg 26 <210> 15 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 15 aatgagtgtg gcagtgtgga tt 22 <210> 16 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 16 atgagacgag aagacgacca gg 22 <210> 17 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 17 aggtagtccc ttatttttcc ctatcc 26 <210> 18 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 18 agaagtcttg gtcctaagat atgtgct 27 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 19 atgtcatgtg gcttgtctgg 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 20 gctgcctatc caagagaacg 20  

Claims (9)

delete delete delete delete delete delete delete SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: DNA extracted from rice using a primer combination for identifying rice varieties including all 15 pairs of primer sets having the nucleotide sequences of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20 Conducting PCR with; Detecting a fluorescent material labeled on the PCR amplification product by an automatic gene analyzer to identify the size of the PCR product in which the fluorescent material is detected; And Identifying the varieties of rice according to the size of the PCR product identified in the step; comprising, rice varieties identification method using a combination of rice varieties identification. The method of claim 8, wherein the identifying the varieties of rice is performed according to the following identification criteria: (1) Nampyeong: PCR amplification products of 284 bp, 473 bp, 484 bp, and 193 bp were detected; (2) alternative: PCR amplification products of 205 bp, 284 bp, 473 bp, and 193 bp were detected; During (3): PCR amplification products of 284 bp, 346 bp, and 193 bp were detected; (4) Dongjin 1: PCR amplification products of 205 bp, 346 bp, 473 bp, 484 bp, and 193 bp were detected; (5) phase: PCR amplification products of 473 bp, 368 bp, and 193 bp were detected; (6) phageization: 484 bp, and 193 bp PCR amplification products detected; (7) Saturn: 284 bp, 473 bp PCR amplification products detected; (8) Sura: PCR amplification products of 284 bp and 193 bp were detected; (9) Shin Dong Jin: PCR amplification products of 205 bp, 473 bp, 484 bp, 368 bp, and 193 bp were detected; (10) False: PCR amplification products of 177 bp, 484 bp and 193 bp were detected; (11) Japan: 284 bp, 346 bp, 473 bp, 484 bp, 193 bp, and 298 bp PCR amplification products were detected; (12) gem: 368 bp, and 193 bp PCR amplification products were detected; (13) Chunam: PCR amplification products of 205 bp, 473 bp, and 193 bp were detected; (14) neutralization: PCR amplification products of 177 bp, 284 bp, 473 bp and 193 bp were detected; (15) Recommendation: PCR amplification products of 284 bp, 473 bp and 193 bp were detected; (16) flower: PCR amplification products of 284 bp, 346 bp, 473 bp, 484 bp, and 193 bp were detected; (17) Mars: 484 bp, 368 bp, and 193 bp PCR amplification products detected; (18) Hwayoung: PCR amplification products of 205 bp, 473 bp, 484 bp, and 193 bp were detected; (19) Daejin: PCR amplification products of 284 bp, 346 bp, 473 bp and 298 bp were detected; (20) Dongjin: PCR amplification products of 205 bp, 284 bp, 484 bp, 193 bp, and 298 bp were detected; (21) three thousand: 473 bp, and 193 bp PCR amplification products detected; (22) Mundo: PCR amplification products of 177 bp, 473 bp, 484 bp, and 193 bp were detected; (23) saliva: PCR amplification products of 177 bp, 284 bp, and 473 bp were detected; (24) Huadong: 473 bp, and 298 bp PCR amplification products detected; (25) sentence: PCR amplification products of 193 bp and 298 bp were detected; (26) Koshihikari: PCR amplification products of 177 bp and 193 bp were detected; (27) Hitomebore: PCR amplification products of 177 bp, 473 bp, 193 bp, and 298 bp were detected; (28) three light: PCR amplification products of 205 bp, 284 bp, 473 bp, and 484 bp were detected; (29) resident: PCR amplification products of 177 bp, 284 bp, and 193 bp were detected; (30) Clouds: PCR amplification products of 205 bp, 346 bp, 473 bp, and 193 bp were detected.

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