KR100713669B1 - The SNP marker for discrimination of Korean and foreign rice cultivar - Google Patents

Abstract

The present invention relates to a varieties identification marker that can be distinguished by the varieties of domestic and foreign rice and varieties identification analysis method using the same, 112 domestic products including 25 varieties of domestic cultivated varieties of limited domestic varieties using the varieties identification marker Can identify varieties and 34 varieties from foreign countries.

Allele-specific polymerase chain reaction, primer, rice cultivar, SNP, insertion and deletion, sequencing

Description

The SNP marker for discriminating domestic and foreign rice varieties {The SNP marker for discrimination of Korean and foreign rice cultivar}

1A is a diagram showing a primer synthesis schematic for allele specific PCR.

FIG. 1B is a diagram showing an example of primer preparation that causes an artificial mutation in the nucleotide sequence with a mismatch at the 3 'end in order to increase the efficiency of allele-specific PCR.

2 is a diagram illustrating a variety identification marker for identifying domestic and foreign rice varieties.

Figure 3 is a diagram showing the identification table for domestically restricted rice varieties (28 pieces) using 20 rice varieties identification markers.

The present invention relates to a varietal identification marker that can identify a single base difference of each type of varieties in the rice nucleotide sequence, and to a varietal identification marker that can be distinguished by the varieties of domestic and foreign rice varieties, and to a rice variety identification method using the same.

Rice is a plant (Gramineae) and genus Oryza, whose chromosomes belong to the n = 12, 2n = 24, and AA genomes. The genome has a size of 430 Mbp, and there are about 60,000 species according to the Int'l Rice Research Institute (IRRI). According to the 2004 Ministry of Agriculture, there are 113 varieties and 195 breeding varieties supplied by the government. There are two species of cultivated rice: Asian rice (O. sativa) and African rice (O. glaberrima). Among them, Asian rice (O. sativa) is more widely cultivated worldwide.

In many societies, rice is closely related to culture as well as to food and economics. Three billion people share culture, tradition and potential (www.fao.org). In 2003, world rice production was about 38 billion tons. More than 50 countries produce more than 100,000 tonnes per year, contributing to the world's total output. Asian farmers produce 91% of the total production, with China and India producing 55% of the total. More than 5% of the world's rice production (23,044 tons in 2003) is traded internationally. The main exporters of rice are Thailand, the United States, Vietnam, Pakistan and India (www.krei.re.kr, www.riceweb.org). Korea's rice production amounted to 3,444 million tons in 2002, and the minimum import volume (MMA) has increased up to 154 tons since 1995, but is gradually decreasing (www.krei.re.kr).

In order to encourage the production of high-quality rice, our country operates a government limited purchase system.

Prior to the Doha Development Agenda for opening up the rice market, the Korean government has implemented a government-limited purchasing system in 2004 to encourage the production of high-quality rice, and selected three varieties by region to purchase 28 varieties. Was carried out.

According to the Ministry of Agriculture and Forestry (RDA), Nampyungbyeo was grown in most areas than Dongjin1byeo, Chuchungbyeo, Junmbyeo, and Ilmibyeo.

In addition, the package grain labeling system, which displays information on rice varieties on packaging, was implemented in January 2004. The Ministry of Agriculture and Forestry promoted the establishment of high quality rice and distribution order through the implementation of 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, identifying rice varieties has been an important issue. Research on rice has recently been carried out by genome sequencing of Nipponbare varieties through the International Rice Genome Sequencing Project (IRGSP), which includes 10 countries. , 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, and 8% have not yet been identified.

Recently, many studies have been conducted to identify rice varieties using various DNA markers. To date, random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP) and microsatellite methods have been used to identify varieties. For example, Wang et al. Identified varieties using restricted fragment length polymorphism (RFLP) markers (Wang, ZY, Tanksley, SD 1989 L. Genome. 32, 1113-1118), and Ryu used protein capillary electrophoresis. Varieties were identified (Ryu, DJ 1998 J. food science and nutrition. 3, 43-347). In addition, 51 varieties of Japonica rice were identified by using six microsatellite markers to confirm their location on the genetic map (Ji, H.S. et al., 1998 Korean J. Breed. 30, 350-360). 40 domestic varieties were identified by RAPD, microsatellite, sequence tagged site (STS), and polymerase chain reaction (PCR) methods, and the advantages and disadvantages of each method were reported (Jeong OY et al., Korean 1998 J Breed. 30, 136-137). We identified 31 rice varieties using RAPD and microsatellite (Kwon, SJ et al., 1999 Korean J Crop Science 44, 112-116), and reported methods for identifying rice varieties using image analysis technology ( Kwon, SJ et al., 1998 Korean J Crop Science 43, 33-34). However, these identification methods and imaging techniques cannot form specific single bands, making it difficult to identify individual varieties from mixed varieties and to discuss the results of the analysis.

The present invention has been proposed in order to solve the problems of the prior art as described above, an object of the present invention is to provide a marker for identifying varieties that can be distinguished by varieties between domestic and foreign rice from each other and a rice variety identification method using the same.

In order to achieve the above object, the present invention is at least one nucleotide sequence selected from the marker for identifying the cultivars represented by SEQ ID NOS: 1 to 40, which can identify the cultivated rice variety, the base sequence from the 3 'end Provided is a marker for identifying a rice variety in which at least one of the bases in positions 2-4 is selected from the modified sequences.

The present invention also provides a marker for identifying a rice variety in which the base is mutated from the 3 'end of the base in the marker for identifying the variety represented by SEQ ID NOS: 1-40.

In addition, the present invention provides a marker for identifying a rice variety consisting of a modified sequence in which any one of the bases 2 to 4 position from the 3 'end in the base sequence is mutated to any one of A, T, G, C. .

In addition, the present invention comprises the steps of separating the genomic DNA (genomic DNA) of rice; At least one nucleotide sequence selected from the marker for identification of the cultivars represented by SEQ ID NOs: 1 to 40, wherein the nucleotide sequence is mutated from bases 2 to 4 from the 3 'end Amplifying each specific region for each variety using at least one rice variety identification marker selected from the modified sequences; And after confirming the specific gene for each amplified variety by electrophoresis, and provides a rice variety identification method comprising the step of identifying each variety through the analysis of the combined bands.

Hereinafter, the content of the present invention in more detail as follows.

In order to develop the marker according to the present invention, SNP positions were searched through sequencing of 80 CAPS for 28 domestic proto-origin varieties, which were purchased as targets, and a modified allele-specific PCR primer was prepared by using the same. SNP markers were developed. According to the analysis, 15 are SNP markers and 5 are InDel markers. Herein, the SNP marker refers to a marker in which a 3'-terminal base is exactly matched to one allele, but is substituted with a base that is mismatched to a specific allele, and an InDel marker is a 3'-terminal. It refers to markers that have bases that match exactly to the allele, but that the 3'-terminal base is inserted or deleted to mismatch the specific allele.

The allele specific primer (or marker) according to the present invention is completely identical to one specific allele, and does not match the 3 'terminal base with 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 in the general agarose gel.

In addition, the marker for identifying a variety according to the present invention may impart a mutation to at least one or more bases so long as it does not affect PCR amplification with the original allele. Preferably, at least one or more selected from the 2-4th base from the 3 'end is mismatched, and more preferably, the third base is mutated from the 3' end.

The nucleotide sequence shown in SEQ ID NOs: 1 to 40 represents the nucleotide sequence of the marker for identifying the rice variety presented as a preferred embodiment of the present invention. Odd sequences all represent anterior primers, even sequences represent posterior primers. These are all 3 'terminal bases are located at the SNP or InDel site of the allele, and the base located 3rd from the 3' end is substituted so as to mismatch in certain alleles. These sequences can be selectively amplified by a PCR process in one allele, but may not be amplified for any other allele. By using these characteristics, it is possible to accurately identify rice varieties using various combinations of the rice variety identification markers according to the present invention.

The marker for identifying the rice variety according to the present invention represented by SEQ ID NOs: 1 to 40 is designed for breed identification for japonica grown rice (rice). Japonica rice varieties are domestic and foreign varieties of subspecies under the scientific name Oryza sativa 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 in 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, which is present in individual rice varieties. Finding the SNP or InDel position, and using this position was prepared as an allele specific primer that can be identified by breed (see Fig. 2).

Hereinafter, the characteristics of each marker represented by SEQ ID NOs: 1 to 40 are as follows.

DK2511: present on chromosome 2, and the site used for sequencing is the OJ1136_C12 clone (SEQ ID NO: 1 (front directional primer), 2 (back directional primer)).

DK6: present on chromosome 11 and site of the site used for sequencing is the AC119073 clone (SEQ ID NOs: 3 (front directional primer), 4 (back directional primer)).

DK17-1: present on chromosome 8 and site of the site used for sequencing is AP003886 clone (SEQ ID NO: 5 (front directional primer), 6 (back directional primer)).

DK17: present on chromosome 8 and site of site used for sequencing is AP003886 clone (SEQ ID NO: 7 (front directional primer), 8 (back directional primer)).

DK50: present on chromosome 11 and site of the site used for sequencing is the AC145367 clone (SEQ ID NO: 9 (forefront primer), 10 (backward primer)).

DK63: located on chromosome 3, the site used for sequencing is the AC135495 clone (SEQ ID NOs: 11 (front directional primer), 12 (back directional primer)).

DK560: present on chromosome 12 and site of the site used for sequencing is BX000560 clone (SEQ ID NO: 13 (front directional primer), 14 (back directional primer)).

DK1361: present on chromosome 10 and site of the site used for sequencing is the AC079888 clone (SEQ ID NOs: 15 (front directional primer), 16 (back directional primer)).

DK1412: present on chromosome 7 and site of site used for sequencing is the AP005198 clone (SEQ ID NO: 17 (front directional primer), 18 (back directional primer)).

DK26: present on chromosome 1, and the site used for sequencing is the AP003022 clone (SEQ ID NO: 19 (front directional primer), 20 (back directional primer)).

DK372: Located on chromosome 1, the site used for sequencing is the AP003432 clone (SEQ ID NOs: 21 (front directional primer), 22 (back directional primer)).

DK2394: present on chromosome 7 and site of site used for sequencing is the AP003866 clone (SEQ ID NOs: 23 (front directional primer), 24 (back directional primer)).

DK2401: present on chromosome 7 and the site used for sequencing is the AP004010 clone (SEQ ID NOs: 25 (forward orientation primer), 26 (forward orientation primer)).

DK2708: located on chromosome 12 and site of the site used for sequencing is AL731784 clone (SEQ ID NO: 27 (front directional primer), 28 (back directional primer)).

DK721: located on chromosome 3, the site used for sequencing is the AC119747 clone (SEQ ID NOs: 29 (front directional primer), 30 (back directional primer)).

DK34: Located on chromosome 1, the site used for sequencing is the AP003209 clone (SEQ ID NOs: 31 (forward primer), 32 (forward primer)).

DK847: present on chromosome 7 and site of the site used for sequencing is the AP005198 clone (SEQ ID NOs: 33 (front directional primer), 34 (back directional primer)).

DK1854: Located on chromosome 4, the site used for sequencing is the AL606616 clone (SEQ ID NOs: 35 (front-side primer), 36 (back-side primer)).

DK601: Located on chromosome 2, the site used for sequencing is the AP005284 clone (SEQ ID NOs: 37 (front directional primer), 38 (back directional primer)).

DK600: Located on chromosome 4, the site used for sequencing is the AL606660 clone (SEQ ID NO: 39 (front directional primer), 40 (back directional primer)).

Each of the primers is used alone or in combination of two or more as a marker for identifying rice varieties, the size of the PCR product obtained by using the primers is designed to about 123 ~ 540bp.

Each primer or rice variety identification method using the same includes the following process.

For identification of rice varieties, genomic DNA is first isolated from rice seed, and genomic DNA is used as a template to amplify specific regions for each variety using markers for identifying rice varieties. After confirming the specific gene for each amplified variety by electrophoresis, each variety can be identified through analysis of the combined bands. In this case, the combined bands may be arranged as a diagram in advance through individual experiments. That is, as shown in Fig. 3, each rice variety can be determined by tabulating the presence or absence of PCR products by markers for identifying each variety of rice varieties and analyzing the results.

In the identification process for the rice variety of the present invention, a specific gene capable of confirming PCR amplification may be used. For example, genomic DNA present at the AB013614 clone can be used as a CCS1 similar to the Cereal Centromeric Sequence in rice as an endogenous gene.

For example, a more detailed example of the breed identification by the rice variety identification marker of the present invention, it is possible to identify the Saechuchungbyeo and Chuchungbyeo varieties by the use of DK560 out of 20 developed SNP markers. In addition, the use of DK6 can discriminate between varieties of Nampyungbyeo and Daeanbyeo. In addition, the use of DK63 and DK560 can distinguish the varieties of Saechuchungbyeo and Surabyeo.

In addition, the use of DK6 and DK17-1 among the SNP markers developed in the present invention can distinguish the varieties of Dongjin 1byeo and Sangmibyeo. In addition, the use of DK63, DK1412, and DK26 can distinguish varieties of Donganbyeo, Dongjin1byeo, and Snagmibyeo.

Also, among the SNP markers developed in the present invention, varieties of Snagmibyeo, Saegaehwabyeo, and Saechuchungbyeo can be distinguished from each other by using DK26, DK721, and DK34.

As in the above example, from two or more combinations of twenty SNP markers, it is possible to identify between each variety of cultivated rice.

In the present invention, as a result of experiments on 28 varieties of varieties limited purchase agent using 20 SNP markers, it was possible to individually identify the 25 varieties except Ilmi rice, Hwabong rice, Yeongnam rice and the content of individual varieties in the mixed sample Could confirm. More specifically, only nine of the 20 SNP markers (DK2394, DK50, DK2401, DK1412, DK17-1, DK34, DK63, DK560, DK2511) can be used to identify the 25 major varieties of government purchase. It was possible.

In addition, in the present invention, individual markers were identified for 146 varieties in a specificity test for 173 rice varieties including 124 domestic native rice varieties and 49 foreign rice varieties using 20 rice varieties for identifying markers. . Among 124 domestic varieties, 112 varieties except 12 varieties were identified, and 49 foreign varieties (1 from Australia, 25 from China, 7 from Japan, 1 from Russia, 1 from Egypt, 1 from India, In Pakistan, 5 Philippines, and 7 unidentified), 34 cultivars except 15 cultivars were identified. Although the ability to identify foreign varieties is lower than that of domestic varieties, the use of the marker according to the present invention developed in domestic varieties is not suitable due to the genetic diversity between domestic and foreign varieties, but there is a possibility for future distinction between domestic and foreign varieties. Showed.

In addition, according to the present invention, the similarity of genes to 28 domestic rice varieties was 99.1 to 100%, and the 28 domestic varieties showed a difference of 0.05% between the respective varieties.

Based on these results, it can be seen that the markers for identifying rice varieties according to the present invention can be used as markers for identifying rice varieties quickly, low cost and effectively, and as markers for distinguishing them from foreign varieties. have. Mixed breeds also have the advantage of identifying individual breed names. According to the present invention, the rice variety identification method using the marker is suggested as a method for stable breed identification by base sequence information, suggesting that it is a useful method for developing a variety identification method for other crops.

Hereinafter, the configuration and effects of the invention will be described in more detail with reference to the following examples. However, the present invention is not limited to these examples.

Example 1 Extraction of Genomic DNA from Rice Seed

1) plant sample

The development of markers for identifying rice varieties and the development of markers for 173 rice varieties (124 varieties in Korea and 49 varieties in foreign countries: 2004 produced in 2004) received from the National Institute of Crop Science (NICS) in the present invention It was used for the test.

Of the 124 varieties in Korea, 28 varieties were used to develop markers, which is the kind of government limited purchase system. Specificity tests were performed with markers developed for the remaining 96 varieties.

2) Extraction of Genomic DNA

0.03g of rice was so small that it was impossible to extract DNA with a DNA extraction kit sold. In order to solve this problem, a CTAB method, which is generally used to extract DNA from plants, was modified and used in the present invention.

CTAB is a method of extracting and purifying DNA from a mixture of cetyl trimethyl ammonium bromide and phenylchloroform (Murray, MG and WF Thompson. 1980 Nuc.Acids Res 8, 4321-4325, Pietsch et al., 1997 Lebensmittel Runds 93, 35-38). This method has the advantage that the purity of DNA is good and the range of use is widely applied.

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) was put in a 1.5 ml tube, 500 µl of CTBA solution was added, mixed well, and soaked in a 65 ° C constant temperature water bath for 30 minutes. The composition of the CTAB solution is as follows: DNA extraction buffer (Sorbitol 63.75g, Tris-base 12.1g, EDTA-Na2 1.68g / 1ℓ) and nuclei lysis buffer (CTAB 20g, 1M Tris (pH 8.0) 200ml 0.25M EDTA 200 mL 5M NaCl 400 mL / 1 L) was mixed 1: 1, and 0.2 volume 10% sarcosyl, 0.2 volume saline, and 1% 2-mercaptoethanol were mixed in a 65 DEG 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 at room temperature. After standing for 5 minutes, centrifugation was performed for 5 minutes at 14,240 × g. The supernatant was transferred to a 1.5 mL tube, and 1 volume of chloroform: isoamyl alcohol (24: 1) was mixed and rotated at room temperature for 5 minutes. The supernatant was transferred to a 1.5 ml tube by centrifugation at 14,240 × g for 5 minutes. The above procedure was repeated until the interface became clear. Add the same amount of isopropanol and mix well for 5 minutes, put in 20 minutes at -20 ℃ and centrifuged for 15 minutes at 4 ℃. The supernatant was discarded and centrifuged at 4 ° C. for 5 minutes by adding 200 μl of a pellet to 70% of a DNA pallet. The supernatant was discarded and the remaining ethanol was well dried by wind or vacuum dryer. To dissolve DNA, 65 μl of sterilized distilled water and 15 μl of RNase A (1 mg / ml) were added and placed in a 37 ° C. water bath for 15 minutes.

Unlike extracting DNA from leaves, it is not easy to extract genomic DNA from rice seeds because it is not possible with a kit to extract DNA from rice grains (0.03g). Purified DNA yielded approximately 100 ng / μl according to some modifications of the CTAB method (Murray et al., 1980 Nuc. Acids Res 8, 4321-4325) to obtain large amounts of purified DNA. The rain was above 1.8.

Example 2 Historically Diverse Varieties of 28 Domestic Rice Varieties

Genetic Diversity Analysis

Twenty-two positions of the DNA sequences of 28 domestic chromosomes were compiled by the DNASTAR computer program, and the sequences were sequenced using the CLUSTAL W (Thompson et al., 1994 Nucleic Acids Res. 22, 4673-4680) program. It was.

 NJ (neighbor-joining) method was used for phylogenetic analysis (Saitou and Nei, 1987). For NJ analysis, the results were first analyzed using a Kimura-2 parametric ranging model, and MEGA version 2.1 was used to draw the NJ tree (Kumar et. Al., 2001 MEGA2 Arizona State University, Tempe, Arizona, USA). ). To determine support for each branch, a bootstrap analysis was conducted with 1,000 replicates.

The similarity of genes to 28 varieties of domestic rice was high from 99.1 to 100%. The bootstrap value is very low at the phylogenetic tree 'branch point. It is generally known that stable grouping is performed when the bootstrap value is 70% or more. As a result, 28 domestic varieties show a difference of 0.05% between the respective varieties, which means that the similarity is very high. This indicates that each variant is not classified into a stable group.

Example 3 Development of Single Nucleotide Polymorphism (SNP) Marker

1) Positioning for sequencing and primer design

The International Rice Genome Sequencing Project (IRGSP) identified the entire sequence of Nippon Barre varieties in 2002 (IRGSP, http // rgp.dna.affrc.go.jp). Syngenta (the world's first agricultural company) analyzed a draft of the entire base of Nippon Barre (Goff et al., 2002). Based on the Nippon Barre sequencing, primers were prepared for sequencing to completely and specifically identify rice.

From the homepage of the rice genome research program (RGP) (http://rgp.dna.affrc.go.jp) information, the CAPS (cleaved amplified polymorphic sequence) and Genebank's analytical clones were selected. Primers were prepared using the web site (http://www.gramene.org) and Primer3 Program (http://frodo.wi.mit.edu). DNASTAR was used to confirm the location of the restriction enzyme and sequence analysis at 80 positions on the chromosome.

2) Construction of Control Gene Specific PCR Primer

The ClustalW method was analyzed using the DNASTAR computer program (www.dnastar.com) to find SNP locations.

InDel markers were analyzed by amplifying DNA fragments containing InDel with two flanking primers. Genotypes were determined by the presence or absence of PCR products on agarose gels. The allele-specific PCR method was modified to produce SNP primers in the present invention (FIG. 1A) (Drenkard, E. et al., 2000 Plant Physiol 124, 1483-1492, Kwok, S. et al., 1990 Nucleic Acids Res 18, 999-1005).

In the present invention, a modification in which any one or two bases of the bases at positions 2 to 5 are modified from the 3 'end to prepare primers for allele-specific PCR for developing SNP markers for identifying rice varieties. Sequences were prepared (FIG. 1A, FIG. 1B). According to this, the modified sequence in which any one of the bases 2 to 4 from the 3 'end of the base sequence was mutated to any of A, T, G, and C was used as a marker for identifying rice varieties. It was confirmed that it is possible to identify with other varieties without disturbing the amplification.

In addition, in developing the marker for identifying rice varieties in the present invention, the SNP or InDel position is the 3 'end, and a sense primer and an antisense primer were prepared to induce artificial inconsistency in the third nucleotide. The binding temperature of the SNP marker was prepared to be 60 ℃, length 123 ~ 540 bp.

In the present invention, 20 markers for identifying rice varieties were made to identify 28 domestic rice varieties (FIG. 2).

In addition, to confirm PCR amplification, a CCS1 primer (SEQ ID NO: 41, 42) similar to the Cereal Centromeric Sequence in rice was prepared as an internal standard, and is present in the AB013614 clone position.

Example 4 Specificity Assay of Markers

1) Polymerase Chain Reaction for Sequencing

For DNA amplification, GeneAmp 9700 (Applied Biosystems) (Kleppe et. Al., 1971: Lawyer et al., 1989; Saiki et al., 1985), HotStart Taq polymerase (Takara Hotstart Taq, 5 units / μl) was used It was. It consists of 10 × PCR buffer (Tris-HCl (pH 8.0) 20 mM, KCl 100 mM and MgCl 2 2 mM), dNTPs (2.5 mM), Taq DNA polymerase. The appropriate concentration of each primer is 1.25 μM. The reaction mixture contained 1 × PCR buffer, 0.2 mM of dNTP, 40 ng / μl template, and 0.025 unit of Taq polymerase in 30 μl of reaction solution.

DNA amplification conditions were as follows. DNA was denatured for 2 minutes and a series of reactions (20 sec at 94 ° C., 20 sec at 60 ° C., 30 sec at 72 ° C.) was repeated 30 times. Cool to 4 ° C. and complete the reaction. To confirm the amplification result, add 1 μl of PCR reaction solution 5 μl loading buffer (0.25% bromophenol blue, 0.25% xylene cyano FF, 15% Ficoll) into a 2% agarose gel and load 100V (5-20V / cm) electrophoresis for 20 minutes. Electrophoresis was stopped when BPB (Bromophenol Blue) reached the midpoint of the gel and photographed with a CCD camera on a UV trans-illuminator. One band was formed and compared to DNA molecular weight markers. If there were two or no bands, no sequencing was done. In addition, it was confirmed whether the PCR amplification using CCS1 as an internal standard in the present invention.

2) Purification of polymerase chain reaction product

For sequencing, PCR products were purified using QIAquick PCR Purification Kit (Quagen.Co.). 5 times PB solution was added to the PCR product and attached to the column. After centrifugation at 14,240 ㅧ g for 1 minute, 700 μl of PE solution was added and centrifuged at 14,240 × g for 1 minute. The column was transferred to a 1.5 ml fresh tube, and 30 µl of sterilized distilled water was kept at room temperature for 1 minute. Elution was carried out by centrifugation at 14,240 × g for 1 minute, which was used as a template for sequencing. The purified PCR product was confirmed by PCR marker on 2% agarose gel by electrophoresis.

3) Sequence Analysis of Rice Varieties

The nucleotide sequence of the domestic varieties was determined from the PCR amplified fragment (approximately 500 bp) using the ABI310 sequencer (ABI Biosystems, USA) with the BigDye Terminator v3.1 Cycle Sequencing kit (ABI 0401041, Foster, CA, USA). . At this time, the appropriate concentration of the primer is 0.16μM, and the reaction mixture contains 20μl of the template DNA 10 ng / μl, 8 units of BigDye terminator. Each PCR product was sequenced in a single or bidirectional manner. To design PCR primers, Nippon Barre genomic DNA sequence data from the Ganmene / Genbank database was used.

4) Specificity Assay of Markers

GeneAmp 9700 (Applied Biosystems), HotStart Taq polymerase (Takara Hotstart Taq, 5 unit / μl) was used to amplify DNA. HotStart Taq polymerase (Takara Hotstart Taq, 5 unit / μl) consists of 10 × PCR buffer (Tris-HCl (pH 8.0) 20mM, KCl 100mM, MgCl ₂ 2mM), dNTPs (2.5mM) and Taq DNA polymerase have.

DNA amplification conditions were as follows. DNA was denatured for 1 minute at 96 ° C. and a series of reactions were repeated 32 times (10 sec at 96 ° C., 10 sec at 60 ° C., 30 sec at 72 ° C.). Cool to 4 ° C. and complete the reaction. Appropriate concentrations of the primers were 1.0 μM each. The composition of the reaction solution was 20 μl of reaction solution, 1 × PCR buffer, 0.2 mM dNTP, 40ng / μl template, and 0.025 unit of Taq polymerase.

In the present invention, PCR was performed using each marker for each rice cultivar using 20 SNP markers using the isolated genomic DNA as a template. As shown in FIG. 3, the presence or absence of each product was prepared in the table | surface, and each rice variety was determined by the analysis of the result.

In the specificity test of all 173 varieties (124 domestic and 49 foreign), 27 varieties could not be identified. The remaining 146 varieties were individually identifiable. According to the analysis of 124 domestic varieties, 12 varieties could not be identified and the remaining 112 varieties could be identified. In addition, according to the analysis results of 49 foreign varieties, 15 varieties could not be identified. The remaining 34 varieties were individually identifiable (FIG. 3).

In addition, according to the present invention, with 9 markers, it was possible to individually identify 25 varieties among 28 domestic rice varieties corresponding to the varieties of the government varieties restricted purchase (Fig. 3). However, the three varieties (Ilmibyeo, Hwabongbyeo, and Youngnambyeo) were indistinguishable because of the same pattern of bands formed. Using only nine markers out of the 20 markers developed in this way, it was confirmed that 25 government purchase varieties could be completely identified.

As described above, the marker for identifying the rice variety of the present invention is capable of identifying various rice varieties including domestic and foreign ones among the rice varieties. It can support and can be usefully used to identify varieties for managing the origin of rice in circulation.

Attach sequence list electronic file  

Claims (6)

A marker for identifying a rice cultivar consisting of any one or more nucleotide sequences selected from markers for identifying a variety represented by SEQ ID NOs: 1-40 In any one or more nucleotide sequences selected from markers for identifying varieties represented by SEQ ID NOs: 1 to 40, any one of the bases at positions 2 to 4 from the 3 'end of the nucleotide sequence is selected from A, T, G, and C. Marker for identification of rice varieties consisting of modified sequences The method of claim 2, wherein the modified sequence is a marker for identifying a variety of rice varieties in which the nucleotide sequence is changed from the 3 'end to the third base in a marker for identifying a variety represented by SEQ ID NOS: 1-40 Separating genomic DNA of rice; At least one nucleotide sequence selected from a marker for identifying a cultivar represented by SEQ ID NOs: 1 to 40, and a nucleotide sequence of the second to fourth positions from the 3 'end of the genome DNA as a template Amplifying each specific region for each variety using at least one rice variety identification marker selected from among the modified sequences; And after identifying the specific gene for each amplified variety by electrophoresis, rice variety identification method comprising the step of identifying each variety through the analysis of the combined bands 5. The method of claim 4, wherein each rice variety is determined by tabulating the presence or absence of PCR products by each marker for each rice variety and analyzing the results. The rice cultivar according to claim 4, wherein CCS1 primers (SEQ ID NO: 41 and SEQ ID NO: 42) similar to the Cereal Centromeric Sequence in rice are used as endogenous genes to confirm PCR amplification. Identification method

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