KR20130091434A

KR20130091434A - Primer for selecting variety resistant to rice stripe disease containing stv-bi gene and the selecting method thereof

Info

Publication number: KR20130091434A
Application number: KR1020120012710A
Authority: KR
Inventors: 박동수; 박수권; 황운하; 이종희; 김상민; 조준현; 곽도연; 윤영남; 여운상; 송유천; 강항원; 권택민
Original assignee: 대한민국(관리부서:농촌진흥청장)
Priority date: 2012-02-08
Filing date: 2012-02-08
Publication date: 2013-08-19

Abstract

The present invention relates to a composition for screening rice streaked leaves resistant rice varieties comprising at least one primer set selected from the group InDel, which is a rice streaked leaves resistant gene. In addition, the present invention comprises the steps of separating genomic DNA from rice samples; Amplifying a target sequence by using the isolated genomic DNA as a template and performing an amplification reaction using at least one primer set selected from the group consisting of SEQ ID NO: 7 to SEQ ID NO: 10; And it relates to a rice streaked leaf blight resistant rice variety selection method and a rice selection kit comprising the step of detecting the amplification product.
By using the method of selecting the rice streaked blight resistant individuals according to the present invention, it is possible to efficiently distinguish streaked blight resistant and susceptible individuals by using a marker that is very closely associated with the location of streaked blight resistant genes. The molecular marker according to has the advantage that it can be useful for rice breeding to improve the streaked leaf blight resistance through marker-assisted selection (MAS).

Description

Primer for Selecting Variety Resistant to Rice Stripe Disease Containing Stv-bi Gene and the Selecting Method

The present invention relates to a primer for the selection of rice varieties having the rice streaked blight resistance gene Stv-bi and a method for analyzing the varieties of rice varieties using the same.

According to the United Nations Food and Agriculture Organization (FAO) statistics, rice (Oryza sativa L.) loses about 25% of its production by plant diseases, pests and weeds. Types of rice-related diseases include white leaf blight, leaf blight blight, stalk blight, stingray disease, frostbite, and streaked blight. Among these, rice stripe disease (rice stripe disease) infected by rice stripe virus (RSV) Is a viral disease mediated by the larvae, a major pest of rice, and causes the most damage to rice in East Asia such as Korea, Japan and China.

Rice stripe leaf blight is particularly common in japonica rice (Falk & Tsai, 8 -1998). In the 1960s, 6.5% of the total paddy area in Korea and 13-19% in Japan was found. (Toriyama, 1995; Chung 1974) In the 2000s, the cultivation area of resistant varieties was reduced to 0.1-2%. In China, it occurred from 600,000 ha in 2000-2003, and increased to 1 million ha in 2004, causing a significant damage of 30-40% of rice yield in the seabed.China (Xie et al. 2005; Zhang et al. 2007, Zhao et al. 2010).

Rice streaks infected with rice streaks are initially formed with light yellow streaks on the leaves, and when the disease progresses, the yellowish white leaves become yellowish yellow. The quantity decreases. Viral strains become hosts of larvae, increasing the density of venom, further increasing viral transmission. Recently, due to global warming, the density of annihilators coming from China has increased, and in 2008, rice streaking leaf blight occurred mainly around the west coast of Korea.

Control of the disease is possible by chemical control of the virus-mediated larvae, but cultivation of varieties resistant to the disease is the most economical and environmentally friendly method. However, when breeding resistant varieties, traditional breeding methods need to determine the resistance of a large number of gene sources or strains through bioassay for the introduction of useful gene sources. Therefore, long time and effort must be accompanied to develop resistant varieties. In addition, in the resistance test, it is difficult to select resistant cultivars because of various degrees of resistance to insect pests depending on various test conditions such as temperature, light intensity, humidity, and plant growth. Therefore, the most effective assay method, to be.

Molecular labeling methods are widely used in molecular breeding systems for the purpose of searching for useful traits, identifying species of organisms, identifying species classifications, and analyzing flexible relationships among populations. Molecular label rice breeding has the advantage of being able to analyze large quantities of resources accurately and quickly because it can search for traits at an early age without being affected by environmental variation. First of all, RFLPs (Restriction Fragment Length Polymorphisms) using the sequence length difference caused by mutations in restriction enzyme recognition sites in the chromosome have been developed. However, this method is cumbersome to use radioisotopes. Then, as a nucleic acid fingerprinting (fingerprinting) using PCR (Polymerase Chain Reaction), RAPD (randomly amplified polymorphic DNA) method and the like have been developed. In the PCR method, a small oligonucleotide (hereinafter referred to as a primer) consisting of about 10 to 20 nucleotides is annealed with DNA or RNA of an organism, and then a synthetic reaction is repeatedly performed by adding a heat resistant DNA polymerase. This is how to lose. This requires only a small amount of DNA (1-50ng) compared to other methods, and has the advantage of being able to confirm the result easily and quickly. Among the methods analyzed by the PCR method, the RAPD method has a disadvantage of inferior reproducibility because the non-specific PCR product is amplified, and the AFLP (Amplified Fragment Length polymorphism) method is spotlighted by high DNA polymorphism detection, but the reproducibility of the band is low. Appearance and analysis is complex, SSR (single sequence repeat) method is a method of preparing a PCR primer based on the nucleotide sequence information of the microsatellite region, which is a DNA repeat sequence to analyze the supersatellite in the individual by the simple base sequence Because the number of repetitions of is different between varieties and individuals, polymorphism occurs when this part is amplified by PCR reaction, and it is actively used for genetic research on animals and plants. There is not enough disadvantage.

In contrast, the InDel (insertion or deletion) marker maintains the advantages of polymorphism when amplified by PCR, and complements the shortcomings of SSR markers, which are not sufficient for high-density genetic mapping. This is a method for preparing PCR primers based on insertion or deletion of nucleotide sequences between varieties.

Rice resistance genes to rice streaks are known to include Stv-a gene on chromosome 6 and Stv-b gene on chromosome 11 (Hayano-Saito et al, 2000; Washio et al. 1968). Indica rice varieties 'Zenith' and Japanese wheat field varieties 'Kuroka' have the Stv-a and Stv-b genes, and indica varieties 'Modan' and 'Mudugo' have the Stv-b gene, the allele of the Stv-b gene. It is known to have bi. In Japan and Korea, the Stv-bi gene of Indica rice variety 'Modan' has been introduced to and used in Japonica rice to develop varieties resistant to the disease. Has the Stv-bi gene.

To develop molecular markers resistant to the disease, Hayano-Saito et al (1998) developed ST-10, which labels the Stv-bi gene, and used it as a molecular marker for breeding resistant varieties. However, the molecular marker ST-10 can only label the Stv-bi gene of Japonica rice and not the indica rice. The molecular marker ST-10 is dominant when polymorphism is examined in agarose gel after PCR (Polymerase Chain Reaction). If the band does not appear as a molecular marker showing a hybridization band, it is difficult to determine whether it is a susceptible variety or a result of an error in the PCR process.

In this context, the present inventors have fostered a resistance near homogeneous gene line (NIL) line of Shinkwang rice, which has the rice streaked blight resistance gene Stv-bi, among the numerous molecular markers present in the rice. The present invention was completed by developing an InDel marker that can be easily analyzed using PCR and agarose gel and selects a resistant strain having Stv-bi into one band.

One object of the present invention is to provide a composition for screening streaked leaf blight resistant rice varieties comprising a primer for detecting the presence of one or more oligonucleotides or fragments thereof selected from the group consisting of InDel.

Another object of the present invention is to isolate genomic DNA from rice samples; Amplifying a target sequence by using the isolated genomic DNA as a template and performing an amplification reaction using one or more primer sets from the group consisting of InDel; And it comprises a step of detecting the amplification product, it is to provide a stripe leaf blight rice varieties selection method.

Another object of the invention is one or more primer sets selected from the group consisting of InDel; Restriction enzymes; And it provides a striped leaf blight resistant rice variety selection kit comprising a reagent for performing the amplification reaction.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention is a primer pair represented by SEQ ID NO: 1 and 2; A pair of primers represented by SEQ ID NOS: 3 and 4; Primer pairs represented by SEQ ID NOs: 5 and 6; A pair of primers represented by SEQ ID NOS: 7 and 8; Primer pairs represented by SEQ ID NOs: 9 and 10; A pair of primers represented by SEQ ID NOs: 11 and 12; A pair of primers represented by SEQ ID NOS: 13 and 14; Primer pairs represented by SEQ ID NOs: 15 and 16; A pair of primers represented by SEQ ID NOs: 17 and 18; Primer pairs represented by SEQ ID NOs: 19 and 20; A pair of primers represented by SEQ ID NOS: 21 and 22;

Provides a primer pair for selecting a rice streaked blight resistant rice varieties selected from the group consisting of.

According to another aspect of the present invention, a primer pair represented by SEQ ID NO: 1 and 2; A pair of primers represented by SEQ ID NOS: 3 and 4; Primer pairs represented by SEQ ID NOs: 5 and 6; A pair of primers represented by SEQ ID NOS: 7 and 8; Primer pairs represented by SEQ ID NOs: 9 and 10; A pair of primers represented by SEQ ID NOs: 11 and 12; A pair of primers represented by SEQ ID NOS: 13 and 14; Primer pairs represented by SEQ ID NOs: 15 and 16; A pair of primers represented by SEQ ID NOs: 17 and 18; Primer pairs represented by SEQ ID NOs: 19 and 20; A pair of primers represented by SEQ ID NOS: 21 and 22;

Provided are molecular markers for selecting rice streaked leaf blight resistant rice varieties selected from the group consisting of:

According to another aspect of the invention, the invention

(i) separating DNA from a rice sample;

(ii) a primer pair represented by SEQ ID NOs: 1 and 2 of the isolated DNA as a template; A pair of primers represented by SEQ ID NOS: 3 and 4; Primer pairs represented by SEQ ID NOs: 5 and 6; A pair of primers represented by SEQ ID NOS: 7 and 8; Primer pairs represented by SEQ ID NOs: 9 and 10; A pair of primers represented by SEQ ID NOs: 11 and 12; A pair of primers represented by SEQ ID NOS: 13 and 14; Primer pairs represented by SEQ ID NOs: 15 and 16; A pair of primers represented by SEQ ID NOs: 17 and 18; Primer pairs represented by SEQ ID NOs: 19 and 20; Amplifying the target sequence by performing an amplification reaction using one or more primer pairs selected from the group consisting of: a primer pair represented by SEQ ID NO: 21 and 22; And

(iii) detecting the amplification product;

It provides a rice stripe leaf blight resistant rice varieties selection method comprising a.

The features and advantages of the present invention are summarized as follows.

(i) Using the method of selecting the rice streaked blight resistant individuals according to the present invention, it is possible to efficiently distinguish streaked blight resistant and susceptible individuals by using markers that are closely related to the location of streaked blight resistant genes. .

(ii) The molecular marker according to the present invention may be useful for rice breeding to improve the resistance to streaked leaf blight through marker-assisted selection (MAS).

1 is a photograph showing the incidence of rice streaked leaf blight of resistant myoemic strains and susceptible varieties.
FIG. 2 shows PCR results of InDel markers for a gem / new BC5F2 generation line.
Figure 3 is a precise genetic map of resistance according to the genotype and the extent of rice streaked leaf blight of markers of the elite / Shinwang BC5F4 generation resistant near homogeneous lineage.
4 is a genotype of rice streaked leaves blight resistance and markers of major domestic rice varieties.

According to another aspect of the present invention, the present invention is a primer pair represented by SEQ ID NO: 1 and 2; A pair of primers represented by SEQ ID NOS: 3 and 4; Primer pairs represented by SEQ ID NOs: 5 and 6; A pair of primers represented by SEQ ID NOS: 7 and 8; Primer pairs represented by SEQ ID NOs: 9 and 10; A pair of primers represented by SEQ ID NOs: 11 and 12; A pair of primers represented by SEQ ID NOS: 13 and 14; Primer pairs represented by SEQ ID NOs: 15 and 16; A pair of primers represented by SEQ ID NOs: 17 and 18; Primer pairs represented by SEQ ID NOs: 19 and 20; A pair of primers represented by SEQ ID NOS: 21 and 22;

More preferably, to provide the best molecular markers for screening rice streaked blight resistant rice varieties of the present invention is a case comprising a primer pair represented by SEQ ID NO: 13 and SEQ ID NO: 14.

The marker provided in the present invention refers to a PCR primer pair, and includes a forward primer and a reverse primer. The primer pair provided in the present invention has two nucleotide sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 22. Preferably InDel 1 (primary pairs represented by SEQ ID NOs: 1 and 2), InDel 3 (primary pairs represented by SEQ ID NOs: 3 and 4), InDel 4 (primary pairs represented by SEQ ID NOs: 5 and 6), InDel 9 (Primary pairs represented by SEQ ID NOs: 7 and 8), InDel 10 (primary pairs represented by SEQ ID NOs: 9 and 10), InDel 11 (primary pairs represented by SEQ ID NOs: 11 and 12), InDel 7 (SEQ ID NO: 13 and Primer pairs represented by 14), InDel 8 (primary pairs represented by SEQ ID NOs: 15 and 16), InDel 5 (primer pairs represented by SEQ ID NOs: 17 and 18), and InDel 6 (primers represented by SEQ ID NOs: 19 and 20) Pairs) and InDel 2 (primary pairs represented by SEQ ID NOs: 21 and 22).

According to another aspect of the present invention,

(i) separating DNA from a rice sample;

(iii) detecting the amplification product;

The extraction of DNA in step (i) is performed by phenol / chloroform extraction, SDS extraction (Tai et al., Plant Mol. Biol. Reporter, 8: 297-303), CTAB separation method (Cetyl Trimethyl) commonly used in the art. Ammonium Bromide; Murray et al., N uc. Res., 4321-4325, 1980) or commercially available DNA extraction kits.

In addition, the term "amplifying step" described herein in step (ii) means a reaction for amplifying a nucleic acid molecule. A variety of amplification reactions have been reported in the art, including polymerase chain reaction (PCR) (US Pat. Nos. 4,683,195, 4,683,202, and 4,800,159), reverse transcription-polymerase chain reaction (RT- , The method of Miller, HI (WO 89/06700) and Davey, C. et al (EP 329,822), the method of ligase chain reaction (Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor Press ligase chain reaction (LCR) (17, 18), Gap-LCR (WO 90/01069), repair chain reaction (EP 439,182), transcription-mediated amplification (TMA) WO 88/10315), self sustained sequence replication (20) (WO 90/06995), selective amplification of target polynucleotide sequences (US Patent No. 6,410,276) , Consensus sequence primed polymerase chain reaction (C P-PCR (US Patent No. 4,437,975), arbitrarily primed polymerase chain reaction (AP-PCR) (U.S. Patent Nos. 5,413,909 and 5,861,245), nucleic acid sequence based amplification (NASBA) (U.S. Patent Nos. 5,130,238, 5,409,818, 5,554,517 and 6,063,603), strand displacement amplification (21,22) and loop- mediated isothermal amplification; (LAMP) 23, but is not limited thereto. Other amplification methods that may be used are described in U.S. Patent Nos. 5,242,794, 5,494,810, 4,988,617 and U.S. Patent No. 09 / 854,317.

PCR is the most well-known nucleic acid amplification method, and many variations and applications thereof have been developed. For example, to improve the specificity or sensitivity of PCR, traditional PCR procedures can be modified to incorporate touchdown PCR (24), hot start PCR (25, 26), and nested PCR (2). ) And booster PCR 27 have been developed. In addition, real-time PCR, differential display PCR (DD-PCR), rapid amplification of cDNA ends (RACE), multiplex PCR, inverse polymerase chain reaction (inverse polymerase) chain reactions (IPCR), vectorette PCR, thermal asymmetric interlaced PCR (TAIL-PCR), and multiplex PCR have been developed for specific applications. For more information on PCR see McPherson, M.J., and Moller, S.G. PCR. BIOS Scientific Publishers, Springer-Verlag New York Berlin Heidelberg, N.Y. (2000), the teachings of which are incorporated herein by reference.

The PCR reaction mixture includes a DNA polymerase, a dNTP, a PCR buffer solution and water (dH ₂ O) in an appropriate amount in addition to the DNA extracted from the rice sample to be analyzed and the primer pair provided in the present invention. The PCR buffer solution includes Tris-HCl, MgCl ₂ , KCl, and the like. At this time, the concentration of MgCl ₂ greatly affects the specificity and quantity of amplification, and may be preferably used in the range of 1.5 to 2.5 mM. In general, if a large excess of Mg ²⁺ to increase the non-specific PCR amplification products and, Mg and the yield of the PCR product was reduced when ² ⁺ is low. The PCR buffer solution may further contain an appropriate amount of Triton X-100 (Triton X-100). PCR also denatures the template DNA at 94-95 ° C., and then denatures it; Annealing; Followed by a cycle of amplification and extension followed by final elongation at 72 ° C. In the above modification and amplification may be performed at 94 ~ 95 ℃ and 72 ℃, respectively. The temperature at the time of binding may vary depending on the type of the primer. Preferably 55 to 60 < 0 > C. The time and number of cycles in each step can be determined according to the conditions commonly practiced in the art. Optimal reaction conditions when performing PCR using a primer pair according to the present invention are as follows. Template denaturation for 5 minutes at 94 ° C., followed by 30 seconds at 94 ° C .; 30 seconds at 55 ° C .; And 72 ° C for 60 seconds, followed by final reaction at 72 ° C for 5 minutes.

In step (iii), DNAs of PCR products may be separated according to sizes according to methods well known in the art. Preferably by agarose gel or polyacrylamide gel electrophoresis or an ABI prism 3100 genetic analyzer-electropherogram. PCR amplification results can be preferably confirmed by polyacrylamide gel electrophoresis, more preferably modified polyacrylamide gel electrophoresis. After electrophoresis, electrophoresis results can be analyzed by silver staining and fluorescence staining. Methods for performing general PCR and analyzing the results are well known in the art.

According to a preferred embodiment of the present invention, the rice sample is derived from the roots, leaves, stems, flowers or ear tissues of rice.

According to a preferred embodiment of the present invention, the rice plant uses YR24982-9-1.

According to another aspect of the present invention,

Primer pairs represented by SEQ ID NOs: 1 and 2 of claim 1; A pair of primers represented by SEQ ID NOS: 3 and 4; Primer pairs represented by SEQ ID NOs: 5 and 6; A pair of primers represented by SEQ ID NOS: 7 and 8; Primer pairs represented by SEQ ID NOs: 9 and 10; A pair of primers represented by SEQ ID NOs: 11 and 12; A pair of primers represented by SEQ ID NOS: 13 and 14; Primer pairs represented by SEQ ID NOs: 15 and 16; A pair of primers represented by SEQ ID NOs: 17 and 18; Primer pairs represented by SEQ ID NOs: 19 and 20; It provides a kit for screening rice streaked leaves resistant rice varieties comprising one or more primer pairs selected from the group consisting of SEQ ID NO: 21 and 22, a restriction enzyme and a reagent for performing an amplification reaction.

In addition, in order to facilitate the PCR reaction, a DNA polymerase and a PCR reaction buffer solution having the composition described above may be further included. In order to confirm whether amplification of the PCR product is amplified, Or identification standards for known varieties may be further included in the kit of the present invention.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are intended to illustrate the invention, and the content of the invention is not limited by the following examples.

(One) Example 1: Plant Preparation for Strip Leaf Blight Bioassay

Resistant near homogeneous gene system YR24982-9-1 using rice seedlings with streaked leaf blight resistance genes at the Stv-b locus, which is a streaked leaf blight resistance locus, as a donor, and cross-breeding with susceptible repeats four times The bioassay, genetic analysis, and molecular markers were performed using the strains of BC5F2 and BC5F4, which were grown using a single rice plant as a model and the resistant near-homogeneous strain YR24982-9-1.

(2) Example 2: Establishment of Striped Leaf Blight-resistant Bioassay Conditions

As a condition for resistance against rice streaked leaf blight, seedlings were grown in plastic barrels 45cm wide and 25cm long, and rice was grown for two weeks. 3 days of inoculation and transplanted to the greenhouse. One month after transplantation, the extent of streaked leaf blight on the plants was examined to determine the resistance of the varieties or strains (FIG. 1, Table 1).

Breed / Line Name Number of plants by disease after inoculation (ratio) Resistance judgment Resistant plant Susceptible plants Disease free Streaks occur Leaf yellowing Suspension or death YR24982-9-1 17 (33%) 27 (53%) 7 (14%) 0 (0%) Resistance A la carte 9 (10%) 5 (16%) 16 (52%) 7 (22%) sensibility

(3) Example 3: streaked leaf blight resistance Association marker primer making

A molecular marker showing a previously developed dominant hybridization band for labeling the Stv-bi gene using the genome sequence information database (http://www.gramene.org) of the japonica rice variety Nipponbare and indica rice variety 93-11 11 insertion or deletion regions were identified between chromosome 17.2Mbp-19.6Mbp, rice 11, a region near the PCR species of ST-10, and primers for PCR amplification for the development of InDel markers were prepared (Table 2).

Primer Sequence (5'to 3 ') Location (Mbp) InDel 1 SEQ ID NO: 1 Forward: CAGCAGCCCTATGGAAACAA 16.20 SEQ ID NO: 2 Reverse: TGGAGGGGAATAGGATTTGG InDel 3 SEQ ID NO: 3 Forward: CAATTTAGATCGATGGCCGA 17.39 SEQ ID NO: 4 Reverse: ATGTGGGCAGATGGCACTTA InDel 4 SEQ ID NO: 5 Forward: CTCAAGCTCGACAAGGTCTG 17.78 SEQ ID NO: 6 Reverse: CCTCCGTCGTCTGCCTTTTT InDel 9 SEQ ID NO: 7 Forward: CGCCGATGGCAATAAATAAA 17.82 SEQ ID NO: 8 Reverse: TGGACCATATCTCTGCGTGG InDel 10 SEQ ID NO: 9 Forward: CTGTTCCGTTGCTGCTCTGT 17.86 SEQ ID NO: 10 Reverse: GGTGAATCTATCGCCTGCAA InDel 11 SEQ ID NO: 11 Forward: AAAATATCGGGGCCATCAAT 17.86 SEQ ID NO: 12 Reverse: TGATCAAGCTGGACCAAGTTCT InDel 7 SEQ ID NO: 13 Forward: AAATTCCAATGCCCAAAACC 17.95 SEQ ID NO: 14 Reverse: TCCTCATGGAGCTCATCCAA InDel 8 SEQ ID NO: 15 Forward: GGCAAATTTGGAAAGGTGGT 17.96 SEQ ID NO: 16 Reverse: TCAACAGCGCCATACATTAAAA InDel 5 SEQ ID NO: 17 Forward: AAAGGAATTAAATTGTACCACAAACA 18.01 SEQ ID NO: 18 Reverse: CATGGAGTTGAGTTGGTTAATGC InDel 6 SEQ ID NO: 19 Forward: CAGGATGAGGCTACAATTTTCAA 18.02 SEQ ID NO: 20 Reverse: GTCTGGATCCACTTCATACCA InDel 2 SEQ ID NO: 21 Forward: ACCGACGGTGATGTAGGGAT 19.60 SEQ ID NO: 22 Reverse: ACTCCACATGGAACACCACG

(4) Example 4: striped leaf blight resistance association Molecular marker Made for development Indel Marker Specificity verification

PCR was performed using the InDel markers InDel 1 to InDel 11 prepared in Example 3 to verify whether an efficient genotyping analysis is possible for the selection of resistance to streaked leaf blight.

① Plant Material

Resistant myofibrils YR24982-9-1 and Ilpumbum were used to grow cultivars of rice varieties resistant to stinging leaf blight, Shingwangpum and Singwangbyeong, and cross-breed the susceptible repeats 4 times. Using the InDel marker and the SSR marker in the neighboring region, BC5F2 460 individuals were grown using a single rice as a model and the resistant near-homogeneous strain YR24982-9-1 as a copy, and confirmed the transfer region of the Shinkwang rice-derived gene fragment by strain. After that, the seed was harvested by introducing homozygous gene fragment derived from Shinkwang rice into homogenous state and harvested seeds. It was.

② From seedlings DNA extraction

In the present invention, DNA was extracted from BC5F2 460 individuals which were grown using a single rice as a model and a resistant YM24982-9-1 as a parent. 0.2 g of the leaf was placed in a mortar and disrupted with liquid nitrogen and placed in a tube together with 1.5 mL of QuickPrep buffer (200 mM Tris-HCl, 250 mM NaCl, 25 mM EDTA, 0.5% SDS) And reacted for 1 hour. After centrifugation at 10,000 × g for 10 minutes, 400 μl of the supernatant was added to a new tube, 300 μl of isopropanol was added, mixed well and reacted at -80 ° C. for 30 minutes. The supernatant was removed by centrifugation at 20,000 x g for 10 minutes, and the supernatant was removed by adding 70% ethanol, followed by drying for 30 minutes. 150 μl of DNA extraction buffer (10 mM NH4OAC, 0.25 mM EDTA) was added and mixed well. After reacting at 60 ° C for 1 hour, the mixture was centrifuged at 10,000 × g for 10 minutes, and 100 μl of the supernatant was placed in a new tube. Using the DNA thus extracted, an experiment related to the present invention was performed.

③ one article / Shin Kwang BC5F2 By object allele type For analysis PCR

The reaction method and conditions for PCR amplification of genomic DNA extracted from the 460 ape / new BC5F2 generation 460 individuals, the resistant near-homogeneous line YR24982-9-1, the albino rice, and the shinkwang rice used in the above process were carried out as follows. PCR was performed using 50 ng of DNA and 10 pmol of SSR marker primer pairs, respectively. PCR amplification was performed using a premix (Cat. No. G2002, Inc., Japan) containing polymerase and PCR buffer, Genet Bio) was used for PCR.

PCR amplification conditions were performed using 50 ng of genomic DNA from each strain, and 30 μl of volume (100 pM of each primer, 200 μM of each dNTPs, 10 mM Tris-HCl pH 9.0, 2 mM MgCl ₂ , 50 mM). KCl, 0.1% Triton X-100 and 0.5 U Taq polymerase) at 94.5 ° C., 5 min, 94 ° C. 30 sec, 55 ° C. 30 sec, 72 ° C. for 1 minute, 40 times, and finally at 72 ° C. After reacting for a minute, the amplified product was added to 0.5% / μg of EtBr in a 3% agarose gel (agarosegel) and confirmed on UV (FIG. 2).

(5) Experiment result 1: one article / Shin Kwang BC5F4 Results of Rice Stripe Blight Bioassay of Selected Lines Indel Marker Confirm consistency with genotype

Gene fragments derived from Shingwangbyeo by strain were investigated by genotype of PCR products using SSR markers near the Stv-b locus and 12 InDel markers developed in this patent, using 460 individuals / new BC5F2 generation 460 individuals by the above-described method. After confirming the translocation area of the plant, 10 strains were selected from which the gene fragments derived from Sinkwang rice were transferred to different positions in the vicinity of the resistance genes. The selected strains were harvested from the seed and subjected to biopsies of streaked leaf blight resistance against F4 plants. Through precision resistance genetic map was constructed (Fig. 3). In FIG. 3, black represents a portion into which the gene of Singwang rice is introduced and gray represents a portion into which the gene of Almop rice is introduced.

Precise / new light BC5F4 generation resistance Genetic map of resistance according to the incidence of rice streaked leaf blight by resistance to allogeneic strains showed resistance genes at 17.78-18.01Mbp from InDel 4 to InDel 6 InDel 7 best labeled the resistance gene.

ST-10, a conventional resistant molecular marker for the disease, can only label Stv-bi genes and cannot label Stv-b genes, which are resistant bi-allelic genes in the same locus. (Polymerase Chain Reaction) When examining polymorphism in agarose gel, it was difficult to determine whether it is a susceptible variety or a result of PCR process if the band does not appear as a molecular marker showing dominant hybridization band.

Marker 7 InDel provided in the present patent can confirm the polymorphism of the PCR product in agarose gel that can be made relatively easily.

(6) Experiment result 2: developed Indel Marker Confirmation of Genotype and Consistency of Striped Leaf Blight Bioassay by Rice Varieties

As a result of confirming the correspondence between the genotype and the resistance of each marker by applying to the domestic rice varieties known for resistance using a marker including InDel 7, which is the final selected co-occurrence marker in Example 5 (Fig. 4) Resistant molecular marker ST-10 was well distinguished for resistance in domestic japonica rice, but could not distinguish resistance of unified rice, whereas InDel No. 7, InDel No. 5 and InDel No. 6 provided in this patent were used for japonica and unified rice. Regardless of whether the resistance is accurately classified. As shown in Example 3, InDel No. 7 was found to be the closest to the resistance gene in InDel 7 when the concordance between the rice stripe leaf blight bioassay and the InDel marker genotype of the Ammonia / Shinwang BC5F3 selection line was confirmed. It was most suitable as a DNA marker for screening rice cultivars with St. bhi-resistant gene streaked leaf blight.

Claims

Primer pairs represented by SEQ ID NOs: 1 and 2; A pair of primers represented by SEQ ID NOS: 3 and 4; A pair of primers represented by SEQ ID NOS: 5 and 6; A pair of primers represented by SEQ ID NOS: 7 and 8; A pair of primers represented by SEQ ID NOS: 9 and 10; Primer pairs represented by SEQ ID NOs: 11 and 12; Primer pairs represented by SEQ ID NOs: 13 and 14; Primer pairs represented by SEQ ID NOs: 15 and 16; Primer pairs represented by SEQ ID NOs: 17 and 18; Primer pairs represented by SEQ ID NOs: 19 and 20; Primer pairs represented by SEQ ID NOs: 21 and 22;
Primer pair for screening rice streaked blight resistant rice varieties selected from the group consisting of.

Primer pairs represented by SEQ ID NOs: 1 and 2; A pair of primers represented by SEQ ID NOS: 3 and 4; A pair of primers represented by SEQ ID NOS: 5 and 6; A pair of primers represented by SEQ ID NOS: 7 and 8; A pair of primers represented by SEQ ID NOS: 9 and 10; Primer pairs represented by SEQ ID NOs: 11 and 12; Primer pairs represented by SEQ ID NOs: 13 and 14; Primer pairs represented by SEQ ID NOs: 15 and 16; Primer pairs represented by SEQ ID NOs: 17 and 18; Primer pairs represented by SEQ ID NOs: 19 and 20; Primer pairs represented by SEQ ID NOs: 21 and 22;
Molecular marker for screening rice streaked blight resistant rice varieties selected from the group consisting of.

Molecular marker for selection of rice streaked blight resistant rice varieties comprising a primer pair represented by SEQ ID NO: 13 and SEQ ID NO: 14.

(i) isolating DNA from the rice sample;
(ii) a primer pair represented by SEQ ID NOs: 1 and 2 of the isolated DNA as a template; A pair of primers represented by SEQ ID NOS: 3 and 4; A pair of primers represented by SEQ ID NOS: 5 and 6; A pair of primers represented by SEQ ID NOS: 7 and 8; A pair of primers represented by SEQ ID NOS: 9 and 10; Primer pairs represented by SEQ ID NOs: 11 and 12; Primer pairs represented by SEQ ID NOs: 13 and 14; Primer pairs represented by SEQ ID NOs: 15 and 16; Primer pairs represented by SEQ ID NOs: 17 and 18; Primer pairs represented by SEQ ID NOs: 19 and 20; Amplifying the target sequence by performing an amplification reaction using one or more primer pairs selected from the group consisting of: a primer pair represented by SEQ ID NO: 21 and 22; And
(iii) detecting the amplification product;
Rice stripe leaf blight resistant rice varieties selection method comprising a.

The method of claim 4, wherein the rice sample is derived from roots, leaves, stems, flowers, or ear tissues of rice.

The selection method according to claim 4 or 5, wherein the rice is YR24982-9-1.

Primer pairs represented by SEQ ID NOs: 1 and 2 of claim 1; A pair of primers represented by SEQ ID NOS: 3 and 4; Primer pairs represented by SEQ ID NOs: 5 and 6; A pair of primers represented by SEQ ID NOS: 7 and 8; Primer pairs represented by SEQ ID NOs: 9 and 10; A pair of primers represented by SEQ ID NOs: 11 and 12; A pair of primers represented by SEQ ID NOS: 13 and 14; Primer pairs represented by SEQ ID NOs: 15 and 16; A pair of primers represented by SEQ ID NOs: 17 and 18; Primer pairs represented by SEQ ID NOs: 19 and 20; A pair of primers selected from the group consisting of SEQ ID NO: 21 and 22; one or more primer pairs selected from the group consisting of, rice stripe leaf blight resistant rice varieties kit comprising a reagent for performing the amplification reaction.