KR101054459B1 - Specific primer and its use for distinguishing rice varieties - Google Patents

Abstract

The present invention relates to a specific primer set for distinguishing rice varieties and its use, and more particularly to distinguishing a rice variety comprising two or more primer sets selected from the group consisting of oligonucleotide primer sets of SEQ ID NOs. A primer set for the purpose, a kit for distinguishing rice varieties comprising the primer set, and a method for distinguishing rice varieties using the primer set.

Rice Variety, Classification, Oligonucleotide, Primer, Kit

Description

Specific primers for discriminating rice cultivars, and uses approximately}

The present invention relates to specific primers and their use for distinguishing rice varieties.

Rice is the world's second-largest food crop after wheat, and more than half of the world's population is rice-based, with more than 90% of its production in Asia, mostly consumed in Asia. Korea, like other Asian countries, uses rice as its staple food, and 15-20 varieties are cultivated and distributed each year, but most of the varieties distributed in Korea are specific varieties (Ipum rice, Junnam rice, Dongjin No. 1, etc.). Most of domestic consumers are unable to identify these varieties. In addition, with the opening and export of imports and exports, some distributors supply imported rice either domestically or in a mixture of domestically imported rice.In order to prevent such illegal distribution, the government institutionalizes the country of origin labeling, but in reality, the distribution channel is The situation is very difficult, such as going through a complex process such as tracking and country of origin verification. In addition, more scientific investigations on suspicions and accusations related to rice varieties, such as theft of crops occurring in some rural areas, are needed. Therefore, rice varieties grown and cultivated in Korea can be quickly and easily identified. The establishment of methods and techniques is urgently needed.

Recently, the genetic resources of rice have been used as a very important material for preserving useful traits and creating new varieties. The rapid identification of species and subspecies of genetic resources collected with the collection of domestic and foreign resources can provide accurate genetic resource management and protection. It is very important to.

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 microcetrite (Kwon, SJ et al., 1999 Korean J Crop Science 44, 112-116), and reported methods for identifying rice varieties using image analysis techniques. (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 difficult to discuss the analysis results.

Korean Patent Registration No. 10-0453568 discloses a microsatellite marker combination for breed identification, and Korean Patent Registration No. 10-0736152 discloses a set of STS markers for discriminating subspecies in rice, but the primer of the present invention. It is different from the set.

The present invention has been devised by the above requirements, and the present invention has a high reproducibility and selects a minimum primer combination that can be easily applied to the determination of most rice varieties, conventional varieties, and some introduced varieties grown in Korea. By applying the classification to the varieties, I want to make clear the distinction between the rice varieties grown in Korea.

In order to solve the above problems, the present invention provides a primer set for distinguishing rice varieties comprising two or more primer sets selected from the group consisting of oligonucleotide primer sets of SEQ ID NOs: 1-8.

The present invention also provides a kit for distinguishing rice varieties comprising the primer set.

The present invention also provides a method for distinguishing rice varieties using the primer set.

According to the present invention, domestic breeding varieties can be distinguished with only at least four primer sets, and PCR time can be shortened through multiplex PCR using fluorescent primers, and the accuracy of genetic analysis can be improved through capillary electrophoresis. .

In order to achieve the object of the present invention, the present invention provides an oligonucleotide primer set of SEQ ID NO: 1 and 2; Oligonucleotide primer sets of SEQ ID NOs: 3 and 4; Oligonucleotide primer sets of SEQ ID NOs: 5 and 6; And it provides a primer set for distinguishing rice varieties comprising two or more primer sets selected from the group consisting of oligonucleotide primer sets of SEQ ID NO: 7 and 8.

The primer set of the present invention preferably comprises at least three primer sets selected from the group consisting of the above primer sets, most preferably four primer sets, ie oligonucleotide primer sets of SEQ ID NOs: 1 and 2; Oligonucleotide primer sets of SEQ ID NOs: 3 and 4; Oligonucleotide primer sets of SEQ ID NOs: 5 and 6; And oligonucleotide primer sets of SEQ ID NOs: 7 and 8. Using the oligonucleotide primer sets of SEQ ID NO: 1 to SEQ ID NO: 8 at the same time can be more efficiently distinguished rice varieties.

The primers may be at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23 in the sequence of SEQ ID NO: 1 to SEQ ID NO: 8, depending on the sequence length of each primer. Oligonucleotides consisting of fragments of at least 24, at least 24, and at least 25 consecutive nucleotides. For example, at least 16 primers, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, and at least 24 within the sequence of SEQ ID NO: 1 Or more, may comprise oligonucleotides consisting of fragments of 25 or more consecutive nucleotides, wherein the primers of SEQ ID NO: 2 are 16 or more, 17 or more, 18 or more, 19 or more, 20 or more within the sequence of SEQ ID NO: 2 , Oligonucleotides consisting of segments of 21 or more consecutive nucleotides. In addition, the primer may include an addition, deletion or substituted sequence of the nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 8.

In the present invention, "primer" refers to a single stranded oligonucleotide sequence that is complementary to the nucleic acid strand to be copied and may serve as a starting point for the synthesis of the primer extension product. The length and sequence of the primer should allow to start the synthesis of the extension product. The specific length and sequence of the primers will depend on the primer utilization conditions such as temperature and ionic strength as well as the complexity of the DNA or RNA target required.

As used herein, oligonucleotides used as primers may also include nucleotide analogues, such as phosphorothioate, alkylphosphothioate or peptide nucleic acid. Or an intercalating agent.

In the oligonucleotide primer set according to an embodiment of the present invention, the rice variety is Junambyeong, Nampyeongbyeong, Odaepyeong, Hwayoungpyeong, Chucheongbyeon, Hwaseongpyeong, Ilpumbyeong, Taebongbyeong, Saesangjubap, Saechucheongbyeong, Dongjin No. 1, Saeggyehwabap, Huangp rice, Hopyeong rice, Alternative rice, Baekjinju No. 1, Sinunbong No. 1, Hyangmi Rice No. 1, Odae No. 1, Heuknam Rice, Namil Rice, Juan No. 1, Chinese Rice, Sura Rice, Heuk Jin Rice, Hyangmi Rice No. 2 It may be two or more selected from the group consisting of rice, crude black sesame, samgwang rice, hyangnam rice, unflavored rice, and black scented rice, but is not limited thereto.

Primer set according to an embodiment of the present invention is preferably used for multiplex (multiplex) PCR. Multiplex PCR refers to a method of PCR amplification in one reaction tube simultaneously using a plurality of primer sets. After performing each PCR reaction using each primer set, it is also possible to perform capillary electrophoresis by combining PCR products, but it is more preferable to perform multiplex PCR to shorten the PCR time and reaction. In general, when performing multiplex PCR, the production efficiency of PCR products is reduced compared to performing each PCR reaction. However, in the case of the primer set of the present invention, four primer sets are put in one tube and multiplex PCR is performed. It can be seen that even if the PCR products are produced properly.

In order to achieve another object of the present invention, the present invention

Oligonucleotide primer sets according to the present invention; And it provides a kit for distinguishing rice varieties, including a reagent for performing an amplification reaction.

In the kit of the present invention, the reagent for performing the amplification reaction may include DNA polymerase, dNTPs, buffers and the like. In addition, the kits of the present invention may further comprise a user guide describing the optimal reaction performance conditions. The guide is a printed document that explains how to use the kit, such as how to prepare a PCR buffer, and the reaction conditions presented. The instructions include brochures in the form of pamphlets or leaflets, labels affixed to the kit, and instructions on the surface of the package containing the kit. In addition, the guide includes information that is disclosed or provided through electronic media such as the Internet.

In order to achieve another object of the present invention, the present invention

Separating genomic DNA from rice;

Amplifying a target sequence by using the isolated genomic DNA as a template and performing an amplification reaction using an oligonucleotide primer set according to the present invention; And

It provides a method for distinguishing rice varieties, comprising the step of detecting the amplification products.

The method of the present invention comprises the step of separating genomic DNA from rice samples. As a method for separating genomic DNA from the sample, a method known in the art may be used. For example, the CTAB method may be used, or a wizard prep kit (Promega) may be used. Using the isolated genomic DNA as a template, an amplification reaction may be performed using an oligonucleotide primer set according to an embodiment of the present invention as a primer to amplify a target sequence. Methods for amplifying target nucleic acids include polymerase chain reaction (PCR), ligase chain reaction, nucleic acid sequence-based amplification, and transcription-based amplification systems. , Strand displacement amplification or amplification via Qβ replication or any other suitable method for amplifying nucleic acid molecules known in the art. Among them, PCR is a method of amplifying a target nucleic acid from a primer pair that specifically binds to the target nucleic acid using a polymerase. Such PCR methods are well known in the art, and commercially available kits may be used.

Amplification of the target sequence or nucleic acid may be performed through multiplex PCR, and more preferably, multiplex PCR using fluorescent primers may be performed. Performing multiplex PCR has the advantage of reducing the PCR time and reaction. Initial denaturation, denaturation, annealing, and extension in the multiplex PCR may be performed according to conventional conditions. However, the final extension reaction is preferably performed at 60 ° C. for 30 minutes.

In the method of the present invention, the amplified target sequence may be labeled with a detectable labeling substance. In one embodiment, the labeling material may be, but is not limited to, a material that emits fluorescence, phosphorescence, or radioactivity. Preferably, the labeling substance is Cy-5 or Cy-3. When amplifying the target sequence, PCR is performed by labeling Cy-5 or Cy-3 at the 5'-end of the primer to allow the target sequence to be labeled with a detectable fluorescent labeling substance. In addition, the label using the radioactive material may add radioactive isotopes such as ³² P or ³⁵ S to the PCR reaction solution when PCR is performed, and the amplification product may be radioactively incorporated into the amplification product while the amplification product is synthesized. The oligonucleotide primer set used to amplify the target sequence is as described above.

The method of the present invention includes detecting the amplification product. Detection of the amplification product may be performed through capillary electrophoresis, DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement or phosphorescence measurement, but is not limited thereto, and preferably is to perform capillary electrophoresis. As one of the methods for detecting amplification products, capillary electrophoresis can be performed. Capillary electrophoresis can use, for example, an ABi Sequencer. In addition, gel electrophoresis may be performed, and gel electrophoresis may use agarose gel electrophoresis or acrylamide gel electrophoresis depending on the size of the amplification product. In addition, in the fluorescence measurement method, PCR is performed by labeling Cy-5 or Cy-3 at the 5'-end of a primer, and a target sequence is labeled with a detectable fluorescent labeling substance, and the labeled fluorescence is measured using a fluorimeter. can do. In addition, radioactivity measuring method is to add a radioactive isotope such as ³² P or ³⁵ S to the PCR reaction solution to label the amplification product when performing PCR, and then radioactive measuring apparatus, for example, Geiger counter or liquid flash The radioactivity can be measured using a liquid scintillation counter.

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

Materials and methods

1. DNA extraction

The rice varieties used in the present invention used the pure rice of the original class received from the Food Crop Science Institute. In order to extract the DNA, the outer and inner shells were removed from the rice, and the brown rice was made. DNA extraction was performed as follows. Seeds or white rice 1-2 granules in a lactic acid paper, finely ground, put into a 1.5ml or 2ml tube and stored frozen. Extraction buffer (350 µl) was added, 2 µl proteinase K (25 µg / µl) was added and mixed well, followed by reaction at 37 ° C for 30 minutes. An equal volume of 2% CTAB (350 μl) was added and mixed well. The mixture was allowed to mix well by inverting lightly every 5 minutes while reacting for 15 minutes in a 65 ° C. constant temperature water bath. Here, 350 μl of a PCI solution (phenol: chloroform: isoamylalcohol = 25: 24: 1) was added thereto, mixed well while inverting for 10 minutes, and centrifuged at 12,000 rpm and 4 ° C. for 10 minutes. Transfer the supernatant to a new tube, add 2/3 volume of isopropanol (500 μl) or 2 volumes of EtOH from the transferred supernatant, mix slowly and condensate the DNA, and place in a -20 ° C freezer to recover as much DNA as possible. After 2 to 3 hours and centrifuged for 10 minutes at 4 ℃, 12,000rpm to precipitate the DNA. After centrifugation, the supernatant was discarded, washed with 70% cold EtOH (300-500 μl), centrifuged at 12,000 rpm for 3-5 minutes, the EtOH was removed, the DNA pellet was dried, and the DNA was dried in 100 μl TE buffer or sterile water. After dissolving, 200 µl of -20 ° C and 95% EtOH was added to lump the DNA, and the DNA was precipitated by centrifugation at 4 ° C and 12,000 rpm for 10 minutes. Discard EtOH, wash DNA mass once in 70% EtOH, dry DNA mass, add 200μl TE buffer or sterile water to dissolve DNA, check DNA amount and dilute to 10-15μg / μl in PCR Used.

2. PCR reaction and fluorescent primer synthesis

The final volume of the PCR reaction was adjusted to 20 μl, and 2 μl of genomic DNA, 2 μl of PCR buffer, 1.6 μl of dNTPs, 0.1 μl of Taq polymerase, and 0.4 μl of four pairs of forward and reverse primers (SEQ ID NOs. 1 to 8), respectively. The rest was supplemented with distilled water. PCR cycles consisted of 95 ° C 5/5 cycles for initial DNA denaturation, DNA amplification at 94 ° C 30 seconds, 55 ° C 30 seconds, 72 ° C 1 minute 35 cycles and final extension at 60 ° C 30min 1 cycle. After the reaction, it was stored at 4 ℃. All PCRs used Px2 Thermal Cycler (ABi).

 The sequence information of primers of OSR20, RM249, RM206, and RM207 is shown in Table 1 below, and synthesized by adding blue, green, yellow, and red fluorescent materials, respectively.

Table 1. Fluorescence and sequence of primers used

Primer (fluorescence) division Sequence (5 '→ 3') OSR20 (Blue) Forward GGAAGAGGAGAGAAAGATGTGTGTCG (SEQ ID NO: 1) Reverse TCAACAGACACACCGCCACCGC (SEQ ID NO: 2) RM249 (Green) Forward GGCGTAAAGGTTTTGCATGT (SEQ ID NO: 3) Reverse ATGATGCCATGAAGGTCAGC (SEQ ID NO: 4) RM206 (Yellow) Forward CCCATGCGTTTAACTATTCT (SEQ ID NO: 5) Reverse CGTTCCATCGATCCGTATGG (SEQ ID NO: 6) RM207 (Red) Forward CCATTCGTGAGAAGATCTGA (SEQ ID NO: 7) Reverse CACCTCATCCTCGTAACGCC (SEQ ID NO: 8)

3. ABi Sequencer Reaction of PCR Products

After completion of the PCR process, the PCR product was diluted 10 times with distilled water. 9 μl of Hi-Di and 0.05 μl of Liz-500 size marker (ABi, USA) were added to the diluted solution, followed by reaction at 94 ° C. for 2 minutes, followed by capillary electrophoresis on an ABi Sequencer (ABi, USA).

Example 1 Design of the Primer Set of the Invention

Sequence information of several markers was obtained from the Internet Database ( http://www.gramene.org/ ), and allele reduced to a number of specific markers while screening the varieties. Gradient PCR tested the annealing at 55 ° C. The sequence was slightly modified using the "Primer-3" program (so that the reaction could occur well at 55 ° C). Four primers were finally selected by fluorescence for each primer.

Example 2: Multiplex PCR Analysis of Rice Varieties Using the Primer Set of the Present Invention

Domestic rice growing using the primer set designed in Example 1 Multiplex PCR was performed on 32 varieties.

Samgwang rice of the graph represented in the ABi Sequencer is illustrated as shown in FIG. It can be seen that the four primer sets mixed with each fluorescent color are displayed at the unique positions of the primers. In FIG. 1, the orange color represents a standard size marker, and the RM206 fluorescently colored in yellow is displayed in black so as to be easily visible on the screen.

The genotyping sizes of the four primers for Samkwang Rice were OSR20 (193bp), RM249 (126bp), RM206 (150bp), and RM207 (119bp).

In the same manner as above, all of the intrinsic genotyping sizes of 32 domestically grown varieties were tested, and the test results are shown in Table 2 below.

Table 2. PCR product size (bp) between varieties by primer

Table 2 (continued)

Table 2 (continued)

Table 2 (continued)

Since there is no interference between these varieties, it is possible to mix and analyze four primer sets at the same time, thereby enabling rapid varieties with reduced time and cost.

1 is a diagram showing the intrinsic genotyping size of Samkwang rice.

<110> Gyeongsangbuk-Do Provincial Agricultural Technology Administration <120> Specific primers for discriminating rice cultivars, and uses          the <130> PN08241 <160> 8 <170> KopatentIn 1.71 <210> 1 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> OSR20 forward primer <400> 1 ggaagaggag agaaagatgt gtgtcg 26 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> OSR20 reverse primer <400> 2 tcaacagaca caccgccacc gc 22 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RM249 forward primer <400> 3 ggcgtaaagg ttttgcatgt 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RM249 reverse primer <400> 4 atgatgccat gaaggtcagc 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RM206 forward primer <400> 5 cccatgcgtt taactattct 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RM206 reverse primer <400> 6 cgttccatcg atccgtatgg 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RM207 forward primer <400> 7 ccattcgtga gaagatctga 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RM207 reverse primer <400> 8 cacctcatcc tcgtaacgcc 20  

Claims (10)

Oligonucleotide primer sets of SEQ ID NOs: 1 and 2; Oligonucleotide primer sets of SEQ ID NOs: 3 and 4; Oligonucleotide primer sets of SEQ ID NOs: 5 and 6; And Junambum, Nampyeongbyeong, Odaepyeong, Hwayoungbap, Chucheongpung, Hwaseongbap, Ungumbap, Taebongbap, Saesangjub, Saechuchungbap, Dongjin No. 1, Sagyehwabap, Joongbap, Hopyeong Rice, Alternative Rice, Baekjinju No. 1, Sinunbong No. 1, Hyangmi Rice No. 1, Odae No. 1, Heuknam Rice, Namil Rice, Juan No. 1, Chinese Rice, Surami Rice, Heukjin Rice, Hyangmi Rice No. 2, Gomyeo Rice, Cho Saeng A primer set for distinguishing two or more rice varieties selected from the group consisting of black wax, samgwang rice, hyangnam rice, unflavored rice, and black rice. delete delete The primer set according to claim 1, which is used for multiplex PCR. An oligonucleotide primer set according to claim 1; And Junambap, Nampyeongbyeong, Odaebyeong, Hwayoungbap, Chucheongbyeong, Hwaseongpyeong, Unbyeongbap, Taebongbyeong, Saesangjub, Saechuchungbyeon, Dongjin No. 1, Saeggyehwabyeon, Huangbap, Hopyeongbap, Alternative Rice, Baekjinju No. 1, Sinunbong No. 1, Hyangmi Rice No. 1, Odae No. 1, Heuknam Rice, Namil Rice, Juan No. 1, Chinese Rice, Sura Rice, Black Jinju Rice, Hyangmi Rice No. 2, Gomung Rice, Josaeng Blackchal, Samgwang Kit for distinguishing two or more rice varieties selected from the group consisting of rice, incense rice, unflavored rice and black rice. The kit of claim 5, wherein the reagent for performing the amplification reaction comprises DNA polymerase, dNTPs, and a buffer. Separating genomic DNA from rice; Amplifying a target sequence by using the isolated genomic DNA as a template and performing an amplification reaction using the oligonucleotide primer set according to claim 1; And Detecting the amplification products, including, junambyeom, Nampyeongbyeong, Odaebyeong, Hwayeongbyeong, Chucheongbyeong, Hwaseongpyeong, a la carte rice, Taebongpyeong, Saesangjubyeong, Saechucheongbyeong, Dongjin No. 1, Saegyehwabyeon, haejangbap, Hopyeongbap, alternative Rice, Baekjinju No. 1, Sinunbong No. 1, Hyangmi Rice No. 1, Odae No. 1, Heuknam Rice, Namil Rice, Juan No. 1, Chinese Rice, Surami Rice, Heukjinju Rice, Hyangmi Rice No. 2, Gomung Rice, Josaeng Blackchal, Samgwang Rice A method of distinguishing two or more rice varieties selected from the group consisting of, Hyangnam rice, Unhyang rice and Black rice. 8. The method of claim 7, wherein the detection of the amplification product is carried out via capillary electrophoresis, DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement or phosphorescence measurement. 8. The method of claim 7, wherein the amplification of the target sequence is performed via multiplex PCR. The method of claim 9, wherein the final extension of the multiplex PCR is performed at 60 ° C. for 30 minutes.

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