KR102283638B1 - Random Amplified Polymorphic DNA primer for discrimination of tobacco cultivars - Google Patents

Abstract

The present invention relates to a method for identifying a tobacco variety by RAPD (Random Amplified Polymorphic DNA) analysis and a primer used in the method, and more specifically, a specific base sequence selected from the group consisting of SEQ ID NOs: 1 to 14 It relates to a RAPD primer having a RAPD primer, a method for detecting DNA polymorphism in tobacco, and a method for identifying a tobacco variety, comprising performing PCR using the same. The RAPD marker for tobacco variety discrimination according to the present invention can be very usefully used to create a tobacco DNA profile by effectively detecting DNA polymorphisms in tobacco. It can effectively perform an analysis of redundancy with existing resources and establish novelty, and has the effect of discriminating the variety of tobacco. In addition, it is possible to clearly distinguish between native varieties (lineages) scattered in Korea, and it is possible to register new varieties of unnamed varieties to protect our precious genetic resources, and to distinguish between domestic and imported varieties. It can contribute to establishing the distribution order in the tobacco market.

Description

RAPD primer for discrimination of tobacco varieties {Random Amplified Polymorphic DNA primer for discrimination of tobacco cultivars}

The present invention relates to a method for identifying a tobacco variety by RAPD (Random Amplified Polymorphic DNA) analysis and a primer used in the method, and more specifically, a specific base sequence selected from the group consisting of SEQ ID NOs: 1 to 14 It relates to a RAPD primer having a RAPD primer, a method for detecting DNA polymorphism in tobacco, and a method for identifying a tobacco variety, comprising performing PCR using the same.

Tobacco ( Nicotiana ) is one of the more than 3,000 Solanaceae crops. tabacum L. ) is used as one of the world's top five favorite products and has great economic value as well as an important model plant in genetics. The size of the genome of tobacco is 4.5×10 ⁹ bp (base pair), which is 1.5 times that of the human genome. North Carolina State University in the United States established the Tobacco Genome Initiative (TGI) and completed the draft of the tobacco genome. is unsupported.

In recent years, along with the radical development of molecular biology, molecular biological methods have been used in various ways to classify plant genus, interspecies, or intraspecies varieties. To this end, nucleic acid fingerprint analysis methods and various DNA markers that enable research on genetic diversity at the nucleic acid (DNA) level have been developed. Differentiation of strains by DNA analysis at the molecular biology level has the advantage of being able to know the quantitative level of the strain as well as a number of characteristics and excluding the influence of the environment. Among them, RFLP (Restriction Fragment Length Polymorphism) is the number or length of DNA fragments generated when a series of DNA chains are treated with restriction enzymes depending on differences in genetic composition between individuals, that is, differences in nucleotide sequence. Based on the fact that they appear different from each other, they are used in various ways. However, RFLP requires a large amount of high-purity DNA, takes a lot of time, effort, and money, and has disadvantages in that the test process is complicated and radioactive isotopes must be used.

As a method to overcome this problem, a Random Amplified Polymorphic DNA (RAPD) method, which is technically simple and easy to track polymorphism, has been developed. The RAPD method yields results quickly, is easy to apply, and does not require sequencing information in advance. As a technology using this technology, Korean Patent Registration No. 10-0264743 discloses “quantitative trait genetic map (QTL) involved in cold tolerance of rice seedlings using marker RAPD and a method for selecting low-temperature resistant rice varieties”, and the Republic of Korea Patent Registration No. No. 10-0215084 discloses “a method for determining the origin of Korean ginseng by RAPD marker and a primer used for the method”, and Korean Patent No. 10-0764561 discloses “a gene for male infertility recovery of onions using a RAPD marker” Determination method” is disclosed.

However, RAPD markers for tobacco varieties have not yet been developed in Korea, and in particular, studies involving genetic resources of domestic tobacco varieties have not been dealt with in foreign countries.

Accordingly, the present inventors produced a total of 80 RAPD primer sets to compare and analyze differences between tobacco varieties through RAPD analysis using molecular markers in a molecular biological approach. Among them, PCR amplification results using specific primers Through the formation of specific bands in five different tobacco varieties (Cheongjuyeop, Hyangcho, NC82, KF118, TA03) cultivated in .

Korean Patent Registration No. 10-0264743 Korean Patent Registration No. 10-0215084 Korean Patent No. 10-0764561

Accordingly, it is an object of the present invention to provide a RAPD primer capable of effectively discriminating varieties of tobacco grown in Korea.

Another object of the present invention is to provide a composition for identifying tobacco varieties comprising the RAPD primer.

It is also to provide a kit for identifying tobacco varieties comprising the composition.

Another object of the present invention is to provide a method for effectively discriminating tobacco varieties by performing PCR using the RAPD primer.

In order to achieve the object of the present invention as described above, the present invention provides a RAPD (Random Amplified Polymorphic DNA) primer for identifying tobacco varieties having a base sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 14.

In one embodiment of the present invention, the tobacco variety may be one selected from the group consisting of Cheongjuyeop, Hyangcho, NC82, KF118 and TA03.

In one embodiment of the present invention, SEQ ID NO: 1 or SEQ ID NO: 14 can discriminate the Cheongjuyeop among tobacco varieties.

In one embodiment of the present invention, SEQ ID NO: 6 or SEQ ID NO: 13 can discriminate the scented herb among tobacco varieties.

In one embodiment of the present invention, one selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 11 can discriminate NC82 among tobacco varieties.

In one embodiment of the present invention, one selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 9 and SEQ ID NO: 12 can discriminate KF118 among tobacco varieties.

In one embodiment of the present invention, one selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 7 can discriminate TA03 among tobacco varieties.

In addition, the present invention provides a composition for identifying tobacco varieties comprising the primer.

In addition, the present invention provides a kit for identifying tobacco varieties comprising the composition.

In addition, the present invention provides a method for determining tobacco varieties, comprising the step of performing PCR on the nucleic acid sample obtained from the individual using the primer.

The RAPD marker for tobacco variety discrimination according to the present invention can be very usefully used to create a tobacco DNA profile by effectively detecting DNA polymorphisms in tobacco. It can effectively perform an analysis of redundancy with existing resources and establish novelty, and has the effect of discriminating the variety of tobacco. In addition, it is possible to clearly distinguish between native varieties (lineages) scattered in Korea, and it is possible to register new varieties of unnamed varieties to protect our precious genetic resources, and to distinguish between domestic and imported varieties. It can contribute to establishing the distribution order in the tobacco market.

1 is a photograph showing the breeding process for each type of tobacco used in the present invention.
Figure 2 shows an electrophoretic picture of the reaction product after PCR amplification using DNA isolated from five tobacco varieties as a template using the primers (SEQ ID NOs: 1 to 14) of the present invention (A: primer OPA17, B: primer OPA19 , C: primer OPA20, D: primer OPB03, E: primer OPB18, F: primer OPC07,lane 1: Cheongjuyeop, lane 2: Hyangcho, lane 3: NC82, lane 4: KF118, lane 5: TA03).

In one aspect, the present invention is characterized by providing a RAPD (Random Amplified Polymorphic DNA) primer for identifying tobacco varieties having a base sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 14.

In the present invention, the tobacco variety may be one selected from the group consisting of Cheongjuyeop, Hyangcho, NC82, KF118 and TA03.

As used herein, the term 'primer' refers to a short nucleic acid sequence capable of forming a base pair with a complementary template and functioning as a starting point for template strand copying. In a specific implementation of the present invention, a primer having one base sequence selected from the group consisting of SEQ ID NOs: 1 to 14 was used.

In one embodiment of the present invention, SEQ ID NO: 1 or SEQ ID NO: 14 can discriminate Cheongjuyeop among tobacco varieties; The SEQ ID NO: 6 or SEQ ID NO: 13 can discriminate the scented herb among tobacco varieties; One selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 11 can discriminate NC82 among tobacco varieties; One selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 9 and SEQ ID NO: 12 can discriminate KF118 among tobacco varieties; One selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 7 can discriminate TA03 among tobacco varieties.

In another aspect, there is provided a composition for discrimination of tobacco varieties comprising the primer (RAPD primer for discrimination of tobacco varieties having a base sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 14).

In another aspect, the present invention provides a kit for identifying tobacco varieties comprising the composition.

Tobacco variety identification kit according to the present invention may include, in addition to the RAPD primer, a reaction buffer, polymerase, dNTP, stabilizer and all biological or chemical reagents, instructions for use, etc. necessary for detecting the tobacco variety in the sample. . Other components of this kit can be appropriately selected by those skilled in the art.

In another aspect, the present invention provides a method for determining a tobacco variety, comprising the step of performing PCR using a RAPD primer on a nucleic acid sample obtained from an individual.

The subject of the present invention is a target tobacco variety plant to be discriminated.

In the PCR of the present invention, the target sequence can be amplified by performing an amplification reaction on a nucleic acid sample obtained from an individual using a RAPD primer for identifying tobacco varieties having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 14. .

In one embodiment of the present invention, PCR is performed at 94°C for 15 minutes after the initial denaturation process, at 94°C for 1 minute (denaturation), at 35°C for 1 minute (annealing), at 72°C for 2 minutes (extension) The liver reaction is carried out 45 times, and as a final extension process, it can be made through the process of reacting at 72 ° C. for 10 minutes.

The target sequence amplified through the PCR may be labeled with a detectable labeling material. As a specific example, the labeling material may be a material emitting fluorescence, phosphorescence, or radiation, but is not limited thereto. Preferably, the labeling material is ethidium bromide (EtBr), Cy-5 or Cy-3. When PCR is performed by labeling Cy-5 or Cy-3 at the 5'-end of the primer during amplification of the target sequence, the target sequence may be labeled with a detectable fluorescent labeling material. In addition, in the case of labeling using a radioactive material, when a ^{radioisotope such as 32} P or ³⁵ S is added to the PCT reaction solution during PCR, the amplification product is synthesized while radiation is mixed into the amplification product, and the amplification product can be labeled with radiation. .

Detection of the amplified product may be performed through a DNA chip, gel electrophoresis, radiation measurement, fluorescence measurement, or phosphorescence measurement, but is not limited thereto. As one of the methods for detecting the amplification product, gel electrophoresis can be performed. 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, when PCR is performed by labeling the 5'-end of the primer with Cy-5 or Cy-3, the target sequence is labeled with a detectable fluorescent labeling material, and the labeled fluorescence is measured using a fluorescence meter. can do. In addition, in the radiation measurement method, a ^{radioisotope such as 32} P or ³⁵ S is added to the PCR reaction solution to label the amplification product during PCR, and then a radiation measuring device, for example, a Geiger counter or liquid scintillation Radiation can be measured using a liquid scintillation counter.

Hereinafter, the present invention will be described in more detail through examples. These Examples are for explaining the present invention in more detail, and the scope of the present invention is not limited to these Examples.

< Example 1>

Suitable for identification of tobacco varieties of the present invention primer Selection

<1-1> Tobacco Varieties Sample Preparation

In order to identify primers for RAOD suitable for identification of actual tobacco varieties, five different tobacco varieties, Cheongjuyeop, Hyangcho, NC82, KF118, and TA03 were bred in a greenhouse for 3 months, and then the leaves were used as samples (materials).

<1-2> DNA extraction

After DNA was extracted from the samples for each tobacco variety prepared in Example <2-1> using the DNeasy Plant Mini Kit (QIAgen), each DNA was quantified at 10 ^{μg/ml with a Qubit TM fluorometer (Invitrogen).}

<1-3> RAPD primer used PCR Perform

In this experiment, PCR analysis was first performed using a total of 80 RAPD primer sets, 20 each for OPA, OPB, OPC, and OPD primers for tobacco variety discrimination. PCR conditions were 45 cycles of pre-denaturation at 94°C for 15 minutes, denaturation at 94°C for 1 minute, annealing at 35°C for 1 minute, and extension at 72°C for 2 minutes, followed by 45 cycles of amplification, followed by final extension at 72°C for 10 minutes.

1.5% agarose gel electrophoresis was performed to confirm the amplified PCR product, and the band was confirmed using a UV Transiluminater.

As a result, as shown in FIG. 2, only 14 primers in a total of 80 RAPD primer sets were identified for specific bands according to tobacco varieties. Specifically, specific bands were identified with primers OPA13 and OPC20 in Cheongjuyeop, specific bands were identified in primers OPB03 and OPC19 in Hyangcho, and specific bands were identified in primers OPA13, OPC04, OPC06, and OPC07 in NC82, For KF118, specific bands were identified in primers OPA19, OPA20, OPC05, and OPC16, and in TA03, specific bands were identified in primers OPA14, OPA17, and OPB18. On the other hand, in the OPD primer set, a specific band for each type was not confirmed.

List of specific molecular markers through RAPD analysis of samples by tobacco variety Sample primer Cheongjuyeop OPA13, OPC20 incense OPB03, OPC19 NC82 OPA13, OPC04, OPC06, OPC07 KF118 OPA19, OPA20, OPC05, OPC16 TA03-A OPA14, OPA17, OPB18

Therefore, through the above results, it was verified through experiments that, in the total 80 RAPD primer sets, there were actually 14 primers for RAPD suitable for discrimination of tobacco varieties, and in the present invention, these 14 primers for RAPD are shown in Table 2 below As shown in SEQ ID NOs: 1 to 14.

Primer for RAPD for identification of tobacco varieties of the present invention primer Nucleotide sequence (5'-3') SEQ ID NO: OPA13 CAGCACCCAC SEQ ID NO: 1 OPA14 TCTGTGCTGG SEQ ID NO: 2 OPA17 GACCGCTTGT SEQ ID NO: 3 OPA19 AGGTGACCGT SEQ ID NO: 4 OPA20 CAAACGTCGG SEQ ID NO: 5 OPB03 CATCCCCCTG SEQ ID NO: 6 OPB18 GGTGACGCAG SEQ ID NO: 7 OPC04 CCGCATCTAC SEQ ID NO: 8 OPC05 GATGACCGCC SEQ ID NO: 9 OPC06 GAACGGACTC SEQ ID NO: 10 OPC07 GTCCCGACGA SEQ ID NO: 11 OPC16 TGGACCGGTG SEQ ID NO: 12 OPC19 CTCACCGTCC SEQ ID NO: 13 OPC20 TGTCTGGGTG SEQ ID NO: 14

So far, the present invention has been looked at with respect to preferred embodiments thereof. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

<110> Chungbuk National University Industry-Academic Cooperation Foundation <120> Random Amplified Polymorphic DNA primer for discrimination of tobacco cultivars <130> PN1412-365 <160> 14 <170> KopatentIn 2.0 <210> 1 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> OPA13 primer <400> 1 cagcacccac 10 <210> 2 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> OPA14 primer <400> 2 tctgtgctgg 10 <210> 3 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> OPA17 primer <400> 3 gaccgcttgt 10 <210> 4 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> OPA19 primer <400> 4 aggtgaccgt 10 <210> 5 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> OPA20 primer <400> 5 caaacgtcgg 10 <210> 6 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> OPB03 primer <400> 6 catccccctg 10 <210> 7 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> OPB18 primer <400> 7 ggtgacgcag 10 <210> 8 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> OPC04 primer <400> 8 ccgcatctac 10 <210> 9 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> OPC05 primer <400> 9 gatgaccgcc 10 <210> 10 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> OPC06 primer <400> 10 gaacggactc 10 <210> 11 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> OPC07 primer <400> 11 gtccccgacga 10 <210> 12 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> OPC16 primer <400> 12 tggaccggtg 10 <210> 13 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> OPC19 primer <400> 13 ctcaccgtcc 10 <210> 14 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> OPC20 primer <400> 14 tgtctgggtg 10

Claims (10)

SEQ ID NO: 1 to RAPD (Random Amplified Polymorphic DNA) represented by the base sequence of SEQ ID NO: 14, comprising a primer, Cheongjuyeop, Hyangcho, NC82, KF118 and TA03 at least one composition for identifying tobacco varieties selected from the group consisting of. delete delete delete delete delete delete delete A kit for identifying tobacco varieties comprising the composition of claim 1. performing PCR using the composition of claim 1 on a nucleic acid sample obtained from an individual;
analyzing the amplified PCR product; and
When the amplified PCR product is amplified by the RAPD primers represented by the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 14, it is determined as a Cheongjuyeop variety,
When amplified by the RAPD primer represented by the nucleotide sequence of SEQ ID NO: 6 and SEQ ID NO: 13, it is determined as a scented plant variety,
When amplified by the RAPD primers represented by the nucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 11, it is determined as a NC82 variety,
When amplified by the RAPD primers represented by the nucleotide sequences of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 9 and SEQ ID NO: 12, it is determined as KF118 variety,
When amplified by the RAPD primer consisting of the nucleotide sequences of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 7, determining the variety as TA03; including, Cheongjuyeop, Hyangcho, NC82, KF118 and TA03 selected from the group consisting of A method for determining one or more tobacco varieties.

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