KR102427121B1 - Molecular marker based on chloroplast genome sequence for discriminating Panax ginseng 'Gowon' cultivar and uses thereof - Google Patents

Abstract

The present invention relates to a chloroplast genome sequence-based molecular marker for discriminating the Panax ginseng Gowon cultivar, and uses thereof. A primer set of the present invention can be used to solve the seed purity problem during cultivation of ginseng and wild ginseng and the unclear cultivar problem during distribution, and can be used as a basis for selecting the cultivar suitable for specific cultivation environments in the cultivation of ginseng and wild ginseng, which are greatly affected by the cultivation environment.

Description

Molecular marker based on chloroplast genome sequence for discriminating Panax ginseng 'Gowon' cultivar and uses thereof}

The present invention relates to a chloroplast genome sequence-based molecular marker for distinguishing ginseng 'plateau' cultivars and their use.

Korean ginseng ( Panax ginseng ) is a perennial plant belonging to the genus Araliaceae. Ginseng has been known as a mysterious elixir since ancient times, and the root is used for medicinal purposes in oriental medicine. Ginseng contains a large amount of high-functional substances such as saponins, ginsenosides, polyacetylenes, acidic polysaccharides, proteins, and phenolic substances, so its pharmacological effect is superior to other crops.

Among the various ginseng varieties, the 'Kowon' variety, which means the best, has flat leaves, light purple stems, purple around the petiole and the base, and a thick body. In addition, Kowon has excellent resistance to spot disease, which occurs most often in the above-ground part of ginseng, and is highly accommodating to environmental changes as it is somewhat resistant to salt and high temperature. It is known that the content of Rd, etc., which promotes the secretion of neocortical hormones, is particularly high.

Wild ginseng, which is artificially cultivated from ginseng seeds or seedlings in the mountains, and cultivated in a form similar to wild ginseng, has superior pharmacological effects compared to ginseng, and belongs to a representative high-income cultivated crop among crops obtained from forests. Currently, domestic wild ginseng farmers are using wild, local, and various varieties of ginseng seeds and seedlings without any special standards, and the cultivation methods are also different depending on the farms. Since ginseng is grown in the same place for 4 to 6 years, it is greatly affected by the cultivation environment and suffers a lot of damage from various diseases, so the survival rate until harvest is very low. Currently, the classification of ginseng seeds used for cultivating wild ginseng is not well done, so it is necessary to check which ginseng seeds to be sown for active and high-yield growth in a specific environment, and whether they are appropriate in terms of growth and yield.

Therefore, various types of molecular markers were developed for ginseng species identification, origin research, variety identification, and efficient molecular breeding. Molecular markers can stably search for and identify genetic variations in all tissues regardless of plant growth using a small amount of DNA, thus supplementing the limitations in morphological and physicochemical discrimination and providing reliability in a short time at relatively low cost. Higher analysis is possible. The InDel (Insertion/Deletion) marker used in the present invention is a method for detecting polymorphism using PCR (Polymerase Chain Reaction) technology, and a relatively simple method can obtain rapid results. InDel markers are molecular markers suitable for breed classification because they exist in various genotypes by using the difference in the nucleotide sequence inserted or deleted from the nucleotide sequence.

On the other hand, Korean Patent No. 1326333 discloses 'SNP primer for distinguishing Korean ginseng yeonpung cultivar and a method for distinguishing yeonpoong cultivar using the same', and Korean Patent No. 1761290 discloses a primer set for distinguishing Korean ginseng cultivar and its use. has been disclosed, but there is no description of a molecular marker based on the chloroplast genome sequence and its use for distinguishing the ginseng 'plateau' variety of the present invention.

The present invention was derived from the above requirements, and the present inventors provided 14 kinds of ginseng (Kowon, K-1, Geumjin, Seonun, Geumseon, Cheongseon, Seonhyang, Cheonryang, Seonwon, Yeonpung, Seonpung, Geumpung, Gopung, Cheonpung) By comparing the chloroplast genome sequence of As a result of performing PCR on 14 species, the present invention was completed by confirming that the primer set of the present invention could accurately distinguish only highland varieties from 14 ginseng species.

In order to solve the above problems, the present invention provides a primer set for distinguishing ginseng 'plateau' varieties comprising the oligonucleotide primer sets of SEQ ID NOs: 1 and 2.

In addition, the present invention provides a kit for distinguishing ginseng 'plateau' varieties including the primer set and reagents for performing the amplification reaction.

In addition, the present invention comprises the steps of isolating genomic DNA from a ginseng sample; amplifying a target sequence by using the isolated genomic DNA as a template and performing an amplification reaction using the primer set according to the present invention; and detecting the product of the amplification step.

The primer set of the present invention can quickly and accurately distinguish 'Kogen' varieties from among various domestic ginseng varieties at the genetic level, so it can be used to solve the seed purity problem that appears during ginseng and wild ginseng cultivation and the unclear variety problem that appears in the distribution process. In the cultivation of ginseng and wild ginseng, which are highly affected by the cultivation environment, it can be used as a basis for selection of varieties suitable for a specific cultivation environment.

1 shows 14 types of ginseng (K-1, Geumjin, Seonun, Geumseon, Cheongseon, Seonhyang, Cheonryang, Seonwon, Gowon, Yeonpung, Seonpung, Geumpung, Gopung, Cheonpung) using the Pg_CpInDel_5 primer set (Table 1) of the present invention. It is the result of performing PCR on the target.

In order to achieve the object of the present invention, the present invention provides a primer set for distinguishing the ginseng 'plateau' variety comprising the oligonucleotide primer sets of SEQ ID NOs: 1 and 2.

The primer may include an oligonucleotide consisting of a fragment of 10 or more, 11 or more, 12 or more, 13 or more, 14 or more consecutive nucleotides in the sequences of SEQ ID NOs: 1 and 2, depending on the sequence length of each primer. have. For example, the primer of SEQ ID NO: 1 (14 oligonucleotides) may include an oligonucleotide consisting of a fragment of 10 or more, 11 or more, 12 or more, 13 or more consecutive nucleotides in the sequence of SEQ ID NO: 1. . In addition, the primer may also include a sequence added, deleted or substituted in the nucleotide sequences of SEQ ID NOs: 1 and 2 capable of binding to the amplification site. The oligonucleotide primer of SEQ ID NO: 1 of the present invention is a forward primer, and the oligonucleotide primer of SEQ ID NO: 2 is a reverse primer.

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

As used herein, the oligonucleotide used as a primer may also contain a nucleotide analogue, for example, a phosphorothioate, an alkylphosphorothioate or a peptide nucleic acid. An intercalating agent may be included.

The present invention also provides a kit for distinguishing ginseng 'plateau' varieties including the primer set and reagents for performing the amplification reaction.

In one embodiment of the present invention, the reagent for performing the amplification reaction may include, but is not limited to, DNA polymerase, dNTPs, and a buffer.

The kit of the present invention may further comprise a user's guide describing optimal conditions for conducting the reaction. A handbook is a printout explaining how to use the kit, eg, how to prepare a PCR buffer, and suggested reaction conditions. Instructions include a brochure in the form of a pamphlet or leaflet, a label affixed to the kit, and instructions on the surface of the package containing the kit. In addition, the guide includes information published or provided through electronic media such as the Internet.

The present invention also

isolating genomic DNA from the ginseng sample;

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

It provides a method for distinguishing ginseng 'plateau' varieties comprising; detecting the product of the amplification step.

The method of the present invention includes isolating genomic DNA from a ginseng sample. A method for isolating genomic DNA from the ginseng sample may use a method known in the art, for example, the CTAB method may be used, or a Wizard prep kit (Promega) may be used. A target sequence may be amplified by using the isolated genomic DNA as a template and performing an amplification reaction using the primer set according to an embodiment of the present invention. Methods for amplifying the target nucleic acid include polymerase chain reaction (PCR), ligase chain reaction, nucleic acid sequence-based amplification, transcription-based amplification system, There are strand displacement amplification or amplification via Qβ replicase 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.

In the method according to an embodiment of the present invention, the ginseng sample may be a plant root, leaf, stem or seed, but is not limited thereto.

In one embodiment of the present invention, the amplified target sequence may be labeled with a detectable labeling substance. The labeling material may be a material emitting fluorescence, phosphorescence, or radioactivity, but is not limited thereto. Preferably, the labeling substance is 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, when a radioactive isotope such as ³² P or ³⁵ S is added to the PCR reaction solution for labeling using a radioactive material, the amplification product is synthesized and radioactivity is incorporated into the amplification product, so that the amplification product can be radioactively labeled. The oligonucleotide primer sets used to amplify the target sequence are as described above.

In one embodiment of the present invention, the method for distinguishing the ginseng 'plateau' variety comprises the step of detecting the amplification product, and the detection of the amplification product is a DNA chip, gel electrophoresis, capillary electrophoresis, or radioactive measurement. , may be performed through fluorescence measurement or phosphorescence measurement, but is not limited thereto. As one of the methods for detecting the amplification product, capillary electrophoresis may be performed. Capillary electrophoresis may use, for example, an ABi Sequencer. In addition, gel electrophoresis can be performed, and agarose gel electrophoresis or acrylamide gel electrophoresis can be used for 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 fluorometer. can do. In addition, the radioactive measurement method includes adding a radioisotope such as ³² P or ³⁵ S to the PCR reaction solution during PCR to label the amplification product, and then a radioactive measuring instrument, for example, a Geiger counter or liquid scintillation. The radioactivity can be measured using a liquid scintillation counter.

In the method according to an embodiment of the present invention, when PCR is performed using the ginseng genomic DNA as a template using the primer sets of SEQ ID NOs: 1 and 2, a band with a size of 248 bp is detected, it is determined as a 'plateau' variety When a band with a size of 231 bp is detected, it can be identified as 'K-1, Geumjin, Seonun, Geumseon, Cheongseon, Seonhyang, Cheonryang, Seonwon, Yeonpung, Seonpung, Geumpung, Gopung and Cheonpung' varieties.

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

Materials and Methods

1. Ginseng Genetic Resource Collection and DNA Extraction

The 14 types of ginseng used in the experiment (K-1, Geumjin, Seonun, Geumseon, Cheongseon, Seonhyang, Cheonryang, Seonwon, Gowon, Yeonpung, Seonpung, Geumpung, Gopung, Cheonpung) were sampled in leaf form by the Rural Development Administration. and stored in a cryogenic refrigerator at -70°C. For DNA extraction, the young leaf samples were rapidly cooled using liquid nitrogen and pulverized using a mortar, and then extracted using Biomedic's Plant gDNA Extraction Kit.

2. InDle marker development

After comparing and analyzing the chloroplast sequences of 14 types of ginseng using the CLC Main Workbewnch program (version 20), a 17 bp sequence (ATTATACTTCTATATTT) was specifically inserted in the genome sequence of the ginseng 'Kogen' variety. A primer set capable of amplifying the region was prepared (Table 1). The InDel polymorphism is described in GenBank accession No. It is present in the trnT-UGU and trnL -UAA intergenic region in OL543607, and the entire fragment site including the amplification site for the InDel polymorphism and the forward and reverse primer sites is located at 48,907~49,137 bp.

Primer set information of the present invention Primer name Sequence (5'→3') (SEQ ID NO:) Pg_CpInDel_5_Fwd GGATTTATCTAGCG (1) Pg_CpInDel_5_Rev GCAGGAATATCTCA (2)

3. DNA amplification using polymerase chain reaction (PCR)

2 μl of DNA (10 ng of DNA) diluted with the extracted ginseng genomic DNA to 5 ng/μl, 1 μl of InDel marker (0.5 μl of 5 μM forward primer, 0.5 μl of 5 μM reverse primer), 5 μl of Taq mixture (0.25 U/μl Taq DNA polymerase, 2x PCR buffer, 0.4 mM dNTPs, 3.2 mM MgCl ₂ , 0.02% bromophenol blue) and 2 μl of distilled water were mixed to make 10 μl of a PCR reaction mixture. PCR process was pre-denaturation 95 ℃ 5 minutes; A total of 34 cycles of denaturation at 95° C. for 30 seconds, annealing at 46.6° C. for 30 seconds, and extension at 72° C. for 1 minute; Final extension (final extension) was performed under the condition of 72 ℃ 10 minutes. PCR amplification products were confirmed by electrophoresis on a 2% agarose gel.

Example 1. Confirmation of discrimination of ginseng 'Kogen' varieties using the primer set of the present invention

14 types of ginseng (Kowon, K-1, Geumjin, Seonun, Geumseon, Cheongseon, Seonhyang, Cheonryang, Seonwon, Yeonpung, Seonpung, Geumpung, PCR was performed using the DNA sample of Gopoong, Cheonpung) as a template.

As a result, a band with a size of 248 bp was confirmed in the 'Kowon' ginseng cultivar, and in the remaining 'K-1, Geumjin, Seonun, Geumseon, Cheongseon, Seonhyang, Cheonryang, Seonwon, Yeonpung, Seonpung, Geumpung, Gopoung, Cheonpung' cultivars, A band with a size of 231 bp was confirmed, and it was found that only the 'Kogen cultivar' could be accurately distinguished from the 14 ginseng species (FIG. 1).

<110> Chungbuk National University Industry-Academic Cooperation Foundation <120> Molecular marker based on chloroplast genome sequence for discriminating Panax ginseng 'Gowon' cultivar and uses thereof <130> PN22144 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 ggatttatt agcg 14 <210> 2 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 gcaggaatat ctca 14

Claims (5)

A primer set for distinguishing ginseng 'Kogen' varieties comprising the oligonucleotide primer sets of SEQ ID NOs: 1 and 2. A kit for distinguishing ginseng 'Kogen' varieties comprising the primer set of claim 1 and reagents for performing an amplification reaction. The kit according to claim 2, wherein the reagents for performing the amplification reaction include DNA polymerase, dNTPs and a buffer. isolating genomic DNA from the ginseng sample;
amplifying a target sequence by using the isolated genomic DNA as a template and performing an amplification reaction using the primer set of claim 1; and
Detecting the product of the amplification step; a method of distinguishing a ginseng 'plateau' variety comprising a. The method according to claim 4, wherein the detection of the product of the amplification step is performed through a DNA chip, gel electrophoresis, radiometric measurement, fluorescence measurement, or phosphorescence measurement.

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