KR101948389B1 - Discriminating method of Codonopsis lanceolata and Adenophora triphylla using SSR marker - Google Patents

Abstract

The present invention relates to a primer set consisting of SEQ ID NOS: 1 and 2; Or a primer set consisting of SEQ ID NOS: 3 and 4, and a primer set for SSR (Simple Sequence Repeat) detection for discriminating four kinds of primers.

Description

{Discriminating method of Codonopsis lanceolata and Adenophora triphylla using SSR marker using SSR markers}

The present invention relates to a primer set for SSR detection and a composition or kit for distinguishing between a primer set and a primer set, and relates to a method for determining the primer set using the primer set.

Codonopsis Lanceolata and Adenophora triphylla are perennial plants belonging to the Lentinaceae family. They are mainly grown in the mountains and mainly use roots. The roots of roe deer have been used as natural medicines in the private sector since ancient times, and have been used as living organisms or cooked food. Dodok has active ingredients such as saponins, phenylpropanoids, alkaloids, triterpenes and flavonoids. It has many saponins as well as ginseng. It is distributed in Korea, Japan and China. It has high vitamin A, C and calcium content. It has saponin and inulin as its main components and has been used for long time as edible and medicinal.

Korea has been using various plants for herbal medicines since ancient times. Doduk and Jigae are both used together as a medicinal herb called 沙 参. Doduck and ginseng are not distinguishable from each other when they are processed as herbal medicines, and they are difficult to distinguish from each other. However, the accurate description of doduk that is described in the Oriental Medicine Dictionary is "Yang Yum" and the precise origin of the saishan is the remainder, and the dodok and the remainder are different in sexuality and efficacy.

In addition to the radical development of molecular biology, molecular biology methods have been widely used to classify plant species, interspecies, or species within a plant. For this purpose, genetic diversity research at the level of DNA (DNA) And a variety of DNA markers are being developed. The discrimination of strains by DNA analysis at the level of molecular biology has the advantage of being able to grasp many characteristics along with the quantitative level of the strains and to exclude the influence of the environment. Among them, Restriction Fragment Length Polymorphism (RFLP) is a restriction fragment length polymorphism (RFLP) in which DNA fragments generated when a series of DNA chains are treated with restriction enzymes are different in their genetic composition, that is, And it is used variously based on the fact that it appears differently. However, RFLP requires a large amount of high purity DNA, is time-consuming, laborious and costly, has a complicated test procedure, and has a disadvantage of using radioactive isotopes.

The SSR (Simple Sequence Repeat) marker is made up of repetitions of short nucleotide sequences, which bind to the plant genome in PCR (Polymerase Chain Reaction) to amplify the DNA. As a result, various sizes of DNA fragments are produced and amplified depending on the plant species or genus. This allows the unique profile to be collected in the SSR reaction by reading the pattern using an electrophoretic method.

1. Korean Patent No. 10-1271330

An object of the present invention is to provide a primer set consisting of SEQ ID NOS: 1 and 2; Or a primer set consisting of SEQ ID NOS: 3 and 4, and a primer set for detecting SSR (Simple Sequence Repeat)

It is still another object of the present invention to provide a composition and a kit for distinguishing four cultivars comprising the primer set.

It is still another object of the present invention to provide a method for distinguishing four species of a variety including a step of performing PCR using the primer set for DNA extracted from a sample.

In order to achieve the above object, the present invention provides a primer set consisting of SEQ ID NOS: 1 and 2; Or a primer set consisting of SEQ ID NOS: 3 and 4, is provided for detecting SSR (Simple Sequence Repeat).

In one embodiment of the invention, the ginseng is selected from the group consisting of Codonopsis lanceolata or Adenophora triphylla .

The term "samsam" of the present invention refers to dodok and its tail, and both dodok and its tail are used in combination as a generic name of samsan.

The term "SSR (Simple Sequence Repeat)" of the present invention is made up of repetitions of short nucleotide sequences and is used to detect DNA polymorphism and analyze genetic diversity.

The term "primer" of the present invention means a short nucleic acid sequence capable of forming a base pair with a template having a base sequence complementary to the sequence and functioning as a starting point for template strand replication. The primer set of the present invention is composed of forward and reverse primers for amplification of the SSR marker.

In addition, the present invention provides a composition for distinguishing four cultivars comprising the primer set.

In one embodiment of the invention, the ginseng is selected from the group consisting of Codonopsis lanceolata or Adenophora triphylla .

In addition, the present invention provides a kit for distinguishing four cultivars comprising the primer set.

In one embodiment of the invention, the ginseng is selected from the group consisting of Codonopsis lanceolata or Adenophora triphylla .

In the present invention, the kit may further include a reaction buffer solution, a polymerase, and dNTP necessary for a PCR reaction in addition to the primer set. In addition, the kit may include a stabilizer and all biological or chemical reagents and instructions for use. Other configurations of such kits may be suitably selected by those skilled in the art.

In addition, the present invention provides a method for distinguishing four species of a variety including a step of performing PCR using the primer set for DNA extracted from a sample.

In one embodiment of the present invention, the method may further comprise analyzing an electrophoresis pattern of the PCR product.

In one embodiment of the present invention, the amplified target sequence may be labeled with a detectable labeling substance. In one specific example, the labeling material can be, but is not limited to, a material that emits fluorescence, phosphorescence, or radiation. Preferably, the labeling substance is Ethidium Bromide (EtBr), Cy-5 or Cy-3. When the target sequence is amplified, PCR is carried out by labeling the 5'-end of the primer with Cy-5 or Cy-3, and the target sequence may be labeled with a detectable fluorescent labeling substance. In addition, if the radioactive isotope such as ³² P or ³⁵ S is added to the PCR reaction solution, the amplification product may be synthesized and the radiation may be incorporated into the amplification product and the amplification product may be labeled with the radiation.

In one embodiment of the present invention, the detection of the amplified product can be performed through DNA chip, gel electrophoresis, radiation measurement, fluorescence measurement or phosphorescence measurement, but is not limited thereto. As one method of detecting the amplification product, gel electrophoresis can be performed. Gel electrophoresis can be performed using agarose gel electrophoresis or acrylamide gel electrophoresis depending on the size of the amplification product. Also, in the fluorescence measurement method, Cy-5 or Cy-3 is labeled at the 5'-end of the primer. When PCR is carried out, the target is labeled with a fluorescent label capable of detecting the target sequence. The labeled fluorescence is measured using a fluorescence meter can do. In addition, in the case of performing the PCR, the radioactive isotope such as ³² P or ³⁵ S is added to the PCR reaction solution to mark the amplification product, and then the radioactivity is measured by a radiation measuring device such as a Geiger counter or liquid scintillation counter The radiation can be measured using a liquid scintillation counter.

In one embodiment of the invention, the ginseng is selected from the group consisting of Codonopsis lanceolata or Adenophora triphylla .

Using the SSR primer set according to the present invention, it is possible to quickly and simply discriminate dodecs and dodecs, which are used in combination with each other, so that they can be usefully used in agriculture.

1 is Codonopsis (Codonopsis using the primer set CLtri0750 lanceolata ) and the adenophora triphylla (electrophoretic pattern).
2 is Codonopsis (Codonopsis using the primer set CLtetra2277 lanceolata ) and the adenophora triphylla (electrophoretic pattern).

Hereinafter, the present invention will be described in more detail with reference to examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.

Example  1. Doduk and From  DNA extraction

Codonopsis lanceolata ) and ginseng ( Adenophora triphylla ) were prepared by harvesting leaves. The prepared dodeca and leaves were extracted by the CTAB method after grinding. Specifically, 1 g of the crushed sample and 1 ml of CTAB solution containing 0.2% 2-mercaptoethanol were added to a microtube, and the mixture was incubated at 65 ° C for 30 minutes Respectively. After the incubation, chloroform and iso-amylalchol were added at a ratio of 24: 1 at a ratio of 700: 1, centrifuged at 12,000 rpm for 10 minutes at 16 ° C, and a supernatant was obtained. After the DNA pellet was identified by inverting after adding isopropanol in a ratio of 1: 1, the resulting supernatant was centrifuged at 12,000 rpm for 5 minutes at 4 ° C, and 200 μl of 70% ethanol was added to the pellet The mixture was centrifuged at 12,000 rpm at 4 ° C for 5 minutes and allowed to stand at room temperature for 10 minutes. Ethanol was volatilized and then 200 μl of 1 × TE buffer was added to dissolve and then 1 μl of RNase (10 mg / ml) C for 30 minutes to confirm the DNA concentration.

Example  2. PCR  DNA amplification using

10 ng of DNA and 10 ng of RAPD primer were added to 5 μl of DNA extracted with CTAB in Example 1, and 20 μl of PCR reaction mixture was prepared. The PCR procedure was as follows: preheating 94 ° C. 300 seconds; denaturation 94 캜 30 sec; annealing 58 ° C for 60 seconds; extension 72 ° C for 60 seconds; Repeat 35 cycles; And post extension 72 ° C for 600 seconds.

Example  3. In PCR  Used SSR primer (primer)

In order to distinguish dodeca from dodec, next generation sequencing was performed to analyze the genomes of dodeca and to develop SSR (Simple Sequence Repeat) markers. Sequence information for the SSR primers used in the PCR to distinguish between dodeca and dodeca is shown in Table 1 below.

primer direction Sequence information CLtri0750 forward 5'-ACTATGGAGGTCTCTCCTCACTC-3 '(SEQ ID NO: 1) reverse 5'-GAAGTATACGCTGTCCCATACG-3 '(SEQ ID NO: 2) CLtetra2277 forword 5'-TAAAGACGTGTGGCCATGT-3 '(SEQ ID NO: 3) reverse 5'-CTATGCAAAGTGTGCTCGTC-3 '(SEQ ID NO: 4)

Example  4. PCR  SSRs showing polymorphism using Primer  Pattern reading

PCR products were analyzed by DNA Fragment Analyzer Automated CE System (Advanced Analytical Technologies, Ankeny, IA, USA). Patterns were read through the bands indicating polymorphism in the dodeca and the remainder.

Using the CLtri0750 primer set (primer set consisting of SEQ ID NOS: 1 and 2), Codonopsis lanceolata and Adenophora triphylla showed that the band of 159 bp was observed in the case of the remainder, while the band of 159 bp in the case of the case of dodeca was formed (Fig. 1).

Further, using the CLtetra2277 primer set (a primer set consisting of SEQ ID NOS: 3 and 4), Codonopsis lanceolata and Adenophora triphylla showed that the band of 395 bp was formed in the remainder, while the band of less than 200 bp was formed in the bands of Adenophora triphylla .

Therefore, it was confirmed that the dodeca can be discriminated by using the CLtri0750 primer set or the CLtetra2277 primer set for SSR markers.

<110> Industry-Academic Cooperation Foundation of Chungbuk University <120> Discriminating method of Codonopsis lanceolata and Adenophora          triphylla using SSR marker <130> PN1610-377 <160> 4 <170> KoPatentin 3.0 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 1 actatggagg tctctcctca ctc 23 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 2 gaagtatacg ctgtcccata cg 22 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 3 taaagacgtg tggccatgt 19 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 4 ctatgcaaag tgtgctcgtc 20

Claims (9)

A primer set for detecting SSR (Simple Sequence Repeat) for distinguishing between dodec and dodecontaining a primer set consisting of SEQ ID NOS: 1 and 2. delete A composition for distinguishing between dodeca and halite, comprising the primer set of claim 1. delete A kit for distinguishing between dodeca and halitosis comprising the primer set of claim 1. delete And performing PCR using the primer set of claim 1 for the DNA extracted from the sample. 8. The method of claim 7,
Wherein the method further comprises analyzing an electrophoresis pattern of the PCR product. delete

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