KR102451456B1 - Primer set for discriminating genetic polymorphism of Codonopsis lanceolata and uses thereof - Google Patents

Primer set for discriminating genetic polymorphism of Codonopsis lanceolata and uses thereof Download PDF

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KR102451456B1
KR102451456B1 KR1020210189215A KR20210189215A KR102451456B1 KR 102451456 B1 KR102451456 B1 KR 102451456B1 KR 1020210189215 A KR1020210189215 A KR 1020210189215A KR 20210189215 A KR20210189215 A KR 20210189215A KR 102451456 B1 KR102451456 B1 KR 102451456B1
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primer
dna
artificial sequence
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조남수
이미선
김주혁
이이
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충북대학교 산학협력단
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    • C—CHEMISTRY; METALLURGY
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Abstract

The present invention relates to a primer set for distinguishing genetic polymorphism of Codonopsis lanceolata, and uses thereof. The primer set of the present invention can analyze the genetic or phylogenetic relationship of Codonopsis lanceolata by effectively detecting DNA polymorphisms of various Codonopsis lanceolata genetic resources. Therefore, the primer set can be usefully used in establishing the origin of Codonopsis lanceolata or in the breeding industry.

Description

더덕의 유전적 다형성을 구별하기 위한 프라이머 세트 및 이의 용도{Primer set for discriminating genetic polymorphism of Codonopsis lanceolata and uses thereof}Primer set for discriminating genetic polymorphism of Codonopsis lanceolata and uses thereof

본 발명은 더덕의 유전적 다형성을 구별하기 위한 프라이머 세트 및 이의 용도에 관한 것이다.FIELD OF THE INVENTION The present invention relates to a set of primers and their use for differentiating genetic polymorphisms in deodeok.

더덕(Codonopsis lanceolata)은 도라지과(Companulaceae)에 속하는 다년생의 쌍자엽 식물로, 한국, 일본, 중국 및 러시아 극동지방 등 습기가 있는 초지, 숲 또는 계곡에 자생하는 식물이다. 더덕은 사삼 또는 백삼이라 불리며, 생약의 사삼은 건조된 뿌리를 칭한다. 건조된 뿌리에는 폴리아세틸렌(polyacetylene), 페닐프로파노이드(phenylpropanoid), 알칼로이드(alkaloid), 트리테르페노이드 (triterpenoid) 또는 다당류 등을 많이 함유하고 있으며, 특히 다량으로 함유되어 있는 사포닌(saponin)은 기침을 멎게 하고 가래를 억제하는 효과, 기관지염, 편도선염, 인후염 등 호흡기질환에 효능이 있다.Deodeok ( Codonopsis lanceolata ) is a perennial dicotyledonous plant belonging to the bellflower family ( Companulaceae ), and is a plant that grows wild in moist grasslands, forests or valleys in Korea, Japan, China and the Russian Far East. Deodeok is called sasam or white ginseng, and sasanam in herbal medicine refers to the dried root. Dried roots contain a lot of polyacetylene, phenylpropanoid, alkaloid, triterpenoid, or polysaccharide, and especially saponin, which contains a large amount of It is effective in stopping coughing and suppressing phlegm, and in respiratory diseases such as bronchitis, tonsillitis, and sore throat.

약용식물의 판별방법으로는 크게 (1) 형태적 판별, (2) 이화학적 판별, (3) DNA 분자마커를 이용한 판별 등으로 나눌 수 있다. 식물의 형태적 판별은 근연속 식물의 종간 구분이 어려우며, 질적형질보다는 양적형질인 경우도 많아 재배 환경에 따른 변이가 일어날 수도 있을 뿐만 아니라, 타식에 의한 혼연종 발생이 빈번하여 식물 종간 형태적 구분이 어렵다. 특히 약용식물은 약용부위에 따라 박피, 절편, 건조 등 다양한 방법으로 가공 유통되므로 전문가가 아니면 구분이 어려운 실정이다. 이화학적 성분분석에 의한 판별의 경우 분석을 위한 시간과 노력이 많이 들며, 약용식물의 재배환경 및 생육조건에 따라 약리적 성분에 변화가 올 수 있어 판별에 한계가 있다. DNA 분석에 의한 분자생물학적 식물판별기술은 위와 같은 문제점을 해결하기 위한 도구로서 인정받고 있는데, DNA 유전체는 환경의 변화에 따른 변화가 없으며, 가공 유통되어 식물 원재료를 쉽게 판별할 수 없는 한약재의 판별에도 유용하게 이용될 수 있다. 분자마커란 중합효소연쇄반응(Polymorphism Chain Reaction) 기술을 사용하여 다형성을 검출하는 방법으로 기본적으로 간단하고 신속하며 재배환경이나 식물의 발육단계, 연구자의 주관성 등 여러 환경적 요인으로부터 결과에 영향을 받지 않는다.The identification methods for medicinal plants can be broadly divided into (1) morphological identification, (2) physicochemical identification, and (3) identification using DNA molecular markers. In the morphological discrimination of plants, it is difficult to distinguish between species of closely-connected plants, and there are many cases of quantitative rather than qualitative traits, which may cause variations depending on the cultivation environment. This is difficult. In particular, medicinal plants are processed and distributed in various ways such as peeling, cutting, and drying depending on the medicinal part, so it is difficult for non-professionals to distinguish them. In the case of discrimination by physicochemical component analysis, it takes a lot of time and effort for analysis, and there is a limit to discrimination because the pharmacological component may change depending on the cultivation environment and growth conditions of medicinal plants. Molecular biological plant identification technology by DNA analysis is recognized as a tool to solve the above problems, but the DNA genome does not change according to changes in the environment, and it is also used for the identification of herbal medicines that are processed and distributed and cannot easily identify plant raw materials. It can be usefully used. Molecular markers are a method of detecting polymorphisms using Polymorphism Chain Reaction technology. They are basically simple and fast, and are not affected by various environmental factors such as the cultivation environment, plant development stage, and the subjectivity of the researcher. does not

식물의 경우 핵, 미토콘드리아, 엽록체 게놈 등 3개의 게놈을 가지고 있다. 식물의 유전정보의 대다수는 핵 게놈에 존재하지만 게놈의 크기가 크고, 많은 인트론(intron)과 엑손(exon)으로 구성되어 있어, 특정 유전자의 염기 서열을 유전체 DNA(genomic DNA)로부터 분석하여 식물종의 보존이나 식별에 이용하는데는 제한적이며, 핵 리보솜 DNA(nuclear ribosomal DNA, nrDNA)의 ITS(internal transcribed spacer) 부위, 알코올탈수소효소(alcohol dehydrogenase)의 특정 인트론 부위 등이 이용된다. 식물 미토콘드리아 게놈의 경우 보통 400kb∼4,000kb 크기로, 동물 미토콘드리아와는 달리 크기가 크고 구조적으로 매우 불안정하며 염기서열이 매우 보존적이기 때문에 속, 종 수준 또는 그 이하의 분류군의 계통관계나 식별에 이용하는 것은 거의 의미가 없다.Plants have three genomes: the nucleus, the mitochondrial genome, and the chloroplast genome. Most of the genetic information of plants exists in the nuclear genome, but the genome size is large and consists of many introns and exons. It is limited in its use for conservation or identification, and the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA) and a specific intron region of alcohol dehydrogenase are used. Plant mitochondrial genomes are usually 400kb to 4,000kb in size, and unlike animal mitochondria, they are large in size, structurally very unstable, and have very conserved base sequences. It makes little sense.

반면에 식물 엽록체는 기원(origin)이나 진화(evolution)와 같은 엽록체 자체 연구뿐만 아니라, 유전학적인 연구에서도 매우 중요시되어왔다. 엽록체 게놈의 크기는 160Kb 내외이고 구조적으로 안정하며, 염기서열의 변이가 큰 비암호화 영역(non coding region)은 고유종의 식별, 아종 및 변종의 식별, 유전적 변이의 비교 분석에도 유용성이 보고되고 있다. 엽록체 게놈에 존재하는 120여개의 유전자 중 84개의 유전자가 단백질로 전사되는데 유전자에 따라서 진화속도가 크게 다르다. 어떤 보존적인 유전자는 고등 분류군의 계통에만 이용될 수 있으나 빨리 진화하는 유전자의 염기서열은 근연속이나 속내의 뚜렷한 종간의 유연관계를 논의하는데도 이용될 수 있다. 따라서 한국 특산속 식물의 타당성, 식별, 보존 전략을 세우는데 엽록체 게놈의 염기서열은 광범위하게 활용이 가능하다.On the other hand, plant chloroplasts have been very important not only in the study of chloroplasts themselves, such as origin and evolution, but also in genetic studies. The size of the chloroplast genome is about 160Kb and it is structurally stable, and the non-coding region with large nucleotide sequence variation is reported to be useful for identification of endemic species, identification of subspecies and variants, and comparative analysis of genetic variations. . Of the 120 genes present in the chloroplast genome, 84 genes are transcribed into proteins, and the rate of evolution varies greatly depending on the gene. Some conserved genes can be used only for lines of higher taxa, but the sequences of fast-evolving genes can also be used to discuss kinship or distinct species kinship within a genus. Therefore, the base sequence of the chloroplast genome can be used extensively to establish the feasibility, identification, and conservation strategies of plants endemic to Korea.

한편, 한국등록특허 제1752658호에는 '더덕 품종 구별을 위한 SSR 분자마커 및 이의 용도'가 개시되어 있고, 한국등록특허 제2010279호에는 더덕 속 식물들 간의 InDel 다형성에 기반한 '더덕 속 식물에서 더덕 종을 구별하기 위한 분자마커 및 이의 용도'가 개시되어 있으나, 본 발명의 더덕의 유전적 다형성을 구별하기 위한 더덕 유전자들 간의 SNP 또는 InDel 다형성에 기반한 KASP용 프라이머 세트에 대해서는 기재된 바가 없다.On the other hand, Korea Patent No. 1752658 discloses 'SSR molecular markers for distinguishing deodeok varieties and their use', and Korean Patent No. 2010279 discloses 'Plants of the genus Deodeok in the genus Deodeok based on InDel polymorphism among plants of the genus Deodeok' is disclosed. Molecular markers and uses thereof for distinguishing 'are disclosed, but there is no description of a primer set for KASP based on SNP or InDel polymorphism between deodeok genes for distinguishing the genetic polymorphism of deodeok of the present invention.

본 발명은 상기와 같은 요구에 의해 도출된 것으로, 본 발명자는 NCBI에 등록된 더덕(Codonopsis lanceolata) 엽록체 서열(MH018574.1)을 포함한 44개 더덕 자원의 엽록체 게놈 서열을 비교하여 233개의 다형성 구간(SNP 190개, InDel 43개)을 탐색한 후 SNP 37개와 InDel 7개에 기반한 KASP(kompetitive allele specific PCR) 마커 44개를 개발하였으며, 상기 개발된 KASP 마커가 78개 더덕 자원의 유전적 다형성을 효과적으로 구별할 수 있음을 확인함으로써, 본 발명을 완성하였다.The present invention was derived from the above needs, and the present inventors compared the chloroplast genome sequences of 44 deodeok resources including the NCBI registered deodeok ( Codonopsis lanceolata ) chloroplast sequence (MH018574.1) to find 233 polymorphic sections ( After searching 190 SNPs and 43 InDels), 44 KASP (kompetitive allele specific PCR) markers were developed based on 37 SNPs and 7 InDels. By confirming that it can be distinguished, the present invention was completed.

상기 과제를 해결하기 위해, 본 발명은 서열번호 1 내지 132로 표시된 올리고뉴클레오티드에서 서열번호 1부터 인접한 3개의 올리고뉴클레오티드가 하나의 프라이머 세트인 것을 특징으로 하는 44개의 올리고뉴클레오티드 프라이머 세트를 포함하는, 더덕(Codonopsis lanceolata)의 유전적 다형성 구별용 프라이머 세트 조성물을 제공한다.In order to solve the above problems, the present invention provides a set of 44 oligonucleotide primers, characterized in that three oligonucleotides adjacent from SEQ ID NO: 1 in the oligonucleotides shown in SEQ ID NOs: 1 to 132 are one primer set, deodeok ( Codonopsis lanceolata ) Provides a primer set composition for distinguishing genetic polymorphisms.

또한, 본 발명은 상기 프라이머 세트 조성물을 포함하는 더덕의 유전적 다형성 구별용 키트를 제공한다.In addition, the present invention provides a kit for distinguishing genetic polymorphisms of deodeok comprising the primer set composition.

또한, 본 발명은 더덕 시료로부터 게놈 DNA를 분리하는 단계; 상기 분리된 게놈 DNA를 주형으로 하고, 상기 프라이머 세트 조성물을 이용하여 증폭 반응을 수행하여 표적 서열을 증폭하는 단계; 및 상기 증폭 단계의 산물을 검출하는 단계;를 포함하는 더덕의 유전적 다형성을 구별하는 방법을 제공한다.In addition, the present invention comprises the steps of isolating genomic DNA from a deodeok sample; amplifying a target sequence by using the isolated genomic DNA as a template and performing an amplification reaction using the primer set composition; and detecting the product of the amplification step; provides a method for distinguishing the genetic polymorphism of deodeok comprising.

본 발명의 프라이머 세트는 다양한 더덕 유전자원들의 DNA 다형성을 효과적으로 검출하여 더덕의 유전적 또는 계통발생학적 관계를 분석할 수 있으므로, 더덕의 기원정립 또는 육종 산업에 유용하게 활용될 수 있을 것으로 기대된다.The primer set of the present invention can effectively detect DNA polymorphisms of various deodeok genetic resources and analyze the genetic or phylogenetic relationship of deodeok, so it is expected to be usefully utilized in the establishment of the origin of deodeok or the breeding industry.

도 1은 본 발명의 KASP용 프라이머 세트(표 2 내지 표 4)를 이용하여 78개 더덕 자원에 대한 계통수(phylogenetic tree)를 나타낸 그림이다.1 is a diagram showing a phylogenetic tree for 78 deodeok resources using a primer set for KASP of the present invention (Tables 2 to 4).

본 발명의 목적을 달성하기 위하여, 본 발명은 서열번호 1 내지 132로 표시된 올리고뉴클레오티드에서 서열번호 1부터 인접한 3개의 올리고뉴클레오티드가 하나의 프라이머 세트인 것을 특징으로 하는 44개의 올리고뉴클레오티드 프라이머 세트를 포함하는, 더덕(Codonopsis lanceolata)의 유전적 다형성 구별용 프라이머 세트 조성물을 제공한다.In order to achieve the object of the present invention, the present invention provides a set of 44 oligonucleotide primers, characterized in that three oligonucleotides adjacent from SEQ ID NO: 1 in the oligonucleotides shown in SEQ ID NOs: 1 to 132 are one primer set , Deodeok (Codonopsis lanceolata) provides a primer set composition for distinguishing genetic polymorphisms.

본 발명의 상기 서열번호 1 내지 132의 프라이머는 각 프라이머의 서열 길이에 따라 18개 이상, 15개 이상, 16개 이상, 17개 이상, 18개 이상, 19개 이상, 20개 이상, 21개 이상, 22개 이상의 연속 뉴클레오티드의 절편으로 이루어진 올리고뉴클레오티드를 포함할 수 있다. 또한, 상기 프라이머는 서열번호 1 내지 132의 염기서열의 부가, 결실 또는 치환된 서열도 포함할 수 있다. The primers of SEQ ID NOs: 1 to 132 of the present invention are 18 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, depending on the sequence length of each primer. , an oligonucleotide consisting of a segment of 22 or more contiguous nucleotides. In addition, the primer may also include an addition, deletion or substitution of the nucleotide sequence of SEQ ID NOs: 1 to 132.

본 발명에 따른 서열번호 1 내지 132의 프라이머는 다양한 더덕 자원의 엽록체 게놈 서열 간의 SNP(single nucleotide polymorphism) 염기 타입 또는 InDel(insertion/deletion) 다형성 영역을 검출하기 위한 KASP용 프라이머 세트로, 연속되는 서열번호 3개의 올리고뉴클레오티드가 하나의 프라이머 세트를 이루며, 2개의 정방향 프라이머와 1개의 역방향 프라이머로 구성되어 있다.The primers of SEQ ID NOs: 1 to 132 according to the present invention are SNP (single nucleotide polymorphism) nucleotide types or InDel (insertion/deletion) polymorphic regions between chloroplast genome sequences of various deodeok resources. A primer set for KASP for detecting a polymorphic region, a continuous sequence Three numbered oligonucleotides constitute one primer set, and are composed of two forward primers and one reverse primer.

구체적으로, 서열번호 1, 2 및 3; 서열번호 4, 5, 및 6; 서열번호 7, 8 및 9; 서열번호 10, 11 및 12; 서열번호 13, 14 및 15; 서열번호 16, 17 및 18; 서열번호 19, 20 및 21; 서열번호 22, 23 및 24; 서열번호 25, 26 및 27; 서열번호 28, 29 및 30; 서열번호 31, 32 및 33; 서열번호 34, 35 및 36; 서열번호 37, 38 및 39; 서열번호 40, 41 및 42; 서열번호 43, 44 및 45; 서열번호 46, 47 및 48; 서열번호 49, 50 및 51; 서열번호 52, 53 및 54; 서열번호 55, 56 및 57; 서열번호 58, 59 및 60; 서열번호 61, 62 및 63; 서열번호 64, 65 및 66; 서열번호 67, 68 및 69; 서열번호 70, 71 및 72; 서열번호 73, 74 및 75; 서열번호 76, 77 및 78; 서열번호 79, 80 및 81; 서열번호 82, 83 및 84; 서열번호 85, 86 및 87; 서열번호 88, 89 및 90; 서열번호 91, 92 및 93; 서열번호 94, 95 및 96; 서열번호 97, 98 및 99; 서열번호 100, 101 및 102; 서열번호 103, 104 및 105; 서열번호 106, 107 및 108; 서열번호 109, 110 및 111;의 프라이머 세트는 SNP에 기반한 KASP용 프라이머 세트이고(표 2 및 3), 서열번호 112, 113 및 114; 서열번호 115, 116 및 117; 서열번호 118, 119 및 120; 서열번호 121, 122 및 123; 서열번호 124, 125 및 126; 서열번호 127, 128 및 129; 서열번호 130, 131 및 132;의 프라이머 세트는 InDel 다형성 영역에 기반한 KASP용 프라이머 세트이다(표 4). Specifically, SEQ ID NOs: 1, 2 and 3; SEQ ID NOs: 4, 5, and 6; SEQ ID NOs: 7, 8 and 9; SEQ ID NOs: 10, 11 and 12; SEQ ID NOs: 13, 14 and 15; SEQ ID NOs: 16, 17 and 18; SEQ ID NOs: 19, 20 and 21; SEQ ID NOs: 22, 23 and 24; SEQ ID NOs: 25, 26 and 27; SEQ ID NOs: 28, 29 and 30; SEQ ID NOs: 31, 32 and 33; SEQ ID NOs: 34, 35 and 36; SEQ ID NOs: 37, 38 and 39; SEQ ID NOs: 40, 41 and 42; SEQ ID NOs: 43, 44 and 45; SEQ ID NOs: 46, 47 and 48; SEQ ID NOs: 49, 50 and 51; SEQ ID NOs: 52, 53 and 54; SEQ ID NOs: 55, 56 and 57; SEQ ID NOs: 58, 59 and 60; SEQ ID NOs: 61, 62 and 63; SEQ ID NOs: 64, 65 and 66; SEQ ID NOs: 67, 68 and 69; SEQ ID NOs: 70, 71 and 72; SEQ ID NOs: 73, 74 and 75; SEQ ID NOs: 76, 77 and 78; SEQ ID NOs: 79, 80 and 81; SEQ ID NOs: 82, 83 and 84; SEQ ID NOs: 85, 86 and 87; SEQ ID NOs: 88, 89 and 90; SEQ ID NOs: 91, 92 and 93; SEQ ID NOs: 94, 95 and 96; SEQ ID NOs: 97, 98 and 99; SEQ ID NOs: 100, 101 and 102; SEQ ID NOs: 103, 104 and 105; SEQ ID NOs: 106, 107 and 108; The primer sets of SEQ ID NOs: 109, 110 and 111; are primer sets for KASP based on SNP (Tables 2 and 3), and SEQ ID NOs: 112, 113 and 114; SEQ ID NOs: 115, 116 and 117; SEQ ID NOs: 118, 119 and 120; SEQ ID NOs: 121, 122 and 123; SEQ ID NOs: 124, 125 and 126; SEQ ID NOs: 127, 128 and 129; The primer sets of SEQ ID NOs: 130, 131 and 132; are primer sets for KASP based on the InDel polymorphic region (Table 4).

또한, 본 발명에 따른 서열번호 1 내지 132의 프라이머에서 2개의 정방향 프라이머의 5' 말단에 FAM 또는 HEX 형광물질이 부착되어 있다. 구체적으로 서열번호 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127 및 130의 올리고뉴클레오티드 프라이머는 형광물질 FAM이 부착된 정방향 프라이머이고, 서열번호 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, 119, 122, 125, 128 및 131의 올리고뉴클레오티드 프라이머는 HEX가 부착된 정방향 프라이머이다. 또한, 서열번호 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129 및 132의 올리고뉴클레오티드 프라이머는 역방향 프라이머이다.In addition, in the primers of SEQ ID NOs: 1 to 132 according to the present invention, FAM or HEX fluorescent substances are attached to the 5' ends of the two forward primers. Specifically, SEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, The oligonucleotide primers of 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127 and 130 are fluorescent FAM attached forward primer, SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, The oligonucleotide primers of 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, 119, 122, 125, 128 and 131 are It is a forward primer with HEX attached. Also, SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, The oligonucleotide primers at 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129 and 132 are reverse primers.

상기 서열번호 1 내지 111의 프라이머 중 정방향 프라이머의 3' 말단에는 SNP 염기가 결합되어 있다(표 2 및 표 3).Among the primers of SEQ ID NOs: 1 to 111, SNP bases are bound to the 3' end of the forward primer (Tables 2 and 3).

본 발명의 용어 "KASP(kompetitive allele specific PCR)"는 PCR(polymerase chain reaction) 기반의 분석 방법 중 하나로, 상동의(homogenous) 그리고 형광(fluorescence) 기반의 유전형 분석 기술이다. KASP는 대립형질(allele)-특이적 올리고 연장(extension) 및 신호생성을 위한 형광공명에너지전이(fluorescence resonance energy transfer)를 기반으로 하는 기술이다.The term "Kompetitive allele specific PCR (KASP)" of the present invention is one of PCR (polymerase chain reaction)-based analysis methods, and is a homogenous and fluorescence-based genotyping analysis technique. KASP is a technology based on fluorescence resonance energy transfer for allele-specific oligo extension and signal generation.

본 발명에 있어서, "프라이머"는 카피하려는 핵산 가닥에 상보적인 단일 가닥 올리고뉴클레오티드 서열을 말하며, 프라이머 연장 산물의 합성을 위한 개시점으로서 작용할 수 있다. 상기 프라이머의 길이 및 서열은 연장 산물의 합성을 시작하도록 허용해야 한다. 프라이머의 구체적인 길이 및 서열은 요구되는 DNA 또는 RNA 표적의 복합도(complexity)뿐만 아니라 온도 및 이온 강도와 같은 프라이머 이용 조건에 의존할 것이다.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 synthesis of the extension product 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.

본 명세서에 있어서, 프라이머로서 이용된 올리고뉴클레오티드는 또한 뉴클레오티드 유사체(analogue), 예를 들면, 포스포로티오에이트(phosphorothioate), 알킬포스포로티오에이트 또는 펩티드 핵산(peptide nucleic acid)를 포함할 수 있거나 또는 삽입 물질(intercalating agent)를 포함할 수 있다. 또한, 프라이머는 DNA 합성의 개시점으로 작용하는 프라이머의 기본 성질을 변화시키지 않는 추가의 특징을 혼입할 수 있다. 본 발명의 프라이머 핵산 서열은 필요한 경우, 분광학적, 광화학적, 생화학적, 면역화학적 또는 화학적 수단에 의해 직접적으로 또는 간접적으로 검출 가능한 표지를 포함할 수 있다. 표지의 예로는, 효소(예를 들어, HRP (horse radish peroxidase), 알칼리 포스파타아제), 방사성 동위원소(예를 들어, 32P), 형광성 분자, 화학그룹(예를 들어, 비오틴) 등이 있다.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. In addition, the primer may incorporate additional features that do not change the basic properties of the primer to serve as the starting point of DNA synthesis. If necessary, the primer nucleic acid sequence of the present invention may include a label detectable directly or indirectly by spectroscopic, photochemical, biochemical, immunochemical or chemical means. Examples of labels include enzymes (eg, horse radish peroxidase (HRP), alkaline phosphatase), radioactive isotopes (eg, 32P), fluorescent molecules, chemical groups (eg, biotin), and the like. .

본 발명은 또한, 상기 프라이머 세트 조성물을 포함하는 더덕의 유전적 다형성 구별용 키트를 제공한다.The present invention also provides a kit for distinguishing genetic polymorphisms of deodeok comprising the primer set composition.

본 발명의 일 구현 예에 있어서, 상기 키트는 증폭 반응을 수행하기 위한 시약을 추가로 포함할 수 있고, 상기 증폭 반응을 수행하기 위한 시약은 DNA 폴리머라제, dNTPs 및 버퍼를 포함할 수 있으나, 이에 제한되지 않는다.In one embodiment of the present invention, the kit may further include a reagent for performing the amplification reaction, and the reagent for performing the amplification reaction may include DNA polymerase, dNTPs and a buffer, but this not limited

본 발명의 일 구현 예에 있어서, 상기 키트는 또한 최적의 반응 수행 조건을 기재한 사용자 안내서를 추가로 포함할 수 있다. 안내서는 키트 사용법, 예를 들면, 역전사 완충액 및 PCR 완충액 제조 방법, 제시되는 반응 조건 등을 설명하는 인쇄물이다. 안내서는 팜플렛 또는 전단지 형태의 안내 책자, 키트에 부착된 라벨, 및 키트를 포함하는 패키지의 표면상에 설명을 포함한다. 또한, 안내서는 인터넷과 같이 전기 매체를 통해 공개되거나 제공되는 정보를 포함한다.In one embodiment of the present invention, the kit may further include a user guide describing optimal conditions for performing the reaction. The handbook is a printout explaining how to use the kit, eg, how to prepare reverse transcription buffer and 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 an electronic medium such as the Internet.

본 발명은 또한,The present invention also

더덕 시료로부터 게놈 DNA를 분리하는 단계;isolating genomic DNA from the deodeok sample;

상기 분리된 게놈 DNA를 주형으로 하고, 본 발명의 프라이머 세트 조성물을 이용하여 증폭 반응을 수행하여 표적 서열을 증폭하는 단계; 및amplifying a target sequence by using the isolated genomic DNA as a template and performing an amplification reaction using the primer set composition of the present invention; and

상기 증폭 단계의 산물을 검출하는 단계;를 포함하는 더덕의 유전적 다형성을 구별하는 방법을 제공한다.It provides a method for distinguishing the genetic polymorphism of deodeok comprising; detecting the product of the amplification step.

본 발명의 일 구현 예에 따른 방법에 있어서, 상기 프라이머 세트 조성물은 전술한 것과 같다.In the method according to an embodiment of the present invention, the primer set composition is the same as described above.

본 발명의 방법은 더덕 시료에서 게놈 DNA를 분리하는 단계를 포함한다. 상기 게놈 DNA를 분리하는 방법은 당업계에 공지된 방법을 이용할 수 있으며, 예를 들면, CTAB 방법을 이용할수도 있고, Wizard prep 키트(Promega 사)를 이용할 수도 있다. 상기 분리된 게놈 DNA를 주형으로 하고, 본 발명의 일 실시예에 따른 올리고뉴클레오티드 프라이머 세트를 프라이머로 이용하여 증폭 반응을 수행하여 표적 서열을 증폭할 수 있다. 표적 핵산을 증폭하는 방법은 중합효소연쇄반응(polymerase chain reaction; PCR), 리가아제 연쇄반응(ligase chain reaction), 핵산 서열 기재 증폭(nucleic acid sequence-based amplification), 전사 기재 증폭시스템(transcription-based amplification system), 가닥 치환 증폭(strand displacement amplification) 또는 Qβ 복제효소(replicase)를 통한 증폭 또는 당업계에 알려진 핵산 분자를 증폭하기 위한 임의의 기타 적당한 방법이 있다. 이 중에서, PCR이란 중합효소를 이용하여 표적 핵산에 특이적으로 결합하는 프라이머 쌍으로부터 표적 핵산을 증폭하는 방법이다. 이러한 PCR 방법은 당업계에 잘 알려져 있으며, 상업적으로 이용가능한 키트를 이용할 수도 있다.The method of the present invention includes isolating genomic DNA from a deodeok sample. As a method for isolating the genomic DNA, 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. The target sequence may be amplified by performing an amplification reaction using the isolated genomic DNA as a template and using the oligonucleotide primer set according to an embodiment of the present invention as a primer. Methods for amplifying a target nucleic acid include polymerase chain reaction (PCR), ligase chain reaction, nucleic acid sequence-based amplification, and transcription-based amplification systems. amplification system), 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.

본 발명의 일 구현 예에 있어서, 상기 증폭된 표적 서열은 검출가능한 표지 물질로 표지될 수 있다. 상기 표지 물질은 형광, 인광 또는 방사성을 발하는 물질일 수 있으나, 이에 제한되지 않는다. 바람직하게는, 상기 표지 물질은 Cy-5 또는 Cy-3이다. 표적 서열의 증폭시 프라이머의 5'-말단에 Cy-5 또는 Cy-3를 표지하여 PCR을 수행하면 표적 서열이 검출가능한 형광 표지 물질로 표지될 수 있다. 또한, 방사성 물질을 이용한 표지는 PCR 수행시 32P 또는 35S 등과 같은 방사성 동위원소를 PCR 반응액에 첨가하면 증폭 산물이 합성되면서 방사성이 증폭 산물에 혼입되어 증폭 산물이 방사성으로 표지될 수 있다. 표적 서열을 증폭하기 위해 이용된 올리고뉴클레오티드 프라이머 세트는 전술한 것과 같다.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 a 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.

본 발명의 일 구현 예에 있어서, 상기 더덕의 유전적 다형성을 구별하는 방법은 상기 증폭 단계의 산물을 검출하는 단계를 포함하며, 상기 증폭 단계 산물의 검출은 DNA 칩, 겔 전기영동, 모세관 전기영동, 방사성 측정, 형광 측정 또는 인광 측정을 통해 수행될 수 있으나, 이에 제한되지 않는다. 증폭 산물을 검출하는 방법 중의 하나로서, 모세관 전기영동을 수행할 수 있다. 모세관 전기영동은 예를 들면, ABi Sequencer를 이용할 수 있다. 또한, 겔 전기영동을 수행할 수 있으며, 겔 전기영동은 증폭 산물의 크기에 따라 아가로스 겔 전기영동 또는 아크릴아미드 겔 전기영동을 이용할 수 있다. 또한, 형광 측정 방법은 프라이머의 5'-말단에 Cy-5 또는 Cy-3를 표지하여 PCR을 수행하면 표적 서열이 검출가능한 형광 표지 물질로 표지되며, 이렇게 표지된 형광은 형광 측정기를 이용하여 측정할 수 있다. 또한, 방사성 측정 방법은 PCR 수행시 32P 또는 35S 등과 같은 방사성 동위원소를 PCR 반응액에 첨가하여 증폭 산물을 표지한 후, 방사성 측정기구, 예를 들면, 가이거 계수기(Geiger counter) 또는 액체섬광계수기(liquid scintillation counter)를 이용하여 방사성을 측정할 수 있다.In one embodiment of the present invention, the method for distinguishing the genetic polymorphism of deodeok comprises detecting a product of the amplification step, and the detection of the product of the amplification step is a DNA chip, gel electrophoresis, capillary electrophoresis , but is not limited thereto, through radiometric, fluorescence, or phosphorescence measurement. 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 fluorescence meter. can do. In addition, the radioactive measurement method is a radioactive isotope such as 32 P or 35 S is added to the PCR reaction solution during PCR to label the amplification product, and then a radioactive measuring instrument, for example, a Geiger counter (Geiger counter) or liquid scintillation The radioactivity can be measured using a liquid scintillation counter.

이하, 본 발명을 실시예를 통해서 상세하게 설명한다. 하기의 특정한 예시 및 실시예는 발명의 일부 구현예를 포함하는 것으로 모든 구현예를 포함하고 있지는 않다는 점에 유의해야 한다. 본 명세서에 의해 개시되는 발명의 내용은 여기에서 설명되는 특정 실시예로 제한되지 않고, 본 발명이 속한 기술 분야에 있어 통상의 기술자가 다양하게 구현할 수 있다는 것을 포함하는 것은 자명하다. 따라서 본 명세서에 개시된 발명의 내용은 여기에 기재된 특정 실시예로 제한되지 않으며, 이에 대한 변형 및 다른 구현예들도 청구범위 내에 포함되는 것으로 이해되어야 한다.Hereinafter, the present invention will be described in detail through examples. It should be noted that the specific examples and examples below are inclusive of some and not all embodiments of the invention. It is apparent that the content of the invention disclosed by the present specification is not limited to the specific embodiments described herein, and includes that those skilled in the art can implement variously. Therefore, it should be understood that the content of the invention disclosed herein is not limited to the specific embodiments described herein, and modifications and other embodiments thereof are also included within the scope of the claims.

재료 및 방법Materials and Methods

1. 더덕 자원의 수집 및 DNA 추출1. Collection of deodeok resources and DNA extraction

본 발명에서 수집된 더덕 자원은 하기 표 1 및 표 2에 정리하였다. 더덕의 게놈 DNA 추출은 액체질소를 이용하여 어린 잎 시료를 급속 냉각하고 막자사발을 이용하여 분쇄한 후 CTAB(Cetyl Trimethyl Ammonium Bromide) 방법을 이용하여 DNA를 추출하여 TE buffer에 용출하였다.The deodeok resources collected in the present invention are summarized in Tables 1 and 2 below. For genomic DNA extraction of deodeok, young leaf samples were rapidly cooled using liquid nitrogen and pulverized using a mortar. Then, DNA was extracted using the CTAB (Cetyl Trimethyl Ammonium Bromide) method and eluted in TE buffer.

Figure 112021151279002-pat00001
Figure 112021151279002-pat00001

Figure 112021151279002-pat00002
Figure 112021151279002-pat00002

2. 더덕 엽록체 게놈 서열 기반 KASP용 프라이머 세트 개발2. Development of primer set for KASP based on the chloroplast genome sequence of Deodeok

43개 더덕(표 3)의 엽록체 게놈을 완성한 후, NCBI에 등록된 더덕 엽록체 게놈 서열(MH018574)을 포함해 총 44개 더덕 자원의 엽록체 게놈 서열을 CLC Main Workbewnch 프로그램(version 20)을 이용하여 비교ㆍ분석하였다. After completing the chloroplast genomes of 43 deodeok (Table 3), the chloroplast genome sequences of a total of 44 deodeok resources, including the chloroplast genome sequence (MH018574) registered with the NCBI, were compared using the CLC Main Workbewnch program (version 20). ㆍAnalyzed.

Figure 112021151279002-pat00003
Figure 112021151279002-pat00003

그 결과, 37개 SNP에 기반한 KASP용 프라이머 세트(표 4 및 표 5)와 7개 InDel에 기반한 KASP용 프라이머 세트(표 6)를 개발하였다.As a result, a primer set for KASP based on 37 SNPs (Tables 4 and 5) and a primer set for KASP based on 7 InDel (Table 6) were developed.

총 44개의 KASP용 프라이머 세트의 표적 유전자는 MH018574(NCBI)에서 찾았으며 ycf1, psbB, psaA, rpl33, trnR-UCU과 인트론(표 4 내지 6의 Locus에 '-'로 표시)에 위치하고 있음을 확인하였다. A total of 44 target genes of the primer set for KASP were found in MH018574 (NCBI), and it was confirmed that they were located in ycf1 , psbB , psaA , rpl33 , trnR-UCU and introns (indicated by '-' at Locus in Tables 4 to 6). did.

본 발명의 KASP용 프라이머 세트는 2개의 정항향 프라이머(Primer Allele X, Primer Allele Y)와 1개의 역방향 프라이머(Primer Common)로 구성되어 있고, Primer Allele X에는 형광물질 FAM이 결합되어 있으며, Primer Allele Y에는 형광물질 HEX가 결합되어 있다. 표 3 및 표 4의 SNP 기반 KASP용 프라이머 세트의 경우 정방향 프라이머의 3' 말단에는 SNP와 같은 염기서열의 차이가 구분되도록 디자인하였으며, InDel 기반 KASP용 프라이머 세트에 관한 표 5의 allele에서 (-)는 결실을 의미한다.The primer set for KASP of the present invention consists of two forward primers (Primer Allele X, Primer Allele Y) and one reverse primer (Primer Common), and Primer Allele X has a fluorescent substance FAM bound, and Primer Allele A fluorescent substance HEX is bound to Y. In the case of the primer sets for SNP-based KASP in Tables 3 and 4, the 3' end of the forward primer was designed to distinguish the difference in nucleotide sequence such as SNP, and in the allele of Table 5 regarding the InDel-based KASP primer set (-) means fruit.

Figure 112021151279002-pat00004
Figure 112021151279002-pat00004

Figure 112021151279002-pat00005
Figure 112021151279002-pat00005

Figure 112021151279002-pat00006
Figure 112021151279002-pat00006

3. KASP 분석3. KASP analysis

PCR 반응물은 genomic DNA 5 ng, 72X KASP Assay mix 및 2X master mix(LGC, 영국)를 각각 농도에 맞게 혼합하였다. 시약 분주와 PCR 반응, 형광 측정은 Array Tape을 이용하는 Nexar system(LGC Douglas Scientific, 미국)에서 수행하였고, PCR 반응은 다음과 같이 수행하였다. 94℃에서 15분간 초기 변성(denaturation)한 후, 94℃ 20초간 denaturation과 61℃~55℃(회당 0.6℃ 감소)에서 1분간 프라이머 결합(primer annealing) 및 신장(elongation)하는 touchdown PCR을 10 cycle을 수행한다. 이후, 94℃ 20초간 denaturation과 55℃에서 1분간 primer annealing 및 elongation을 26 cycle간 수행한다. PCR 반응이 끝난 이후에 각 웰의 FAM과 HEX 형광값을 측정하여 SNP 와 InDel genotyping을 수행하였다. For PCR reaction, 5 ng of genomic DNA, 72X KASP Assay mix and 2X master mix (LGC, UK) were mixed according to each concentration. Reagent dispensing, PCR reaction, and fluorescence measurement were performed in a Nexar system (LGC Douglas Scientific, USA) using Array Tape, and PCR reaction was performed as follows. After initial denaturation at 94°C for 15 minutes, 10 cycles of denaturation at 94°C for 20 seconds and touchdown PCR for 1 minute at 61°C to 55°C (decrease by 0.6°C per cycle) and elongation (primer annealing) carry out Thereafter, denaturation at 94°C for 20 seconds and primer annealing and elongation at 55°C for 1 minute were performed for 26 cycles. After the PCR reaction was completed, SNP and InDel genotyping were performed by measuring the FAM and HEX fluorescence values of each well.

4. 더덕 자원의 유연관계 분석4. Analysis of the relationship between deodeok resources

Araya(LGC Douglas Scientific, 미국)로 형광 측정한 결과를 Intellics software(LGC Douglas Scientific)를 이용하여 유전적 다양성 분석을 수행하였다. PowerMarker software version 3.25(Liu and Muse, 2005)을 이용하여 야생 더덕 자원 78개에 대해 본 발명의 더덕 엽록체 게놈 서열 기반 KASP용 프라이머 세트 44개를 적용한 데이터를 바탕으로 Genetic distance를 SharedAllele distance method of PowerMarker software version 3.25로 계산하였고, MEGA program version 7.0.26를 사용하여 UPGMA based phylogenetic tree를 나타내었다.The result of fluorescence measurement with Araya (LGC Douglas Scientific, USA) was analyzed for genetic diversity using Intellics software (LGC Douglas Scientific). Based on the data of applying 44 primer sets for KASP based on the chloroplast genome sequence of the present invention to 78 wild deodeok resources using PowerMarker software version 3.25 (Liu and Muse, 2005), the Genetic distance was calculated using the SharedAllele distance method of PowerMarker software. It was calculated using version 3.25, and the UPGMA based phylogenetic tree was shown using MEGA program version 7.0.26.

실시예 1. 본 발명의 KASP용 프라이머 세트를 이용한 더덕 자원들간의 유연관계 분석 결과Example 1. Results of analysis of relationship between deodeok resources using the primer set for KASP of the present invention

수집된 더덕 자원의 유전적 거리를 분석하기 위해, 본 발명의 KASP용 프라이머 세트를 이용한 형광 측정 결과를 토대로, 78개 더덕 자원에 대한 계통수(phylogenetic tree)를 작성하였다(도 1).In order to analyze the genetic distance of the collected deodeok resources, based on the fluorescence measurement results using the primer set for KASP of the present invention, a phylogenetic tree for 78 deodeok resources was prepared (Fig. 1).

이를 통해, 본 발명의 44개 KASP용 프라이머 세트를 이용하면 다양한 더덕 자원의 유전적 다형성을 구별하여 이들의 유전적 관계를 비교할 수 있음을 알 수 있었다.Through this, it was found that by using the 44 primer sets for KASP of the present invention, the genetic polymorphisms of various deodeok resources can be distinguished and their genetic relationships can be compared.

<110> Chungbuk National University Industry-Academic Cooperation Foundation <120> Primer set for discriminating genetic polymorphism of Codonopsis lanceolata and uses thereof <130> PN21488 <160> 132 <170> KoPatentIn 3.0 <210> 1 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 acaccacaca gaaagacaaa ttatttgat 29 <210> 2 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 caccacacag aaagacaaat tatttgag 28 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 gataatgttt gataaaaatc tcctcgggta 30 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 ccttgaatcg atttgtttct ttattgt 27 <210> 5 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 ctccttgaat cgatttgttt ctttattgg 29 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 ttctttcagc taccgtccgg gaaaa 25 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 ggattattcc 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gaaatggaat ggccttttct ttttatttc 29 <210> 17 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 gttgaaatgg aatggccttt tctttttatt ta 32 <210> 18 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 cccatcgtag cacattctat gaaatcaaa 29 <210> 19 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 ggagaagagt tgagtaaatt gaaactct 28 <210> 20 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 gagaagagtt gagtaaattg aaactcc 27 <210> 21 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 ggtgcttttt ggccatgaat ctcctt 26 <210> 22 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 acaagaaaga ggaaagtcaa aatatgaaaa att 33 <210> 23 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 caagaaagag gaaagtcaaa atatgaaaaa tc 32 <210> 24 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 24 ctatccatta gacaatggac gcttttcat 29 <210> 25 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 25 ctcttaacca gcccatcaaa taagta 26 <210> 26 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 26 ctcttaacca gcccatcaaa taagtg 26 <210> 27 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 27 gcagggtaat gtttcgcagt ttaatgaat 29 <210> 28 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 28 ggttctttat agttttctct tttttaatgg 30 <210> 29 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 29 ctggttcttt atagttttct cttttttaat gt 32 <210> 30 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 30 ccagattaat ccagatttta ctaattatat 30 <210> 31 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 31 aataattcaa atgaatgact cgcaattcg 29 <210> 32 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 32 caataattca aatgaatgac tcgcaattca 30 <210> 33 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 33 ctctcctacg taatgattat gacccaaa 28 <210> 34 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 34 gtcagatata ttgggtcata tgggattta 29 <210> 35 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 35 cagatatatt gggtcatatg ggatttc 27 <210> 36 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 36 gatctctcga ccggggagag aa 22 <210> 37 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 37 gtctggtctt ggatcataaa taatgac 27 <210> 38 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 38 atgtctggtc ttggatcata aataatgat 29 <210> 39 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 39 cggatcgatt aagtagccga attctaaa 28 <210> 40 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 40 gggacctatt atgggacaga cac 23 <210> 41 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 41 atgggaccta ttatgggaca gacaa 25 <210> 42 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 42 gttgaagcgt aatgatcata cgtctgta 28 <210> 43 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 43 ggatcggagt attttattaa tattcgattt c 31 <210> 44 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 44 gggatcggag tattttatta atattcgatt ta 32 <210> 45 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 45 ctctgtccat ttcgtttttt ggtctcatt 29 <210> 46 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 46 ctatttaaca ttatgtagca ttcttttcg 29 <210> 47 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 47 cctctattta acattatgta gcattctttt ct 32 <210> 48 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 48 ggagacctaa gataggttag agaaactaa 29 <210> 49 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 49 tatgcaaatg gaacactttc gcg 23 <210> 50 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 50 atcttatgca aatggaacac tttcgca 27 <210> 51 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 51 ccccgaaatt cttttcgaga atgtctaat 29 <210> 52 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 52 cccaataaaa tggcttggta acagag 26 <210> 53 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 53 cccaataaaa tggcttggta acagat 26 <210> 54 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 54 cggaacgaat gggacgcgct tt 22 <210> 55 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 55 atgcattcgt aatagactat catgact 27 <210> 56 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 56 ctatgcattc gtaatagact atcatgaca 29 <210> 57 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 57 gtgccatgct cccccattac tatat 25 <210> 58 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 58 aaccaataac tactcgacat acgtt 25 <210> 59 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 59 aaccaataac tactcgacat acgtc 25 <210> 60 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 60 attcaacgag gactgattga aaggagaaa 29 <210> 61 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 61 ataatcagtt gtaacatcga aatctacg 28 <210> 62 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 62 ctataatcag ttgtaacatc gaaatctact 30 <210> 63 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 63 atgaacagag ataggtcttt gagattcttt 30 <210> 64 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 64 cggctcatgt catacacagc c 21 <210> 65 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 65 ctcggctcat gtcatacaca gct 23 <210> 66 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 66 taaactctac gggtggaaaa atagtggat 29 <210> 67 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 67 agaaccagga acggagagct tta 23 <210> 68 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 68 gaaccaggaa cggagagctt tc 22 <210> 69 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 69 ttaagaccaa agggtcggcg gaaaa 25 <210> 70 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 70 aaaagaactt ttgcttaata aaaaatggaa tcg 33 <210> 71 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 71 aaaagaactt ttgcttaata aaaaatggaa tct 33 <210> 72 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 72 tcctcaagtt aggctagtta atcaaagaaa 30 <210> 73 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 73 aaataaattt gactttagtc tttttgtgtc ct 32 <210> 74 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 74 ataaatttga ctttagtctt tttgtgtccg 30 <210> 75 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 75 aaactagtgc tagttgatga gagtgactt 29 <210> 76 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 76 cttggattgg attagtgatt ccttg 25 <210> 77 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 77 gtcttggatt ggattagtga ttccttt 27 <210> 78 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 78 cctatcctct attctctcgt catattcaa 29 <210> 79 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 79 cattttgttt ttagaaaccc caggtatttt 30 <210> 80 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 80 ttgtttttag aaaccccagg tatttc 26 <210> 81 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 81 ccgaagccgc ttatactata ttccattaa 29 <210> 82 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 82 tcctagtctt cgacacaaga aaagt 25 <210> 83 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 83 cctagtcttc gacacaagaa aagg 24 <210> 84 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 84 cgacacaaga agggggtttg gaaaa 25 <210> 85 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 85 ggaattggtt ctataaccga tgcaga 26 <210> 86 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 86 gaattggttc tataaccgat gcagg 25 <210> 87 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 87 cgtgagaaat tatttgtaaa atggctccat 30 <210> 88 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 88 aattcgacta tagttttcgg taacgtatt 29 <210> 89 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 89 cgactatagt tttcggtaac gtatc 25 <210> 90 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 90 actcgcagca tgggtctagc ttatt 25 <210> 91 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 91 acctgtggtc tcaactaaga agag 24 <210> 92 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 92 tacctgtggt ctcaactaag aagaa 25 <210> 93 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 93 ccagaaaatc cagggaatga ttggataat 29 <210> 94 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 94 attcgaaatt caactccaag aatcattcaa 30 <210> 95 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 95 cgaaattcaa ctccaagaat cattcat 27 <210> 96 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 96 tttaactatt cactcccgac tgcgaatt 28 <210> 97 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 97 gaaaaattgg ctctatatcc aacagc 26 <210> 98 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 98 tgaaaaattg gctctatatc caacagt 27 <210> 99 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 99 ctagagggcg aaccaaaaca aaatacttt 29 <210> 100 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 100 ggaagataaa tctggtaagg aagaacaa 28 <210> 101 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 101 gaagataaat ctggtaagga agaacac 27 <210> 102 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 102 ggagggcgtc acctcttttt cttaa 25 <210> 103 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 103 aaatattcat attgccctct gacaaaaatc 30 <210> 104 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 104 ataaaatatt catattgccc tctgacaaaa ata 33 <210> 105 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 105 gcaagtcgag aaatagatgg ttgaatcaa 29 <210> 106 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 106 ccaacttatc acttatttgt ttattgcaac 30 <210> 107 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 107 aaccaactta tcacttattt gtttattgca aa 32 <210> 108 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 108 ctctttctac cgggaagtca aaaagtttt 29 <210> 109 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 109 tgaaagagag atgtcctgaa ccg 23 <210> 110 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 110 cttgaaagag agatgtcctg aacca 25 <210> 111 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 111 taagcaagta tgcccccatc gtcta 25 <210> 112 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 112 aaaaaaggaa aaattaattg gaactctact cta 33 <210> 113 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 113 aaaggaaaaa ttaattggaa ctctactctg 30 <210> 114 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 114 gtctcaaagg gcccggtatt atgta 25 <210> 115 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 115 tttttattcc cccgtatccg gc 22 <210> 116 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 116 cttttttatt cccccgtatc cggt 24 <210> 117 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 117 caattcaata aatccacttc taaaaaattc ctt 33 <210> 118 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 118 ccatgagatg gtagaatccc cttgtt 26 <210> 119 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 119 aattcaataa atccacttct aaaaaattcc tc 32 <210> 120 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 120 gcttgtgttt cgtcttcccc gatta 25 <210> 121 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 121 gtacggttgt gcaattctat ttttttt 27 <210> 122 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 122 ctgtacggtt gtgcaattct atttttttc 29 <210> 123 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 123 cgcatgatga agggaagata aaagaagaa 29 <210> 124 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 124 ttctcccccc cccccc 16 <210> 125 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 125 ctttctcccc ccccccca 18 <210> 126 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 126 gcgctttcac atcttcttaa cccgaa 26 <210> 127 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 127 attattattt ccttaaataa attctcatga gac 33 <210> 128 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 128 attatttcct taaataaatt ctcatgagag 30 <210> 129 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 129 tgtacaattt ctcaaaggat tcgggtcaa 29 <210> 130 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 130 aattaaatta agggattagt cttttttttt ttt 33 <210> 131 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 131 aattaaatta agggattagt cttttttttt tta 33 <210> 132 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 132 gcgttaatat gtgctctaag gtttcaaata 30 <110> Chungbuk National University Industry-Academic Cooperation Foundation <120> Primer set for discriminating genetic polymorphism of Codonopsis lanceolata and uses thereof <130> PN21488 <160> 132 <170> KoPatentIn 3.0 <210> 1 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 acaccacaca gaaagacaaa ttatttgat 29 <210> 2 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 caccacacag aaagacaaat tatttgag 28 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 gataatgttt gataaaaatc tcctcgggta 30 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 ccttgaatcg atttgtttct ttattgt 27 <210> 5 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 ctccttgaat cgatttgttt ctttattgg 29 < 210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 ttctttcagc taccgtccgg gaaaa 25 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220 > <223> primer <400> 7 ggattattcc gggcg ggttc g 21 <210> 8 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 attccgggcg ggttca 16 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 cgctattcca tccccgttat ccat 24 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 agttagtcct atacatgtgg ccc 23 <210 > 11 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 cagttagtcc tatacatgtg gcct 24 <210> 12 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 cccaccatca tttagctatt gcaattctt 29 <210> 13 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 actcaagaca agaagacgga tattga 26 <210> 14 <211 > 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 ctcaagacaa gaagacggat attgc 25 <210> 15 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 ctcaatatcc gtcttcttgt cttgagtat 29 <210> 16 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 gaaatggaat g gccttttct ttttatttc 29 <210> 17 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 gttgaaatgg aatggccttt tctttttatt ta 32 <210> 18 <211> 29 <212> DNA <213 > Artificial Sequence <220> <223> primer <400> 18 cccatcgtag cacattctat gaaatcaaa 29 <210> 19 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 ggagaagagt tgagtaaatt gaaactct 28 <210> 20 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 gagaagagtt gagtaaattg aaactcc 27 <210> 21 <211> 26 <212> DNA <213> Artificial Sequence < 220> <223> primer <400> 21 ggtgcttttt ggccatgaat ctcctt 26 <210> 22 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 acaagaaaga ggaaagtcaa aatatgaaaa att 33 <210> 23 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 caagaaagag gaaagtcaaa atatgaaaaa tc 32 <210> 24 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 24 ctatccatta gacaatggac gcttttcat 29 <210> 25 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 25 ctcttaacca gcccatcaaa taagta 26 <210> 26 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 26 ctcttaacca gcccatcaaa taagtg 26 < 210> 27 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 27 gcagggtaat gtttcgcagt ttaatgaat 29 <210> 28 <211> 30 <212> DNA <213> Artificial Sequence <220 > <223> primer <400> 28 ggttctttat agttttctct tttttaatgg 30 <210> 29 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 29 ctggttcttt atagttttct cttttttaat gt 32 <210> 30 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 30 ccagattaat ccagatttta ctaattatat 30 <210> 31 <211> 29 <212> DNA <213> Artificial Sequence <220> <223 > primer <400> 31 aataattcaa atgaatgact cgcaattcg 29 <210> 32 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 32 caataattca aatgaatgac tcgcaattca 30 <210> 33 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 33 ctctcctacg taat gattat gacccaaa 28 <210> 34 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 34 gtcagatata ttgggtcata tgggattta 29 <210> 35 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 35 cagatatatt gggtcatatg ggatttc 27 <210> 36 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 36 gatctctcga ccggggagag aa 22 < 210> 37 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 37 gtctggtctt ggatcataaa taatgac 27 <210> 38 <211> 29 <212> DNA <213> Artificial Sequence <220 > <223> primer <400> 38 atgtctggtc ttggatcata aataatgat 29 <210> 39 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 39 cggatcgatt aagtagccga attctaaa 28 <210> 40 < 211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 40 gggacctatt atgggacaga cac 23 <210> 41 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 41 atgggaccta ttatgggaca gacaa 25 <210> 42 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> p rimer <400> 42 gttgaagcgt aatgatcata cgtctgta 28 <210> 43 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 43 ggatcggagt attttattaa tattcgattt c 31 <210> 44 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 44 gggatcggag tattttatta atattcgatt ta 32 <210> 45 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer < 400> 45 ctctgtccat ttcgtttttt ggtctcatt 29 <210> 46 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 46 ctatttaaca ttatgtagca ttcttttcg 29 <210> 47 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 47 cctctattta acattatgta gcattctttt ct 32 <210> 48 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 48 ggagacctaa gataggttag agaaactaa 29 <210> 49 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 49 tatgcaaatg gaacactttc gcg 23 <210> 50 <211> 27 <212> DNA <213 > Artificial Sequence <220> <223> primer <400> 50 atcttatgca aatggaacac tttcgca 27 <210> 51 <21 1> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 51 ccccgaaatt cttttcgaga atgtctaat 29 <210> 52 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 52 cccaataaaa tggcttggta acagag 26 <210> 53 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 53 cccaataaaa tggcttggta acagat 26 <210> 54 <211> 22 < 212> DNA <213> Artificial Sequence <220> <223> primer <400> 54 cggaacgaat gggacgcgct tt 22 <210> 55 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 55 atgcattcgt aatagactat catgact 27 <210> 56 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 56 ctatgcattc gtaatagact atcatgaca 29 <210> 57 <211> 25 <212> DNA < 213> Artificial Sequence <220> <223> primer <400> 57 gtgccatgct cccccattac tatat 25 <210> 58 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 58 aaccaataac tactcgacat acgtt 25 <210> 59 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 59 aaccaataac tactcg acat acgtc 25 <210> 60 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 60 attcaacgag gactgattga aaggagaaa 29 <210> 61 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 61 ataatcagtt gtaacatcga aatctacg 28 <210> 62 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 62 ctataatcag ttgtaacatc gaaatctact 30 < 210> 63 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 63 atgaacagag ataggtcttt gagattcttt 30 <210> 64 <211> 21 <212> DNA <213> Artificial Sequence <220 > <223> primer <400> 64 cggctcatgt catacacagc c 21 <210> 65 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 65 ctcggctcat gtcatacaca gct 23 <210> 66 < 211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 66 taaactctac gggtggaaaa atagtggat 29 <210> 67 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 67 agaaccagga acggagagct tta 23 <210> 68 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> pri mer <400> 68 gaaccaggaa cggagagctt tc 22 <210> 69 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 69 ttaagaccaa agggtcggcg gaaaa 25 <210> 70 <211> 33 < 212> DNA <213> Artificial Sequence <220> <223> primer <400> 70 aaaagaactt ttgcttaata aaaaatggaa tcg 33 <210> 71 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400 > 71 aaaagaactt ttgcttaata aaaaatggaa tct 33 <210> 72 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 72 tcctcaagtt aggctagtta atcaaagaaa 30 <210> 73 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 73 aaataaattt gactttagtc tttttgtgtc ct 32 <210> 74 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 74 ataaatttga ctttagtctt tttgtgtccg 30 <210> 75 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 75 aaactagtgc tagttgatga gagtgactt 29 <210> 76 <211> 25 <212> DNA <213 > Artificial Sequence <220> <223> primer <400> 76 cttggattgg attagtgatt ccttg 25 <210> 77 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 77 gtcttggatt ggattagtga ttccttt 27 <210> 78 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer < 400> 78 cctatcctct attctctcgt catattcaa 29 <210> 79 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 79 cattttgttt ttagaaaccc caggtatttt 30 <210> 80 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 80 ttgtttttag aaaccccagg tatttc 26 <210> 81 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 81 ccgaagccgc ttatactata ttccattaa 29 <210> 82 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 82 tcctagtctt cgacacaaga aaagt 25 <210> 83 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 83 cctagtcttc gacacaagaa aagg 24 <210> 84 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 84 cgacacaaga agggggtttg gaaaa 25 < 210> 85 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 85 ggaattggtt cta taaccga tgcaga 26 <210> 86 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 86 gaattggttc tataaccgat gcagg 25 <210> 87 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 87 cgtgagaaat tatttgtaaa atggctccat 30 <210> 88 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 88 aattcgacta tagttttcgg taacgtatt 29 < 210> 89 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 89 cgactatagt tttcggtaac gtatc 25 <210> 90 <211> 25 <212> DNA <213> Artificial Sequence <220 > <223> primer <400> 90 actcgcagca tgggtctagc ttatt 25 <210> 91 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 91 acctgtggtc tcaactaaga agag 24 <210> 92 < 211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 92 tacctgtggt ctcaactaag aagaa 25 <210> 93 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 93 ccagaaaatc cagggaatga ttggataat 29 <210> 94 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 94 attcgaaatt caactccaag aatcattcaa 30 <210> 95 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 95 cgaaattcaa ctccaagaat cattcat 27 <210> 96 <211> 28 < 212> DNA <213> Artificial Sequence <220> <223> primer <400> 96 tttaactatt cactcccgac tgcgaatt 28 <210> 97 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 97 gaaaaattgg ctctatatcc aacagc 26 <210> 98 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 98 tgaaaaattg gctctatatc caacagt 27 <210> 99 <211> 29 <212> DNA < 213> Artificial Sequence <220> <223> primer <400> 99 ctagagggcg aaccaaaaca aaatacttt 29 <210> 100 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 100 ggaagataaa tctggtaagg aagaacaa 28 <210> 101 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 101 gaagataaat ctggtaagga agaacac 27 <210> 102 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 102 ggagggcgtc acctcttttt cttaa 25 <210> 103 <211> 30 <2 12> DNA <213> Artificial Sequence <220> <223> primer <400> 103 aaatattcat attgccctct gacaaaaatc 30 <210> 104 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 104 ataaaatatt catattgccc tctgacaaaa ata 33 <210> 105 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 105 gcaagtcgag aaatagatgg ttgaatcaa 29 <210> 106 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 106 ccaacttatc acttatttgt ttattgcaac 30 <210> 107 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 107 aaccaactta tcacttattt gtttattgca aa 32 <210> 108 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 108 ctctttctac cgggaagtca aaaagtttt 29 <210> 109 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 109 tgaaagagag atgtcctgaa ccg 23 <210> 110 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 110 cttgaaagag agatgtcctg aacca 25 < 210> 111 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> p rimer <400> 111 taagcaagta tgcccccatc gtcta 25 <210> 112 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 112 aaaaaaggaa aaattaattg gaactctact cta 33 <210> 113 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 113 aaaggaaaaa ttaattggaa ctctactctg 30 <210> 114 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400 > 114 gtctcaaagg gcccggtatt atgta 25 <210> 115 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 115 tttttattcc cccgtatccg gc 22 <210> 116 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 116 cttttttatt cccccgtatc cggt 24 <210> 117 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 117 caattcaata aatccacttc taaaaaattc ctt 33 <210> 118 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 118 ccatgagatg gtagaatccc cttgtt 26 <210> 119 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 119 aattcaataa atccacttct aaaaaattcc tc 32 <210> 120 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 120 gcttgtgttt cgtcttcccc gatta 25 <210> 121 <211> 27 <212> DNA <213> Artificial Sequence <220> < 223> primer <400> 121 gtacggttgt gcaattctat ttttttt 27 <210> 122 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 122 ctgtacggtt gtgcaattct atttttttc 29 <210> 123 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 123 cgcatgatga agggaagata aaagaagaa 29 <210> 124 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> primer < 400> 124 ttctcccccc cccccc 16 <210> 125 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 125 ctttctcccc ccccccca 18 <210> 126 <211> 26 <212> DNA < 213> Artificial Sequence <220> <223> primer <400> 126 gcgctttcac atcttcttaa cccgaa 26 <210> 127 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 127 attattattt ccttaaataa attctcatga gac 33 <210> 128 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 128 attatttcct taaataaatt ctcatgagag 30 <210> 129 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 129 tgtacaattt ctcaaaggat tcgggtcaa 29 <210> 130 <211> 33 <212> DNA <213 > Artificial Sequence <220> <223> primer <400> 130 aattaaatta agggattagt cttttttttt ttt 33 <210> 131 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 131 aattaaatta agggattagt cttttttttt tta 33 <210> 132 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer<400> 132 gcgttaatat gtgctctaag gtttcaaata 30

Claims (4)

서열번호 1 내지 132로 표시된 올리고뉴클레오티드에서 서열번호 1부터 인접한 3개의 올리고뉴클레오티드가 하나의 프라이머 세트인 것을 특징으로 하는 44개의 올리고뉴클레오티드 프라이머 세트를 포함하는, 더덕(Codonopsis lanceolata)의 유전적 다형성 구별용 프라이머 세트 조성물.In the oligonucleotides shown in SEQ ID NOs: 1 to 132, three oligonucleotides adjacent from SEQ ID NO: 1 are one primer set, comprising a set of 44 oligonucleotide primers, deodeok ( Codonopsis lanceolata ) For distinguishing genetic polymorphisms Primer set composition. 제1항의 프라이머 세트 조성물을 포함하는 더덕(Codonopsis lanceolata)의 유전적 다형성 구별용 키트.A kit for distinguishing genetic polymorphisms of deodeok ( Codonopsis lanceolata ) comprising the primer set composition of claim 1 . 더덕 시료로부터 게놈 DNA를 분리하는 단계;
상기 분리된 게놈 DNA를 주형으로 하고, 제1항의 프라이머 세트 조성물을 이용하여 증폭 반응을 수행하여 표적 서열을 증폭하는 단계; 및
상기 증폭 단계의 산물을 검출하는 단계;를 포함하는 더덕(Codonopsis lanceolata)의 유전적 다형성을 구별하는 방법.isolating genomic DNA from the deodeok sample;
amplifying a target sequence by using the isolated genomic DNA as a template and performing an amplification reaction using the primer set composition of claim 1; and
Detecting the product of the amplification step; Deodeok ( Codonopsis lanceolata ) A method of distinguishing a genetic polymorphism comprising a. 제3항에 있어서, 상기 증폭 단계의 산물의 검출은 DNA 칩, 겔 전기영동, 방사성 측정, 형광 측정 또는 인광 측정을 통해 수행되는 것을 특징으로 하는 방법.The method according to claim 3, 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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KR20170051866A (en) * 2015-11-03 2017-05-12 주식회사 바이오메딕 SSR molecular markers for discriminating of Codonopsis lanceolata cultivars and uses thereof
KR101954673B1 (en) * 2017-10-27 2019-03-06 충북대학교 산학협력단 Molecular marker for discriminating Codonopsis lanceolata cultivars and uses thereof
KR102010279B1 (en) * 2018-10-31 2019-08-13 충북대학교 산학협력단 Molecular marker for discriminating Codonopsis lanceolata among genus Codonopsis and uses thereof
KR102298751B1 (en) * 2020-06-10 2021-09-07 충북대학교 산학협력단 Molecular marker based on chloroplast genome sequence for discriminating Codonopsis sp. and uses thereof

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KR20170051866A (en) * 2015-11-03 2017-05-12 주식회사 바이오메딕 SSR molecular markers for discriminating of Codonopsis lanceolata cultivars and uses thereof
KR101954673B1 (en) * 2017-10-27 2019-03-06 충북대학교 산학협력단 Molecular marker for discriminating Codonopsis lanceolata cultivars and uses thereof
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