한국산 나문재속 식물에 대한 계통학적 유연관계를 밝히고, 분자계통학적 연구를 통해 나문재속 종간 유연관계를 확인할 수 있는 분자마커를 찾아내기 위해 연구를 수행하였다. 핵 리보솜 DNA ITS와 엽록체 DNA matK, psbA-trnH 그리고 trnL-trnF를 분자마커로 사용하였다. ITS 영역은 칠면초와 해홍나물 그리고 해홍나물과 방석나물을 구분하지 못하였다. psbA-trnH와 trnL-trnF 영역의 염기서열은 칠면초와 방석나물을 구분하지 못하였다. 그러나 4종의 분자마커 영역을 조합하여 분석한 결과 나문재속 식물 5종이 각각 독립적인 계통을 형성하는 것을 확인하였다. 따라서 나문재속 계통관계 분석을 위해서 여러 개의 분자마커 조합이 유용할 것으로 판단된다. 나문재속 내 분류군 간의 계통관계를 명확히 밝히기 위해 차후에 좀 더 많은 생태학적, 형태학적 자료를 조사해야 할 것으로 보인다.

벼의 엽록체 small HSP의 고온 스트레스 하에서의 기능을 밝히기 위하여 Oshsp21 cDNA를 pET 발현 vector에 도입하였다. 형질전환된 대장균 배양액에 IPTG를 첨가하여 단백질 발현을 유도시킨 다음 고온 stress 하에서의 생존율을 대조구와 비교하였다. 그 결과 대조균주의 경우 에서의 생존율이 크게 감소하였으나 Oshsp21이 발현된 대장균의 경우 70% 이상의 생존율을 나타내었다. 또한 대장균 단백질을 에서 30분간 열처리한 후, 대장균 단백질을 가용성과 비가용성 단백질로 분획한 다음, 각각의 비율을 조사한 결과, 대조균주의 경우 총 단백질의 약 60%가 비가용성 단백질로 변성되었으나, Oshsp21을 발현시킨 대장균의 경우 총 단백질의 약 35%만이 비가용성 단백질로 나타났다. 이러한 결과는 벼의 엽록체 small HSP는 세포내에서 분자 chaperone으로 기능하여 고온내성을 부여할 수 있음을 나타낸다.

엽록제 small HSP의 기능을 조사하기 위하여 항상적으로 발현하는 형질전환 식물체를 작성하였다. 고온 스트레스 하에서의 형질전환 식물체의 고온내성을 chlorophyll 형광으로 측정하였다. Leaf disc를 고온조건에서 5분간 처리한 후, 광화학계 II의 불활성화를 나타내는 Fo 값의 증가 또는 Fv 값의 감소치를 조사하였다. 형질전환 식물체는 고온 스트레스 하에서의 이들 값의 증감율이 현격히 감소하였다. 또한 유식물체를 에서 45분간 처리한 후, 에서 계속적으로 배양하였을 때, 비형질전환 식물체는 전부 고사하였으나, 형질전환 식물체의 약 80%는 생존하였다. 이러한 결과는 엽록체 small HSP가 고온 스트레스 하에서 광합성기구를 보호하는데 있어서 중요한 기능을 담당하고 있음을 나타낸다.

고등식물에 있어서 엽록체에 존재하는 저 분자량 heat shock protein (smHSP)은 식물의 내열성 획득에 있어서 필수유전자임이 mutant를 이용한 유전학적인 연구에 의해 보고된 바 있다. 고온내성이 강한 작물인 벼로부터 엽록체 smHSP cDNA를 분리하고자 벼의 잎에서 분리한 mRNA로 작성한 cDNA library로부터 screening하였다. 선발된 smHSP cDNA는 1,026 bp의 염기로 구성되어 있었으며, 239개의 아미노산

Dysphania ambrosioides (L.) Mosyakin & Clemants which belongs to Chenopodiaceae/Amaranthaceae sensu in APG system has been known as a useful plant in various fields as well as an invasive species spreading all over the world. To understand its phylogenetic relationship with neighbour species, we completed chloroplast genome of D. ambrosioides collected in Korea. Its length is 151,689 bp consisting of four sub-regions: 83,421 bp of large single copy (LSC) and 18,062 bp of small single copy (SSC) regions are separated by 25,103 bp of inverted repeat (IR) regions. 128 genes (84 protein-coding genes, eight rRNAs, and 36 tRNAs) were annotated. The overall GC content of the chloroplast genome is 36.9% and those in the LSC, SSC and IR regions are 34.9%, 30.3%, and 42.7%, respectively. Distribution of simple sequence repeats are similar to those of the other two Dysphania chloroplasts; however, different features can be utilized for population genetics. Nucleotide diversity of Dysphania chloroplast genomes 18 genes including two ribosomal RNAs contains high nucleotide diversity peaks, which may be genus or species-specific manner. Phylogenetic tree presents that D. ambrosioides occupied a basal position in genus Dysphania and phylogenetic relation of tribe level is presented clearly with complete chloroplast genomes.

Background : Angelica species are representative medicinal plants and it has been used in traditional medicinal methods, especially, in the traditional Asian medicine. The Angelica species used in conventional medicine varies by country according to specific regulations, i.e. A. gigas Nakai in Korea, A. sinensis Diels in China, and A. acutiloba Kitagawa in Japan. Because of the similarity between the names among Angelica, they can be confused in the market. Methods and Results : In this study, twenty-four chloroplast insertion or deletion (cpInDel) markers were developed from chloroplast DNA of A. gigas Nakai and tested for the classification of Angelica species. Primer sets were designed from flanking sequences of the discovered InDel loci from chloroplast DNA of A. gigas Nakai using CLC Main Workbench with the following parameters : primer length = 18 - 26 bp (Opt. 23 bp); GC% = 50 - 70% (Opt. 60%); Ta = 55 - 62℃ (Opt. 58℃); product size range = 120 - 300 bp. Polymorphism and genotype analysis of 13 Angelica species were performed using the developed cpInDel markers. Conclusion : The 24 cpInDel markers developed in this study could be used for genetic diversity analysis and classification of Angelica species.

Background: In the herbal medicinal industry, Angelica gigas Nakai, Angelica sinensis (Oliv.) Diels. and Angelica acutiloba (Siebold & Zucc.) Kitag. are often confused, because the roots of the three species can not be distinguished by their appearance. This confusion can cause serious side effects. In this study, we determined the origins of Angelica roots distributed in the Korean market using the simple sequence repeat (SSR) markers developed based on the A. gigas chloroplast DNA sequence. Methods and Results: We collected twenty seven A. gigas and three A. acutiloba samples from the Seoul, Daegu, and Cheongju herbal medicinal markets. Fifty sections of one collection were mixed and ground to make a powder, which was used for DNA extraction using the cetyl trimethylammonium bromide (CTAB) method. Chloroplast based SSR markers were applied to the DNA for the determination of the species. In addition, polymorphism was found in eight samples. The phylogenetic analysis showed that the A. gigas roots collected from herbal medicinal markets were clearly discriminated from A. sinensis and A. acutiloba even though they were grouped into four clusters. Conclusions: This study showed that chloroplast based SSR markers would help the discrimination of Angelica roots in the Korean herbal medicinal industry and the markers are useful to prevent confusion between Angelica roots.

다양한 환경에서 수집한 국내 민들레속 유전자원 수집종의 엽록체 DNA 영역(trnL-trnF와 rps16-trnK) 염기서열을 이용하여 종내․ 간 변이 및 배수성을 구명하여 유전자원 육성의 기초 자료룰 제공하고자 수행하였다. 민들레속 유전자원의 배수성은 털민들레, 서양민들레, 붉은씨서양민들레가 3배체이고, 흰 민들레와 흰노랑민들레는 4배체였다. 염기서열의 길이는 trnLtrnF 영역에서 자생종류인 털민들레, 흰민들레, 흰노랑민들레 가 931 bp에서 935 bp, 서양민들레는 910 bp, 붉은씨서양민들레 는 975 bp로 종간 차이를 나타내었고, 종 특이적 염기서열 88개, 자생종 및 귀화종 특이적 염기서열 41개가 검출되었다. rps16- trnK 영역은 털민들레 882∼883 bp, 흰민들레 875∼881 bp, 흰 노랑민들레는 878∼883 bp 서양민들레 874∼876 bp, 붉은씨서 양민들레는 847∼848 bp로 37개 종특이적 염기서열이 검출되 었다. 염기서열의 유사도는 trnL-trnF 영역에서 0.860∼1.000 사이로 평균 0.949이며, rps16-trnK 영역의 유사도는 0.919∼ 1.000 사이로 평균 0.967이었다. 염기서열을 바탕으로 유연관계를 분석한 결과, trnL-trnF 영역은 크게 자생종류와 귀화종 류로 구분되었으며, 서양민들레와 붉은씨서양민들레는 같은 종 간에 유집되었고, 자생종류는 분리되지 않았으며, rps16-trnK 4개 그룹과 유집되지 않은 5개체로 나뉘었다. 흰노랑민들레는 두 영역 모두 흰민들레와 동일 계통군을 형성하였고, 염기서열 상 두 종간 뚜렷한 차이가 없었다. 유연관계에서 모두 독립적으로 존재한 흰민들레 No. 10 (조계산)과 털민들레 1번(광양)은 민들레 유전자원 육성소재로 활용이 기대된다.

Background : Codonopsis lanceolata is a flowering perennial climber. The roots are used as medicinal materials or vegetables. C. lanceolata is distributed in India and East Asia such as China, Japan as well as Korea. Recently, demand for C. lanceolata is increasing as a healthy food. In South Korea, this plant is widely cultivated in Gangwon-do province. Although, C. lanceolata is one of the most important medicinal plants in Korea, it is easy to be confused with other species of the same genus. Simple sequence repeat (SSR) marker is a powerful tool for distinguish specific species. In addition, there are many studies that show species-specific polymorphisms in chloroplasts SSR. In this study, we developed chloroplast SSR markers that can distinguish C. lanceolata from 6 Codonopsis species. Methods and Results : We collected 6 Codonopsis species include C. lanceolata. and extrated DNA using CTAB method. The DNA was diluted to 10 ng/㎕ and kept at –20℃. We designed the primer sets using CLC Main Workbench based on chloroplast DNA SSR region of C. lanceolata. PCR was performed using three independent plants for each species. Conclusion : We designed six primer sets from six SSR regions of C. lanceolata cpDNA. All of the primer sets amplified the amplicon effectively. Two of the 6 primer sets had polymorphism. We could distinguish C. lanceolata from 6 Codonopsis species using two primer sets.

Background : Cudrania tricuspidata Bureau is a widely used medicinal perennial woody plant. Obtaining information about the genetic diversity of plant populations is highly important for conservation and germplasm utilization. In this study, we developed single nucleotide polymorphism (SNP) markers derived from chloroplast genomic sequences to identify distinct Korean-specific ecotypes of C. tricuspidata via amplification refractory mutation system (ARMS)-PCR analyses. We performed molecular authentication of twelve C. tricuspidata ecotypes from different regions using DNA sequences in the chloroplast TrnL-F intergenic region. Methods and Results : SNPs were identified based on the results of nucleotide sequence for the intergenic region of TrnL-TrnF gene (chloroplast). Molecular markers were designed for those SNPs with additional mutations on the second base from SNPs for amplification refractory mutation system-polymerase chain reaction (ARMS-PCR). HRM pattern analyses were performed using the Mx3005P QPCR System (Agilent Technologies, CA, USA). Conclusion : We collected 12 individual lines of C. tricuspidata from various region in South Korea and China. Based on the nucleotide sequence in the trnL-trnF intergenic region of these lines, six SNPs and a deletion of 12 bps were identified and 12 individual lines were able to be grouped in one Korean ecotype and two different ecotypes of chinese lines, chinese line 1 and 2. The SNP markers developed in this study are useful for rapidly identifying these specific C. tricuspidata ecotypes collected from different regions.

Background : Angelica gigas is a monocarpic perennial plant. A. gigas, also called DangGui or Korean Angelica, is a major medicinal herb used in Asian countries such as Korea, Japan and China. In Korea, we are using the roots of A. gigas. but, Chinese using Angelica sinensis and Japanese using Angelica acutiloba with the same name 'DangGui'. The biggest problem in the use of A. gigas is the confusion with A. acutiloba or A. sinensis. This confusion can cause an medical accident or lack of pharmacological ingredients. In this study, we developed chloroplast InDel markers that can distinguish A. gigas, A. acutiloba or A. sinensis. Methods and Results : We collected 14 Angelica plant samples including A. gigas, A. acutiloba and A. sinensis and extrated DNA using CTAB method. The DNA was diluted to 10 ng/㎕ and kept -20℃. We designed the primer sets using CLC Main Workbench based on chloroplast DNA InDel region of between A. gigas and A. acutiloba. PCR were performed on the 14 Angelica plant samples including A. gigas, A. acutiloba and A. sinensis (5 repeats each). Electrophoresis was performed using fragment analyzer automated CE system. We designed 6 InDel primer sets and the primer sets amplified the amplicons effectively. Three of the 6 primer sets showed polymorphism. Conclusion : We could distinguish A. gigas, A. acutiloba, and A. sinensis using 2 newly developed InDel markers.

Background : In the herbal medicine market, Angelica gigas, Angelica sinensis, and Angelica acutiloba are all called "Danggui" and used confusingly. We aimed to assess the genetic diversity and relationships among 14 Angelica species collected from different global seed companies. Toward this aim we developed DNA markers to differentiate the Angelica species. Methods and Results : A total of 14 Angelica species, A. gigas, A. acutiloba, A. sinensis, A. pachycarpa, A. hendersonii, A. arguta, A. keiskei, A. atropurpurea, A. dahurica, A. genuflexa, A. tenuissima, A. archangelica, A. taiwaniana, and A. hispanica were collected. The genetic diversity of all 14 species was analyzed by using five chloroplast DNA-based simple sequence repeat (SSR) markers and employing the DNA fragment analysis method. Each primer amplified 3 - 12 bands, with an average of 6.6 bands. Based on the genetic diversity analysis, these species were classified into specific species groups. The cluster dendrogram showed that the similarity coefficients ranged from 0.77 to 1.00. Conclusions : These findings could be used for further research on cultivar development by using molecular breeding techniques and for conservation of the genetic diversity of Angelica species. The analysis of polymorphic SSRs could provide an important experimental tool for examining a range of issues in plant genetics.

Background : The P. ginseng breeding line G07006, was selected for salt tolerance through salinity screening of mature leaves at the NIHHS of the RDA in 2014-2016. However, it is difficult to maintain a genetically stable breeding line of cross-pollinating crop in the field. Therefore molecular marker required to identify and maintain breeding line G07006. Methods and Results : DNA was extracted following the CTAB DNA extraction protocol (Doyle and Doyle, 1987) with modifications. A pair-end (PE) library was constructed and sequenced using an Illumina MiSeq platform by Lab Genomics, Inc. (Seongnam, Korea). Approximately 4.0 Gb of sequencing data were obtained, and de novo assembled by a CLC genome assembler(v. beta 4.6, CLC Inc., Rarhus, Denmark). The complete chloroplast(CP) genome size is 156,356 bp, including two inverted repeats (IRs) of 52,060 bp, separated by the large single-copy (LSC 86,174 bp) and small single-copy (SSC 18,122 bp) regions. This CP genome encodes 114 unigenes (80 protein-coding genes, four rRNA genes, and 30 tRNA genes), in which 18 are duplicated in the IR regions. Conclusion : This complete chloroplast DNA sequence will provide conducive to discriminate line G070006 (salt-tolerant) and further enhancing genetic improvement program of this important medical plant.