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 : The Codonopsis genus belongs to the Campanulaceae, and it is recorded that there are four species of Codonopsis genus in Korea, such as Codonopsis lanceolata, Codonopsis pilosula, Codonopsis minima, and Codonopsis ussuriensis. C. lanceolata has been proved to be safety and efficacy, and has been widely used for medicinal and edible purposes for a long time in East Asian countries including Korea, China and Japan. However, little genetic research has been done. Methods and Results : Ten species of Codonopsis plants were collected and DNA was extracted using CTAB (cetyl trimethylammonium bromide) method. The extracted DNA was diluted to 5 ng/㎕ for the PCR (polymerase chain reaction) process. C. lanceolata genome was used to develop molecular markers by searching insertion and deletion regions (InDel) in the chloroplast sequence. The developed markers were applied to 4 individuals per Codonopsis species. PCR amplification was carried out using a denaturation at 94℃ for 30 sec, annealing at 58℃ for 30 sec and extension at 72℃ for 30 sec, repeated for 35 total cycles. The PCR products were separated in a 4% agarose gell at 100 V for 40 min. Conclusion : Using the molecular markers developed in this study, genetic diversity of Codonopsis genus was tested, and at the same time, a specific molecular marker was developed to differentiate C. lanceolata from the Codonopsis plants.

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.

Background : Codonopsis lanceolata is a perennial plant of Campanulaceae and mainly distributed in East Asia such as Korea, China, and Japan. C. lanceolata has a unique taste and aroma, and it is rich in minerals such as phosphorus and calcium, and vitamin B1 and B2, so our ancestors used the plant as medicinal herb and edible vegetable. However, systematic cultivation and development of varieties have not been achieved compared to demand or high added value. The genetic diversity and relationship analysis of the plants help to increase the efficiency of breeding through genetic variation. Methods and Results : Ten species of Codonopsis plants were used as materials and DNA was extracted from each 4 individuals per species and quantified at a concentration of 10 ng /㎕. The extracted DNA was pooled by species and PCR was performed using the EST-SSR marker developed based on C. lanceolata in the previous study. PCR amplification was carried out using a denaturation at 94℃ for 30 sec, annealing at 58℃ for 30 sec and extension at 72℃ for 30 sec, repeated for 35 total cycles. The PCR products were separated in a 4% agarose gell at 100 V for 40 min. Conclusion : In this study, C. lanceolata collections was determined among several Codonopsis species using these molecular marker. It is expected that the data of this study can be used as reference for genetic polymorphism analysis and related gene studies of Codonopsis species.

Background : Codonopsis lanceolata is a perennial plant of Campanulaceae with characteristic flavor and aroma and this plant has saponin, flavonoid, and inulin, which are reported to have physiological activity and antioxidant activity. In contrast, breeding or study of C. lanceolata varieties had not been done for a long time. Genetic polymorphism and phylogenetic relationship analysis of the plants by region of the crops can help the collection of genetic backgroud data for variety development. Methods and Results : In this study, we collected 26 C. lanceolata lines (95 individual plants) from 26 regions in Korea. We genotyped the collected lines using SSR markers developed in the previous study and analyzed the population structure based on the results. Population structures were analyzed using model-based STRUCTURE software (version 2.3.4) using the following parameters: Number of clusters (K) set = 1 to 12; Number of Iterations = 5; Length of Burning Period = 100,000; Number of MCMC (Markov Chain Monte Carlo) Reps after Burnin = 100,000. As a result, Of the 26 collections, were genetically grouped into 6 or 7 groups. Conclusion : The 26 C. lanceolata collections (95 individual plants) were genetically grouped but not grouped by collected regions. These results suggest that C. lanceolata has diverse genetic backgrounds and this data could be used as a basis for genetic polymorphism analysis of Codonopsis species.

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, an elite, inbred line or a variety has not been developed yet. Simple sequence repeat (SSR) marker is a powerful tool for analysis of genetic relationships. In addition, it is a useful tool for studying the non-reference plant genome, due to its even distribution throughout the genome, as well as its high polymorphism between individuals. Methods and Results : We constructed microsatellite-enrichment libraries using C. lanceolata genomic DNA, and obtained a total of 226 non-redundant contig sequences. Routine PCR was performed using gDNA as templates for the polymorphic markers screening. Finally, total 15 polymorphic SSR markers based on C. lanceolata genomic sequences were successfully developed. These markers were applied to 53 C. lanceolata collected from Korea. 103 alleles of the 15 SSR markers ranged from 3 to 19 alleles at each locus, with an average of 6.87. The average of observed heterozygosity and genetic diversity were 0.42 and 0.62, respectively. The average of polymorphism information content (PIC) value was 0.57. The genetic distance value ranged from 0.73 to 0.93, and there was no observed distinct group according to the collecting areas. Conclusion : We developed 15 novel SSR markers from C. lanceolata genomic sequences for further genetic studies. Also, we concluded that the lineage of C. lanceolata collected in Korea has not been established systematically.

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 : Medicinal crop has been used in the traditional Asian medicinal methods. From ancient times, various kinds of medicinal crop are being cultivated in East Aisa including Korea, China and Japan. In Korea, they used a variety of medicinal plants in folk medicine and oriental medicine since ancient times. Molecular markers can be widely used in a variety of settings such as effective-loci estimation, genetic-diversity characterization, allelic-effect studies, gene-flow studies, quantitative-trait locus (QTL) mapping, and evolutionary studies. The genetic analyses of crops require large numbers of useful molecular markers for genetic or QTL identification, comparative mapping and breeding. Studying the genetic diversity and genetic relationship of crops can assist breeders. Crop genetics within a breeding program enable the economic and effective cultivar development. We tried to develop a variety of molecular markers from Angelica gigas genomic sequences for genetic studies and breeding. Methods and Results : A. gigas resources cultivated in Republic of Korea were collected. Fresh leaves were ground with liquid nitrogen and gDNA was extracted using a DNeasy Plant Mini kit (Qiagen, Valencia, CA, USA). We sequenced the whole genomes of five A. gigas accessions using Illumina HiSeq 2500 platform and identified genomic Simple Sequence Repeat (SSR) and InDel markers. DNA amplification was conducted using the PCR system (Bio-Rad T-100 Thermal Cycler). PCR products were separated by capillary electrophoresis on the ABI 3730 DNA analyzer (Applied Biosystems) and Fragment analyzer automated CE system (Advanced Analytical Technologies, Ankeny, IA, USA). Conclusion : We developed novel SSR and InDel markers from A. gigas genomic sequences for further genetic studies and breeding.

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 : Codonopsis is a flowering plants belong to the family Campanulaceae, and has many kinds of medicinal properties. As currently recognized, two other groups, Campanumoea and Leptocodon, are included in the Codonopsis. The enlarged genus Codonopsis is distributed in Eastern, Southern, Central, and Southeastern Asia. C. lanceolata, C. clematidea and C. pilosula has many kinds of medicinal properties and this plants are used as medicinal and edible plants. C. ovata and C. mollis are distributed in Pakistan Kashmir and Himalaya mountains at an altitude of about 3,000 m, and flowers bloom in July to August. Methods and Results : In this study, we analyzed the genetic diversity of 5 Codonopsis species using 8 SSR markers base on C. lancelolata genomic sequences. Samples were obtained from fresh leaves of 5 plants from each species and genomic DNA was extracted using CTAB method. PCR was performed in total 20μl reaction volume containing 20 ng of DNA template and 5 pmole of primers. PCR conditions composed pre-denaturation at 95℃ for 5 min, then 35 cycles of 95°C for 30 sec, 60°C for 30 sec and 72°C for 30 sec, and a final extension at 72℃ for 30 min. The amplified band sizes ranged from 74 to 301 bp and clearly showed single or doble bands in eletrophoresis. From the phylogenetic analysis, C. lanceolata was grouped together, but the others were not grouped together according to the species. Conclusion : We concluded that C. lanceolata cultivated in Korea is different from the other species, and the eight SSR markers used in this study are able to distinguish C. lanceolata from the other species.

Background : Codonopsis lanceolata is used as a natural medicine or vegetables. It originates in East Asia such as Korea, Japan and China. C. lanceolata roots contain various chemical compounds including saponins like Panax ginseng. Although C. lanceolata are cultivated in different regions of South Korea, no variety has been developed. Therefore, it is necessary to develop discriminating methods such as molecular markers in C. lanceolata species. Methods and Results : To find simple sequence repeat (SSR) markers, we sequenced C. lanceolata genomic DNA using Illumina HiSeq 2000 System. A total of 250,455 putative SSR loci were obtained, and 26,334 non-redundant primers were designed to amplify these SSRs. Di-nucleotied repeats were the most abundant SSR reapeats, accounting for 89.53% (23,578) of primer designed SSRs. Tri-nucleotide, tetra-nucleotide and penta-nucleotide accounted for 8.44% (2,223), 1.3%, (348) 0.2% (55), respectively. Tri-, tetra-, and penta-nucleotide (total of 2,626 SSRs) were investigated in silico to identify polymorphism between individuals. Consequently, 573 SSRs showed polymorphism. Forty genomic SSR markers were tested in 16 C. lanceolata plants for determination of PCR amplification and polymorphism. From these primers, 27 (67.5%) amplified products and the average polymorphism information content (PIC) value was 0.52. Conclusion : We development 27 SSR markers from C. lanceolata using NGS, and it could be used for breeding of new varieties in the future.

β amyloid protein (Aβ) plays a critical role in the pathogenesis of Alzheimer's disease (AD) and possibly in Aβ -induced mitochondrial dysfunction and oxidative stress. Aβ can directly cause reactive oxygen species (ROS) production. Overproduction of ROS is considered to be involved in the pathogenesis of neurodegeneration of AD. Here, we investigated 9 kinds of ramie (Boehmeria nivea, (L.) Gaud., BN; hereafter denoted as BN) for their protective action against oxidative stress in a cellular system using C6 glial cells. We observed loss of cell viability and high levels of ROS generation after treatment with hydrogen peroxide (H2O2) and Aβ25-35. However, treatments with BN extracts led to an increase in cell viability and decrease in ROS production induced by H2O2 and Aβ25-35. In particular, the extracts of BN-01 (seobang variety from Seocheon) and BN-09 (local variety from Yeonggwang) showed excellent anti-oxidative properties. This indicates that BN extracts could prevent neurodegeneration by reducing oxidative stress in cells.

In this study, genetic diversity of wild Codonopsis lanceolata collected in Korea were analysed using SSR makers. Wild C. lanceolata roots were collected in Jeollanam-do Jangheung-gun Choentae Mountain as in roots. The wild C. lanceolata plants were cultivated in Chungbuk National University greenhouse and the leaves were sampled from 36 plants. The genomic DNA of C. lanceolata was extracted using CTAB. PCR was performed using a program of 35 cycles at 94℃ for 30 sec, 60℃ for 30 sec, and 72℃ for 30 sec with an pre-denaturation of 94℃ for 5 min and a final extension of 72℃ for 30 min. The PCR reaction mixture contains 5 pmole of primers and 20 ng of DNA template in a 20 μL reaction volume. The genotype of the analyzed samples were very different. Therefore, the wild C. lanceolata collected in Korea look genetically diverse.

Codonopsis lanceolata is a perennial climber. The roots are used as medicinal materials or vegetables. Recently, demand for C. lanceolata is increasing as a healthy food. C. lanceolata is distributed in India and East Asia such as China, Japan as well as Korea. In South Korea, this plant is widely cultivated in Gangwon-do province. No C. lanceolata varieties were developed in Korea. The objective of this study is to analyze genetic diversity of C. lanceolata cultivated in Korea using SSR makers. C. lanceolata roots were collected in each region were cultivated in Chungbuk National University greenhouse. Samples were obtained from fresh leaves of 5 plants from each collection region. The genomic DNA was extracted using CTAB. Genetic diversity was analysed using 4 sets of C. lanceolata SSR makers. PCR was performed in total 20 μL reaction volume containing 20 ng of DNA template, 5 pmole of primers. The genotypes of the analyzed samples were very similar. That means that the genetic diversity of C. lanceolata cultivated in Korea is very low.

Citrus canker caused by Xanthomonas citri is a notorious disease affecting a decrease in fruit productivity and quality. Citrus export to USA is also prohibited by the disease. Therefore, development of citrus canker resistant variety is essential and exploitation of markers for molecular breeding is urgent. To develop DNA molecular markers, we performed whole genome resequencing for 8 varieties: 4 citrus canker resistant varieties including C. hybrid ‘Kioymi’ and 4 citrus canker susceptible varieties including C. iyo ‘Miyauchiiyokan’. In total, 642 polymorphic SNPs were detected between resistant and susceptible varieties. Of the 642 SNPs, 50 SNPs were preferably selected based on integrative genomics viewer. To apply the markers in a broad range of citrus variety, we performed genotyping with 6 other varieties very well known as citrus canker resistant and susceptible varieties in addition to previous mentioned 8 varieties. Three of the 50 SNPs were identified as a marker to distinguish citrus canker resistant varieties from susceptible varieties. Secondly, we developed molecular markers to apply for F1 lines crossed by ‘Kiyomi’ and ‘Miyauchiiyokan’. Of the 50 SNPs, we identified 2 SNP markers to distinguish between F1 resistant and susceptible lines. One of them is a resistance gene that plays a role in plant defense mechanism. In this study, we developed 5 molecular marker candidates possible to apply for molecular breeding to develop citrus canker resistant variety. We are working on development of candidate markers related to citrus canker.

Ramie (Boehmeria nivea L.) is a hardy perennial herbaceous plant of the Urticaceae family and has been grown as a fiber crop in several countries including Korea for many centuries. Ramie leaves also have been traditionally used as a major ingredient of a type of rice cake called ‘Song-pyun’ in the Southwest area of Korea, especially Yeong-Gwang province. Despite its economic importance, the molecular genetics of ramie have not been studied in detail yet. Genetic resources of ramie were widely collected from domestic local sites by Bioenergy Crop Research Center (RDA) and Yeong-Gwang Agricultural Technology Center. For the systematic and efficient management of the genetic resources, we developed SSR (simple sequence repeat) markers of ramie. To do this, we generated microsatellite-enriched genomic DNA libraries using magnetic bead hybridization selection method. 247 non-redundant contigs containing SSR motif were generated using nucleotide sequences of 376 clones from the libraries. Primer sets were designed from the flanking sequences of the repeat motif. Finally, we selected 10 SSR markers, possibly showing polymorphism among the genetic resources. Results on the genotype analysis of the ramie genetic resources using the SSR markers will be presented.

Content analysis of loliolide in the leaves of Boehmeria nivea (Bn) collected from different region during four months (June, July, August, and September) was conducted by HPLC. The content of loliolide was detected in the leaves of B. nivea from Bns-2, -7, -10, -23, -38, -41, -67, -76, and -90 in June (5.02, 6.35, 6.93, 5.89, 4.31, 4.24, 4.91, 5.12, and 5.46 mg/g, respectively), July (4.32, 6.42, 7.72, 7.97, 4.05, 4.32, 5.65, 6.67, and 5.39 mg/g, respectively), August (3.52, 5.17, 3.90, 4.27, 3.26, 4.72, 3.82, 3.30, and 3.31 mg/g, respectively), and September (7.04, 7.25, 7.43, 7.86, 6.76, 6.38, 7.60, 6.79, and 4.77 mg/g, respectively). Among them, the highest content of loliolide was found in Bn-23 and in September. These results may be useful in determining the optimal harvest time at which phytochemical reaches a maximum level.

Citrus canker caused by Xanthomonas citri pv. citri is one of economically important diseases in the citrus industry. The devastating bacterial disease results in unattractive quality and a significant reduction in fruit production. Citrus growers and industry in Korea has been struggling with the serious disease causing the prohibition of export market. Korea also became the top import market for oranges. The development of markers linked to citrus canker resistance is strongly needed. In this study, we investigated molecular markers between ‘Kiyomi’ (Citrus unshiu ｘ C. sinensis), a resistant cultivar, and Natsudaidai (C. natsudaidai), a susceptible cultivar. To develop markers, we focused on structural variation (copy number variation, CNV, and presence/absence variation, PAV). It has been well documented that CNV and PAV of defense-related genes are associated with resistant cultivars. Using a read depth approach following next-generation sequencing, we performed genome-wide analysis of CNV and PAV in two varieties. As a result, 633 genes showing at least two times difference between the mapping reads from two varieties and 61 genes showing presence of the mapping reads in only either one of them were screened. Visual inspection using the Integrative Genomics Viewer (IGV) was performed and experimental validation is being investigated. Interestingly, one of PAV candidates showed polymorphism in ‘Kiyomi’ and ‘Natsudaidai’ as well as other resistant and susceptible cultivars. Our results suggest a necessity for the detection of structural variation and indicate that the candidates may be useful for molecular breeding for citrus canker resistance and understanding disease resistance mechanism.