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.