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: Adenophora triphylla var. japonica (Regel) H. Hara shows vegetative growth with radical leaves during the first year and shows reproductive growth with cauline leaves and bolting during the second year. In addition, the shape of the plant varies within the same species. For this reason, there are limitations to classifying the species by visual examination. However, there is not sufficient genetic information or molecular tools to analyze the genetic diversity of the plant. Methods and Results: Approximately 34.59 Gbp of raw data containing 342,487,502 reads was obtained from next generation sequencing (NGS) and these reads were assembled into 357,211 scaffolds. A total of 84,106 simple sequence repeat (SSR) regions were identified and 14,133 primer sets were designed. From the designed primer sets, 95 were randomly selected and were applied to the genomic DNA which was extracted from five plants and pooled. Thirty-nine primer sets showing more than two bands were finally selected as SSR markers, and were used for the genetic relationship analysis. Conclusions: The 39 novel SSR markers developed in this study could be used for the genetic diversity analysis, variety identification, new variety development and molecular breeding of A. triphylla.

Background: Panax ginseng C. A. Meyer is wood-cultivated ginseng (WCG) in Korea which depends on an artificial forest growth method. To produce this type of ginseng, various P. ginseng cultivars can be used. To obtain a WCG similar to wild ginseng (WG), this method is usually performed in a mountain using seeds or seedlings of cultivated ginseng (CG) and WG. Recently, the WCG industry is suffering a problem in that Panax notoginseng (Burk.) F. H. Chen or Panax quinquefolium L. are being sold as WCG Korean market; These morphological similarities have created confusion among customers. Methods and Results: WCG samples were collected from five areas in Korea. After polymerase chain reaction (PCR) amplification using the primer pair labeled with fluorescence dye (FAM, NED, PET, or VIC), fragment analysis were performed. PCR products were separated by capillary electrophoresis with an ABI 3730 DNA analyzer. From the results, WCG cultivated in Korea showed very diverse genetic background. Conclusions: In this study, we tried to develop a method to discriminate between WCG, P. notoginseng or P. quinquefolium using simple sequence repeat (SSR) markers. Furthermore, we analyzed the genetic diversity of WCG collected from five cultivation areas in Korea.

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 : Panax ginseng C.A. Meyer is a perennial herb belongs to the family Araliaceae. Wild-cultivated ginseng (WCG) is a specific type of ginseng in Korea which cultivated on artificial forest cultivation method. To obtain a WCG which is similar to wild ginseng (WG), this method usually performed in a mountain using seeds or seedlings of cultivated ginseng (CG) and WG. WCG is very expensive because it is difficult to cultivate. However, systematic cultivation method have not yet been developed compared to high added value. Furthermore, very high price of WCG caused the problem that Panax notoginseng or Panax quinquefolium are sold as WCG in Korean market. In this study, we analyzed the genetic diversity of WCG collected from five areas in Korea using SSR markers. Methods and Results : WCG samples were collected from five areas in Korea (Bucheon, Cheongju, Hoengseong, Judeok and Ulsan). DNA extraction was performed using CTAB method. SSR markers were collected from the published papers. After test PCR using the markers, one of the primer pair was labeled with fluorescence dye (FAM, NED, PET, or VIC) and GeneScan analysis were performed. DNA amplification was conducted using T-100 Thermal Cycler (Bio-Rad). PCR products were separated by capillary electrophoresis on the ABI 3730 DNA analyzer (Applied Biosystems). Conclusion : Eight SSR markers were collected from the published literature and used for the analysis. From the 8 tested SSR markers, 7 SSR markers showed polymorphism between varieties. GenScan analysis were performed using the selected SSR markers to analyze the phylogenetic relationship of WCG. From the results, WCG cultivated in Korea showed that they have a very diverse genetic background.

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 : 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 : Wild-cultivated ginseng (WCG) prices are very different according to root ages. Generally, two methods are used to discriminate the root ages of Panax ginseng C.A. Meyer. The first method is the yearly determination method by the ring dyeing method, and the second method is the confirmation the number of stem vestiges in the rhizome. In this study, we analyzed the agronomic and growth characteristics of the WCG cultivated in Korea. In this study, to determine the appropriate root ages discrimination method for the determination of the root ages of WCG, the root ages of WCG and cultivated ginseng was examined. Methods and Results : We examined the cultivated ginseng (CG) and WCG that was collected and sold by regional groups at the Korean market. WCG does not form annual rings, which are clear and regular in wild ginseng. Therefore, it is impossible to identify the age of WCG by using the annual growth rings staining method. However, the age can be estimated by determining the number of stem vestiges in the rhizome. Conclusion : From the results of the Study on identification of root age for quality evealuation in WCG in Korea. Appropriate root ages discrimination method of WCG was confirmation the number of stem vestiges in the rhizome.

Background : Panax ginseng C.A. Meyer is a representative medicinal plants and it has been used in traditional medicine because the plant have many effective component such as saponins. To obtain a wild-cultivated ginseng (WCG) which is similar to wild ginseng (WG), this method usually performed in a mountain using seeds or seedlings of cultivated ginseng (CG) and WG. WCG is very expensive because it is difficult to cultivate. However, systematic cultivation method have not been developed compared to their high added value. Furthermore, very high price of WCG caused the problem that Panax notoginseng or Panax quinquefolium are sold as WCG in Korean market. This is concerned as a serious problem to consumers. In this study, we analyzed the agronomic and growth characteristics of the WCG cultivated in Korea. Methods and Results : We examined the WCG that was collected and sold by regional groups at the Korean market. The root age, growth conditions, and quality level of the cultivated WCG were confirmed. WCG samples were collected from five areas in Korea (Bucheon, Cheongju, Hoengseong, Judeok and Ulsan). The main root diameter, root shape index, rhizome length, and root weight showed high level of variation and they did not form annual rings. Conclusion : Agronomic and growth characteristics of WCG showed high variations according to cultivating regions.

Background : Adenophora triphylla var. japonica (Regel) H. Hara shows vegetative growth by radical leaf until 1 year after sowing and shows reproductive growth during the second year and there is a characteristic of bolting by turning into cauline leaf. In addition, the phenotypes of plants varies even though they are belonging to the same species. For this reason, there is a limit for the classification of the species by the method of visual examination. Methods and Results : Simple sequence repeat (SSR) markers were developed based on the genomic sequence of A. triphylla using next generation sequencing to prepare the basis of molecular breeding and analyze the genetic diversity. Ninety-five primer sets including tri-, tetra- and penta-nucleotide motif types were randomly selected and they were applied to mixed genomic DNA and finally 39 primer sets showing from two to four bands were selected and used for genetic relationship analysis. Conclusions : Using the next generation sequencing, 39 polymorphic SSR markers were developed.

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 : 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 : Wild-cultivated P. ginseng (WCG) is a specific ginseng in Korea which depends on artificial forest growth method. To obtain a WCG which is similar to wild ginseng (WG), this method usually performed in a mountain using seeds or seedlings of cultivated ginseng (CG) and WG. Recently, very high price of WCG caused the problem that Panax notoginseng or Panax quinquefolium are sold as WCG in Korean market. This is concerned as a serious problem to consumers. In this study, we tried to develop a method to discriminate WCG, CG or WG using simple sequence repeat (SSR) markers and phylogenetic analysis. Methods and Results : WCG samples (3, 5, or 6-years old) were collected in Hoengseong, Gangwondo. DNA extraction was performed using CTAB method. SSR markers were collected from the published papers. After test PCR using the markers, one of the primer pair was labeled with fluorescence dye (FAM, NED, PET, or VIC) and Gene Scan analysis were performed. NTsys-PC program was used for the phylogenetic analysis of the data. Eight SSR markers were collected from the published literature and used for the analysis. From the 8 tested SSR markers, 7 SSR markers showed polymorphism between varieties. GenScan analysis were performed using the selected SSR markers to analyze the phylogenetic relationship of WCG. Conclusion : Phylogenetic analysis showed the relationship between WCG and P. ginseng cultivars and the seven SSR markers used in this study are able to distinguish Wild-cultivated P. ginseng.