Panax ginseng C.A. meyer (family: Araliaceae) is a perennial crop that has been widely used as a traditional medicine in Korea. Various P. ginseng cultivars exhibit a range of morphological and physiological traits as well as genetic diversity. To elucidate the differences of primary metabolism underlying such genetic diverstiy, we performed primary metabolite profiles in adventitious roots from five Panax ginseng cultivars using gas chromatography-mass spectrometry (GC-MS). The GC-MS analysis revealed eight primary metabolites as biomarkers and allowed us to classify the five cultivars into three groups. We selected three cultivars to represent each group and analyzed their transcriptomes by Illumina sequencing. We inspected 100 unigenes involved in seven primary metabolite biosynthesis pathways and found that 21 unigenes encoding 15 enzymes were differentially expressed among the three cultivars. Integrated analysis of transcriptomes and metabolomes revealed that the ginseng cultivars differ in primary metabolites as well as in the putative genes involved in the complex process of primary metabolic pathways. Our data derived from this integrated analysis provide insights into the underlying complexity of genes and metabolites that co-regulate flux through these pathways in ginseng.

Panax Ginseng is a perennial medicinal plant originated from North-east asia. Because of its well-known tonic effects mainly from ginsenosides, various types of processed ginseng products have been distributed around the world. Here, we analyzed secondary metabolite profiling of adventitious roots of 5 korean ginseng cultivars, Chunpoong (CP), Sunhyang (SH), Gopoong (GO), Sunun (SU), and Cheongsun (CS). At the same time, the profiles of relative gene expressions related to ginsenoside biosynthesis pathway were compared among ginseng cultivars. Secondary metabolite profiles were revealed by UPLC/Q-TOF-MS from extracts of bioreactor derived adventitious roots of five ginseng cultivars. Using principal component analysis, secondary metabolite profiles of ginseng cultivars were categorized into three groups. Metabolites with high VIP values were annotated using known database and standards compounds. Relative gene expression of ginsenoside related gene were analyzed using realtime PCR. The three groups had distinct metabolite contents. Furthermore, gene expression profiles related to ginsenoside were also different, which might contribute diverse secondary metabolite composition of ginseng cultivars. Further integrated analysis would provide a relationship between genetic background of ginseng cultivars and secondary metabolite profiles.

The generation and analysis of genomic resources information are essential to understand genomic features of crops. Even though medicinal component and its effect of Panax ginseng was well studied, the genomic study has been recently started. The ginseng genome has been known to undergo two rounds of whole genome duplication (WGD), therefore we investigated an evidence of WGD in ginseng draft sequence for understanding current ginseng genome structure. Four paralogous gene-rich genome blocks were found, consisted by eight scaffolds, using about 3.0 Gb whole genome draft sequence and 48,821 unigenes of P. ginseng generated by whole genome shotgun sequencing. The eight scaffold sequences were ordered and connected into four genomic blocks, using zig-zag extension within scaffold sequences recently duplicated. The paralogous scaffold pairs that were recently duplicated showed high sequence conservation in genic and non-genic regions. However, paleo duplicated paralogue scaffold sequences showed little conservation only in genic regions. Finally, a total of 110 paralogous gene pairs and its expression were identified from recently and paleo duplicated scaffold pairs, which were co-linear among four genomic blocks. This study provides the first insight into duplicated genome structure of ginseng and will be a valuable information for further ginseng genomics including improvement of draft sequence quality, chromosome anchoring of scaffolds, and genetic mapping.

Clubroot is a devastating disease caused by Plasmodiophora brassicae and results in severe losses of yield and quality in Brassica crops including Brassica oleracea. Therefore, it is important to identify resistance gene for CR disease and apply it to breeding of Brassica crops. In this study, we applied genotyping-by-sequencing (GBS) technique to construct high resolution genetic map and mapping of clubroot resistance (CR) genes. A total of 18,187 GBS markers were identified between two parent lines resistant and susceptible to the disease, of which 4,103 markers were genotyped in all 78 F2 plants generated from crossing of both parent lines. The markers were clustered into nine linkage groups spanning 879.9 cM, generating high resolution genetic map enough to refine reported reference genome of cabbage. In addition, through QTL analysis using 78 F2:3 progenies and mapping based on the genetic map, two and single major QTLs were identified for resistance of race 2 and race 9 of P. brassicae, respectively. These QTLs did not show collinearity with CR loci found in Chinese cabbage (Brassica rapa) but roughly overlapped with CR loci identified in cabbage for resistance to race 4. Taken together, genetic map and QTLs obtained in this study will provide valuable information to improve reference genome and clubroot resistance in cabbage.

Chloroplasts are plant-specific organelles, which have their own genome. Most of the plant chloroplast genomes (CP genome) are highly conserved in terms of its gene contents and genome structures, and they exist in cells with abundant copy numbers. Because of numerous copy numbers, the complete chloroplast sequence assembly pipeline with small amount of whole genome resequencing data, produced by NGS technique, was established in our laboratory. From 14 accessions of cabbage (Brassica oleracea L.) resequencing data produced by Illumina Hi-seq 2000, CP genomes were assembled and compared to each other. 18 sequence variance regions were detected, and 6 HRM(High Resolution Melting curves) markers were developed. Approximately 1 Gb of whole genome sequencing data of 10 Brassica rapa and 2 Brassica napus were also obtained from Institute of Vegetables and Flowers, Chinese Academy of Agricultural Science. With these resequencing data, all CP genomes from these accessions were assembled. Total 27 complete CP genomes of B.oleracea, B.rapa, B.napus, and brassico-raphanus which is a novel allotetraploid species between B.rapa and Raphanus sativus, were compared in sequence level. Phylogenetic analysis based on the comparison revealed that B.rapa could be the maternal species when rapeseeds and brassico-raphanus became allotetraploid species. Additionally, CP genome of B.napus cv.M083 is closer to B.rapa accessions than the other B.napus accessions, thus B.napus could have several different origins.

Mitochondria are essential organelles of eukaryotic cells and plant cells contain varying numbers of mitochondrial genome sequences. Sizes and shapes of mitochondria differ within a tissue or in the same cells. Previously sequenced complete mitochondrial genome (NC_016118) of Brassica oleracea size was 360,271 bp, where segmental duplication (repeat block) was 141,800 bp. In this study, we resequenced this whole mitochondrial genome by using WGS (whole genome sequencing) and assembled organelles genome method (unpublished). Newly sequenced mitochondrial genome length was 219,975 bp and circle form. A new sequence segment of approximately 4,800 bp was obtained compared to the previous genome sequence without any large repeat block. Newly obtained mitochondria genome sequence was compared with recently reported mitochondria genome sequences of various species (B. oleracea, B. juncea, B. rapa, B. napus and B. carinata) and subspecies (cabbage, cauliflower, brussels sprouts, kohlrabi, broccoli and kale) by PCR using primers specifying different region of genome sequences. PCR analysis results have also confirmed the variation between previous and newly sequenced mitochondrial genome circles form. Thus, the results suggest new B. oleracea mitotype, including evolutionary events such as inheritance, rearrangement, genome compaction, and diversity

There is a growing number of plant genomes that are being sequenced, but most of these available assemblies do not cover the entire genome mainly due to the highly repetitive sequences found in most plant genomes. Nevertheless, these repeats, although a challenge in assembly algorithms, provide relevant information about a genome’s history that could help explain its structure and complexity. Here, we cytogenetically mapped previously and presently characterized major repeats of Panax ginseng genome, including several LTR retrotransposons (PgDel2, PgDel3, PgTat1, PgTat2, PgTork) and one tandem repeat, PgTR Fluorescence in situ hybridization (FISH) results showed differential accumulation of Ty3/gypsy LTR retrotransposons into different chromosomal regions or subgenomes, suggesting a non-random preferential amplification of retrotransposons in these regions and an allopolyploid origin of P. ginseng. In silico analysis based on 1x whole genome sequence reads suggests that PgTR is the most abundant tandem repeat in ginseng, which was further corroborated by FISH analysis. More importantly, its unique distribution pattern among the 24 ginseng chromosomes, coupled with the non-random distribution of LTR retrotransposons and rDNA arrays, allowed us to discriminate and characterize each individual ginseng chromosome. These different newly characterized cytogenetic markers allowed reorganization of previously reported ginseng karyotype with better resolution, demonstrating the irutility in ginseng chromosome identification. These information give us insight about the genomic structure of P. ginseng, and should be useful for future comparative cytogenetics studies among closely related species to unravel its genomic history. This work was supported by the Next-Generation BioGreen21 Program (No. PJ008202), Rural Development Administration, Republic of Korea.

Tongil (IR667-98-1-2) rice, developed in 1972, is a high-yielding rice variety derived from a three-way cross between indica and japonica. Tongil contributed to staple food self-sufficiency of Korea, an achievement that was termed the ‘Korean Green Revolution’. In this study, we analyzed the nucleotide-level genome structure of Tongil rice and compared it to those of the parental varieties. A total of 17.3 billion Illumina Hiseq reads, 47× genome coverage, were generated from Tongil rice. Three parental accessions, two indica and one japonica types, of Tongil rice were also sequenced for approximately 30x genome coverage. A total of 2,149,991 SNPs were detected between Tongil and Nipponbare; the average SNP frequency of Tongil was 5.77 per kb. Genome composition based on the SNP data by comparing with the three parental genome sequences on sliding window of Nipponbare genome sequence revealed that 91.8% of the Tongil genome originated from the indica parents and 7.9% from the japonica parent, different from the theoretical expectation in a three-way cross, i.e., 75% indica and 25% japonica parental origins on average. Copy number of SSR motifs, ORF gene distribution throughout the whole genome, gene ontology (GO) annotation, yield-related QTLs or gene locations, and polymorphic transposon insertions were also comparatively analyzed between Tongil and parents using sequence-based tools. The results indicated that each genetic factor was transferred from parents into Tongil in proportion to the whole-genome composition. The Tongil rice is the first successful superior cultivar derived from indica × japonica hybridization in Korea. Defining of genome structure demonstrates that the Tongil genome is composed mostly of the indica genome with a small proportion of japonica genome introgression. This work was supported by a grant from the Next-Generation BioGreen 21 Program (Plant Molecular Breeding Center No. PJ008125), Rural Development Administration, Republic of Korea.

Genome duplication is an abundance phenomenon and in plant kingdom and consequently formed paralogous region. Korean ginseng (Panax ginseng C.A. Meyer) has a possibility of tetraploid by comparing chromosome numbers of relative species. During development of EST-SSR markers in Korean ginseng, most of primer sets have produced multiple bands in gel electrophoresis. In this study, for identifying origin of multiple bands, five EST-SSR markers showing multi-band were selected and two bands around expected size were sequenced. Sequence comparison classified the multiple bands into individual loci. Two bands can be identified by SNP or InDel variation with number of SSR units. Sequencing result represented that paralogous loci with high similarity were existence caused by recent duplication. One clear band were amplified with newly designed locus specific primer picked from SNP variation. SNP and InDel polymorphism between paralgous loci were useful for identifying each locus. This study will provide better understanding of ginseng genome and will be helpful for development of DNA markers.

Ginseng (Panax ginseng C.A. Meyer) is the most famous medicinal herb in East Asia. Although medicinal components and their functions have been widely investigated, ginseng has been regarded as an underdeveloped crop in genetics and genomics research areas. This study was conducted to elucidate the structure and evolution of the ginseng genome by analyses of expressed sequence tags (ESTs) and bacterial artificial chromosome (BAC) sequences. The EST analysis based on the calculation of synonymous substitutions per synonymous site (Ks) in paralog and ortholog pairs revealed that two rounds of polyploidy events occurred in the common ancestor of ginseng and American ginseng (P. quinquefolius L.) and subsequent divergence of the two species. The sequence analysis of repeat-rich BAC clones characterized the major component of the ginseng genome, long terminal repeat retrotransposons (LTR-RTs). The LTR-RTs were classified into five main families, of which three (PgDel, PgTat, and PgAthila) belonged to Ty3/gypsy and the other two (PgTork and PgOryco) to Ty1/Copia. High abundance of the LTR-RTs were revealed by whole genome shotgun (WGS) read mapping and fluorescence in situ hybridization (FISH) analysis. Particularly, the most abundant PgDel family have played major roles in expanding heterochromatic regions as well as remodeling euchromatic regions. Biased intensity of the PgDel2 FISH signals on half the total chromosomes demonstrates the allopolyploid feature of ginseng genome. Insertion time estimation of each LTR-RT implied that LTR-RTs have proliferated after the recent polyploidization of ginseng. These results suggest that the ginseng genome of the present day has been expanded and evolved by two rounds of polyploidization and accumulation of LTR-RTs.

The C-repeat/dehydration-responsive element binding transcription factors (CBF/DREBs) are involved in an important pathway for abiotic stress-response in plants. We have identified CBF/DREB1 gene family from Brassica rapa whole genome sequence and designated them as BrDREB1s. They contain conserved nucleus localization signal, AP2/EREBP domain, and CBF/DREB1 signature, as other known plant CBF/DREB1s. By comparative genomics, we found that nine of ten BrDREB1 genes were present in seven macro-synteny blocks co-linear to four Arabidopsis counterpart blocks and also genomic organizations of their flanking regions were very similar to those for co-linear Arabidopsis CBF/DREB1 genes. In particular, three genes, BrDREB1A, BrDREB1B1, and BrDREB1C1, were closely located within a 59 kb genomic sequence, which was similar to that of their Arabidopsis counterpart genes. However, the genomic regions of those BrDREB1 genes contained additional sequences, compared to their co-linear regions in A. thaliana. The expression of BrDREB1 genes under abiotic stresses were examined by searching microarray database and by RT-PCR analysis. All of eight genes tested were highly up-regulated during cold treatment and some of them were also responsive to salt, drought, and ABA treatment. Taken together, these results indicate that CBF/DREB1-mediated stress signaling pathway is also functioning in B. rapa. On the other hand, differences in genomic organization and gene number for CBF/DREB1 are thought to cause different response to stress between B. rapa and A. thaliana. In this presentation, we will introduce more detailed results for CBF/DREB1 gene family in B. rapa.

Next generation sequencing (NGS) approaches can also be useful tool for characterization of organelle genomes. We generated chloroplast (CP) genome sequences of two Korean ginseng cultivars, Chunpoong and Yunpoong, based on reference-guided assembly using whole genome NGS data. We used 0.5x of P. ginseng genome NGS reads to assemble CP genome. Of the NGS reads used, about 6% were mapped to the reference CP genome with mean coverage of 94x due to high copy number of CP genome in plant cell. CP genomes of the two cultivars were predicted to be 156,248 bp and 156,355 bp in length and showed about 0.1% differences at nucleotide level, compared to reference CP genome sequenced from P. ginseng (Acc.no. NC_006290), whereas difference between CP genomes of the two cultivars is very rare. In this study, we developed the molecular marker to perform taxon identification and also to elucidate phylogenetic relationship among Korean ginseng cultivars. Now, we are analyzing the CP genomes of other P. ginseng cultivars together with other Panax species including American ginseng and Panax related species.

Panax ginseng is a well-known herbal plant originated from North-east asia for its various tonic effects. However, production of ginseng roots takes long time in field condition, usually five through six years until harvest. Additionally, ginseng is very susceptible to many kinds of biotic and abiotic stresses, for example, Rhizoctonia solani, which causes damping-off, or high temperature. To overcome these limits, induction of adventitious roots has been studied for more than 10 years and also adventitious roots are widely used materials for genetic research of P.ginseng. In this study, we induced adventitious roots from registered Korean ginseng cultivars and cultured them in bioreactor condition. Induction rate of adventitious roots from nine Korean ginseng cultivars was evaluated and growth pattern of four cultivars in bioreactor scale was also studied. Furthermore, genes that are related to biosynthesis of saponins in ginseng, ginsenoside, were discovered in ginseng whole-genome shotgun sequences for genetic research.

Miniature inverted- repeat transposable elements are expected to play vital role in evolution of genes and genome of major eukaryotic organisms. However, there have been little reports on MITEs in B. rapa, a polyploidy model genome. We identified 13 novel MITE families in B. rapa genome by computational approach. Out of 13 MITEs families three, eight and two were classified under stowaway-like, tourist-like and hAT super families based on their unique structural characteristics. We characterized the members of 13 MITE families from the available 256 Mbp from whole genome draft sequences of B. rapa. We found ech MITE has high copiy number ranges from 14 to 977 which are distributed randomly along all the chromosomes. We also found more than 40% of the MITE members were associated with genes and gene rich regions. Furthermore, the polymorphism due to insertion and non-insertion of MITEs analysis suggest that MITEs are active in the genome. As, such the newly identified MITEs will provide a foundation for the further analysis of roles of MITEs in gene and genome evolution.

Genetic map provides basic and important informations for breeding. Therefore, genetic map construction is a essential process in plant research. Panax ginseng is one of the most famous medical plant in the world. However, genetic informations of this medical plant for breeding are not enough. Because of little informations, genetic map construction of panax ginseng provides very useful information for breeding. Using Solexa next generation sequencing (NGS) technology, we have been produced a lot of expressed sequence tags (ESTs) and whole genome sequences from Chunpoong (368 Gb) and Yunpoong (6 Gb) cultivar. To develop large amount of DNA markers and thus construct high resolution genetic map, we inspect large scale of SSR motif and putative SNP sites which can be used as dCAPs markers using produced ginseng’s sequence data. As a result, we can find a number of DNA markers that have polymorphism between Yunpoong and Chunpoong cultivar. These developed DNA markers were analyzed for F2 population of Yunpoong x Chunpoong to find markers showing Mendelian segregation ratio 1:2:1.