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.

Molecular characterization of crops improved through biotechnology has traditionally been conducted using Southern blot analysis which has been used to determine T-DNA copy number, the presence or absence of backbone (sequence outside of the T-DNA) and to demonstrate generational stability of the T-DNA insert. The advancement of high-throughput DNA sequencing (HTS) technology allows efficient characterization of the transgene incorportated into the genome of the plant by rapidly sequencing the entire plant genome. By combining NGS (Next Generation Sequencing) technologies with bioinformatic methods that identify the T-DNA insert derived from the plasmid vector and genome-T-DNA junction sequences, it has been shown that conclusions equivalent to those of a Southern blot are readily obtained. NGS is done at sufficient coverage depth (>75x) across the entire genome. By mapping the sequence reads to the plasmid vector, and identifying the number of unique junctions, we can confirm insert number, copy number, absence of backbone, across multiple generations. With the widespread availability of NGS and steadily decreasing costs it is likely that academia and industry will fully transition to NGS-based molecular characterizations in the near future.

Anthocyanin is known for positive health beneficial effects that including reduces age related oxidative stress and inflammatory responses. It was produced by vegetable crops and a lettuce is one of the crops. The general pathway of anthocyanin expression is well defined but it is not clear how environments effects on anthocyanin accumulation in a lettuce. Therefore we initiated to study interaction between anthocyanin expression and environment factors. Frist, we applied RGB leaf images in a lettuce to calculate anthocyanin areas in a leaflet with two different cultivars, different developmental stages, and different environments. Later, we attempted to capture RNA expression level with next generation sequence (NGS) RNA sequencing method called RNA-seq. As a result, combined two technologies showed that quantitate phenotypic data help to understand the gene expression of anthocyanin in lettuce cultivars.

The transcriptomes of four ginseng accessions such as Cheonryang (Korean ginseng cultivar), Yunpoong (Korean ginseng cultivar), G03080 (breeding line of Korean ginseng), and P. quinquefolius (American ginseng) was characterized. As a result of sequencing, total lengths of the reads in each sample were 156.42 Mb (Cheonryang cultivar), 161.95 Mb (Yunpoong cultivar), 165.07 Mb (G03080 breeding line), and 166.48 Mb (P. quinquefolius). Using a BLAST search against the Phytozome databases with an arbitrary expectation value of 1E-10, over 20,000 unigenes were functionally annotated and classified using DAVID software, and were found in response to external stress in the G03080 breeding line, as well as in the Cheonryang cultivar, which was associated with the ion binding term. Finally, unigenes related to transmembrane transporter activity were observed in Cheonryang and P. quinquefolius, which involves controlling osmotic pressure and turgor pressure within the cell. The expression patterns were analyzed to identify dehydrin family genes that were abundantly detected in the Cheonryang cultivar and the G03080 breeding line. In addition, the Yunpoong cultivar and P. quinquefolius accession had higher expression of heat shock proteins expressed in Ricinus communis. These results will be a valuable resource for understanding the structure and function of the ginseng transcriptomes.

The advent of Next Generation Sequencing (NGS) technology has changed the research paradigm and become an essential tool for recent biological and medical study. In today’s market, there are several sequencing platforms which have specific sequencing principle, and the result of each sequencing platform has different characteristics among them. Hence, each sequencing method became more specialized for specific research purpose, and researchers who consider NGS analysis have to understand the very basic characteristics of each NGS platform. NGS is used in various studies and they are usually classified into 5 categories (Re-sequencing, RNA-seq, de novo assembly, Metagenomics and Epigenomics) of analysis. In this session, we will introduce the characteristics of sequencing platforms and examples of recent research on each of the 5 analysis categories. In addition, we will talk about the benefit of NGS study compared to the traditional study and how these NGS technologies can be applied in developmental biology research.

Common buckwheat (F. esculentum) and tartaryan buckwheat, also called as bitter buckwheat (F. tartarycum) grain and leaves (buds) are used in various dietary preparations and as leafy vegetable. The cultivated area of buckwheat is increased based on its nutritional value. Particularly bitter buckwheat is a rich source of rutin compared to common buckwheat which helps in reducing intra-vascular cholesterol, high blood pressure, diabetes and is also reported to have a crucial role in pharmaceutical research. With this functional characteristics of bitter buckwheat, the cultivation is now highly increased. But a few genetic and genomic research of tartari buckwheat are published until now. Here we described the complete full chloroplast genome sequence with NGS. Tartary buckwheat complete chloroplast genome is composed of a total sequence length of 159,272 bp which is 327 bp lesser than common buckwheat genome of 159,599 bp. Large single copy region (LSC) is comprised of 84,398 bp in tartary and 84,888 bp in common buckwheat whereas small single copy region (SSC) is 13,292 bp and 13,343 bp and the size of inverted repeat region (IR) is 61,532 bp and 61,368 bp in tartary and common buckwheat respectively. Total RNA bases were 11942 and 11950 and overall GC-content in tartary and common buckwheat is almost similar which is 37.9% and 38% with a GC skew of -0.016 and 0.02 respectively. Total repeat bases accounted for 1,056 bp and 804 bp with an average repeat length of 48 bp and 45 bp and the length of an average intergenic distance was 495 bp and 502 bp in tartary and common buckwheat respectively. F. tarataticum cp genome has a total of 104 genes including 82 protein coding genes, 29 transfer RNA genes and 4 ribosomal RNA genes. Protein coding genes include photosynthesis related genes majorly in addition to transcription and translation related genes. LSC region has 62 protein coding genes and 22 tRNA genes whereas SSC region contains 11 protein coding genes and one tRNA gene. The nucleotide and amino acid sequences of protein coding genes in LSC, SSC and inverted repeat regions in F.tartaricum and F.esculentum are highly similar with a total average identity of 98.8 and 98.3% respectively.

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.

Currently, the type of short insertions and deletions (InDels) polymorphisms are increasingly focused in genomic research. InDels have been known as a source of genetic markers that are widely spread across the genome. Genetic relationship among Korean pear cultivars compared with their parents was also identified that they are closely related P. pyrifolia, P. ussuriensis and/or hybrids between two species. Lack of genetic resources including molecular markers has made it difficult to study pears severely. Recently developed next generation sequencing (NGS) platforms offer opportunities for high-throughput and inexpensive genome sequencing and rapid marker development. The main goal of this study was to develop polymorphic InDel markers in ‘Whangkeumbae’ and ‘Minibae’, which were chosen as the representative cultivars of P. pyrifolia and P. ussuriensis × pyrifolia in each among Korean pears using genomic sequences generated by NGS technology. In this study, more than 18.6 Gbp and 15.8 Gbp sequences were obtained from NGS of ‘Whangkeumbae’ and ‘Minibae’, respectively. ‘Whangkeumbae’ contained 197,210 InDels and 197,272 InDels in ‘Minibae’. In InDels validations between ‘Whangkeumbae’ and ‘Minibae’, the number of polymorphic InDels were 149,338 and non-polymorphic InDels were 122,572. For InDel primer set designing, 11,308 of primers were designed from polymorphic InDels and 10,919 of InDel primers were recommended. The study shows that the utility of NGS technology to design amount of efficient InDels and the developed InDel primers will be used for genetic mapping, breeding by marker assisted selection (MAS) and QTL mapping of Korea native pear as well as further genetic studies.

Single nucleotide polymorphisms (SNPs) are the most frequent type among variations found in genomic regions and are valuable markers for genetic mapping, genetic diversity studies and association mapping in plants. There are three basic species known as Korean native which are Pyrus ussuriensis, P. pyrifolia, and P. fauriei. Genetic relationship among Korean pear cultivars compared with their parents was identified that they are closely related P. pyrifolia, P. ussuriensis and/or hybrids between two species. Lack of genetic resources, including molecular markers to study pears are very severe. Recently developed next generation sequencing (NGS) platforms offer opportunities for high-throughput and inexpensive genome sequencing and rapid marker development. The objective of this study was to develop polymorphic SNP markers in ‘Whangkeumbae’ and ‘Minibae’, which were chosen as the representative cultivars of P. pyrifolia and P. ussuriensis × pyrifolia in each among Korean pears, using genomic sequences generated by NGS technology. In this study, more than 18.6 Gbp and 15.8 Gbp sequences were obtained from NGS of ‘Whangkeumbae’ and ‘Minibae’, respectively. ‘Whangkeumbae’ and ‘Minibae’ contained 2,712,288 and 2,747,224 SNPs, respectively. In SNPs validations between ‘Whangkeumbae’ and ‘Minibae’, the number of polymorphic SNPs were 2,516,438 and non-polymorphic SNPs were 1,179,391. For HRM primer design, 2,125,479 HRM candidate primers were obtained from polymorphic SNPs and 343,731 SNP primers were developed. This study shows that the utility of NGS technology to discover efficiently a large number of SNPs and SNP primers can provide valuable information in the genome study of Pyrus spp.

최근 급속하게 발달한 차세대 유전체분석기술을 기반으로 밀양23호와 기호벼의 유전체 서열을 분석하고, 새로운 CAPS 마커를 개발하였다. NGS를 통해 Nipponbare 유전체 길이의 60 배수만큼 염기서열을 결정하였고, CDS 안에서 두 품종간 특이적으로 나타나는 SNP를 CAPS 마커로 이용하였다. 새롭게 개발된 146개 CAPS 마커와 기존의 보고된 219개 마커를 통합하여 총 365개의 마커로 밀양23호/기호벼의 재조합자식 유전집단에 대해 분자 유전지도를 작성하였다. 벼의 줄기굵기와 간장 그리고 수장에 관한 QTL을 탐색한 결과, 총 19개의 유의성이 있는 QTL을 찾을 수 있었다. 이 중에 4개 줄기굵기 형질 관련 QTL과 2개 간장 형질 관련 QTL이 기존에 보고되지 않은 새로운 QTL이었다. 그 줄기굵기 QTL 중 가장 큰 LOD값을 갖는 qI1D5는 5번 염색체에서 탐색되었으며, 1절굵기 표현형 변이는 8.99%였다. 또한, 간장관련 QTL 중 가장 큰 LOD 값을 갖는 qCL5은 5번 염색체에서 탐색되었고, 이 QTL의 간장 표현형 변이는 4.24%였다. 재염기서열을 통해 밝혀진 SNP 중 소수만이 본 연구에 사용되었다. 향후 본 연구에서 밝혀진 SNP 정보를 이용한다면 더 많은 마커를 개발하여, 고밀도 유전지도 작성이 가능할 것이다. 더 나아가 MGRIL을 이용하여 농업적으로 유용한 형질에 대해 더 정확한 QTL 분석과 유용유전자의 개발이 가능하게 될 것이다.

The genome sequencing of plants is of critical importance in the race to improve plant breeding. Next generation sequencing technologies have brought about great improvements in sequencing throughput and cost, and it has revolutionized plant research, allowing to decode the whole genomes of many species. Next generation sequencing also enables to detect functional genes and markers of important traits to facilitate molecular breeding and improve agriculture production and conservation.

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.

Mutagenesis approach in combination with whole genome sequencing has become an import role in genetic and molecular biological study and breeding of crop plants. In this study, we screened the fast neutron M4 10,000 soybean mutant plants based on morphological phenotypes of agronomically important traits and characterized the mutant of interest using resequencing. Fast neutron radiation has been known to be a very effective mutagen to cause large deletion in genome. The screened mutant showed abnormal phenotypes in plant heights, seed sizes, color of leaves, number of leaves, maturity and number of branches etc. Among them, the mutant displaying short plant height and bush type of growth habit was selected for identification of the altered genomic regions. Analysis of deletion sites of genome in interesting soybean mutant was performed using next generation sequencer Illumina Hi-seq. Mutant sequence reads generated by paired-end shotgun library were mapped on a draft soybean reference soybean (G. max cv. Williams 82). The paired-end DNA sequences of 21.6 Gb produced by Illumina Hi-seq produced 21 fold sequence depth. Among the predicted deletion sites, total 3 deletion regions confirmed by PCR. Glyma03g02390 gene and Glyma03g03560 gene were involved in the deletion regions. Glyma03g02390 gene was related to AMP binding, catalytic activity, cofactor binding and metabolic process of cell growth and Glyma03g03560 gene was concerned to oxygen binding, defense response to bacterium, and especially process of indole acetic acid (IAA) biosynthesis. These genes detected in this mutant will be studied about their molecular function in stunted phenotype.