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.

The biological wastewater treatment plant, which uses microbial community to remove organic matter and nutrients in wastewater, is known as its nonlinear behavior and uncertainty to operate. Therefore, operation of the biological wastewater treatment process much depends on observation and knowledge of operators. The manual inspection of human operators is essential to manage the process properly, however, it is impossible to detect a fault promptly so that the process can be exposed to improper condition not securing safe effluent quality. Among various process faults, equipment malfunction is critical to maintain normal operational state. To detect equipment faults automatically, the dynamic time warping was tested using on-line oxidation-reduction potential (ORP) and dissolved oxygen (DO) profiles in a sequencing batch reactor (SBR), which is a type of wastewater treatment process. After one cycle profiles of ORP and DO were measured and stored, they were warped to the template profiles which were prepared already and the distance result, accumulated distance (D) values were calculated. If the D values were increased significantly, some kinds of faults could be detected and an alarm could be sent to the operator. By this way, it seems to be possible to make an early detecting of process faults.

Blueberry (Vaccinium spp.) is a member of the Ericaceae and eleven varieties have been registered at the Korea Seed & Variety Service for Plant Variety Protection (PVP). This study was to develop simple sequence repeat (SSR) markers next generation sequencing (NGS) analysis and to analysis genetic relationship of blueberry 31 varieties. Highbush blueberry ‘Camellia’ and rabbiteye blueberry ‘Alapaha’ varieties were used as sequencing materials. Out of total 987 SSR primers detected between ‘Camellia’ and ‘Alapaha’, 148 SSR primers were initially applied to select SSR markers for identification of blueberry varieties. Fourteen SSR markers showed polymorphism between 8 varieties. Seven SSR markers showed reproducibility and clear peak among 14 SSR markers. Genetic relationships of 31 blueberry varieties were analyzed and identified using 7 SSR markers. A total of 30 polymorphic SSR alleles were obtained and two to seven alleles were detected for each locus with an average of 4.3 alleles per locus. Average polymorphism information content was 0.556, ranging from 0.374 to 0.714. Genetic distance of clusters ranged from 0.38 to 0.93 by unweighted pair-group method with arithmetical average based on Jaccard’s distance coefficients. These newly developed SSR markers indicate usefulness for variety identification related to seed dispute and distinctness, uniformity and stability (DUS) test for blueberry.

Pepper (Capsicum spp.) germplasm shows diverse phenotypic variations including fruit size, color, pungency, and many other horticultural traits. Traditional markers including SSR, AFLP, and RFLP have been used to construct genetic maps using biparental populations. However to assess the genetic diversity of large number of germplasm, a robust and rapid marker development and genotyping approach is needed. We used six pepper accessions including C. annuum, C. chinense, C. baccatum and C. frutescens and performed genotyping-by-sequencing (GBS). To select the most appropriate condition, eight different 2 bp selective nucleotides were used to make GBS libraries. Selective nucleotide ‘OO’ showed the largest number of reads in all samples, and 11,026 to 47,957 high-quality SNPs were called in six accessions. When C. annuum ‘CM334’ genome sequence was used as a reference, C. annuum showed the smallest number of SNPs, while C. baccatum which was known to be a different Capsicum clade showed the largest number of SNPs. Pepper core collection chosen to represent the genetic diversity of whole germplasm will be genotyped by high-density SNPs developed from GBS. We will perform genome-wide association study (GWAS) using genetic and phenotypic variation to identify the functional genetic loci controlling horticultural traits.

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.

Although the overall structure of the chloroplast genome is generally conserved, a number of sequence variations have been identified, which are valuable for plant population and evolutionary studies. Here, we constructed a chloroplast variation map of 30 landrace rice strains of Korean origin, using the Oryza rufipogon chloroplast genome (Genbank: NC_017835) as a reference. Differential distribution of single nucleotide polymorphisms (SNPs) and indels across the rice chloroplast genome is suggestive of a region-specific variation. Population structure clustering revealed the existence of two clear subgroups (indica and japonica) and an admixture group (aus). Phylogenetic analysis of the 30 landrace rice strains and six rice chloroplast references suggested and supported independent evolution of O. sativa indica and japonica. Interestingly, two “aus” type accessions, which were thought to be indica type, shared a closer relationship with the japonica type. One hypothesis is that “Korean aus” was intentionally introduced and may have obtained japonica chloroplasts during cultivation. We also calculated the nucleotide diversity of 30 accessions and compared to six rice chloroplast references, which shown a higher diversity in the indica and aus groups than in the japonica group in lower level substitution diversity.

Chloroplast (cp) genome sequences provide a valuable source for DNA barcoding. Molecular phylogenetic studies have concentrated on DNA sequencing of conserved gene loci. However, this approach is time consuming and more difficult to implement when gene organization differs among species. Here we report the complete re-sequencing of the cp genome of Capsicum pepper (Capsicum annuum var. glabriusculum) using the Illumina platform. The total length of the cp genome is 156,817 bp with a 37.7% overall GC content. A pair of inverted repeats (IRs) of 50,284 bp were separated by a small single copy (SSC; 18,948 bp) and a large single copy (LSC; 87,446 bp). The number of cp genes in C. annuum var. glabriusculum is the same as that in other Capsicum species. Variations in the lengths of LSC, SSC and IR regions were the main contributors to the size variation in the cp genome of this species. A total of 125 simple sequence repeat (SSR) and 48 insertions or deletions variants were found by sequence alignment of Capsicum cp genome. These findings provide a foundation for further investigation of cp genome evolution in Capsicum and other higher plants.

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.

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.

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.

GM벼 OsCK는 벼 유래의 OsCK1 유전자를 벼에 형질전환 하여 벼흰잎마름병 및 벼도열병에 대한 저항성을 높게 한벼로 농촌진흥청에서 개발하였다. 형질전환 벡터의 구성은 양쪽 border (LB, RB) 상간에 2개의 MAR 염기서열이 서로 마주보는 형태로 위치하고 있으며, 제초제 저항성 유전자 PAT는 CaMV 35S promoter에 의하여 발현이 유도되고, 목표 유전자인 choline kinase (OsCK)는 actin promoter에 의하여 발현이 조절되며 left border 기준으로 역방향으로 배치되었다. 도입유전자 확인을 위하여 adaptor ligation PCR을 수행하였는데, MAR 영역에 위치하는 제한효소로 GM벼 genomic DNA를 절단한 후 adaptor를 붙였다. 염기서열 분석을 위하여 T-DNA의 양 말단에서 primer를 제작한 후 sequence 분석을 하였다. 분석한 결과, T-DNA의 right border 인근의 MAR sequence가 벼 genome의 10번 염색체 129971번 염기와 연결되어 있음을 확인하였다. Left 영역의 삽입위치는 이후 실시한 Illumina NGS 시스템을 이용하여 확인할 수 있었으며, GM 벼에는 2개의 T-DNA가 도입되었음을 알 수 있고, 첫 번째 T-DNA는 벼 10번 염색체 BAC클론 OSJNBa0014J14의 128947번째 염기와 129970째 염기에 위치하고 벼 genome 염기 1024 bp가 결실됨을 확인하였다. 이 과정에서 첫 번째 T-DNA left border와 첫 번째 MAR sequence 일부(370 bp)가 결실되었고 right border와 두 번째 MAR 영역 199 bp가 결실되었음도 확인하였다. 두 번째 T-DNA는 right border가 결실된 형태로 첫번째 T-DNA의 35S promoter 중간에 삽입되었음을 확인하였다.

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.

such as small plant size, small genome, short life cycle, self-fertility, and etc. Moreover, Brachypodium standard line Bd21 had already been sequenced and the data of which now have been available to the public. The development of next-generation sequencing technologies has allowed the discovery of large numbers of genome-wide DNA polymorphisms. Brachypodium standard line Bd21 was exposed to chronic gamma radiation. M2 1376-1 line was less stained with phloroglucinol compared to wild-type, which indicated reduced lingin content. Also, it’s filial generations showed dwarf phenotype. Genomic DNA was extracted from the M3 plant and was used to construct a whole-genome re-sequencing library for using Illumina HiSeq2500. The trimmed reads were aligned to the Brachypodium reference genome sequence (http:// www.phytozome.net). SICKLE, BWA, and Picard were used for accurate variant detection. More than 110 M reads were generated and 96.53% of them were mapped. This represents ~35 fold coverage. As a result, mutant specific SNVs, Insert/Deletions, and non-synonymous mutations were obtained. Moreover, non-synonymous mutations were identified from 5 lignin biosynthesis genes (Bradi1g31320, Bradi3g52350, Bradi5g21550, Bradi3g22980, Bradi5g14720). The obtained results will be incorporated in development of biofuel crops.

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.