Cold stress, which includes chilling (<20℃) and/or freezing (<0℃) temperatures, adversely affects the growth and development of plants and significantly constraints the spatial distribution of plants and agricultural productivity. Cold signal in plants is transmitted to activate C-repeat/drought-responsive elements-binding factor (CBF)-dependent and independent transcriptional pathway. In Arabidopsis, cold-regulated genes have been estimated to constitute ~4% to 20% of the genome. Chinese cabbage (Brassica rapa ssp. pekinensis), like Arabidopsis, is a member of the Cruciferae family. With expectation that Chinese cabbage has similar cold-responding process as Arabidopsis, transcriptome profiles were examined in two comparable DH lines, Chiifu and Kenshin, using 24K microarray and expression of some of genes was analyzed by RT-PCR. Hundreds genes showed over two fold change upon freezing treatment, but only four genes specific for each line. Most of examined CBF-dependent and -independent pathway related genes have a similar expression patterns between Chiifu and Kenshin, except BrICE1 (inducer of CBF expression 1), MYB15, BrRAP2.1 and BrRAP2.6 (ethylene-responsive transcription factor 2.6). The BrICEL (inducer of CBF expression 1 like) showed specifically expression in Kenshin. The expression levels of vernalization related genes (BrRTV1, BrVRN5, BrVIN3L, VIP3 and VIP5) showed no difference between Chiifu and Kenshin and did not response to freezing treatment in our experiment. We will discuss more detailed expression data on poster.

Most indica rice varieties show a low efficiency of transformation because of difficulties in callus formation and low-regeneration frequencies in conventional culture such as MS16 or N6 medium. Recently, some improved methods were reported for Agrobacterium-mediated transformation using mature elite indica seeds however, these procedures take a long time (5–7 months) to obtain transgenic plants and still with significantly low efficiency. In this study, we provide additional improvements in the indica rice transformation protocol introducing new selection method by tosoflavin/tflA which was based on bacterial photosensitizer and its degrading enzyme pair. We introduced direct in planta transformation using early stage of germinating rice seeds instead of usual embryogenic callus. Methods that use embryos as a starting material for inoculation with Agrobacterium are also used for comparison with the new protocol using rice seeds. Transformed cells proliferated from rice seeds obtain toxoflavin resistance, and transgenic plants are eventually regenerated from those proliferated tissues. However, we found out that tissue proliferation from indica seeds and shooting and rooting are very sensitive to minor salt nutrients in the media and need to pay attention to use indica rice specific nutrient media. The use of naturally occurring photosensitizers such as toxoflavin as selection agents appears to give rapid and unambiguous selection results owing to their unique phytotoxic mode of action. In particular, the toxoflavin/tflA selection system might be useful for generating transgenic indica rice cultivar where high false-positive backgrounds with current selection marker systems are problematic.

Brown leaf spot, caused by necrotrophic Cochliobolus miyabeanus (imperfect; Bipolaris oryzae), is one of the devastating disease in rice (Oryza sativa). Especially, recommended agricultural system such as diminishing fertilizer and environmental alteration like temperature increment result in the favorable conditions for the outbreak of this disease. Lack of water supply also requires drought-tolerant rice cultivar. We hypothesized that regulation of programmed cell death (PCD) should be a common solution conferring resistance and tolerance against above biotic and abiotic stresses at the same time. Among 17 CYCLIC NUCLEOTIDE-GATED ION CHANNEL in rice (OsCNGCs), over expression of a CNGC resulted in lesion mimic phenotypes in Dongjin background. Further, knock out of a CNGC resulted in enhanced resistance against rice brown spot in the field. These results indicate that selected OsCNGC should be involved in the PCD regulation and fungal infection-specific regulation of OsCNGC expression might induce resistance against rice brown spot because of pathogen’s necrotrophic nature. Vitamin E, tocopherol, is involved in the accumulation inhibition of reactive oxygen species involving superoxide and hydrogen peroxide. Tocopherol cyclase in Nicotiana benthamiana (NtTC), which is included in tocopherol systhesis, conferred tolerance against drought stress to rice. We already have settled down the recombination system effectively removing selection markers in vector. Based on these systems, we will define fungal secretome and genome, confirmation of PAMPs/effectors, identification of rice pattern recognition receptor, and functional characterization of these rice genes in the respect of PCD and disease resistance. We will also develop marker-free transgenic rice tolerant to drought and/or salinity stresses. These works should give us a bundle of rice genes conferring resistance and tolerance against biotic and abiotic stresses and amount of information useful for the analyses of common cross talk points between disease resistance and stress-tolerance.

벼 재배에 있어 강피(Echinochloa oryzicola)와 물피(E. crus-galli)는 가장 경합력인 큰 문제 잡초로 알려졌는데 최근 제초제 저항성 피의 증가로 피의 효과적인 방제가 새로운 화두가 되고 있다. 저항성 피의 효과적인 방제를 위해서는 제초제 저항성 여부의 조기진단을 통한 대체 제초제를 이용한 방제가 필요하다. 따라서 본 연구는 제초제 저항성 피의 효과적인 진단을 위해 피노믹스 기반의 비파괴 조기진단법을 개발하고자 수행되었다. Acetolactate synthase (ALS) 저해제, acetyl CoA carboxylase (ACCase) 저해제, photosystem II (PSII) 저해제에 저항성 및 감수성인 각각의 강피, 물피, 열대피(E. colona)를 3-4엽기까지 온실조건에서 생육한 후 해당 제초제를 다양한 약량으로 경엽살포한 후 일정한 시간간격으로 잎의 형광반응을 조사하였다. 그 결과 PSII 저해제 저항성 피는 제초제 경엽처리후 3-4일 이내 저항성 피의 진단이 가능하였으며, ALS 저해제 및 ACCase 저해제 피의 경우는 7-8일 이내 저항성 피의 진단이 가능하였다. 따라서 가장 보편적으로 저항성 잡초의 진단에 사용하는 whole plant assay의 소요시간 20-30일에 비해 잎의 형광반응 측정을 통해 저항성 잡초의 진단 소요시간을 혁신적으로 단축할 수 있었다.

PCR-based assays with co-dominant or dominant allele-specific STS (sequence tagged sites) markers and sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) have been used in this study to discriminate Glu-1 and Glu-3 alleles in Korean wheat cultivars. Three alleles were identified at each high molecular weight glutenin (Glu-1) homoeologous locus and 10 alleles were identified at low molecular weight glutenin (Glu-3) homoeologous locus in Korean wheat cultivars. At Glu-1 loci, wheat cultivars contained Glu-A1a, b and c alleles amplified a 1093, 1063 and 920bp fragments, respectively, Glu-B1b, c and f alleles discriminated with 707(Glu-B1b and f) and 662bp(Glu-B1c) fragments and Glu-D1a, d and f alleles amplified a 576(Glu-D1d) and 612bp(Glu-D1a and f) fragments, respectively. At Glu-3 loci, wheat cultivars contained Glu-A3c, d and e alleles amplified a 573, 967 and 158bp fragments, respectively. Glu-B3d, g, h and i alleles gave a 662, 853, 1022 and 621bp, respectively and Glu-D3a, b and c alleles amplified a 884(Glu-D3a and b) and 338bp(Glu-D3c) fragments. A multiplex-PCR assay was also established which permitted the discrimination of the major Glu-1 and Glu-3 to determine glutenin compositions in a single PCR reaction and agarose gel assay because these systems could be useful to select DNA-based identification of wheat lines carrying good breadmaking performance in Korean wheat breeding programs. Three multiplex-PCR for Glu-1 and Glu-3, Glu-A1c + Glu-B1acf + Glu-D1d, Glu-A3ac + Glu-A3d + Glu-A3e and Glu-B3d + Glu-B3fg + Glu-B3h were established in this study.

Chilling stress affects growth and yield of warm-climate crops such as soybean (Glycine max L.) that is susceptible to low temperature (10-18℃). A comparative proteomic approach was employed to explore the mechanisms underlying soybean response to chilling stress. Soybean seedlings were germinated for 3-4 days and exposed to low temperature (10℃) for 3 days, and the proteins were extracted from seedling leaves. Protein separation by SDS-PAGE followed by liquid chromatography electro-spray ionization tandem mass spectrometry (LC-ESI MS/MS) was effective approach to identify proteins, based on the number of peptides reliably identified. A total of 77 proteins out of 704 proteins were identified in the presence of chilling stress. Most proteins identified had functions related to cell signaling, metabolism, energy and transport, protein biosynthesis and degradation, cytoskeleton, and were involved in regulating reactions and defending against stress. It is therefore likely that the response of soybean plant’s proteome to chilling stress is complex, and that the identification proteins may play an important role in regulating adaptation activities following challenge to chilling stress to facilitate cellular homeostasis. Furthermore, our result suggest that new ways of engineering stress-tolerant plants responding climate change by providing outline for agriculturally important chilling stress.

In order to cope with the recent global warming and climate change that is projected to have a grave impact on agriculture worldwide, we will direct our focus on developing crops tolerant to multiple abiotic stresses including drought, cold and heat, with the following research activities conducted by three different research groups including an international research team at IRRI. 1) Development of heat/cold tolerant rice variety : Major genes conferring heat tolerance and cold resistance in rice will be identified by comparative transcriptome analyses and new molecular markers will be developed based upon these data. EMS mutagenesis and proteomics analyses will accompany this approach to supplement this gene identification and marker development efforts. Once reliable markers are obtained in this way, new varieties of heat/cold tolerant japonica rice will be bred through introgression of these genes. 2) Development of drought/heat tolerant rice variety : Through QTL mapping conducted on RILs between a drought resistant line and an elite line, genes conferring drought/heat tolerance will be identified and molecular markers will be developed using SNP/GBS genotyping methods. Using these markers, new rice varietis with drought/heat tolerance will be bred by employing marker assisted selection (MAS) as well as marker assisted backcross (MABC). 3) Identification of genes involved in multiple stress responses in ginseng and brassica : Molecular breeding of stress tolerance traits in ginseng and brassica is not well established to date. Taking advantage of the ginseng whole genome sequence data information and other comparative genomics approaches, members of the stress-response transcription factor family CBF/DREB will be identified and their functional analyses will be performed in ginseng and brassica using transcriptome profiling of both wild type and transgenic plants including the adventitious root-derived transgenic ginseng. Major Publications: - Lee, J., W. Jiang, et al. (2011). “Shotgun proteomic analysis for detecting differentially expressed proteins in the reduced culm number rice.” Proteomics 11(3): 455-468. - Ji, H., S. R. Kim, et al. (2010). “Inactivation of the CTD phosphatase-like gene OsCPL1 enhances the development of the abscission layer and seed shattering in rice.” The Plant journal 61(1): 96-106. - Chin JH, Gamuyao R, Dalid C, Bustamam M, Prasetiyono J, Moeljopawiro S, Wissuwa M, Heuer S (2011) Developing rice with high yield under P-deficiency: Pup1 sequence and application. Plant Physiology 156: 1-15. - Hong-Il Choi, Nam Hoon Kim et al. (2011) Development of Reproducible EST-derived SSR Markers and their application for genomics and breeding of Panax ginseng Journal of Ginseng Research 35(4): 399-412.

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.

Whole-genome expression profile data are useful to approach genome system biology with quantification of expressed RNAs from a whole-genome transcriptome, depending on the tissue samples, such as control and exposed tissue. The hybrid mRNA sequences from Rohce/454 and Illumina/Solexa are more powerful to find novel genes through de novo assembly in any whole-genome sequenced species. The 20x and 50x coverage of the estimated transcriptome sequences using Roche/454 and Illumina/Solexa, respectively, is effective to create novel expressed reference sequences. However, only an average 30x coverage of a transcriptome with short read sequences of Illumina/Solexa is enough to check expression quantification, compared to the reference EST sequence. In any NGS application, the transcriptome expression information would be more useful than complete genome information research with the lowest sequencing budget for biologists to better understand gene regulation of related genetic phenotypes with the in silico method. The study of target genes related to specific traits including disease resists is available pathway analysis through comparing RNA sequencing among the genotypes.

Soybean is one of the most important crop plants used for seed protein and oil content that has undergone substantial phenotypic and physiological changes during domestication. Thanks to the advent of the next-generation sequencing platforms, genome sequences of many major crop plants including soybean and maize have been unraveled. We have resequenced the genomes of 10 cultivated soybean and 6 accessions of their wild progenitors (Glycine soja) selected from the Korean soybean germplasm to >15 × raw data coverage. We have investigated genome-wide variation patterns in soybean and obtained millions of high-quality single nucleotide polymorphisms (SNPs). Further analyses of the extracted SNPs including population structure analysis, introgressions, linkage disequilibrium, and reduction of diversity are ongoing in order to provide an unprecedented opportunity to finely resolve the domestication history of cultivated soybean. At the same time, we have conducted a comparison study between the Williams 82 soybean reference genome sequence and a genetic map. Here, I will present our current analysis status of the soybean genome resequencing data. Then, I present our recent progresses in the understanding of dynamic genetic features of soybean chromosome revealed by comparison of genetic and sequence-based physical maps in which we have used a portion of our resequencing data to substantiate putative introgression region detected during the construction of a genomewide soybean genetic map.

Progress in next-generation sequencing technologies have enabled discovery of massive amount of genome-wide DNA polymorphisms, single nucleotide polymorphisms (SNPs) and insertion-deletion (InDels), which are an invaluable resource to analyze genetic diversity in a population. We performed whole-genome resequencing of ten Korean rice accessions including six cultivars and four mutant lines. A total of 2,448 million raw reads was generated with 58-fold coverage and uniquely mapped to 87.5% of the Nipponbare as a reference genome. We identified 3,240,025 DNA polymorphisms including 2,867,878 SNPs, 151,845 insertions and 220,302 deletions between the Korean rice accessions and Nipponbare. We observed that in ten Korean rice accessions, the frequency of potential SNPs was estimated to be one per 2.1kb on Nipponbare (382Mb). According to annotation of DNA polymorphisms, 634,617 SNPs were found in gene region, and only 169,738 SNPs were occurred in coding region. Altogether, 86,251 non-synonymous SNPs were located on 76,891 genes. We also examined the cultivar-specific SNPs to select candidate SNPs which would have possibility of being associated with unique phenotype or agronomical trait of each cultivar. It was estimated that the portion of cultivar specific SNPs is 1~12% of the total SNPs. These DNA polymorphisms obtained from our result will provide an invaluable resource to identify molecular markers and genes associated with diverse traits of agronomical importance.

Rice is one of the most important crop in the world and the genome sequences of a rice cultivar, Nipponbare has been used not only for rice research, but also as the model reference genome sequences in monocotyledon species among the crops. With the development of the next generation sequencing(NGS) techniques producing high order of coverage and with the need for epigenomic analysis, the need for resequencing of the domestic rice cultivars with the reference “Nipponbare” genome sequence at the de novo level. According the simulation of Nipponbare reference genomes with Eulerian methods, the target size of maximal contig and N50 of ones of the domestic cutivar rice were estimated to be 10M and 1M, respectively. To achieve the target size, various mate-paired libraries were constructed and sequenced. With the high order of coverage obtained from domestic rice cultivar, Ilmi, with NGS technology, the effect of trimming and error correction on reads quality profiles and size distribution of contigs were analyzed. Also the computational parameters for validation of assembled contigs were analyzed. For the analysis of epigenomic methylation modification of the genome sequences, the methyl binding microarray technology was developed. The various methyl-CG binding proteins were characterized. The binding and scanning of methyl-CG domain to promoter binding microarray and their downstream genes were analyzed. Also rice mutants related to methylation were selected to understand the effect of methylation on gene expresson and its effect on phenotypes.