MicroRNAs (miRNAs) are a class of non-coding RNAs approximately 21-nt in length which play important roles in regulating gene expression in plants. Although many miRNA studies have focused on a few model plants, miRNAs and their target genes remain largely unknown in hot pepper (Capsicum annuum), one of the most important crops cultivated worldwide. We here employed high-throughput small-RNA and degradome sequencing to comprehensively identify small-RNAs and their targets in pepper. From these, we identified several novel targets of miRNAs, including the major de novo methylation enzyme involved in RNA-directed DNA methylation in plants. Furthermore, we identified several highly abundant 22-nt miRNA families that target conserved domains in NB-LRRs and trigger the production of phased secondary siRNAs. We showed that transient co-expression of can-miR482 with Rpi-blb1, one of the potato NB-LRRs, resulted in the attenuation of the hypersenstive responses in Nicotiana benthamiana, suggesting that interaction between miR-482 family and disease resistance proteins is likely to serve as a conserved trigger for defense mechanism in Solanaceae. This work provides the first reliable draft of the pepper small RNA transcriptome that offers an expanded picture of miRNAs in relation to NB-LRR regulation, providing a basis for understanding the functional roles of miRNAs in disease resistance pepper.

Platycodon grandiflorum A. is a perennial plant belongs to Campanulaceae family. This plant has been used herbal medicine ingredient in East Asia. Because of the high saponin content, it is an economically important medicinal plant in Korea. It has been reported that saponins of P. grandiflorum were mainly synthesized in root tissues. The studies about root growth of the plant were few. Expansin is an important protein playing a role in root growth of plants, and is known as a nonenzymatic protein. Expansins are novel plant cell wall loosening proteins leading to turgor-driven cell extension. Expansin encoding genes exist in multigene family, and there are more than 30 genes in Arabidopsis thaliana. and more than 50 genes in Oryaza sativa. Therefore, identification of the genes was difficult in P. grandiflorum because of the lack of genome sequence. Recently, the development of next generation sequencing (NGS) technologies make it possible to obtain the target genes sequences rapidly and precisely. In this study, to identify the expansin encoding genes in P. grandiflorum, we used RNA-seq analysis with Illumina HiSeq platform. We analyzed whole transcriptome of P. grandiflorum through the RNA-seq analysis based on next generation seuqencing. CLC Genomics Workbench software (Clc Bio inc.) was used for assembly. We assembled 122,663 contigs and search 123 contigs were identified from the search using 61 expansin gene

Pre-harvest sprouting (PHS) is the precocious germination condition of grains while the spike is still in the mother plant. Because PHS in wheat drastically reduced the quality and economic value of wheat grain, the improving PHS wheat is one of the most important breeding goal in Korean wheat breeding program In this study, we evaluated PHS and germination index (GI) in 33 Korean wheat cultivars, and performed transcriptome analysis between Keumkang (susceptible) and Woori (tolerance). A total of 33 Korean wheat cultivars were used for PHS (28 cultivars) and GI assessment in greenhouse. The DAF (Day After Fertilization) 35 of keumkang and Woori spikes were harvested to perform transcriptome analysis using RNA-sequencing. Each transcriptome was compared with PHS or ABA treated DAF 35 Keumkang and Woori spikes. The PHS in 28 Korean cultivars and GI in 33 cultivars were ranged from 1.33% to 87.44% and from 0.01% to 2.41%, respectively. Woori was demonstrated the second lowest PHS and the lowest GI, however, Keumkang was 23th of 28 cultivars in PHS and 13th of 33 cultivars in GI analysis. Six cDNA library from the DAF 35 of Keumkang and Woori wheat grain, PHS treated DAF 35 of Keumkang and Woori, and ABA treated DAF 35 of Keumkang and Woori were constructed and sequenced. A total of 53.37 Gb of high-quality reads were obtained using HiSeq 2500. The average mapping rate of assembled transcripts were 88.98%. The differentially expressed genes (DEG) revealed total 332 DEG (105 annotated) were upregulated in DAF 35 Woori library, total 5694 DEG (4623 annotated) were upregulated in PHS treated DAF 35 Keumkang library in comparison with DAF 35 Keumkang library. A total of 86 DEG (51 annotated) were upregulated in PHS treated DAF 35 Woori library in comparison with PHS treated DAF 35 Keumkang library. The Gene ontology and further analysis will be discussed.

Flowering time is a important agronomic trait for grain production in rice. So the control of flowering time is a critical step. In Arabidopsis, expression of certain key flowering gene such as FLOWERING LOCUS C (FLC) is known to be epigenetically regulated by chromatin modification through Enhancer of Zeste[E(z)], a histone methyltransferase, that core component of repressive complex, polycomb repressive complex2(PRC2). However, the chromatin mechanism involved in the regulation of rice flowering genes is presently not well known. Here we show that predict coding region of a intronic LncRNA[termed rice COLDAIR(OsCOLDAIR)], which is expected to associate with a component of PRC2, is predicted at rice FLC gene. And additionally we suggest interaction of histone methyltransferase and E3 SUMO ligase that indicate possibility of interaction with rice E(z) gene and rice E3 SUMO ligase. Our study contribute to control of rice flowering time by observing two factor that can regulate expression of related of rice FLC gene.

FLOWERING TIME CONTROL PROTEIN, FPA gene encode RNA Recognition Motif (RRM) domain protein and plays important roles in flowering time control in Arabidopsis. Floral transition is significant for reproductive products in all flowering plants. However, little is known about the functions of Medicago autonomous pathway gene. We had cloned the FPA gene on Medicago based on the sequence similarity of Arabidopsis FPA sequence. The RT-qPCR analysis of MtFPA expression patterns showed that the MtFPA transcripts accumulated ubiquitously in roots, leaves, stems, flowers, and pods. When fused to the green fluorescence protein, MtPFA-GFP was localized in the nucleus as speckle pattern of protoplast from Arabidopsis. To examine the function of MtFPA, 35S::MtFPA transgenic plants were generated in Arabidopsis late flowering mutant background, fpa-2. Overexpression of MtFPA specifically caused early flowering under long day conditions compared with non-transgenic plants. In MtFPA transgenic lines, AtFLC expression were down-regulated whereas the floral integrators, AtFT and AtSOC1 were up-regulated as compare with control plant. As these results, MtFPA suggest that is a functional ortholog of the Arabidopsis and may play an important role in the regulation of flowering transition in Medicago.

Small RNAs, such as microRNAs (miRNAs) and small interfering RNAs (siRNAs), play crucial roles in post-transcriptional gene silencing (PTGS) in eukaryotes. Small RNAs function cell-autonomously as well as non-cell-autonomously. It has been well characterized that pathogenic fungi secrete some effector molecules facilitating their infection into plants. However, it is unclear whether molecules produced in plant cells are able to move into fungal cells during infection. To test if small RNAs generated from plant cells can move to fungal cells during infection, we generated transgenic Arabidopsis and rice plants expressing siRNAs targeting GFP gene generated from double-stranded RNA interference (dsRNAi) constructs for GFP gene. And then these transgenic plants were inoculated with transgenic rice blast fungus, Magnaporthe oryzae, expressing GFP transgene. Here, we showed that ectopic expression of siRNAs targeting GFP gene in transgenic plants significantly suppressed GFP expression in rice blast fungi inoculated, indicating that small RNA molecules generated in plant cells can move into infected fungal cells and efficiently degrade fungal GFP transcripts. Our results would provide a new small RNA-based strategy for the development of resistant crops against fungal pathogens.

Platycodon grandiflorum is a herbal flowering perennial plant belongs to Campanulaceae family. The saponins derived from P. grandiflorum were termed platycosides and platycodin D, which is the most abundant saponin in the plant and pharmacologically active component, was intensively studied. Platycodin D is synthesized from triterpenoids by several enzymes including cytochrome P450. Cytochrome P450 is known to exist in superfamily in plant kingdom and essential roles in saponin biosynthetic pathway by hydroxylation or oxidation of triterpene skeletons. However, the key genes of P450 involved in biosynthesis of saponin was not identified because of its low conservation rate in amino acid sequence level among plant species and gene superfamilies. Recently, next generation sequencing (NGS) technology is rapidly developed as a method to discover target genes. In this study, we tried to identify P450 genes involved in saponin biosynthetic pathway from the various tissues of P. grandiflorum using RNA-seq analysis. The sequencing was performed using Illumina Hi-Seq platform after cDNA library preparation. The produced reads were assembled using CLC Genomics Workbench software (CLC Bio, Inc.). We obteined 122,663 contigs and found out 191 putative P450 genes. The phylogenetic relationship was analyzed and putative genes related to platicoside biosysthesis were selected and cloned for further analysis.

Platycodon grandiflorum is a species of herbaceous flowering perennial plant of the family Campanulaceae. The major ingredients are platycosides, terpenoid saponins. It contains 1-4 % of the dry weight and there are about 20 types of platycosides. Among them, platycodin D have various pharmacological effects on cough and cold. Platycosides are synthesized from oleanane by mevalonic acid pathway and cytochrome P450s and UGTs are important enzymes in the saponin biosynthesis. UGT is glucose transfer enzyme and act on the final step of the secondary metabolite biosynthesis. In this study, we tried to identify UGT genes involved in saponin biosynthetic pathway from the various tissues of P. grandiflorum and non germinated seeds using RNA-seq analysis. The sequencing was performed using Illumina Hi-Seq platform after cDNA library preparation. The produced reads were assembled using CLC Genomics Workbench software (CLC Bio, Inc.). We obtained 122,663 contigs and found 137 putative UGT genes. The phylogenetic relationship was analyzed and putative genes related to platicoside biosysthesis were selected and cloned for further analysis.

In Brassica as matter of seedling manner, they have the bilocular ovary and 20~28 seeds per silique after fertilization. Rarely some of B. juncea and yellow sarson (Brassica rapa ssp, tricolaris) have multilocular ovary. In this stdudy, the LP8 (YS-033, CGN06835) is shown tetralocular ovary as well as high seed yields. As microscope study for the different size of immature bud sections and we have known the floral meristem with already four locules in immature buds less size than 1mm of LP8. To identify of determining of tetralocular ovary formation, RNA-seq was carried out on the isolated RNA from less than 1mm and from 1mm of bud size respectively. By contrast tetralocular ovay and bilocular ovary, Chiifu is used. A total of 994 differentially expressed genes(DEGs) are detected in only LP8. Among the DEGs, we identify 18 DEGs in only immature buds of less size than 1mm. The expression patterns of 18 DEGs are validated by real time quantitative PCR and these genes are cloned and the sequence analyzed. At present, 12 candidated gene are analyzed by sequencing and there are detected by large fragment insertion as well as SNPs in sequence comparison to Chiifu. We will perform the genetic transformation of these DEG genes in Arabidopsis for relation between genes and tetralocular ovary. Our results will be helpful in understanding for mechanisms of tetraovular ovary in Brassica rapa.

Water-deficiency is one of the most serious challenges which restrict crop production. Root is the primary tissues exposed to water limitation in soil. Although a number of transcriptome data under water limitation have been produced in rice, but most of them have analyzed the effect of leaf or shoot. Thus, understanding of relating molecular mechanism is still limited. To get global view of the effect on water deficiency in rice root, we carried out RNA-Seq experiment. To do this, we compared the RNA-Seq transcriptome data of 3 day samples under water deficiency with those of unstressed rice roots with unstressed control. As a result, we identified 1,098 genes upregulated in water stress condition for 3 days. Gene ontology (GO) enrichment analysis revealed that 18 GO terms are overrepresented. Of them, valyl-tRNA aminoacylation, transcription from RNA polymerase II promoter, glycine catabolic process, and L-phenylalanine catabolic process are more significant, indicating that transcription of new transcripts, control of translation fidelity, and reuse of primary and secondary metabolites can be activated during water stress.

Genome editing that allows targeted mutagenesis in higher eukaryotic cells and organisms is broadly useful in biology, biotechnology, and medicine. We have developed zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and Cas9 RNA-guided engineered nucleases (RGENs), derived from the type II CRISPR/Cas prokaryotic adaptive immune system, to cleave chromosomal DNA in a targeted manner, producing DNA double-strand breaks in cells, the repair of which via endogenous systems gives rise to targeted genome modifications. The Cas9 protein, when complexed with small guide RNAs (sgRNAs), recognizes and cleaves target DNA sequences complementary to the guide RNAs in vivo, inducing targeted genome modifications at high frequencies in cultured cells and whole organisms. Despite broad interest in RNA-guided genome editing, RGENs are limited by off-target mutations. Here, we show that off-target effects of RGENs can be reduced below the detection limits of deep sequencing by choosing unique target sequences in the genome and modifying both guide RNA and Cas9. Furthermore, we deliver purified recombinant Cas9 protein complexed with sgRNAs (RGEN ribonucleoproteins (RNPs)) to animal embryos and cultured human cells including hard-to-transfect pluripotent stem cells to achieve highly efficient RNA-guided genome editing in cells and whole organisms. RGEN RNPs cleave chromosomal DNA almost immediately after delivery and are degraded rapidly in cells, reducing off-target effects and mosaicism.

MicroRNAs (miRNAs) are a class of non-coding RNAs approximately 21-nt in length which play important roles in regulating gene expression in plants. Although many miRNA studies have focused on a few model plants, miRNAs and their target genes remain largely unknown in hot pepper (Capsicum annuum), one of the most important crops cultivated worldwide. We here employed high-throughput sequencing to comprehensively identify small RNAs and their targets in pepper. From these, we identified several novel targets of miRNAs, including the major de novo methylation enzyme involved in RNA-directed DNA methylation in plants. Furthermore, we identified several highly abundant 22-nt miRNA families that target conserved domains in NB-LRRs. We showed that transient co-expression of the miRNA with NB-LRRs, resulted in the attenuation of the hypersenstive responses in Nicotiana benthamiana, suggesting that interaction between miRNA family and disease resistance proteins is likely to serve as a conserved trigger for defense mechanism in Solanaceae. This work provides the first reliable draft of the small RNA transcriptome in pepper that offers an expanded picture of miRNAs in relation to NB-LRR regulation, providing a basis for understanding the functional roles of miRNAs in disease resistance.

Legume and rhizobia symbiosis plays an important role in conversion of atmospheric dinitrogen to ammonia. On a global scale, this interaction represents a key entry point for reduced nitrogen into the biosphere, and as a consequence this symbiosis is important in both natural and agricultural systems. Symbiotic development of nodule organ is triggered by chito-oligosaccharide signals (Nod factors) from the bacterium which are perceived by the legume root. Understanding the molecular and cellular processes that underlie Nod factor perception is one focus of legume biology. Although forward genetics has proved to be an important tool to identify key players in Nod factor perception, we still know relatively little regarding the functional networks of genes and proteins that connect the earliest steps of Nod factor perception to immediate downstream outcomes. To elucidate genes and proteins that link Nod factor perception to cellular and physiological responses we are taking a discovery-based strategy based on whole transcriptome profiling using RNA-seq analysis in the roots of Medicago truncatula in response to Sinorhizobium meliloti. Functional characterization of a number of candidate genes is currently in progress to further examine their role in nodulation such as generating transgenic plants

The newly developed varieties, Jayoung (violet flesh color) and Hongyoung (red flesh color) that harboring various anthocyanins and flavonoids in flesh colored potato are highly increase their interesting not only for food but also functional characteristics such as anti-inflammatory effects. Up to date, most of the molecular markers developed in potato are linked to disease resistance including late blight and PVY, nematode. A few markers linked to economically important functional materials such as anthocyanin biosynthesis are published. With the low cost and high throughput of NGS (Next Generation Sequencing) technology, numerous molecular markers are highly increased in may crops. Among the molecular markers, SNPs (Single nucleotide Polymorphisms) are most useful markers owing to their large numbers in inter and intra varieties in potato. Here we reported SNPs discovery from transcriptome sequencing data acquired from colored flesh potato cultivars, Jayoung and Hongyoung with white flesh color Atlantic. Total RNA was isolated from shoot in tuber after breaking dormancy about 2cm length. Short read sequence data were obtained form Illumina Hiseq2000 and the raw dat set were trimmed with Q socore over 20. Sequencing data were align to reference genome (Solanum tuberosum v4.03, http://potatogeomics.plantbiolgy.msu.edu). About 70% of sequence read were mapped int to reference genome. 139,050, 140,976 and 146,429 total SNPs were discovered in Hongyoung, Jayoung and Atlantic, respectively. All SNPs are mapped into the psedomolecules in reference genome by chromosome. SNPs are also analyzed with homozygous and heterozygous SNPs and genic and intergenic region. SNPs are compared with Potato Infinium 8K Chip data. SNPs found in candidate genes of anthocyanin biosynthesis were discovered. These SNPs information of flesh colored potato will be further analyzed for the allele mining for anthocyanin syhthesis and control region

In some plant species, prolonged exposure to low temperature during the winter season is necessary to acquire the competence to flower in the following spring. This process, known as vernalization, is an epigenetic change in which a mitotically stable change of the developmental potential of the meristem (competence to flower) is maintained even in the absence of the inducing signal (prolonged cold exposure). In Arabidopsis, vernalization results in stable epigenetic repression of a potent floral repressor, FLOWERING LOCUS C (FLC). Increased enrichment of Polycomb Repressive Complex 2 (PRC2) and trimethylated Histone H3 Lys 27 (H3K27me3) at FLC chromatin is necessary for the stable maintenance of FLC repression by vernalization. A long intronic noncoding RNA (termed as COLDAIR) is required for the vernalization-mediated epigenetic repression of FLC. COLDAIR physically associates with a component of PRC2 and targets PRC2 to FLC. COLDAIR is required for establishing stable repressive chromatin at FLC through its interaction with PRC2. In addition, floral integrator genes are targets of PRC2 complex, resulting in delayed flowering time through repression mechanism of PRC2 complex. Recently another long non-coding RNA was isolated from floral integrator gene and characterized the function of this long non coding RNA.

Environmental stresses including drought, extreme temperatures, and high salinity are major factors that severely limit crop productivity worldwide. To overcome yield loss due to these environmental stresses, a large number of researches have been conducted to understand how plants respond to and adapt these environmental stresses. Posttranscriptional regulation as well as transcriptional regulation of gene expression is recognized as a key regulatory process in plant stress responses, and these cellular processes are regulated by diverse RNA-binding proteins (RBPs). Over the last years, we have extensively investigated the functional roles of RBPs that harbor an RNA-recognition motif at the N-terminal half and a glycine-rich region at the C-terminal half (glycine-rich RNA-binding proteins, GRPs), zinc finger-containing GRP, and cold shock domain proteins (CSDPs) in Arabidopsis thaliana, rice (Oryza sativa), wheat (Triticum aestivum), and rapeseed (Brasicca napus) under stress conditions. Our comparative analysis demonstrated that certain family members display RNA chaperone function during stress adaptation process in monocotyledonous plants as well as in dicotyledonous plants. These findings point to the importance of the regulation of mRNA metabolism in plant response to environmental stresses and shed new light on the practical application of these RBPs to develop stress-tolerant transgenic crops.