Rice (Oryza sativa L.) is one of the most important staple crops in the world, providing main energy source for more than half of the world’s population. It is even closely associated with economic and political stability in many developing countries of Asia and Africa. These days, moreover, amount of land suitable for the agriculture is shrinking due to a variety factors, such as rapid climate changes and industrializations, while rice eating human populations keeps growing. To meet the nutritional and socio-economic demands worldwide, dedicated efforts in developing superior rice varieties need to be reinforced, accumulating and combining beneficial alleles as much as possible from rice germplams. Here, we propose a pipeline for genome assisted breeding where new genomic technologies including GWAS, omics and evolutionary studies together with follow-up breeding programs are integrated. Once pinpointing candidates genes, the integrated genomics approach allows informed choice of parents for the following breeding program based on the haplotype information, in addition to providing precise molecular marker information. We also conducted proof-of-concept analysis, using various agriculturally important phenotypes for rice improvements.

We have generated 383 independent transgenic lines for genetically modified (GM) rice that contained PsGPD (Glyceraldehyde-3-Phosphate Dehydrogenase), ArCspA (Cold Shock Protein), BrTSR15 (Triple Stress Resistance 15) and BrTSR53 (Triple Stress Resistance 53) genes over-expression constructs under the control of the constitutive (CaMV 35S) promoter. TaqMan copy number assay was determined inserted T-DNA copy number. Also flanking sequence tags (FSTs) analysis was isolated from 203 single copy T-DNA lines of transgenic plants and sequence mapped to the rice chromosomes. In analyzing single copy lines, we identified 157 flanking sequence tags (FSTs), among which 58 (36%) were integrated into genic regions and 97 (62%) into intergenic regions. About 27 putative homozygous lines were obtained through multi-generations of planting, resistance screening and TaqMan copy number assay. To investigate the transgene expression patterns, quantitative real-time PCR analysis was performed using total RNAs from leaf tissue of single copy, intergenic region of T-DNA insertion and homozygous T2 plants. The mRNA expression levels of the examined transgenic rice were significantly increased in all of the transgenic plants. In addition, myc-tagged 35S:BrTSR15 and 35S:BrTSR53 transgenic plants were displayed higher levels of transgene protein. These results may be useful for producing of large-scale transgenic plants or T-DNA inserted mutants in rice.

The oriental melon (C. melo var. makuwa), called ‘Chamoe’ in Korean, is a popular fruit crop cultivated mainly in Asia and a high–market value crop in Korea. To provide a genomic resource as a reference genome for the Cucurbitaceae crop improvement, we performed whole genome sequencing of Korean landrace, Gotgam chamoe. We used Illumina HiSeq2000 sequencing platform to generate 89 Gb (205X) of paired and mate pair sequence reads. The pre-processed reads were de novo assembled resulting in 4,764 scaffolds with a N50 scaffold length of 249kb. This assembly represented 379.8Mb which was 84.7% of the 448Mb of the whole genome. The assembled draft was predicted 26,634 genes of which 80% were predicted by known protein or C. melo unigene homology. Approximately 20% of predicted genes were hypothetical. A total of 1,885 non-coding RNA was detected including rRNA. The transposable elements were accounted for 21% (71.6Mb) of the total assembly. All the marker candidates including SSR, INDEL, SNP were mined and presented. The draft genome will provide a useful platform for genomic research and improvement for Cucurbitacea crops.

As sessile organisms, plants have evolved mechanisms that allow them to adapt and survive periods of various environmental stresses including high salinity and drought. The ubiquitin-proteasome system (UPS) is an integral player in plant response and adaptation to various abiotic stresses. Understanding UPS function has centered mainly on defining the role of E3 ubiquitin ligases, which are the substrate-recruiting component of the ubiquitination pathway. Here, we report on Ring finger E3 ligase, Oryza sativa salt- and drought-induced RING finger protein1 gene (OsSDRFP1) in defense responses to osmotic stresses. Results of qRT-PCR and In vitro ubiquitination assay demonstrated that OsSDRFP1 act as an E3 ligase in response to salt and drought stresses. in this study, Subcellular localizations showed that the OsSDRFP1 was observed in cytosol (66%) and nucleus (34%) under non-treated conditions. However, the florescence signals of rice protoplasts after salt treatments detected in nucleus (60%) higher than in cytosol (30%). The Arabidopsis plants overexpressing OsSDRFP1 clearly exhibited hypersensitive responses to salt stress. whereas, OsSDRFP1-overexpressing plants were more tolerant to both drought- and ABA-stresses than the wild-type plants. These results might suggest that OsSDRFP1 has a dual function as a regulator of high salt- and drought-stresses.

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.

Map-based cloning is a basic method for identifying the mutated gene in plants. We selected the gametophytic mutant, named as AP-26-09, in activation-tagging pool. Mutant plant showed various kinds of pollen phenotype, such as the different number of nucleus or abnormal shapes. For the map-based gene cloning, we conducted phenotypic analysis of F2 mapping population through the screening of DAPI-stained pollen using fluorescence microscopy. Genomic DNA of F2 plants is prepared from leaves of approximately 1000 plants. In order to define chromosomal region where mutation is located, we designed SSLP markers and performed PCR amplification. In this study, we characterized gametophytic mutant and determined the chromosomal location using map-based approach.

Cabbage head splitting can greatly affect both the quality and commercial value of cabbage (Brassica oleracea). To detect the genetic basis of head-splitting resistance, a genetic map was constructed using an F2 population derived by crossing “748” (head-splitting-resistant inbred line) and “747” (head-splitting-susceptible inbred line). The map spans 830.9cM and comprises 270 markers distributed in nine linkage groups, which correspond to the nine chromosomes of B. oleracea. The average distance between adjacent markers was 3.6cM. A total of six quantitative trait loci (QTLs) conferring resistance to head splittingwere detected in chromosome 2, 4, and 6. Two QTLs, SPL-2-1 and SPL-4-1, on chromosomes 2 and 4, respectively, were detected in the experiments over 2 years, suggesting that these two potential loci were important for governing the head-splitting resistance trait. Markers BRPGM0676 and BRMS137, which were tightly linked with head-splitting resistance, were detected in the conserved QTL SPL-2-1 region using bulked segregant analysis. Synteny analysis showed that SPL-2-1 was anchored to a 3.18Mb genomic region of the B. oleracea genome, homologous to crucifer ancestral karyotype E block in chromosome 1 of Arabidopsis thaliana. Moreover, using a field emission scanning electron microscope, significant differences were observed between the two parental lines in terms of cell structures. Line “747” had thinner cell wall, lower cell density, larger cell size, and anomalous cell wall structure compared with the resistant line “748”. The different cell structures can provide a cytological base for assessing cabbage head splitting.

The ubiquitin-26S proteasome system is important in the quality control of intracellular proteins. The ubiquitin-26S proteasome system includes the E1 (ubiquitin activating), E2 (ubiquitin conjugating) and E3 (ubiquitin ligase) enzymes. U-box proteins are a derived version of RING-finger domains, which have E3 enzyme activity. Here, we present the isolation of a novel U-box protein, OsUPS, from rice (Oryza sativa).The cDNA encoding the O.sativa U-box protein(OsUPS) comprises 1338bp, with an open reading frame of 445 amino acids. The open reading frame of the OsUPS protein is comprised of notable domains: a single ~70-amino acid domain and a GKL domain that contains conserved glycine, lysine/ arginine residues and leucine-rich feature. We found that full-length expression of OsUPS was up-regulated in both rice plants and cell culture in the absence of inorganic phosphate (Pi). A self-ubiquitination assay indicated that the bacterially expressed OsUPS protein had E3 ligase activity, and subcellular localization results showed that OsUPS was located in the chloroplast. Suppression of OsUPS resulted in servre signs of toxicity caused by the over-accumulation of Pi. These results support the notion that OsUPS plays an important role in the Pi signaling pathway through the ubiquitin-26S proteasome system.

Fibroin silk proteins from spider or silkworm are attractive biomaterials that are of particular biotechnological interest for industrial and medical purposes because of their unique physical and mechanical properties. In this study, we generated and characterized the transgenic rice plant expressing a spider silk protein. Spider silks have great potential as biomaterials with extraordinary properties. Here, we report the cloning and characterization of the major ampullate silk protein gene from the spider Araneus ventricosus. A cDNA encoding the partial major ampullate silk protein (AvMaSp) was cloned from A. ventricosus. An analysis of the cDNA sequence shows that AvMaSp consists of a 240 amino acid repetitive region and a 99 amino acid C-terminal non-repetitive domain. The peptide motifs that were found in the spider major ampullate silk proteins, (A)n, (GA)n, and (GGX)n, were conserved in the repetitive region of AvMaSp. Phylogenetic analysis further confirmed that AvMaSp belongs to the spider major ampullate spidroin family of proteins. Recombinant AvMaSp-R was degraded abruptly by trypsin. However, AvMaSp-R was stable at 100 °C for at least 30 min. Additionally, the AvMaSp-R was stable at pH values from 2 to 12 for at least 1 h. Taken together, our findings describe the molecular structure and biochemical properties of the A. ventricosus major ampullate silk protein and demonstrate its potential as a biomaterial.

We used an efficient system to create rice mutant by Ac/Ds transposon insertion mutagenesis, such as selected homozygous mutant in dwarf phenotypes. We reported here the identification of function of dwarf OsGASD gene(Oryza sativa Gibberellin Acid Sensitive Dwarf). OsGASD gene encodes a 344 amino acid polypeptide and no homology proteins in Gene Bank. The osgasd mutnat was sensitive to exogenous gibberellic acid(GA) level. We performed experiment to controlled expression the OsGASD gene, its role in plant development, a quantitative analysis of endogenous GA content and sensitivity to GA. The osgasd mutant includes smaller amount of active GAs than wild-type. osgasd mutant plant of GA biosynthesis pathway causes GA deficiency and dwarf plants, and endogenous GA suppliance can restore the wild type phenotype in this mutant. There result indicated that OsGASD gene regulated the elongation of shoot, stem and plant height. The increased expression of OsGASD gene dramatically induces expression of the factors associated with GA biosynthesis, whereas osgasd mutant suppression of the factors associated with GA biosynthesis, loading to dwarf phenotypes. That applied GA3 at the plant development stage to survey the response of OsGASD gene to GA3. We suggest that OsGASD gene is related to factors of GA biosynthesis pathway regulating rice internodes development.

R2R3 MYB transcription factors play regulatory roles in plant responses to various environmental stresses and nutrient deficiency. In this study, we isolated MYB-like gene respond to phosphorus deprivation in rice and designated OsMYB4P, an R2R3 MYB transcription factor, from rice under low-phosphate conditions. OsMYB4P is 993bp long and encodes a 330 amino acid polypeptide. OsMYB4P was localized in the nucleus and acted as a transcriptional activator. Transcriptional levels of OsMYB4P in cell suspension, shoots, and roots of rice increased under low phosphate conditions. Shoots and roots of OsMYB4P overexpressing plants grew well in high and low phosphate conditions. In addition, root system architecture was altered considerably as a result of OsMYB4P overexpression. Under both phosphate sufficient and deficient conditions, more Pi accumulated in shoots and roots of OsMYB4P overexpressing plants than in the wild type. Overexpression of OsMYB4P led to greater expression of Pi transporter-family proteins OsPT1, OsPT2, OsPT4, OsPT7, and OsPT8 in shoots, and to decreased or unchanged expression of these proteins in roots, with the exception of OsPT8. These results demonstrate that OsMYB4P may be associated with efficient utilization of Pi in rice.

Establisment of rice library is an essential approach for rice functional genomics study. Utilizaing maize transposable element Ac/Ds is a promising method to construct insertional mutagenesis library of rice. Ac/Ds tagging system has received extensive application in rice during the past several years. The maize Ds element is one of the main tagging vehicles used in rice. Narrow leaf mutant have short height, narrow leaf width and large angle. To compare with wild type and narrow leaf mutant in detail, we observed the leaves under microscope. In specific portion(large and small vein), no significantly reduce cell size and number of cell. Knock-out of the OsNLR(narrow leaf ribokinase) gene inhibits internodes, panicles, angle(between leaf and stem), leaf, seed. OsNLR was shown to specifically expressed on leaf. In real time PCR analysis with mature leaf of wild type and mutant, there might be a functional association between OsAGO7, NRL1, NAL1 and NAL7 in regulating leaf development. We tested on the experimental field using wild type and mutant plants. In agricutural traits that contain leaf and seed related traits(except angle) significantly reduce in mutant plants. These results demonstrate that OsNLR gene may be associated with leaf development.

Serotonin N-acetyltransferase (SNAT), the penultimate enzyme in melatonin biosynthesis, catalyzes the conversion of serotonin into N-acetylserotonin. Plant SNAT is localized in chloroplasts. To test SNAT localization effects on melatonin synthesis, we generated transgenic rice plants overexpressing a sheep (Ovis aries) SNAT (OaSNAT) in their chloroplasts and compared melatonin biosynthesis with that of transgenic rice plants overexpressing OaSNAT in their cytoplasm. To localize the OaSNAT in chloroplasts, we used a chloroplast targeting sequence (CTS) from tobacco protoporphyrinogen IX oxidase (PPO), which expresses in chloroplasts. The purified recombinant CTS:OaSNAT fusion protein was enzymatically functional and localized in chloroplasts as confirmed by confocal microscopic analysis. The chloroplast-targeted CTS:OaSNAT lines and cytoplasmexpressed OaSNAT lines had similarly high SNAT enzyme activities. However, after cadmium and butafenacil treatments, melatonin production in rice leaves was severalfold lower in the CTS:OaSNAT lines than in the OaSNAT lines. Notably, enhanced SNAT enzyme activity was not directly proportional to the production of N-acetylserotonin, melatonin, or 2-hydroxymelatonin, suggesting that plant SNAT has a role in the homeostatic regulation of melatonin rather than in accelerating melatonin synthesis.

Although melatonin biosynthetic genes from plants have been cloned, the melatonin catabolism mechanisms remain unclear. To clone the genes responsible for melatonin metabolism, we ectopically expressed 35 fulllength cDNAs of rice 2-oxoglutarate-dependent dioxygenase (2-ODD) in Escherichia coli and purified the corresponding recombinant proteins. In vitro 2-ODD assays showed four independent 2-ODD proteins that were able to catalyze melatonin into 2-hydroxymelatonin, exhibiting melatonin 2-hydroxylase (M2H). These M2H proteins had peak activities at pH 8.0 and 30°C. The Km ranged from 121 μM to 371 μM with the Vmax ranging from 1.7 to 18.5 pkat/mg protein, respectively. The M2H enzyme activities were dependent on cofactors such as α-ketoglutarate, ascorbate, and Fe2+, similar to the 2-ODD enzymes. M2H activity was inhibited by prohexadione-Ca, an inhibitor of 2-ODD, in a dose-dependent manner. M2H activity was high in the roots of rice seedlings, concurrent with high transcription levels of 2-ODD 21, suggesting that 2-ODD 21 was a major gene for M2H activity. Analogous to the high M2H activity in the roots, 2-hydroxymelatonin was found in large quantities in roots treated with melatonin. These results suggest that melatonin was metabolized into 2-hydroxymelatonin by the M2H genes in plants, but the physiological significance of 2-hydroxymelatonin remains to be examined in the future.

Brassica rapa subspecies show morphological variability, containing vegetable types and oilseed types. The yellow sarson types(Brassica rapa ssp, tricolaris) have distinct morphology, yellow seeded and contain some lines with very unique character of tetralocular ovary. For genetic studies on tetralocular ovary related to high seed yields, we produced genetic segregation population with F2 and double haploid(DH) population. The yellow sarson LP8 (YS-033, CGN06835) with character of tetralocular ovary used as a maternal plant and crossed by LP21 of turnip rape type with bilocular ovary as paternal plant. We took on the microspore cultures on immature bud which is collected on sizing from 2mm to 3.2mm for DH population. The regenerations DH plants are analyzed by ploidy determination using flow cytrometer and selected on diploid plants. These regenerated DH and F2 plants are doing bud pollination and measuring the phenotype traits. Also, these populations will be used for identify of genetic locus relate to tetralocular ovary using genotyping by sequencing.

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.

Plants have evolved elaborate innate immune systems against invading pathogens, such as bacteria, fungi, oomycetes, viruses and insects. Among them, intracellular immune receptors known as nucleotide-binding site and leucine-rich repeat (NB-LRR) play critical roles in effector-triggered immunity (ETI) regarding to plant defense. Here, we identified potential NB-LRR coding sequences from pepper genome using bioinformatics analysis and performed comparative analysis with Solanaceae plants. As a result, we identified 267, 443, and 755 NBS-encoding genes in the genome of tomato, potato, and pepper, respectively. These may indicate that the Solanaceae NB-LRRs were evolved through species-specific unequal-duplication event. Further phylogenetic and clustering analyses revealed that Solanaceae NB-LRRs were classified into the 14 subgroups with 1 TNL and 13 CNL types. We found that the genes in CNL-G1 and CNL-G2 subgroup were highly expanded compared to other subgroup showing a large portion of NB-LRR in pepper genome. Among 755 NB-LRRs in pepper genome, 623 were physically mapped on all 12 pepper chromosome pseudomolecules. Furthermore, a number of NB-LRRs in the same group were physically clustered by tandem array in the specific chromosome. Genome-wide identification of pepper NB-LRR family and their evolutionary analysis could provide an important resource for identification and characterization of genes for breeding of disease resistance crops.

Panax ginseng C.A Meyer is commonly used in Asian traditional medicine to treat a variety of diseases. Ginsenosides are glycosylated triterpenes, referred to saponins, have been especially noted as active compounds contributing to the various efficacy of ginseng. In this study, we are trying to select high saponin content of ginseng lines from the gamma irradiated adventitious roots. Recently, we have generated several mutant ginseng lines improving ginseniside content by gamma radiation. The mutant lines were selected by phenotypes and ginsenoside content (HPLC analysis) of the irradiated adventitious root lines. However, the ginsenoside content of the mutant lines was not sufficient for commercial use and the selection method was not suitable for large scale of mutant line selection. In this study, we are testing Fourier transfer infrared spectroscopy (FT-IR) as a new selection method of mutant lines in Panax ginseng. About 5,000 pieces of Panax ginseng adventitious roots were exposed to gamma radiation (60Co). Irradiation dosages were 0, 25, 50 and 70Gy. Survival rate of the irradiated samples was evaluated by counting the number of survival main roots after 5 weeks culture in the solid MS medium with NAA, IAA and 5% sucrose. In present, we are collecting the survived adventitious root lines (about 900 lines) from the gamma irradiated ginseng roots for FT-IR and HPLC analysis. After analysis of FT-IR and HPLC, we will assess the suitability of the FT-IR as a screening method for the preparation of mutant lines in ginseng.

High temperature is one of major environmental stress. Some of molecular markers related heat stress or tolerance have been reported by many researchers. Heat tolerance managing is difficult through the phenotypic selection, so marker assistant selection (MAS) using molecular markers like as RAPD, SSR ect. was tried to selection of useful traits for heat tolerance. Fourteen SSR markers reported by previous research were selected for this research. These markers were linked to important traits including grain filling duration, HIS (Heat susceptibility index) grain filling duration. In this study, we tried to evaluate 14 SSR markers for MAS using 31 useful wheat resources including 24 crossing line from Turkey and six Korean wheat cultivars using 14 SSR markers. The average of the number of alleles and PIC values in this study were 6.14 and 0.63, respectively. Two major clades and six sub clades were grouped by phylogenetic tree using UPGMA program. Six Korean wheat cultivars were distinct from other Turkey resources in the phylogenetic dendrogram. From the results, we expected that these markers were able to adapt to screening wheat genotyping for heat tolerance.

It is necessary to carry out a risk assessment to determine the consequences of releasing a particular plant species containing specific transgenes before transgenic plants can be grown under filed conditions. Gene flow from transgenic plants to wild closely related species has raised concern recently. Since transgenic crops were released in 1996, the global area of transgenic crops has been increasing rapidly. The transgene introgression from transgenic crops to their wild relatives is unavoidable in some species. Transgene introgression is of concern because the crop–wild plant hybrids might be conferred with a selection advantage to increase their performance, which could result in negative ecological consequences to natural ecosystems. The genus Brassica has 159 species, including a number of wild species that are of great importance to the economy. Most transgenic Brassica gene flow research has focused on the most successful cross between transgenic oilseed rape Brassica napus and its wild relatives Brassica rapa, a widely distributed weed in the farming system in Europe and America, since the hybridization can spontaneously happen and the generations can backcross to B. rapa easily in the wild conditions. In this study, we aimed to characterize transgene introgression, segregation, and expression in backcrossed generations between tramsgenic B. napus and B. rapa. These results will contribute to the environmental risk assessment and assist in biosafety management.