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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

We recently reported rice promoters that are active in late stages of pollen development. However, rice promoters that allow manipulation of gene expression at earlier stages of pollen development are still very limited to date. In this study, we have chosen 10 putative microspore promoters, OsMSP1 through OsMSP10, based on publicly available transcriptomic datasets in rice (Oryza sativa L.). Sequence analysis of these promoter regions revealed some cis regulatory elements involved in pollen-specific expression. We also examined promoter activities using the promoter-GUS reporter constructs in both transgenic rice and Arabidopsis. In rice, all of the 10 promoters directed GUS signals from the microspore stage throughout the all stages of pollen development. In addition, while GUS signals from 4 promoters, OsMSP2, OsMSP7, OsMSP9 and OsMSP10, seem to be expressed preferentially during pollen development, those from other six promoters were observed in vegetative tissues such as leaves, stems, and roots of seedlings. Similarly, in Arabidopsis, all of the 10 promoters directed GUS signals during pollen development. In detail, 8 promoters, OsMSP1 ~ OsMSP8 directed GUS signals from the microspore stage, whereas 2 promoters, OsMSP9 and OsMSP10, exhibited GUS signals from tricellular stage. Furthermore, seven promoters, except for OsMSP1, OsMSP2 and OsMSP10, showed GUS signals in shoot apical region or root tissues of seedlings. Furthermore, we verified microspore activity of four promoters, OsMSP1, OsMSP2, OsMSP3 and OsMSP6, by complementation analysis of the sidecar pollen (scp) mutant which displays microspore-specific defects. Currently, further analyses are underway for GUS expression of T2 generation in transgenic rice and scp complementation with remaining promoters.

To identify genes that play critical roles during male gametogenesis in Arabidopsis, we have isolated several pollen morphological mutants from a mutagenized seed pool generated with a T-DNA activation vector. In this study, we have focused on a mutant plant producing ~50% abnormal pollen grains including high levels of collapsed pollen at maturity. The pollen developmental analysis showed that the mutant pollen phenotype was first observed at tricellular stage. Interestingly, the mutation was only maintained as a heterozygote due to the severely reduced genetic transmission through both sexes. TAIL PCR analysis led to the identification of the responsible gene which encodes a conserved oligomeric golgi complex component-related protein (COGCC). RT-PCR analysis showed predominant expression of the gene in reproductive organs including developing spores. The gene identity was confirmed by the result that mutant plants harboring a T-DNA containing corresponding wild type gene produced less level of mutant pollen grains. Furthermore, confocal laser scanning microscopy using mature pollen expressing COGCC-RFP driven under the native promoter showed small punctate signals, which are likely to be from the Golgi complex. Further experiments for co-localization of the COGCC-RFP with the Golgi markers are underway.

The next-generation sequencing(NGS) technology is being used for more effective genetic mapping. In previous study, we obtained 60x coverage of sequence from Milyang23 and Gihobyeo on average comparing with Nipponbare reference genome. Also, we developed new derived cleaved amplified polymorphic sequence(dCAPS) markers based on the single nucleotide polymorphisms(SNPs) in coding region sequence(CDS) between these varieties. Totally, 1,726,798 SNPs between Milyang23 and Gihobyeo were detected. Among them, 146 SNP were selected for making dCAPS markers and located on genetic map with previously reported 219 PCR-based DNA markers. The map was applied to the detection of quantitative trait loci(QTLs) for stem internode diameters, culm length and panicle length within MGRIL population, and six QTLs with relatively high LOD score were found at three chromosomes; culm length and stem diameter including the first internode diameter, third and fourth internode diameter. This study showed that the NGS allowed the rapid discovery of a large number of SNPs for dCAPS marker. So, we tried to find out more single nucleotide polymorphisms(SNPs) which were located on the whole genome sequence, such as un-translated region(UTR), intron, Inter-region and coding region sequence(CDS) between Milyang23 and Gihobyeo varieties. And we collected phenotypic information about culm length, panicle length, four stem internode diameters and panicle number in rice MGRIL population for QTLs. Furthermore, results of QTL analysis described above will shows relevance of molecular markers in mapping genes for useful breeding.

Rice is a staple food crop for more than half of the world population. Severe losses of rice production was caused by various environmental conditions such as cold, heat and flooding annually. Rice is a highly sensitive to low temperature below 15-20 ℃ because of originating from tropical or subtropical climates. Especially, seedling of rice is easily damaged to low temperature and result in seedling yellowing, growth retardation, reduced tillering and yield losses at last. We used a recombinant inbreeding lines (RIL) population of 384 individuals derived from a cross between Hanareum 2, a highly cold sensitive variety and Unkwang, a cold tolerant variety for molecular mapping of QTLs related to cold tolerance. Seedling discoloration of each lines and parents caused by cold response were investigated in field condition after transplanting. And leaf samples of RIL population were collected for evaluation of chlorophyll content using 80% acetone extraction. The seedling of each lines and parents was subjected to low temperate by 5~13 ℃ during 14 days. The cold recovery score (CRS) of RILs was recorded after 4 days recovery period according to standard evaluation system (SES, IRRI). Total of eight QTLs were detected on chromosome 1, 7, 8, 10, 11 and 12 using cold tolerance traits, chlorophyll content, seedling discoloration and cold recovery score in 384 RILs. The qCRS12, which detected on chromosome 12 between two flanking markers id12002113, id12002563 (1.1 Mbp) showed 25 LOD score with 26% of phenotypic variation of cold recovery score in RILs population. The positive allele contributing to cold tolerance came from the cold tolerant parent Unkwang. The result may provide useful information for a marker-assisted breeding program to improve cold tolerant in rice.

Asterales are dicotyledonous flowering plants and are one of the Asterid clade, incuding many species as well as Codonopsis and Platycodon. Here, we have determined the complete chloroplast genome sequences of C. lanceolata and P. grandiflorus by using the targeted denovo assembly method of short reads derived from whole genome resequencing. The total lengths of each chloroplast genome sequence are 156,180 bp for C. lanceolata and 155,453 bp for P. grandiflorus. In their chloroplast genomes, 106 genes (75 protein-coding genes, 4 rRNA genes, 23 tRNA genes, and 4 hypothetical chloroplast open reading frames [ycfs]) exhibited the relatively similar positions. Also, 7 protein-coding genes commonly showed to contain introns in both C. lanceolata and P. grandiflorus chloroplast genome, while psaA gene contain intragenic regions only in P. grandiflorus chloroplast genome. In further analysis, we identified the codon usage bias to A or T and found the different simple sequence repeat (SSR) loci of each chloroplast genome (18 SSR loci of C. lanceolata and 16 SSR loci of P. grandiflorus). In the phylogenetic trees based on 72 protein-coding genes, C. lanceolata is more closely related to P. grandiflorus than the other plant species order Asterales. Also, we found the highest sequence diversities of 12 protein-coding genes in small single copy (SSC) region than in the inverted repeat (IRs) and large single copy (LSC) region, and 3 genes such as rpoC2 (LSC region), ndhB (IRs region), and ndhF (SSC region) showed the highest number of segregating sites in each region. Additionally, we developed the molecular markers for phylogenetic applications of C. lanceolata and P. grandiflorus chloroplast genome.

Recently, the increased consumption of mixed-grain flour products have led to improved human health in busy modern life. For this reason, the verification of commercial food authenticity is one of important subjects. The development of DNA techniques such as real-time PCR has led to the increasing efficiency of illegal food product detection. Here, we have developed a comprehensive method for detecting the grain flour of various rice cultivars in commercial food products derived from different plant species. In the genetic variation analysis of different protein coding genes on various chloroplast genomes, we found the high numbers of segregating sites in rpoB and rpoC2 more than in other genes. Thus, we have attempted to develop chloroplast DNA (cpDNA) markers, which were Os_m_rpoB in rpoB, and Os_m1_rpoC2 and Os_m2_rpoC2 in rpoC2. To assess the applicability of three cpDNA markers, we have identified the appropriate statistical measurements of each marker in various mixed-grain flour samples derived from rice cultivars and different plant species by real-time PCR, In addition, the three cpDNA markers successfully applied for detecting of nonexistent rice flour in different commercial food products.

In this study, we sought to identify primary pears species and Korean native pears, without the use of morphological characteristics. In addition, this study was to establish pear DNA fingerprinting data for Korean native pears using 12 microsatellite markers, and to accurately classify a database for management of the Korean pear collection. Forty two pear accessions (7 primary pears, 5 Asian pears, 29 Korean pears, and 2 reference pears) were analyzed with twelve primers covering whole pear genome. In the present study, all pear accessions were successfully classified along with their pedigrees, and the distribution of primary pears was parallel to those of the previous taxonomic results. Korean pears were divided into 3 groups. Group I was characterized by Pyrus calleryana, and included Korean pea pears. Group II was characterized by P. pyrifolia, and was classified into 2 small groups. The first small group comprised of ‘Najucheongbae’, ‘Sunchanggulimdolbae’, ‘Andongmookbae’, ‘Andongdangsilri’, and ‘Najucheongbae’ and was presumed to be cultivars of P. pyrifolia. The second small group consisted of ‘Cheongdangrori’ and ‘Pyeongchangsuhyangri’. These two accessions were assumed to be a hybrid of P. pyrifolia and the other cultivar. Group III was characterized by P. ussuriensis. ‘Goesanhwangbae’, ‘Andongcheongsilri’, ‘Gongjucheongsilri’, and ‘Yecheoncheongbae’ were assumed to be cultivars of P. ussuriensis. Contrary to ‘Ulreungdocheongbae A’, ‘Ulreungdocheongbae B’ was classified as belonging to the P. ussuriensis group. It is possible that this is a consequence of, P. ussuriensis genes being transferred into ‘Ulreungdocheongbae B’. The result of this research reaffirmed the efficiency of a standard set of microsatellite markers and provides data, which will be useful for developing a core collection of pears.