To develop a strong root-specific gene expression system, six gene promoters were investigated by using transgenic Arabidopsis and a GUS:GFP reporter gene. These promoters were initially selected from Arabidopsis genes which are specifically expressed only in roots, based on the TIGR information. The GUS activity of these promoters was measured in several tissues of Arabidopsis by using both histochemical and fluorimetric GUS assays. The results showed that the activity of these promoters was strongly detected only roots. This was also confirmed by RT-PCR analysis. Therefore, these six promoters could be used for utilization of a root-specific expression of target genes.

Cucumber mosaic virus (CMV), which has the broadest host range among plant viruses, is a very destructive pathogen in pepper production. Various resistance sources against CMV have been identified by plant breeders. One of them is Capsicum annuum ‘Bukang’, which contains a single dominant resistance gene, Cmr1. C. annuum ‘Bukang’ is resistant to CMV-P0 strains (CMV-Kor and CMV-Fny), but susceptible to CMV-P1. CMV-P1 is a new strain recently identified in South Korea. Previously we showed that CMV-P1 RNA1 helicase domain is responsible for overcoming Cmr1 and may be play a role in viral replication and systemic infection. To identify the plant host factors involved in CMV-P1 replication and movement, we utilized a yeast two-hybrid system derived from C. annuum ‘Bukang’ cDNA library as a prey. A total of 78 potential host genes interacting with the CMV-P1 RNA1 helicase domain were isolated in the first screening, and PCR confirmation and sequencing analysis narrowed the candidates to ten genes. The candidate genes have found to be encoding acireductone dioxygenase, ADP-glucose pyrophosphorylase, ADP-ribosylation factor 1, ADP-ribosylation factor, calreticulin-3 precursor, cysteine synthase, formate dehydrogenase, histone-H3, phosphomannomutase and polyubiqutin 6PU11. Previous studies showed that these genes were involved in virus infection, replication or virus movement. To elucidate the function of these genes, VIGS and coimmumo precipitation assay is being done.

Rice seed storage proteins (SSPs) are accumulated in storage organelles of the endosperm during seed maturation. The SSPs from the rice seeds consist of glutelins as a major SSP, and prolamins and globulins comprise about the rest 20 % of the SSPs. To improve the nutritional quality of rice seeds or processing properties of rice flour, we are attempting to change the composition of the SSPs in rice seeds. For this purpose, we generated many transgenic rice plants, which show the altered levels of the SSPs, by using the RNA interference (RNAi). Accumulation of glutelins was 76% reduced in the GluA-RNAi lines. The Pro-RNAi lines revealed the reduced levels of prolamins to 36%. The protein level of globulins was 61% reduced in the Glb-RNAi lines. Interestingly, an obvious reduction of glutelins, prolamins, and globulins was not examined in the GluA:Pro:Glb-RNAi lines. This suggests that a reduction of a few SSPs could be compensated by the increases of other SSPs at the protein levels. We are also attempting to generate transgenic rice plants expressing both a high-molecular-weight (HMW) glutelin subunit and a low-molecular-weight (LMW) glutelin subunit. These manipulations of rice SSPs might be an important contribution on improving the functional properties of rice seeds.

Cold stress at the seedling stage is a major threat to rice production. Cold tolerance is controlled by complex genetic factors. We used an F7 recombinant inbred line (RIL) population of 123 individuals derived from the cross of a cold-tolerant japonica and a cold-sensitive indica cultivars, for QTL mapping. Phenotypic evaluation of the parents and RILs in an 18/8oC (day/night) cold-stress regime showed continuous variations for cold tolerance or sensitivity. Six QTLs for seedling cold tolerance were identified on chromosomes 1, 2, 4, 10, and 11 with percent phenotypic variation (R2) ranging from 6.1% to 16.5%. Three main-effect QTLs (qSCT1, qSCT4, and qSCT11) were detected in all cold-tolerant RILs which explained high sum of phenotypic variation (SPV) ranging from 27.1% to 50.6%. Two QTLs (qSCT1 and qSCT11) on chromosomes 1 and 11 were fine mapped. The marker In1-c3 from ORF LOC_Os01g69910 of the BAC clone B1455F06 encoding calmodulin-binding transcription activator (CAMTA) and another marker, In11-d1 from ORF LOC_Os11g37720 (Duf6 gene) of the BAC clone OSJNBa0029K08, co-segregated with seedling cold tolerance. These two InDel markers amplified 241-bp and 158-bp alleles, respectively, in cold-tolerant RILs, and in the cold-tolerant donor Jinbu, which were absent in cold-sensitive parent BR29 and cold-sensitive RILs.

The objective of this study were to identify QTLs for agronomic traits using a set of introgression lines carrying wild rice (Oryza rufipogon) segment in cultivated rice (ssp. japonica cv. Hwaseongbyeo). Ninety-six ILs were evaluated for seven agronomic traits, amylose and protein contents. The proportion of the recurrent genome in ILs ranged from 87.8 to 100%, with an average of 96.7%. The mean number of homozygous and heterozygous donor segments were 2 (ranging 0-7) and 1.7 (ranging 0-6), respectively, and the majority of these segments had size less than 10 cM. A total of 22 quantitative trait loci were identified for 9 traits and each QTL explained 7.2% to 56.6% of the phenotypic variance. Some QTLs were clustered in a few chromosomal regions. A first cluster was located near RM527 on chromosome 6 with QTLs for culm length, panicle length, days to heading, 1000-grain weight and protein content. Three ILs with high spikelets per panicle compared to the recurrent parent were selected to detect and fine map the wild segments responsible for this variation. The results will be discussed.

Recent release of whole genome draft sequences in legume species have led comparative genome studies among legume plants including Glycine max, G. soja, Cajanus cajan and Medicago truncatula. The majority of comparative genomic researches have been conducted based on synteny of coding sequences and coding sequence variations may be one of major forces for speciation and evolution. However, non-coding sequences have been also reported to be important phenotypic regulators. Especially, since short sequence motifs in the promoter regions are highly conserved, they are suggested to be another resources of interests in comparative studies. In this study, we predicted the conserved short sequence motifs by BLASTN algorithm using dicot promoter database from Softberry (http://www.softberry.com). A total of 37,396 conserved short sequence motifs were identified onto 2 kb upstreams of 46,367 high confident gene model of G. max (cv. Williams 82). Meanwhile, whole genome of 7 soybean landraces (G. max) and 7 wild soybean genotypes (G. soja) were sequenced at low depth of less than ten using Illumina Hiseq 2000. Among these genotypes, nucleotide variations were identified in predicted conserved regulatory motifs by mapping of short reads to the reference genome sequence using the Samtools program (http://samtools.sourceforge.net/). Fifteen and two genes, which have SNPs in regulatory motifs and no SNP in coding sequence, were identified by comparisons of inter-species and intra-species, respectively. qRT-PCR experiments are in progress for investigating differences of these 17 genes expressions at transcriptional level.

An Arabidopsis small GTPase, RabG3b, was previously characterized as a component of autophagy and as a positive regulator for xylem development in Arabidopsis. In this work, we assessed whether RabG3b modulates xylem-associated traits in poplar in a similar way as in Arabidopsis. We generated transgenic poplars (Populus alba x P. tremula var. glandulosa) overexpressing a constitutively active form of RabG3b (RabG3bCA) and performed arrange of morphological, histochemical, and molecular analyses to examine xylogenesis. RabG3bCA transgenic poplars showed increased stem growth due to enhanced xylem development. Autophagic structures were observed in differentiating xyelm cells undergoing programmed cell death (PCD) in wild type poplar, and were more abundant in RabG3bCA transgenic poplar plants and cultured cells. Xylogenic activation was also accompanied by the expression of secondary wall-, PCD-, and autophagy-related genes. Collectively, our results suggest that Arabidopsis RabG3b functions to regulate xylem growth through the activation of autophagy during wood formation in Populus, as does the same in Arabidopsis.

MYB-like domain (MLD) gene is a transcription factor that plays a diverse role in plant development and in response to abiotic stresses. In this study, we isolated and developed CaMV35S::OsMLD rice lines and determined its expression pattern under abiotic stresses. It has Myb_CC_LHEQLE superfamily similar to most transcription factor genes but with a very unique binding domain of SHLQKYR in the C-terminal region. Overexpressing rice lines showed enhanced tolerance to salinity with elevated mRNA transcript. Additionally, mRNA transcripts were up-regulated by ABA, H2O2 and dehydration stresses. Further investigation in the enhanced tolerance to salinity showed an increased accumulation of proline and a decreased in malondialdehyde contents indicating that OsMLD gene may be involved in the regulation of proline and osmolytes during abiotic stresses. These results showed that OsMLD gene could be used in the development of rice intended for soil with salinity-related problem.

UDP-glucose 4-epimerase (UGE) catalyzes the reversible conversion of UDP-glucose to UDP-galactose. The gene, named BrUGE1, isolated from a Chinese cabbage had a total length of 1,328 bp that contains a single open reading frame (ORF) of 1,056 bp which encodes a polypeptide of 351 amino acid residues with a calculated mass of 39.0 kDa. Sequence analysis of BrUGE1 protein has the characteristic of an active site tetrad and NAD-binding motif (typically TGXXGXXG) of the extended short chain dehydrogenase/ reductase (SRD) superfamily. Expression analysis showed that BrUGE1 is tissue specific and highly expressed in stem of rice plant. Interestingly, BrUGE1 mRNA was highly accumulated by drought stress with significantly higher amount of soluble sugar. Morphological evaluation showed an increase in yield by 27%. Panicle length, number of productive tillers/hill, and filled spikelets were significantly increased by 17~20% compared to the wild type Gopum. Moreover, the growth of the wild type Gopum seedlings on galactose was increasingly inhibited with a decrease in UDP-glc epimerase 1 expression compared to the transgenic rice lines. In the Ubi-1::BrUGE1 lines, the increase of UDP-glc epimerase 1 expression was apparently sufficient to overcome the toxic effects of galactose. Taken together, the Ubi-1::BrGUE1 rice lines increased yield probably by increasing the rate of filled grains. The enhanced drought tolerance may be due to the induction of soluble sugar which may act as osmolyte to compensate dehydration during drought stress.

The genus Rubus belongs to the Rosaceae family and is comprised of 600-800 species distributed worldwide. Understanding the genetic relationships and genetic structure in Rubus species is important for enabling efficient management, conservation, characterization and utilization of the species. However, as a minor crop, genetic research foundation was limited to explore genetic diversity and relationships in Rubus species. The present study shows the results of application SSR markers that were developed from SSR-enriched libraries of the one Rubus species (Rubus coreanus Mique.) in our previous study. We used 34 polymorphic microsatellite markers to analysis of genetic diversity within the Rubus species, including redraspberry, blackraspberry, blackberry and mountainberry. All the 34 SSR primers pairs produced 483 polymorphic and reproducible amplification fragments. The largest number of alleles per primer pair was confirmed at GB-RC-167, GB-RC-100, GB-RC-076 and GB-RC-245, which contained 26, 25, 23 and 21, respectively. An average value of polymorphic information contents (PIC) were 0.74 with a range of 0.36 to 0.92. Population structure and phylogenetic analyses showed that all Rubus species formed three largely distinct clusters, which were confirmed by principal coordinate analysis (PCoA). We obtained the results that the developed SSR markers showed a substantial degree of genetic diversity in the various Rubus species distributed in Korea.

Regulations in the EU, Japan, Korea, etc. require that foods and feeds made of or derived from genetically modified organisms (GMOs) should be approved and labeled according to a threshold. Recently, disease resistant transgenic rice was developed in Korea, which resulted from the transformation events involving choline kinase gene, OsCK1. In order to monitor unintended release of the developed GM rice in the near future, as well as to meet GM-labeling requirements, the development of a reliable method for detection of disease resistant GM rice is requisite. Here, specific primer pairs for the detection of GMO was designed on the basis of a introduced gene and the flanking junction sequences between a plant DNA and a integrated gene construct, and also SPS gene was used as an endogenous reference material. Specificities of all designed primers were tested through qualitative PCRs. Clearly, target specific amplicons could be detected from disease resistant GM rice event. In addition, the limits of detection (LOD) using the event-specific primers were approximately 0.1% for the disease resistant GM rice line. This result indicated that the developed detection method is suitable for the traceability of disease resistant GM rice, because of using the primer specifically corresponded to the junction site between plant genomic DNA and inserted DNA. Keywords: genetically modified organisms, disease resistant GM rice, PCR detection, event-specific primer

Transgenic cymbidium plants containing drought and salt stresses tolerance genes were produced by using a highly efficient Agrobacterium-mediated transformation system. The gene (AtSZF2) is salt and drought stresses tolerant gene and transferred into cymbidium plants. These transgenic cymbidium plants are investigated for gene introduction by PCR and analyzed by salt and drought stresses to check its gene expression. To investigate the gene expression of AtSZF2, leaves of transgenic cymbidium plants were soaked in salt solution (200 mM NaCl). Also, transgenic cymbidium plants were kept under no watering for 6 weeks to check the expression of drought stress tolerance. As a result, wild type plants showed more damage than transgenic plants under salt treatment. Further, transgenic cymbidium plants retained green color and healthy status, while wild type plants showed no tolerance after 6 weeks of no-watering treatment.

For purposes of studying intron structures and predicting consensus splice motifs, a total of 102 legume species were used to isolate introns across the family. Of 196 gene-targeted PCR primer pairs, we successfully amplified 118 intron-containing genes (60.2%) and obtained a total of 1,870 introns with an average size of 143 nucleotides. Species-based compilation of 5’- and 3’-splicing motifs showed lineage-specific conservation in each splicing motif. Compilation of the entire intron set permitted prediction of the consensus sequences of splicing signal motifs in legumes, AYGWGTABABGH and TVNC/TAGGHTV for the 5’- and 3’-splicing motifs, respectively. Interestingly, these consensus motifs are very similar to the corresponding splicing signals of two model systems, Arabidopsis and rice. This result is suggestive of conservation of pre-mRNA splicing mechanisms in higher plants. Multiple alignments of CALTL introns demonstrated that the region from the branch point to 3’ splice site was relatively more conserved than the region from5’ splice site to the branch point. Phylogenetic analysis demonstrated that each of three splicing motifs, 5’-splice sites, 3’-splice sits, and branch site, was relevant to evolutionary divergence of species and phylogenetically informative, suggesting that splice signal sequences would be useful as a potential tool for the molecular phylogenetic analysis.

For genetic mapping studies, biparental segregating populations are often useful, however recombination is limited, giving rise to large genomic regions under QTL, and one can only study alelles present in both parents. In Wageninegn UR, a core collection is being developed representing all Brassica rapa morphotypes and geographic origins. As most B. rapa accessions are heterozygous and heterogeneous, we started a project to fix the collection through microspore culture. The resulting Diversity Fixed Foundation Set will be an interesting resource for association mapping studies, which have as advantage that they present the allelec variation present in the collection, and for mapping studies recombination is increased. Nineteen accessions of eight subspecies of Brassica rapa were used for microspore culture to developdoubled haploid lines. Eight morphotypes were represented: 3 Chinese cabbage, 2 Chinese turnip cabbage, 3 Pak choi, 5 Turnip, 3 Broccolleto, 1 Mizuna, 1 Komatsuna and 1 Turnip greenfrom the 19acessions examined, embryos were obtained for 13, representing six subspecies (Komatsuna and Turnip Green had no response). The embryo yields differed significantly between these 13 accessions. We regenerated normal plants from 10 accessions that survived more than 4 weeks in the soil using microspore culture. Nine accessions flowered after 4 weeks vernalization at 5℃ and seeds were harvested from 5 accessions. From a Mizuna, we obtained 3791 seeds from one plant and total 7318 seeds were harvested from 5 accessions representing 4 subspecies(Chinese cabbage, Chinese turnip cabbage, Pak choi, Mizuna). At present, we carry out experiment for obtain more seeds and induce embryos from the other plant materials.

Glycolysis is responsible for the conversion of glucose into pyruvate and for supplying reducing power and several metabolites. Fructose-1,6-bisphosphate aldolase (AtFBA1), a central enzyme in the glycolysis pathway, was isolated by functional complementation of the salt-sensitive phenotype of a calcineurin (CaN)-deficient yeast mutant. Under high salinity conditions, aldolase activity and the concentration of NADH were compromised. However, expression of AtFBA1 maintained aldolase activity and the NADH level in yeast cells. AtFBA1 shares a high degree of sequence identity with known class I type aldolases, and its expression was negatively regulated by stress conditions including NaCl. The fusion protein GFP-AtFBA1 was localized in the cytosol of Arabidopsis protoplasts. The seed germination and root elongation of AtFBA1 knock-out plants exhibited sensitivity to ABA and salt stress. These results indicate that AtFBA1 expression and aldolase activity is important for stress tolerance in yeast and plants.

Soybean seed contains a wide range of secondary metabolite compounds such as isoflavones, phyto-sterols, lecithins and saponins. The secondary metabolites are diverse in chemical structure and property. Therefore, it is not easy to analyze simultaneously the diverse metabolites. We assessed LC-MS profiling analysis to evaluate seed component diversity in 33 soybean cultivars and to identify diverse substances according to their fragmentation patterns. The 33 cultivars were divided clearly into two groups according to PCA of the profile data of seed components. The soluble extracts from hypocotyle as well as cotyledon in Group 1 were characterized by the presence of a compound with 969.5 m/z, while the extracts in Group 2 were characterized by the presence of a compound with 980.6 m/z. The two cultivars Williams 82 and Enrei were selected from each group, and then subjected to further analyses. PMF (Peptide MS Fingerprint) data generated by the Q-TOF analysis and MASCOT database search identified the compounds composed of 37 amino acids as the 4-kDa peptide (Albumin 1b). Substitution of three amino acids was found between the two groups. Three candidate genomic sequences were distributed on soybean genome. Expression analysis by RT-PCR indicated one of the three sequences encodes the 4-kDa peptide and expressed in developing seed. In this study, we confirmed the comprehensive analysis with LC-MS is a powerful tool to elucidate metabolite diversity in plant materials including soybean seed.

Rice stripe disease, caused by rice stripe virus (RSV), is one of the major virus diseases in east Asia. The objective of this study was conducted to identify new resistance genetic source to rice stripe virus (RSV) disease. Genetic diversity of 155 rice cultivars was evaluated using 9 co-dominant InDel markers and STS marker ST10. These cultivars were classified into two groups by cluster analysis based on Nei`s genetic distances. The marker showed different band pattern among RSV resistance or susceptible cultivar. In comparison with bioassay for RSV resistance and genotyping using SSR markers showed that Stv-bi and InDel 7 marker observed recombination value within 3.8% and RSV resistance gene was closely related to InDel 7. Also InDel 7 divided as resistance type alleles and susceptible type alleles except for some varieties. Interestingly, 02428, Daw dam, Erguailai, Padi Adongdumarat, PERVOMAJSZKIJ, and Tung Ting Wan Hien 1 showed Japonica type in InDel 7 marker. However, these cultivars revealed resistant to RSV bioassay. These results indicate that those cultivar can be able to get the different gene resistance with Stv-bi gene. Newly identified resistance gene is considered useful for improving RSV resistance in japonica rice. Therefore, we will progress the allelism test and genetic analysis for identification of new gene source.

Phomopsis seed decay (PSD), primarily caused by Phomopsis longicolla, is a major contributor to poor soybean seed quality and significant yield loss, particularly in early maturing soybean genotypes. However, it is not yet known whether PSD resistance is associated with early maturity. This study was conducted to identify quantitative trait loci (QTLs) for resistance to PSD and maturity time using a recombinant inbred line (RIL) population derived from a cross between the PSD-resistant Taekwangkong and the PSD-susceptible SS2-2. Based on a genetic linkage map incorporating 117 simple sequence repeat markers, QTL analysis revealed two and three QTLs conferring PSD resistance and maturity time, respectively, in the RIL population. Two QTLs (PSD-6-1 and PSD-10-2) for PSD resistance were identified in the intervals of Satt100-Satt460 and Sat_038-Satt243 on chromosomes (Chrs) 6 and 10, respectively. These QTLs do not overlap with any previously reported loci for PSD resistance in other soybean genotypes. Two QTLs explained phenotypic variances in PSD resistance of 46.3% and 14.1%, respectively. Among three QTLs for maturity time, two (Mat-6-2 and Mat-10-3) were located at positions similar to the PSD resistance QTLs. The identification of the QTLs linked to both PSD resistance and maturity time indicates a biological correlation between these two traits. The newly identified QTLs for resistance to PSD associated with maturity time in Taekwangkong will help improve soybean resistance to P. longicolla.

Panax ginseng C.A. Meyer, commonly known as Korean or Asian ginseng, is a perennial herb which is native to Korea and China. Its roots are highly prized for several medicinal properties. Therefore, Ginseng has been a top-ranked subject of many fields of scientific research worldwide. However, very limited number of research work has been published on species authentication using DNA marker system. In this study, 22 simple sequence repeat (SSR) markers were used to analyze the genetic diversity and population structure of 167 ginseng cultivars from 11 regions and 10 breed varieties. A total number of 111 alleles were detected, with an average of 5.05 per locus. The average expected heterozygosity and polymorphism information content (PIC) for SSR locus were 0.35 and 0.30, respectively. The model-based structure analysis revealed that 66.5% of all cultivars could be grouped into three populations with inferred value (allele shared >70%) membership. More than 33% of tested cultivars derived from two ancestries, which was basically consistent with clustering based on genetic distance. Almost all of the cultivars shared the ancestry with S1 and S2 except 1 China Jilin and 3 USA cultivars. The result indicated that most of Korean ginsengs are closely interrelated between the two ancestors but USA ginsengs are totally different from Asian cultivars.

Next generation sequencing (NGS) approaches can also be useful tool for characterization of organelle genomes. We generated chloroplast (CP) genome sequences of two Korean ginseng cultivars, Chunpoong and Yunpoong, based on reference-guided assembly using whole genome NGS data. We used 0.5x of P. ginseng genome NGS reads to assemble CP genome. Of the NGS reads used, about 6% were mapped to the reference CP genome with mean coverage of 94x due to high copy number of CP genome in plant cell. CP genomes of the two cultivars were predicted to be 156,248 bp and 156,355 bp in length and showed about 0.1% differences at nucleotide level, compared to reference CP genome sequenced from P. ginseng (Acc.no. NC_006290), whereas difference between CP genomes of the two cultivars is very rare. In this study, we developed the molecular marker to perform taxon identification and also to elucidate phylogenetic relationship among Korean ginseng cultivars. Now, we are analyzing the CP genomes of other P. ginseng cultivars together with other Panax species including American ginseng and Panax related species.