The Asteraceae/Compositae family is one of the biggest families in flowering plants and has more than 23000 species including the economically important lettuce, sunflower, and chicory as well as the agronomic weeds. With its significance and the progress in sequencing technology, its species have been subjected to the genome sequencing project worldwide. Although chrysanthemum is an important plant in the floricultural industry, however, it has been less studied at the level of genomics, compared with other species in the Asteraceae. There were only several reports on comparative analysis of transcriptome for chrysanthemum. Actually, the genome of Chrysanthemum species is known to be gigantic and complex with diverse status ranging from diploid to decaploid. Since the cultivated and commercial chrysanthemum exhibits hexaploid genome, we decided to select the diploid species with smaller genome as a material for reference genome sequencing. Thus, we launched a genome sequencing project with C. boreale which was previously reported to be diploid by cytogenetic analysis. We constructed sequencing libraries with insert size 300bp and 500bp and sequenced them from the paired end in 100bp read length with Illumina’s HiSeq platform. After quality checking, we preprocessed raw reads by removing duplicated reads and trimming reads with low quality value. Kmer frequency analysis with the cleaned reads showed that the genome is heterozygous, highly repetitive and gigantic, ranging from 2.9Gb to 5.8Gb. The cleaned reads were further subjected to error correction and primary assembly with SOAPdenovo2. Here, we’ll report the result of Kmer frequency analysis and genome assembly.

Understanding how crops interact with their environments is increasingly important in breeding program, especially in light of highly anticipated climate changes. A total of 150 recombinant inbred lines (RILs) of F12 generation derived from Dasanbyeo (Indica) x TR22183 (Japonica) were evaluated at Suwon 2010, Shanghai 2010, IRRI 2010 wet season, Suwon 2011, Shanghai 2011, IRRI 2011 dry season, and IRRI 2011 wet season as a total of seven diverse environments. Traits evaluation included eight important agronomical traits such as days to heading (DTH), culm length (CL), panicle length (PL), panicle number per plant (PN), spikelet number per panicle (SN), spikelet fertility (SF), 100-grain weight (GW), and grain yield (GY). As a result of genotyping using 384-plex GoldenGate oligo pool assay (OPA) set (RiceOPA3.1), the linkage map for 235 SNP markers covering a total of 926.53 cM with an average interval of 4.01 cM was constructed and a total of 44 main-effect quantitative trait loci (QTL)s and 35 QTLs by environment interaction (QEI) were detected for all eight traits using single environment and multi-environments analysis, respectively. Of these, fourteen putative QTLs for DTH, CL, PN, SN, GW and GY found in single environment analysis had the similar position to QEI for those traits, suggesting that these same QTLs from both single-and multi-environments are major and stable for certain traits. To the best of our knowledge, 12 QTLs consisted of four QTLs for CL (qCL2, qCL8.1, qCL8.2, and qCL8.3), six QTLs for GW (qGW3.1, qGW3.2, qGW7, qGW8, qGW10.1, and qGW10.2), one QTL for GY (qGY3) and one for SF (qSF4) out of 44 QTLs obtained from single environment analysis were considered to be novel since no overlapping QTL was reported from previous studies. In addition, 12 out of 35 QTLs obtained from multi-environments analysis were also novel.

Molecular characterization of crops improved through biotechnology has traditionally been conducted using Southern blot analysis which has been used to determine T-DNA copy number, the presence or absence of backbone (sequence outside of the T-DNA) and to demonstrate generational stability of the T-DNA insert. The advancement of high-throughput DNA sequencing (HTS) technology allows efficient characterization of the transgene incorportated into the genome of the plant by rapidly sequencing the entire plant genome. By combining NGS (Next Generation Sequencing) technologies with bioinformatic methods that identify the T-DNA insert derived from the plasmid vector and genome-T-DNA junction sequences, it has been shown that conclusions equivalent to those of a Southern blot are readily obtained. NGS is done at sufficient coverage depth (>75x) across the entire genome. By mapping the sequence reads to the plasmid vector, and identifying the number of unique junctions, we can confirm insert number, copy number, absence of backbone, across multiple generations. With the widespread availability of NGS and steadily decreasing costs it is likely that academia and industry will fully transition to NGS-based molecular characterizations in the near future.

Leaf mold disease in tomato (Solanum lycopersicum) is caused by Cladosporium fulvum, a fungal leaf pathogen. One of effective ways to control leaf mold is to breed disease-resistant tomato cultivars. Cf-4 and Cf-9 resistance (R) genes encode proteins that carry a leucine rich repeat domain and are located in plasma membrane. They trigger hypersensitive response following recognition of corresponding Avr4 and Avr9 proteins of C. fulvum, respectively. Cf-4 and Cf-9 genes are originated from wild tomato species S. habrochaites and S. pimpinellifolium and have been introgressed into commercial tomato cultivars. These two highly homologous orthologs exist as a cluster with four highly homologous paralogs. Due to this reason, development of genetic markers to distinguish these two functional R genes from their orthologs and paralogs is difficult. In this study, we tried to develop single-nucleotide polymorphism (SNP) markers to select tomato cultivars carrying resistant Cf-9 genotype. The genomic sequences of resistant Cf-4 and Cf-9 alleles, susceptible cf-9 alleles, and their paralogs were obtained from the GenBank database, and two functional SNPs causing non-synonymous substitution were found among them. Based on two SNPs, the Cf-9_2-SNP-F/R primer set for high resolution melting (HRM) analysis was developed. HRM analysis with this primer set could successfully distinguish tomato cultivars carrying resistant Cf-9 allele among 30 commercial tomato cultivars, which were characterized with the gene-based marker. These indicate that the SNP marker developed in this study is useful to trace Cf-9 genotype efficiently in marker-assisted selection in tomato.

Proline has been shown to accumulate in plant under various type of stresses. In our previous study, changes in cold hardiness and proline content showed contrasting patterns during a constant deacclimation. This study was performed to investigate the proline accumulation and related gene expression in response to repeated deacclimation and reacclimation in peach cultivar ‘Daewol’. Proline content was analyzed using the ninhydrin method and related gene expressions were examined using quantitative real-time RT-PCR. Proline contents of ‘Daewol’ increased during the repeated deacclimation treatments. Interestingly, during the twice deacclimation, expressions of P5CS (Δ1-pyrroline-5-carboxylatesynthase) constantly decreased, whereas expressions of P5CR (Δ1-pyrroline-5-carboxylatereductase) increased. Expressions of OAT (ornithine-δ-aminotransferase) indicated up- and down- pattern in response to repeated deacclimation and reacclimation. Our results indicated that proline responds positively to higher temperature in the shoots of peach cultivar ‘Daewol’ and expressions of both P5CS and P5CR genes could show contrasting patterns during the deacclimation. Moreover, our results suggest that ornithine pathway could serve as an alternative pathway in proline synthesis process during deacclimation in peach.

Tomato spotted wilt virus (TSWV) causes one of the most destructive viral diseases that threaten tomato (Solanum lycopersicum) worldwide. So far, eight TSWV resistance genes, Sw1a, Sw1b, sw2, sw3, sw4, Sw-5b, Sw-6, and Sw-7 have been identified and Sw-5b has been incorporated into tomato for prevention of TSWV. The objectives of this research are first to discover single nucleotide polymorphisms (SNPs) in Sw-5 alleles and then to develop SNP markers to distinguish resistant genotypes against TSWV for marker-assisted breeding in tomato. First, DNA sequences of Sw-5b alleles from both resistant and susceptible cultivars amplified using known Sw-5 gene-based marker was analyzed. The single functional SNP (G→A) was detected as non-synonymous substitution because this SNP causes change of arginine (Arg599) to glutamine (Gln599). Next, the primer pair for high resolution melting analysis (HRM) was designed around this SNP. To determine accuracy of this SNP marker to distinguish resistant Sw-5b genotypes against TSWV, genotypes of 32 commercial tomato cultivars were checked. The newly developed SNP marker could select six cultivars carrying resistant Sw-5b genotype, which was 100% correlated with genotypes based on the gene-based marker. These results indicate that the SNP maker developed in this study could be useful for better tracking resistance to TSWV in tomato breeding.

Genetically modified (GM) crops have never been cultivated commercially in Korea, it is necessary for a thorough assessment of the risks associated with their environmental release. We determined the frequency of pollen mediated gene flow from disease resistant GM rice (OsCK1) to non-GM rice (Nagdongbyeo) and weedy rice (R55). A total of 449,711 or 164,604 seeds were collected from non-GM and weedy rice, respectively which were planted around OsCK1. Resistance of the hybrids was determined by repeated spraying of herbicide and DNA analysis using specific primer to confirm hybrids. Though non-GM rice and weedy rice have similar flowering time, the hybrids were found only in non-GM rice and out-crossing ranged from 0.018% at 0.3 m to 0.013% at 0.6 m. All of hybrids were located within 0.6 m distance from the GM rice plot in southerly direction. The meteorological factors including temperature and relative humidity during flowering time were found to be the most important factors for determining rice out-crossing. It should be considered many factors like the local weather condition and flowering time to set up the safety management policy to prevent pollen mediated gene flow between GM and conventional crop.

The β-carotene biofortified transgenic rice was developed by transforming rice cv. Nakdongbyeo with phytoene synthase (Psy) and carotene desaturase (Crt I) genes isolated from Capsicum and Pantoea. The aim of this study was to perform molecular characterization of rice transformants of T5-T7 generation harboring Psy and Ctr I genes driven by endosperm specific globulin promoter for biosafety evaluation of β-carotene biofortified transgenic rice. The structure and sequence of T-DNA in the transformation vector and the insertion sites, flanking sequences and generational stability of inserted T-DNA in transgenic rice lines were analyzed. The transformation vector consisted of right border, MAR gene, carotenogenic genes unit, herbicide resistance selectable marker unit, MAR gene and left border in sequential order. T-DNA was introduced at the position of 30,363,938-30,363,973 bp of chromosome No. 2 by adaptor-ligation PCR. Stable integration of T-DNA and stable expression of bar gene was confirmed in T5 to T7 generations. It was also confirmed that the backbone DNA of transformation vector containing antibacterial gene was not present in the genome of β-carotene biofortified transgenic rice. HPLC analysis confirmed that carotenoids were consistently detected through T5-T7 generations.

Proline (Pro) accumulation is a common physiological reaction in response to abiotic stresses in many plants. Accumulation of Pro is believed to play the important role in protecting cellular components from dehydrating effects due to such stresses. The study was performed to investigate the relationship between cold hardiness and Pro content or expression of related genes in peach cultivars during a constant experimental deacclimation. Changes in cold hardiness were determined using electrolyte leakage method in the shoots of 10 peach cultivars (Prunus persica ‘Aikawanakajima’, ‘Chiyomaru’, ‘Daewol’, ‘Janghowon Hwangdo’, ‘Kiraranokiwami’, ‘Mihong’, ‘Misshong’, ‘Soomee’, ‘Suhong’, and ‘Sun Gold’). Pro content was analyzed using the ninhydrin method and related gene expressions were examined using quantitative real-time RT-PCR. While cold hardiness of 10 peach cultivars decreased, Pro contents of those increased during the deacclimation. Notably, at the same time, expression of P5CS (Δ1-pyrroline-5-carboxylatesynthase) decreased in 10 peach cultivars, whereas expressions of P5CR (Δ1-pyrroline-5-carboxylatereductase) and OAT (ornithine-δ-aminotransferase) increased. Our results demonstrate that Pro responds positively to higher temperature in the shoots of 10 peach cultivars and expression of both P5CS and P5CR genes could show contrasting patterns during the deacclimation. Furthermore, our results suggest that ornithine pathway, which has been suggested to be important during seedling development, could serve as an alternative pathway in Pro synthesis process during the deacclimation in peach.

In here, we screened drought tolerant varieties with modified leaf water loss rate assay and visual drought tolerant phenotype in the greenhouse conditions with more than 800 varieties. Among these varieties, Samgang, Gumei4 and Apo showed the lowest of leaf water loss rate and strong drought tolerant phenotype. To identify drought QTLs with Samgang variety, we developed the doubled-haploid (DH) population consist of 101 lines derived from a cross the drought tolerant cultivar Samgang and the drought sensitive cultivar Nagdong. To score the drought phenotype degrees of this population, we withheld water for 6 weeks and treated the watering for 7 days. After watering, visual phenotype was observed 1 to 9 degree according to the standard evaluation system for rice, IRRI. Drought sensitive parent Nagdong was almost died and was scored as 9 degree, while tolerant parent Samgang showed slightly leaf tip drying phenotype and was scored as 3 degree in our experimental conditions. Three main QTLs were detected on chromosome 2, 6, and 11 with this visual phenotype. We also measured relative water contend of these population under drought stress conditions, and got one main QTL on chromosome 11. The QTL loci on chromosome 11 with flanking markers RM26755-RM287 has a function for visual phenotype and relative water content under drought conditions.

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.

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.

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.

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.