It is necessary to alleviate environmental and economic disadvantages of fossil fuels for global warming. Among the conceivable options, the use of plant biomass for the production of bioethanol is considered as a potential alternative for fossil fuels. Plant biomass that contains lignocellulose for bioethanol production has recently emerged as biofuel feedstock because of its sustainable and environment-friendly properties. However, lignin inhibits the hydrolysis process and the lignin recalcitrance in ethanol conversion remains in a problem. The attempt for down-regulating enzymes involved in lignin biosynthesis is one of attractive strategy to reduce the lignin contents. Recently, Brachypodium distachyon has been proposed as an alternative monocotyledon model species. The close phylogenetic relationship of Brachypodium with other grasses suggests that the Brachypodium may be useful for structural and functional genomic studies in these species. Brachypodium, standard line Bd21, was subjected to irradiation at doses of 50, 100, 150, 200, and 250 Gy. Phenotypes were investigated using M0:2 population. Through histochemical analysis using phloroglucinol, 25 M2 putative lignin deficient mutants were selected. Depend on the phenotypic and histochemical data, mutants were selected and used for measuring lignin content. Total lignin content was measured using the acetyl bromide (AcBr). Mutant #142-3-1 contains 16.9 (mg/g dry cell wall) of total lignin and the lignin level was significantly reduced (87.9%) compared to wild-type (19.23 mg/g dry cell wall). Additionally, Mutant line #2259-1-2 reduced lignin level at 94.4% (18.15 mg/g dry cell wall) in comparison to wild-type. The enzymatic hydrolyses in lignin deficient lines have been performing with the time courses. Lignin composition, cell wall carbohydrates, and genetic analysis in mutant lines will be discussed.

Gliadins are the main class of wheat seed storage proteins. Since gliadins show a high level of polymorphism as well as genetically fixed, it can be used as a marker for the genetic identification. Gliadin subunit diversity information can be useful for wheat quality breeding programs. Tunisia is a country in the North Africa bordered with the Mediterranean Sea to the north and the east but with the Sahara desert to the south, which represent extremely different growth climate. Therefore, there may be a numerous variation in Tunisian common wheat and durum wheat. Total 48 lines of wheat consisted of 32 common wheat (16 Korean wheat and 16 Tunisian common wheat) and 16 Tunisian durum wheat were incorporated in this study. Gliadins were extracted with 70% ethanol and fractionated by acid polyacrylamide gel electrophoresis (A-PAGE) at 8% in aluminum lactate buffer (pH 3.1). The gel was stained with 0.25% Coomassie Brilliant Blue (CBB) R-250. The presence of each gliadin subunit band was scored and cluster analysis was carried out. The cluster showed that wheat varieties were classified into some groups and their genetic distance could be identified. The obtained information will be helpful to the future breeding program of tetraploid durum wheat as well as hexaploid common wheats. Acknowledgement: This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2012K1A3A1A09028123) and carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Development (Project title: Development of high yielding wheat with stress tolerance via molecular breeding strategies, Project No. PJ008031)”, Rural Development Administration, Republic of Korea.

Brachypodium distachyon is a temperate annual grass that has a short life cycle, a small genome size, self fertility, and a small physical stature. The relationship with major cereal crop including wheat, Brachypodium is considered as a monocot model plant. Recently, the cell wall composition of Brachypodium is reported closely related with maize and Miscanthus giganteus. Therefore, Brachypodium is emrging as a powerful model plant for bioethanol production. Here, Brachypodium was chronically irradiated with the doses of 50 Gy, 100 Gy, 150 Gy, 200Gy, 250Gy, and 300 Gy. Plant height and fresh weight were observed dosage-dependent negative effect. However, tiller number and internode diameter were found to be increased their value as compared to control. The cell wall yield showed a decreased tendency with dosage-dependent negative, but cell wall yield of 50 Gy and 200 Gy were detected higher than control. The lignin content of irradiated Brachypodium stem was reduced with dosage incease The ratios of lignin content to control were 97.6% (50 Gy), 91.9 (100 Gy), 87.3% (150 Gy), 89.4% (200 Gy), 81.6% (250 Gy), 85.2% (300 Gy). SEM image analysis demonstrated that cell size of 300 Gy plant was decreased by 45% of control. RT-PCR was performed to analyze transcript accumulation of lignin pathway related genes with irradiated Brachypodium stem. CCR, PAL, C4H, and 4CL were detected at least 2 times higher expression than control at 150 Gy, 200 Gy, 250 Gy. The preteatment and enzyme hydrolysis will be discussed for bioethanol production.

Brachypodium has been focused as new model plant for grass species. Like small size, small room requirement, and fast growth, Brachypodium shows numerous advantages as a model plant. Brachypodium is a typical grass at the genome level, which also exhibits an overall similarity of gene content and gene families when compared with rice (Oryza sativa) and sorghum (Sorghum bicolor) genomes. Brachypodium is an excellent material for structural and functional genomic studies in grass species. Targeting-Induced Local Lesions IN Genomes (TILLING) is a high-throughput technique and an approach for reverse genetics study. Moreover, it has been wildly utilized to find induced mutation. Bradi3g45515 is orthologue of the cellulose synthase-like HvCslF8 in barley. For TILLING library construction, 384 M2 Brachypodium mutants induced by chronic-gamma irradiation were used. Single nucleotide polymorphysm (SNP) and small deletion in Bradi3g45515 were searched through TILLING analysis. Template DNA for PCR reaction were prepared according to two dimensional pooling (eightfold) strategy. Heteroduplex DNA was digested by SURVEYOR nuclease (TRANSGENOMIC) and the DNA fragment was detected using polyacrylamide gel electrophoresis. Positive signal appeared at polyacrylamide gel from more than 4 lines and their Bradi3g45515 region were sequenced. SNP(s) were identified in 509-2 and 677-3 mutant line. Cellulose content and/or cell wall materials content will be measured using these mutants.

Mutant analysis is one of most optimized genome-wide approach towards acquiring utile phenotypes and defining related genes. Gamma-irradiation, an acknowledged way of mutant-generating method, was applied to gain sets of mutant line in Brachypodium distachyon. B. distachyon is a model plant, commonly used in genus of Gramineae for the research of structure genomics and functional genomics. B. distachyon contribute to rapid and easy analysis because of its small size and quick growth. Mutant population was generated by different doses of gamma-irradiation (0, 50, 100, 150, 200, 250 Gy) in the gamma field phytotron. Distance from the source gives same irradiation duration for each plant. Plant growth parameters such as plant height, tiller number, leaf length & width, internode number & diameter, maturity and yield components (ear number biomass) were scored on M0 plants. Plant responses to different doses of radiation are evaluated and the effective radiation dosages to generate mutant using gamma-phytotron are suggested. Chronic irradiation using gamma-phytotron is useful tool to generate mutants for genomic variations such as SNP or INDEL as well as suitable for functional study of genes in Gramineae.

Rye has important genes for biotic and abiotic stress resistance. Introduction of these genes to wheat by breeding wheat-rye translocation have been intensively used in wheat breeding program. Rye chromatin 1RS and/or 2RL show superior performance in unfavorable environments. In order to develop high yielding wheat, we applied various molecular breeding strategies. To develop EST-derived 1RS specific markers, we used comparative genomics with public sequence databases of Poaceae family. Putative rye chromatin specific sequences were used to design 1RS specific markers. To identify genes related to water deficiency, cDNA AFLP analysis was used in PEG treated seedlings of 1RS RILs. For functional analysis of identified genes and markers, we used Brachypodium distachyon, as a new model plant of temperate grasses. B. distachyon were recently applied for transformation and we constructed Agrobacterium-mediated transformation system. Integration of those strategies and conventional breeding method would enhance the usefulness of rye chromatins for wheat improvement.

Brachypodium distachyon is rapidly emerged in biological study and has been currently used as a model system for genetics and functional studies for crop improvement and biofuel production. Phosphinothricin (PPT) has been widely used as a selectable agent, which raises ammonium content and induces toxicity in non-transformed plant cells. However PPT selection is not much effective on Brachypodium callus consequently reducing transformation efficiency. In order to identify the efficient conditions of PPT selection, calli obtained from mature seeds of Brachypodium (PI 254867) were cultured on the callus inducing medium (CIM) or regeneration medium (ReM) containing serial dilutions of the PPT (0, 2, 5, 10, and 15 mg/l) in dark or light condition. Callus growth and ammonium content of each treatment were measured 2 weeks after the treatment. Although callus growth and ammonium content did not show much difference in CIM, slow callus growth and increased ammonium accumulation were found in ReM. No significant difference of ammonium accumulation in response to PPT was found between dark and light conditions. In order to identify major factors affecting increased ammonium accumulation, callus was cultured on the media in combined with phytohormones (2,4-D or kinetin) and carbon sources (sucrose or maltose) containing with PPT (5 mg/l). The highest ammonium content in callus was found in the kinetin and maltose media.

Glutamine synthetase (GS) plays important roles in plants like assimilation of ammonium and detoxification of the ammonium released from many metabolic processes such as amino acid degradation or photorespiration. Using ATP, ammonia is combined with glutamate to yield glutamine by the action of GS. Phosphinothricin (PPT) is widely used as a herbicide because it competes with glutamate to bind the active site of GS. PPT has been used to produce transgenic Brachypodium distachyon callus and plants as a selectable agent. PPT treatment raises ammonium content and induces toxicity in non-transformed cells. To find out efficient condition for selecting transformed callus, ammonium content were measured in this study. Non-transformed callus were derived from mature seeds of Brachypodium distachyon (Bd21). The callus were cultured on the callus inducing media (CIM) or regeneration media (RM) containing serial dilutions of the PPT (2, 5, 10 and 15 mg/l) with or without light. Ammonium content was measured 2 weeks after PPT application. Ammonium toxicity associated with PPT treatment was dose-dependent on RM whereas PPT treatment was not significantly influenced on CIM. There is no influence on dark or light condition. Additionally, callus were cultured on the media containing phytohormones combined with PPT (5 mg/l) and the most affecting element causing increased ammonium content has been identified. Acknowledgements: This work was supported by a grant (No. 20070301-034-016-007) from BioGreen 21 Program, RDA, Republic of Korea.

Rye (Secale cereale L.) chromatins have been used to introduce agronomically important traits into wheat (Triticum aestivum L.). Wheat-rye translocations in the form of 1RS.1AL, 1RS.1BL, 2BS.2RL have been developed for an important genetic source of disease and pest resistance. The long arm of rye chromosome 2 (2RL) has valuable genes that confer resistance to pests such as biotype L of Hessian fly, powdery mildew, leaf and stem rust. Here, we report the generation and analysis of expressed sequence tags from Hessian fly infested wheat-rye translocation. RNAs were isolated from young seedlings infested by Hessian fly. cDNA library was constructed using Clontech cDNA library construction kit. Random sequencing of candidate clones were performed. The EST clones might be useful to clone target gene sequences and would provide clues on molecular interaction between wheat and Hessian fly.

barley grain and malt is highly related to beer quality, especially hordein is known to be a more significant factor in malting process than albumin. In this study, we proposed selection criteria for high quality malting barley with aid of grain and malt quality parameter scores and storage protein subunit profile informations. Albumin and hordein were extracted and denatured protein subunits were evaluated with malt and grain quality parameters. Total 13 local adaptability test (LAT) lines were planted in four locations (Naju, Iksan, Jeju, and Jinju) and evaluated for malt and beer making qualities. Seventeen germplasms (world collections for high or low seed storage protein content) were also evaluated for biochemical genetic marker. Denatured seed storage protein subunits of albumin and hordein of all tested lines and germplasms were evaluated using 12% 1D SDS-PAGE. Scored data of protein subunit's presence or absence was applied to Agglomerative Hierarchical Clustering (AHC) for statistical analysis. Subunits fractionated within specific molecular weight ranges (97.4-31.0, 66.2-31.0, and 45.0-31.0 kDa) were highly correlated with agricultural characteristics. Several LAT lines showing good performance in agricultural characteristics were clustered in dendrogram constructed by biochemical-genetic assay using XLSTAT. Specific band pattern showed in good performance LAT lines were also observed in some germplasms of world collection having low protein contents which are known to have superior quality in malting. The results would provide selection criteria for high quality malting barley in the malting barley breeding program.

UNUSUAL FLORAL ORGAN (UFO), a novel gene, is involved in controlling flowering initiation and development. In Arabidopsis, UFO is required for floral organ identity in the second and third whorls. However, the mode of expression and function of TaUFO have not been studied yet. The cDNA sequence of TaUFO is comprised of 1344 bp open reading frame which encodes 50.82 KDa polypeptide consisting amino acid residues. F-box protein, the components of TaUFO, plays an important regulatory role in a wide diversity of developmental and physiological responses. In almost all F box proteins, the N terminus of the protein contains the F-box motif, and the rest of the protein contains the protein-protein interaction domains required for target protein binding. In order to elucidate the function of the TaUFO, various phytohormones and abiotic stresses were applied on young seedlings (14 day after germination) and its transcripts were evaluated. TaUFO:GFP fusion construct was transformed into onion epidermal cells by particle bombardment to elucidate the subcellular localization of the TaUFO protein. The function of the F-box protein is to interact with target proteins. With the use of a yeast two-hybrid screen to isolate proteins interacting with the TaUFO (F box protein), we identified potential TaUFO interactive protein in wheat spikelet library.