Plant & Animal Genomes XIV Conference

January 14-18, 2006
Town & Country Convention Center
San Diego, CA

Development Of Traitexplorer^Tm, A High-Throughput Process Of in vivo Screening Of Genomic Fragments From Higher Plants

Yoshimitsu Takakura¹ , Satoru Usami¹ , Yuko Hiei¹ , Yuji Ishida¹ , Toshiyuki Komori¹ , Toshiki Mine¹ , Masakazu Kashihara¹ , Ichiro Oka¹ , Naoki Takemori¹ , Jun Ueki¹ , Tomoaki Kubo² , Norio Katoh ¹ , Toshihiko Komari¹

¹  Plant Innovation Center, JAPAN TOBACCO INC., 700 Higashibara, Iwata, Shizuoka, 438-0802, JAPAN

²  National Institute of Crop Science, National Agriculture and Bio-oriented Research Organization, 2-1-18 Kannondai, Tsukuba, Ibaraki, 305-8518, Japan

Importance of non-protein-coding sequences as well as of alternative splicing of protein-coding sequences have been drawing considerable attention in studies of eukaryotic genomes, highlighting the value of functional analysis of genomic fragments. We have initiated a trial to screen genomic fragments of higher plants that cause phenotypic changes when introduced into plants. Genomic libraries were constructed from various Gramineae species, such as Oryza rufipogon, Rhodes grass, sorghum, Sudan grass, Italian millet and teosinte, using plant-transformation-competent cosmid vectors. The size of inserts was between about 30 kb and 40 kb. The clones were individually transferred from Escherichia coli to Agrobacterium tumefaciens LBA4404. Japonica rice cultivar Yukihikari was transformed individually with the A. tumefaciens strains, which carried the large genomic fragments. The steps of handling of clones and bacteria, rice transformation, high-density cultivation of transgenic rice in glasshouses and evaluation of plants have all been optimized to make the total throughput very high. The transfer of large genomic segments from E. coli to A. tumefaciens and from A. tumefaciens to rice was confirmed by PCR and Southern blot analysis. In the case of a genomic library of O. rufipogon, 5310 clones were introduced into rice. Thus, a given DNA segment in O. rufipogon may be found in this population of transgenic rice at a probability of 0.4. This screening system was designated as TraitExplorer^TM because, once agronomically useful phenotypes are identified, such characteristics may be exploited as new traits in crop improvement.