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A collection of 50,000 Tos17-induced mutant rice lines carrying about 250,000 independent insertions was generated. DNA pools derived from 50,000 lines have been produced for polymerase chain reaction (PCR)–based reverse genetics screening. For in silico screening of mutants of genes of interest, a large-scale analysis of the mutants by sequencing the genomic DNA sequence flanking Tos17 insertions is in progress. To facilitate the functional analysis, the database on phenotypes covering all the mutant lines has been developed. About half of the mutant lines exhibited at least one phenotype. About 5–10% of the mutations were shown to be caused by insertion of Tos17, whereas the rest of the mutations were deletions, possibly caused by double-strand break repair and point mutations. These deletion mutations can be detected by the PCR-based screening method, providing a new resource for functional analysis of genes. Considering gene redundancy in rice and the availability of a large number of full-length cDNAs, we have begun producing a new type of activation tagged lines in which 15,000 independent normalized full-length cDNAs are overexpressed under the control of the ubiquitin promoter.

We have generated 47,932 T-DNA tag lines in japonica rice using activation tagging vectors that contain tetramerized 35S enhancer sequences. To facilitate use of those lines, we isolated the genomic sequences flanking the inserted T-DNA via inverse polymerase chain reaction. For most of the lines, we performed four sets of amplifications using two different restriction enzymes toward both directions. In analyzing 41,234 lines, we obtained 27,621 flanking sequence tags (FSTs), among which 12,505 were integrated into genic regions and 15,116 into intergenic regions. Mapping of the FSTs on chromosomes revealed that T-DNA integration frequency was generally proportional to chromosome size. However, T-DNA insertions were nonuniformly distributed on each chromosome, that is, higher at the distal ends and lower in regions close to the centromeres. In addition, several regions showed extreme peaks and valleys of insertion frequency, suggesting hot and cold spots for T-DNA integration. The density of insertion events was somewhat correlated with the expressed, rather than the predicted, gene density along each chromosome. Analyses of expression patterns near the inserted enhancer showed that at least half the test lines displayed greater expression of the tagged genes. Although in most of the increased lines expression patterns after activation were similar to those in the wild type, thereby maintaining the endogenous patterns, the remaining lines showed changes in expression in the activation tagged lines. In this case, ectopic expression was most frequently observed in mature leaves. Currently, the database can be searched with the gene locus number or location on the chromosome at www.postech.ac.kr/life/pfg/risd. Upon request, seeds of the T₁ or T₂ plants will be provided to the scientific community.