Tissue culture–induced mutations and overexpression of full-length cDNAs as a tool for functional analysis of rice genes

Abstract

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.