Development of Transposon Tagged Insertional Mutant Library in Tomato for Functional Genome Analysis

Tomato (Lycopersicon esculentum), an important crop plant enriched with wide range of genetic resources and fully sequenced genome, is a model system for fruit ripening and disease resistance studies in plants. Although TILLING population is available in tomato for functional analysis, but T-DNA and/or transposon tagged knockout lines, enhancer trap lines etc. are equally important genetic resources for deciphering gene function. In addition to the development of TILLING population, we initiated to develop a genome wide transposon tagged insertional mutant library in tomato for functional analysis. To accomplish this we are exploiting Ac-Ds transposon elements of maize, which have been earlier shown to work in tomato. The constructs used for rice insertional mutagenesis were suitably modified using a dicot promoter and kanamycin selection gene to efficiently function in tomato. Our approach involves making an efficient Ac transposase tagged line and large number of Ds transposon tagged lines. The insertion of Ds transposon into different locations would be achieved by crossing the Ac transposase line with a library of Ds transposon tagged starter lines and F₂ progeny would be selected for lines that have a Ds transposon tag in the genome but are free of Ac transposase.  Transposase and transposon tagged lines would be screened with the help of GFP and RFP reporter genes. The gene loci bearing insertion site would be identified by TAIL-PCR.  The long term goal of this work is to make a genome resource that can be used by tomato community for functional validation of genes. The current status of above strategy regarding generation of Ac transposase and Ds transposon tagged lines would be presented.