In the frame of a multi institutional effort (Cirad, Cnrs, Ird, Inra) supported by Genoplante a library of 30,000 T-DNA insertion lines has been generated through Agrobacterium co-culture of embryogenic calli in the standard cultivar Nipponbare, -the genome of which has been used for the international project of sequencing (IRGSP 2005)-(Sallaud et al. 2004). These lines are being field evaluated in a collaborative effort with the international centre CIAT (Centro Internacional de Agricultura Tropical based in Cali, Colombia) for seed amplification and detection of phenotypes while other mutant screens (e.g. pathogens and grain filling) and validation of gene functions are underway. A collection of 20,000 Flanking Sequence Tags has been generated from the left borders of T-DNA inserts in the library.

As the rice transformation procedure includes short duration tissue culture, the 30,000 lines of the Genoplante library contain an estimated total of 90,000 novel Tos17 insertions (3.2 on average per line). A novel method of high-throughput selective amplification of flanking regions of newly transposed Tos17 - called TOSTRAP - has been developed. Using the TOSTRAP protocol we amplified flanking sequences from at least one newly-transposed copy of Tos17 in 20,000 lines. We typically generated PCR fragments of size ranging from 300 bp to 2,000 bp (average size 700 bp) that contain a TAG sequence that enable the use of one primer to sequence all the generated PCR products. The TOSTRAP protocol enables to amplify multiple PCR products from the same DNA sample in 25% of the analysed rice lines. This implies that 75% of our products are single PCR products and 25% multiple PCR products (in most cases 2 PCR products).

The project aims at sequencing 20,000 Tos17 flanking regions for readily providing the scientific community with additional tags for reverse genetics and improving the insert coverage in the mutant library.

The Genoplante insertion library is used first by both the French and international communities dealing with rice and cereal functional genomics. In France a well structured rice community of ca. 30 scientists (INRA, IRD, CIRAD, CNRS) is concentrated in the Languedoc-Roussillon region and has established functional genomics programs on disease response (UMR BGPI 385), grain development and filling and embryogenesis (UMR GDP 5096), plant development in adaptation to stress (UMR PIA 1096). Aside this, the wheat (INRA) and maize (BIOGEMMA) public and private research communities part of the Genoplante consortium are using a comparative genomics approach with pivotal rice genome sequence and associated resources. A growing interest also exists in the Arabidopsis scientific community for validating the function of their favourite genes in rice. The library is also a central resource for international projects such as those of the Generation Challenge Program (CEREALIMMUNITY, and STRESS MUTANT) as well as for projects funded by the European commission (CEDROME). The publicly released Tos17 FST generated will provide helpful additional tags for all these projects and communities.