Sandrine Balzergue a, Véronique Brunaud a, Stéphanie Chauvin a, Sébastien Aubourg a, Franck Samson a, Marie Daty a, Morgane Boutillon d, Richard DeRose d, Corinne Cruaud e, Jean Weissenbach e, Cécile Cognet c, Matthieu Simon c, Alain Lecharny a, Michel Caboche a, Bertrand Dubreucq b, Georges Pelletier c and Loïc Lepiniec b. a- Unité de Recherche en Génomique Végétale, INRA, FRE CNRS, 2 rue G.Crémieux, CP 5708, F Evry Cedex, France b- Laboratoire de Biologie des Semences, INRA-URGV, Centre de Versailles, Route de St Cyr, F Versailles Cedex, France c- Station de génétique et Amélioration des plantes, Centre de Versailles, Route de St Cyr, F Versailles Cedex, France d- RhoBio, 2 rue G. Crémieux, CP 5707, F Evry Cedex, France e- GENOSCOPE, 2 rue G. Crémieux, CP 5707, F Evry cedex, France. FST resource : Production reports and T-DNA lines integrations studies Protocol of FST production : High-throughput PCR Walking samples to be sequenced Sequencing genomic DNA Arabidopsis seedlings 96 wells plate genomic DNA extraction (Lyophilisation+homogenei- -sation+CTAB) FLAGdb ++ integration Informatic treatment of data Visual isolation of amplified band + ’purification’ PCR Enzymatic treatment + PCR I/II PCR plate-96 samples Loading on agarose gel Systematic identification of the integration site of the T-DNA insertion lines provided by INRA-SGAP Versailles. Recovery of the Flanking Sequence Tag (FST). References: Bechtold N et al. (1993). In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. C.R. Acad. Sci. Paris, Sciences de la vie; 316: Bouchez D et al. (1993). A binary vector based on Basta resistance for in planta transformation of Arabidopsis thaliana. C.R. Acad. Sci. Paris, Sciences de la vie; 316: Balzergue S., Dubreucq B. et al. (2001). Improved PCR-walking for large scale isolation of T-DNA borders. Biotechniques; 30(3):496-8, 502, 504. Samson F et al. Nucleic Acids Res Jan 1;30(1):94-7. “ FLAGdb/FST : a database for mapped flanking insertion sites (FSTs) of Arabidopsis thaliana T-DNA transformants”. Brunaud V. et al. “T-DNA integration into the Arabidopsis genome depends on sequences of pre-insertion sites.” EMBO Rep Dec;3(12): Production reports ( ) FST number currently in Génoplante FLAGdb ++ : FST FST number currently in public FLAGdb ++ : FST T-DNA lines number distributed (since July 2000): 708 lines NOBIO ANNOTATION Bi S. Aubourg EUGENE Bi C. Gaspin PREDOTAR Bi I. Small GENES ORPHELINS BiAf A. Lecharny-P. Perez GABI-GENOPLANTE B. Weisshaar-A. Lecharny DISTRIBUTED LINES 708 INRA-Versailles 3 PUBLICATIONS Biotechniques 2001 Nucleic Acids Res EMBOreports 2002 FST NOBIO G.Pelletier-L. Lepiniec GENOPLANTE-INFO RHOBIO-INF FST ‘Génoplante’ FST Publiques DEPOT EMBL FST Gene and FST densities along the 5 chromosomes of A. thaliana Involved model for T-DNA integration mechanism Many thancks for all participants to the FST projects : Alexandra Avon, Chantal Arar, Nicole Bechtold, Florence Catonnet, Stéphanie Durand, Amandine Freydier, Naïma Kebdani, Françoise Le-Boulaire, Isabelle Le-Clainche, Pierre Libeau, Virginie Pellouin, Stéphanie Pateyron, David Rouquié, Valérie Sourice, Glenn Ulrici, Sophie Villatoux A model of T-DNA integration process. (1) A T-rich region [Tn] is a preferential site of entry of T-DNA LB 3’ end. (2) T-DNA scans the plant DNA until it finds a microsimilarity downstream of T-rich region. (3) degradation of the 3’ end portion of T-DNA downstream of the duplex. (4) A nick is generated in the host genome. (5) The right end of T-DNA is ligated to the bottom strand of host DNA. Frequently pairing with a G (6) The top strand of host DNA is degraded between the two similarities. May result in a deletion of variable length in host DNA In average, there is 1 FST every 16Kb except towards the centromere where FST are progressively less frequently observed. About 40% of the integrations are in a gene (regions defined by the AGI-predicted genes + 200bp on each sideof them) and covering 54% of the A. thaliana genome. The highest density of FST is observed on the 5’ UTR of gene (200bp before the start codon) perhaps because this region is more accessible to any intervention of foreign structure such as DNA or enzyme. From FLAGdb ++ In order to optimize highthroughput production of FSTs, adaptations of the original technique have been made in all steps (extraction,restriction, ligation and PCR) and espacially reactions were performed in 96-well plates. Finally, a protocol to isolate a single PCR product from each sample (i.e. individual line) has been setting-up. Our data suggest that a fully exploitable FST can be obtained for 60% of the insertion lines processed. This includes lack of primary amplification (16%), sequencing problems (16%) and tandem inserts (8%). To date, FST have been obtained and inserted in a FLAGdb ++ database FST are already available in a public database. Moreover, since July 2000, 708 T-DNA lines was distributed to 26 different laboratories (264 requests). About 150 public T-DNA lines were also distributed through the FLAGdb ++ public database. The FLAGdb++ database allows to access to the insertion sites of Génoplante FST but also to the other collection of mutants (GABI, SIGnAL). All together, the T-DNA lines collection, FST sequences and FLAGdb ++ constitute a powerful tool for functional genomics in Arabidopsis. The study of the FST insertions sites on the genome allows to observe a statistically significant difference in base composition 10 bases around the insertion site compare to the rest of genome. The results demonstrate a model of insertion with microsimilarities between host genome and T-DNA sequence. A rapid estimation of the quality of FST production with a questionnaire send to the laboratories who requesting lines, revealed that 89% of researchers find the right FST in their lines (on 207 reply obtained). Moreover, FST insert in the database always have a residual T-DNA sequence to check the right origin of the FST. The Génoplante FST project will be fully achieved in July Recognition of a T-rich region might be a common feature in the integration of foreign DNA in eucaryotic genomes. 5’UTR3’UTRintronexonintergénic Number of FST/Mb ATG