FST resource : Production reports and T-DNA lines integrations studies

1. URGV - + Protocol of FST production : High-throughput PCR Walking 96 samples to be sequenced Arabidopsis seedlings 96 wells plate Sequencing DISTRIBUTED LINES 708 INRA-Versailles genomic DNA extraction (Lyophilisation+homogenei- -sation+CTAB) 3 PUBLICATIONS Biotechniques 2001 Nucleic Acids Res. 2002 EMBOreports 2002 genomic DNA EUGENE Bi1999077 C. Gaspin Enzymatic treatment + PCR I/II Informatic treatment of data FST NOBIO 2001-007 G.Pelletier-L. Lepiniec GENOPLANTE-INFO RHOBIO-INF 40557 FST ‘Génoplante’  14658 FST Publiques PREDOTAR Bi1999030 I. Small PCR plate-96 samples Loading on agarose gel ANNOTATION Bi2001071 S. Aubourg FLAGdb++ integration DEPOT EMBL 12 286 FST GABI-GENOPLANTE 10 < NO2001090 > 16 B. Weisshaar-A. Lecharny Visual isolation of amplified band + ’purification’ PCR GENES ORPHELINS BiAf2001038 A. Lecharny-P. Perez Number of FST/Mb 121 77 77 77 43 33 ATG intergénic 5’UTR exon intron 3’UTR intergénic 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: 1194-9 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: 1188-1193 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. 2002 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. 2002 Dec;3(12):1152-1157. FST resource : Production reports and T-DNA lines integrations studies NOBIO 2001-007 Sandrine Balzerguea, Véronique Brunauda , Stéphanie Chauvina, Sébastien Aubourga, Franck Samsona, Marie Datya, Morgane Boutillond, Richard DeRosed, Corinne Cruaude, Jean Weissenbache, Cécile Cognetc, Matthieu Simonc, Alain Lecharnya, Michel Cabochea, Bertrand Dubreucqb,Georges Pelletierc and Loïc Lepiniecb. a- Unité de Recherche en Génomique Végétale, INRA, FRE CNRS, 2 rue G.Crémieux, CP 5708, F-91057 Evry Cedex, France b- Laboratoire de Biologie des Semences, INRA-URGV, Centre de Versailles, Route de St Cyr, F-78026 Versailles Cedex, France c- Station de génétique et Amélioration des plantes, Centre de Versailles, Route de St Cyr, F-78026 Versailles Cedex, France d- RhoBio, 2 rue G. Crémieux, CP 5707, F-91057 Evry Cedex, France e- GENOSCOPE, 2 rue G. Crémieux, CP 5707, F-91057 Evry cedex, France. Systematic identification of the integration site of the T-DNA insertion lines provided by INRA-SGAP Versailles. Recovery of the Flanking Sequence Tag (FST). Production reports (2000 - 2003) FST number currently in Génoplante FLAGdb++ : 40557 FST https://genoplante.infobiogen.fr FST number currently in public FLAGdb++: 14658 FST http://flagdb-genoplante-info.infobiogen.fr/projects/fst T-DNA lines number distributed (since July 2000): 708 lines 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,40557 FST have been obtained and inserted in a FLAGdb++ database. 14658FST 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. 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 2003. Gene and FST densities along the 5 chromosomes of A. thaliana Involved model for T-DNA integration mechanism 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. From FLAGdb++ • A model of T-DNA integration process. • 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. Recognition of a T-rich region might be a common feature in the integration of foreign DNA in eucaryotic genomes. 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. 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