Sandrine Balzerguea, Vronique Brunauda , Stphanie Chauvina, Sbastien Aubourga, Franck Samsona, Marie

1
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/pr
ojects/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. 14658 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.
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