Transcript Document

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).
Protocol of FST production : High-throughput PCR Walking
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
96 samples to be sequenced
Arabidopsis
seedlings
96 wells plate
Production reports (2000 - 2003)
T-DNA lines number distributed (since July 2000): 708 lines
Sequencing
DISTRIBUTED LINES
708
INRA-Versailles
genomic DNA extraction
(Lyophilisation+homogenei-sation+CTAB)
3 PUBLICATIONS
Biotechniques 2001
Nucleic Acids Res. 2002
EMBOreports 2002
EUGENE
Bi1999077
C. Gaspin
genomic DNA
Enzymatic treatment
+ PCR I/II
Informatic
treatment of data
PREDOTAR
Bi1999030
I. Small
PCR plate-96
samples
Loading on agarose gel
FST
NOBIO 2001-007
G.Pelletier-L. Lepiniec
ANNOTATION
Bi2001071
S. Aubourg
FLAGdb++ integration
Visual isolation
of amplified band
+ ’purification’ PCR
GENOPLANTE-INFO
RHOBIO-INF
40557 FST ‘Génoplante’
14658 FST Publiques
DEPOT EMBL
12 286 FST
GABI-GENOPLANTE
10 < NO2001090 > 16
B. Weisshaar-A. Lecharny
GENES ORPHELINS
BiAf2001038
A. Lecharny-P. Perez
+
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
From FLAGdb++
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.
Number
of FST/Mb
121
77
77
33
77
5’UTR
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
43
Recognition of a T-rich region might be a common feature in the integration of
foreign DNA in eucaryotic genomes.
ATG
intergénic
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.
exon
intron
3’UTR
intergénic
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
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.