Plant Molecular Biology 53: 247–259, 2003.
© 2003 Kluwer Academic Publishers. Printed in the Netherlands.
An Arabidopsis thaliana T-DNA mutagenized population (GABI-Kat) for
ﬂanking sequence tag-based reverse genetics
Mario G. Rosso, Yong Li, Nicolai Strizhov
, Bernd Reiss, Koen Dekker and Bernd Weisshaar
GABI-Kat, Max Planck Institute for Plant Breeding Research, Carl-von-Linn´e-Weg 10, 50829 Köln, Germany;
Max Planck Unit for Structural Molecular Biology, Notkestrasse 85, 22607 Hamburg, Ger-
Institute for Genome Research, Bielefeld University, Universitätsstrasse 25, 33594 Bielefeld, Germany
author for correspondence; e-mail email@example.com)
Received 4 July 2003; accepted in revised form 25 September 2003
Key words: FST, gene function, insertion mutagenesis, knock-out, reverse genetics
The GABI-Kat population of T-DNA mutagenized Arabidopsis thaliana lines with sequence-characterized insertion sites is
used extensively for efﬁcient progress in plant functional genomics. Here we provide details about the establishment of the
material, demonstrate the population’s functionality and discuss results from quality control studies. T-DNA insertion mutants
of the accession Columbia (Col-0) were created by Agrobacterium tumefaciens-mediated transformation. To allow selection
of transformed plants under greenhouse conditions, a sulfadiazine resistance marker was employed. DNA from leaves of T
plants was extracted and used as a template for PCR-based ampliﬁcation of DNA fragments spanning insertion site borders.
After sequencing, the data were placed in a ﬂanking sequence tag (FST) database describing which mutant allele was present
in which line. Analysis of the distribution of T-DNA insertions revealed a clear bias towards intergenic regions. Insertion
sites appeared more frequent in regions in front of the ATG and after STOP codons of predicted genes. Segregation analysis
for sulfadiazine resistance showed that 62% of the transformants contain an insertion at only one genetic locus. In quality
control studies with gene-speciﬁc primers in combination with T-DNA primers, 76% of insertions could be conﬁrmed. Finally,
the functionality of the GABI-Kat population was demonstrated by exemplary conﬁrmation of several new transparent testa
alleles, as well as a number of other mutants, which were identiﬁed on the basis of the FST data.
Abbreviations: BLAST, Basic Local Alignment Search Tool; CDS, coding sequence; CDSi, genomic DNA se-
quence from translation start to stop, including introns; FST, ﬂanking sequence tag, genomic sequences adjacent
to a DNA insertion; IAA, isoamyl alcohol; KO, knockout; MAtDB, MIPS Arabidopsis thaliana Database; ORF,
open reading frame; sul, sulfadiazine; T-DNA, transfer DNA; WT, wild type
The availability of the Arabidopsis thaliana genome
sequence (Arabidopsis Genome Initiative, 2000) en-
FST sequence data have been submitted to the
EMBL/GenBank/DDBJ Nucleotide Sequence Databases and
are also available from http://www.mpiz-mpg.de/GABI-Kat/.
The GenBank accession numbers for KO lines mentioned in
this paper are: agamous 001H07 (AY262006), agamous 146G09
(AY268088), terminal ﬂower 217E02 (AY333421) terminal ﬂower
221D07 (AY271513), terminal ﬂower 228A07 (AY268091);
terminal ﬂower 404F07 (AY275835), wax2 086A07 (AY271515),
wax2 177D09 (AY271516), dihydroﬂavonol 4-reductase 295C10
(AY323491) chalcone synthase 304D03 (AY271514), chalcone
isomerase 176H03 (AY268089), ﬂavanone 3-hydroxylase 292E08
ables us to identify each A. thaliana gene by sequence
and genome position. However, for only some 620
of the estimated 29 000 genes has a description of
the gene-phenotype relationship been made public
(Meinke et al., 2003). In order to characterize the func-
tion of each gene of this model plant and to determine
the biological signiﬁcance of each gene product by the
year 2010 (Chory et al., 2000; Somerville and Dangl,
2000), an easy access to knockout (KO) mutants in
pre-selected genes for reverse genetics, and saturation
mutagenesis at a genomic level, is required.
(AY268090), ﬂavonoid 3
-hydroxylase-like 349F05 (AY323492),
bHLH042 transcription factor/tt8 241D05 (AY300160).