Rapid identification of Arabidopsis insertion mutants by non-radioactive detection of T-DNA tagged genes.
To assist in the analysis of plant gene functions we have generated a new Arabidopsis insertion mutant collection of 90 000 lines that carry the T-DNA of Agrobacterium gene fusion vector pPCV6NFHyg. Segregation analysis indicates that the average frequency of insertion sites is 1.29 per line, predicting about 116 100 independent tagged loci in the collection. The average T-DNA copy number estimated by Southern DNA hybridization is 2.4, as over 50% of the insertion loci contain tandem T-DNA copies. The collection is pooled in two arrays providing 40 PCR templates, each containing DNA from either 4000 or 5000 individual plants. A rapid and sensitive PCR technique using high-quality template DNA accelerates the identification of T-DNA tagged genes without DNA hybridization. The PCR screening is performed by agarose gel electrophoresis followed by isolation and direct sequencing of DNA fragments of amplified T-DNA insert junctions. To estimate the mutation recovery rate, 39 700 lines have been screened for T-DNA tags in 154 genes yielding 87 confirmed mutations in 73 target genes. Screening the whole collection with both T-DNA border primers requires 170 PCR reactions that are expected to detect a mutation in a gene with at least twofold redundancy and an estimated probability of 77%. Using this technique, an M2 family segregating a characterized gene mutation can be identified within 4 weeks.
Ríos
Gabino
G
Max-Planck Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, D-59829 Köln, Germany.
Lossow
Andrea
A
Hertel
Britta
B
Breuer
Frank
F
Schaefer
Sabine
S
Broich
Melanie
M
Kleinow
Tatjana
T
Jásik
Ján
J
Winter
Jochen
J
Ferrando
Alejandro
A
Farrás
Rosa
R
Panicot
Mireia
M
Henriques
Rossana
R
Mariaux
Jean-Baptist
JB
Oberschall
Attila
A
Molnár
Gergely
G
Berendzen
Kenneth
K
Shukla
Vijaya
V
Lafos
Marcel
M
Koncz
Zsuzsanna
Z
Rédei
George P
GP
Schell
Jeff
J
Koncz
Csaba
C
D000465:Algorithms; D017360:Arabidopsis / Q000235:genetics* / Q000254:growth & development; D001665:Binding Sites / Q000235:genetics; D015139:Blotting, Southern; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics*; D018744:DNA, Plant / Q000737:chemistry / Q000235:genetics; D018745:Genome, Plant; D008957:Models, Genetic; D016254:Mutagenesis, Insertional; D009154:Mutation; D030821:Plants, Genetically Modified; D016133:Polymerase Chain Reaction; D012231:Rhizobium / Q000235:genetics; D016324:Sequence Tagged Sites; D013698:Templates, Genetic
D017931:DNA Primers; D004269:DNA, Bacterial; D018744:DNA, Plant; C036483:T-DNA