Targeted gene inactivation in petunia by PCR-based selection of transposon insertion mutants.
Establishment of loss-of-function phenotypes is often a key step in determining the biological function of a gene. We describe a procedure to obtain mutant petunia plants in which a specific gene with known sequence is inactivated by the transposable element dTph1. Leaves are collected from batches of 1000 plants with highly active dTph1 elements, pooled according to a three-dimensional matrix, and screened by PCR using a transposon- and a gene-specific primer. In this way individual plants with a dTph1 insertion can be identified by analysis of about 30 PCRs. We found insertion alleles for various genes at a frequency of about 1 in 1000 plants. The plant population can be preserved by selfing all the plants, so that it can be screened for insertions in many genes over a prolonged period.
Koes
R
R
Department of Genetics, Vrije Universiteit, Amsterdam, The Netherlands.
Souer
E
E
van Houwelingen
A
A
Mur
L
L
Spelt
C
C
Quattrocchio
F
F
Wing
J
J
Oppedijk
B
B
Ahmed
S
S
Maes
T
T
D000483:Alleles; D001483:Base Sequence; D017931:DNA Primers; D004251:DNA Transposable Elements; D018506:Gene Expression Regulation, Plant / Q000235:genetics; D017343:Genes, Plant; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010944:Plants / Q000235:genetics*; D016133:Polymerase Chain Reaction
D017931:DNA Primers; D004251:DNA Transposable Elements