Targeted gene inactivation in petunia by PCR-based selection of transposon insertion mutants.

Abstract

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 dTphl. Leaves are collected from batches of 1000 plants with highly active dTphl 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 dTphl 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prolonged period.offspring and found to be due to insertions of dTphl elements (E.S., A.v.H., D.K., J.N.M.M., and R.K., unpublished data). Thus, the high incidence of mutations in the W138 line is mainly due to transposition of dTphl elements.We show how plants with a dTphl insertion in a specific gene can be identified in a large plant population by a polymerase chain reaction (PCR) assay. Such plants are heterozygous for the insertion allele unless the insertion occurred in a previous generation. After selfing, progeny homozygous for the insertion allele are obtained that can be analyzed for an altered phenotype. The screened plant population is maintained by selfing all plants, so that semipermanent libraries are obtained that can be screened at any time. These libraries are open to the scientific community.

AtDMC1, the Arabidopsis homologue of the yeast DMC1 gene: characterization, transposon‐induced allelic variation and meiosis‐associated expression

“…Identification of an Atdmcl mutant, either by comparison of the AtDMC1 mapping position with the mapping positions of the mutations affecting meiosis in Arabidopsis, or by PCRbased selection of transposon insertion mutants (Koes et aL, 1995) and their further complementation with the AtDMC1 genomic clone, will be helpful in further refining our understanding of the functions of the DMCI homoIogue in plants.…”

A petunia ABC protein controls strigolactone-dependent symbiotic signalling and branching

“…4e). Insertion of dTph1 in the coding region of a gene frequently results in a complete loss of function 19 .The W138 line with the mutant pdr1 gene (W138-pdr1) was compared directly with W138 and was crossed with P. hybrida W115 for further segregation analysis. Five homozygous pdr1 mutant lines (W115 3 W138-pdr1) and wild-type lines (W115 3 W138) were derived from the F 2 generation (Supplementary Fig.…”

“…4e). Insertion of dTph1 in the coding region of a gene frequently results in a complete loss of function 19 .…”

ODORANT1Regulates Fragrance Biosynthesis in Petunia Flowers

“…Derivatized samples were analyzed and quantified by gas chromatography-mass spectrometry as described for the emitted volatiles. Flavonols in the ethyl acetate extract were separated on silica-TLC plates containing F254 as described by Koes et al (1995) and visualized under UV light (254 nm). Three independent experiments were performed.…”