A PCR-based assay to detect hAT-like transposon sequences in plants.

Despite their potential as endogenous tools for forward and reverse genetics, members of the hobo, Ac, Tam3 (or hAT) superfamily of transposable elements have been characterized in but a limited number of plant species. To expedite their isolation, we developed a PCR-based assay for the detection of hAT-like transposon sequences in plants which was applied to isolate and initially characterize such sequences from Petunia hybrida, Phaseolus vulgaris, Bambusa vulgaris, Brassica napus and Rhododendron simsii.

AFLP maps of Petunia hybrida: building maps when markers cluster.

AFLP mapping in Petunia hybrida was undertaken with the intention of building a high-density genetic map suitable for applications such as map-based gene cloning. In total five maps were constructed from two mapping populations, with placement of more than 800 markers. Despite the large number of markers the resulting map is roughly ten-fold smaller than those of other plant species, including the closely related tomato. Low levels of recombination are reflected in clusters of tightly linked markers, both AFLPs and RFLPs, in all the maps. Clustering patterns vary between mapping populations, however, such that loci tightly linked in one population may be separable in another. Combined with earlier reports of aberrant meiotic pairing and recombination, our results suggest that, for species like petunia, map-based cloning may be more complex than in model species such as arabidopsis and tomato.

Analysis by Transposon Display of the behavior of the dTph1 element family during ontogeny and inbreeding of Petunia hybrida.

Transposon Display is a high-resolution method that was used here to visualize simultaneously individual members of the dTph1 transposable element family of Petunia hybrida. The method provides a tool for accurate analyses of copy numbers and insertion frequencies, and a means to study the behavior of a family of elements as a whole. Somatic insertion events can be identified and insertion events in a cell of the L2 apical lineage can be distinguished unequivocally from those in a cell outside this lineage. In sublines of the high-copy-number line W137, an average insertion rate equivalent to transposition of 10% of the total number of element copies in each generation was measured, copy number increases of over 20% in four generations were recorded, and element position turnover was analyzed. Insertion events are detected essentially randomly both in time and space. The general applicability of the technique for the analysis of the transpositional behavior of element systems is discussed.

Plant tagnology.

Transposable elements have been used as an effective mutagen and as a tool to clone tagged genes. Insertion of a transposable element into a gene can lead to loss- or gain-of-function, changes in expression pattern, or can have no effect on gene function at all, depending on whether the insertion took place in coding or non-coding regions of the gene. Cloning transposable elements from different plant species has made them available as a tool for the isolation of tagged genes using homologous or heterologous tagging strategies. Based on these transposons, new elements have been engineered bearing reporter genes that can be used for expression analysis of the tagged gene, or resistance genes that can be used to select for knockout insertions. While many genes have been cloned using transposon tagging following traditional forward genetics strategies, gene cloning has ceased to be the rate-limiting step in the process of determining sequence-function relations in several important plant model species. Large-scale insertion mutagenesis and identification of insertion sites following a reverse genetics strategy appears to be the best method for unravelling the biological role of the thousands of genes with unknown functions identified by genome or expressed sequence tag (EST) sequencing projects. Here we review the progress in forward tagging technologies and discuss reverse genetics strategies and their applications in different model species.

Transposon Display identifies individual transposable elements in high copy number lines.

The dTph1 transposable element family of Petunia hybrida line W138 consists of between 100 and 200 members. A strategy that allows simultaneous detection of individual elements is described. Sequences flanking dTph1 elements are amplified by means of a ligation-mediated PCR. The resulting fragments are locus-specific and can be analysed by polyacrylamide gel electrophoresis. One of the applications of Transposon Display is the isolation of dTph1-tagged genes. Fragments that co-segregate with a mutant phenotype can be extracted from the gel and reamplified, providing access to tagged genes, as demonstrated in a reconstruction experiment. Data on the molecular identification of a phenotypic mutant, isolated in a random tagging experiment is also presented. Upon sequencing, the obtained candidate fragment was found to be identical to part of the previously identified Fbp1 gene.