Insertion mutagenesis and study of transposable elements using a new unstable virescent seedling allele for isolation of haploid petunia lines

Abstract

The new unstable virescent seedling (vis) allele of a petunia mutant, that has green leaves but white cotyledons with green revertant spots, was used to identify spontaneously occurring haploid petunia lines with active transposable elements. Endogenous transposons were trapped into the single petunia nitrate reductase structural gene (nia) using chlorate selection on haploid protoplasts. In two mutant lines, the dTph1-like transposable element dTph1-3 was inserted at almost the same position but in opposite orientations in the first exon of the nia gene. In a third mutant, a different transposable element was integrated into the fourth exon. This element, called dTph4, is 787 bp long and has 13 bp terminal inverted repeats of which 12 bp are identical to those of dTph1. Insertion of dTph1-3 and dTph4 results in an 8 bp duplication of the target site, as already described for dTph1. In contrast to dTph1-like elements, dTph4 is present at low copy number in the petunia genome. This can facilitate its use for gene tagging in petunia. The dTph1-3 and dTph4 elements excise frequently, as transposon footprints were found in most of the insertion mutants. The data demonstrate that haploid petunia is an excellent system for gene tagging and for the study of transposable elements.

Toward the Analysis of the Petunia MADS Box Gene Family by Reverse and Forward Transposon Insertion Mutagenesis Approaches: B, C, and D Floral Organ Identity Functions Require SEPALLATA-Like MADS Box Genes in Petunia

“…The shape and size of the petals were nearly as in the wild type ( Figure 3A). Close inspection of fbp2-1 and fbp2-2 petals using scanning electron micros- (Renckens et al, 1996) that shares extensive identity with dTph1 and has identical terminal inverted repeats. Therefore, the dTph1-derived transposon primer also may recognize the more rare dTph4 insertion events.…”

Molecular Analysis of the anthocyanin2 Gene of Petunia and Its Role in the Evolution of Flower Color

“…Closer inspection of the 4-bp insertion sequence shows that it has precisely the structure predicted for the insertion and subsequent excision of a transposon, strongly suggesting that the ϩ4-bp and Ϫ1-bp an2 Ϫ alleles were generated by two independent excisions of the same transposon (Figure 7). The petunia transposons dTph1 (Gerats et al, 1990), dTph2 (van Houwelingen et al, 1998, dTph3 (Kroon et al, 1994), dTph4 (Renckens et al, 1996;Alfenito et al, 1998), and dTph5 (C. Spelt and R. Koes, unpublished data) all belong to the Activator superfamily and generate an 8-bp target site duplication upon insertion. After excision, a footprint is left behind that consists of remains of the target site duplication (у7 bp of each target site duplication; cf.…”

Molecular Analysis of the anthocyanin2 Gene of Petunia and Its Role in the Evolution of Flower Color

“…Closer inspection of the 4-bp insertion sequence shows that it has precisely the structure predicted for the insertion and subsequent excision of a transposon, strongly suggesting that the ϩ4-bp and Ϫ1-bp an2 Ϫ alleles were generated by two independent excisions of the same transposon (Figure 7). The petunia transposons dTph1 (Gerats et al, 1990), dTph2 (van Houwelingen et al, 1998, dTph3 (Kroon et al, 1994), dTph4 (Renckens et al, 1996;Alfenito et al, 1998), and dTph5 (C. Spelt and R. Koes, unpublished data) all belong to the Activator superfamily and generate an 8-bp target site duplication upon insertion. After excision, a footprint is left behind that consists of remains of the target site duplication (у7 bp of each target site duplication; cf.…”