Transgene repeats in aspen: molecular characterisation suggests simultaneous integration of independent T-DNAs into receptive hotspots in the host genome.

Abstract

Rearrangements of T-DNAs during genetic transformation of plants can result in the insertion of transgenes in the form of repeats into the host genome and frequently lead to loss of transgene expression. To obtain insight into the mechanism of repeat formation we screened 45 transgenic lines of aspen and hybrid aspen transformed with six different gene constructs. The frequency of T-DNA repeat formation among randomly screened transgenic lines was found to be about 21%. In ten transgenic lines direct repeats were detected. An inverted repeat was found in one other transgenic line. Sequencing of the junctions between the T-DNA inserts revealed identical residual right-border repeat sequences at the repeat junctions in all ten transgenic lines that had direct repeats. Formation of "precise" junctions based on short regions of sequence similarity between recombining strands was observed in three transgenic lines transformed with the same plasmid. Additional DNA sequences termed filler DNAs were found to be inserted between the T-DNA repeats at eight junctions where there was no similarity between recombining ends. The length of the filler DNAs varied from 4 to almost 300 bp. Small filler DNAs--a few base pairs long--were in most cases copied from T-DNA near the break points. The large filler sequences of about 300 bp in two transgenic lines were found to be of host plant origin, suggesting that transgene repeat formation occurred as a result of the simultaneous invasion of a receptive site in the host genome by two independent T-DNA strands. On the basis of the results obtained, and in the light of previous reports on T-DNA/plant DNA junctions in aspen and other crop plants, a mechanistic model for transgene rearrangement and filler formation is suggested.