Distribution of insertion sites of the maize transposon, Ds, in the tomato genome

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The Ac and Ds transposable elements of maize have been demonstrated to function in a variety of heterologous plant species (1-5). In transgenic tomato, Ac transposition retains several features that are characteristic of its behavior in maize; including the tendency to transpose, with approximately equal frequency, to sites that are linked or unlinked to its original location (6). In this report, we examine the distribution of sites of Ds insertion in tomato. We also describe the isolation of immobile derivatives of Ac that are capable of activating Ds transposition.

We examined the distance over which Ds transposes in transgenic tomato by determining both the genomic location of the startpoint of transposition, that is, the T-DNA which carries Ds, as well as the locations of transposed Ds elements. To generate material for mapping studies, three independent transformants bearing Ds were crossed to plants containing Ac. Our Ds element carries a selectable kanamycin resistance gene and is positioned within a hygromycin resistance gene that serves as an excision marker. Therefore, selection for resistance to both hygromycin and kanamycin enriches for progeny in which transposition of Ds has taken place. Sequences flanking the T-DNAs and transposed Dss were recovered from selected plants, by either IPCR or lambda cloning, and were placed on the tomato RFLP map.

Preliminary results suggest that, like Ac, Ds transposition generates clusters of linked insertions in tomato. In one Ac X Ds family, genomic locations of the Ds-bearing T-DNA and of eight different Ds insertions have been determined (Fig. 1). The T-DNA of this family resides on chromosome 9, in close proximity to the TMV resistance gene, Tm2a. Three tightly linked pairs of Ds insertions originating from this T-DNA have been identified on chromosomes 6, 9, and 11, while single Ds insertions have been identified on chromosomes 1 and 2. The paired insertions comprise small clusters, with each member showing linkage to its partner within a 1-15 cM range. Southern analysis has shown that at least three of these mapped Dss represent germinal transposition events which transmit stably to subsequent generations. In a second Ac X Ds family, we have determined that the Ds-bearing T-DNA maps to chromosome 8 and we are currently identifying the genomic locations of transposed Ds elements.

Fig.1 RFLP map of transposed Ds elements

Unexpectedly, in one plant family we detected two different, apparently immobile, derivatives of the autonomous Ac element that had been utilized in our studies to activate Ds transposition. These stable Ac elements (st-Ac) appear as distinct Ac-hybridizing restriction fragments on Southern blots that are transmitted to progeny in a Mendelian manner. There has been no detectable transposition of either st-Ac for five generations. To test the ability of the Ac derivatives, designated st-Ac2 and st-Ac3, to trans-activate Ds, plants carrying each element (but no other Ac-hybridizing sequence) were crossed to a Ds-bearing line. Southern analysis was performed on DNA extracted from F1 progeny of crosses that had been selected for hygromycin and kanamycin resistance. Nearly all of eighteen individuals examined showed evidence of Ds excision. Furthermore, approximately 20%, of st-Ac2 X Ds, and 30%, of st-Ac3 X Ds, progeny showed new integrations of Ds. We have isolated portions of Ac2 and Ac3 by PCR. Sequencing of the isolates is underway to identify the lesions which render these elements immobile. We anticipate that st-Ac2 and st-Ac3 may prove useful as Ds trans-activators in future transposon mutagenesis schemes in tomato.

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