SITE-SELECTED INSERTIONAL MUTAGENESIS OF TOMATO WITH MAIZE AC AND DS ELEMENTS

Site-selected insertion (SSI) is a PCR-based technique which uses prim ers located within the transposon and a target gene for detection of t ransposon insertions into cloned genes. We screened tomato plants bear ing single or multiple copies of maize Ac or Ds transposable elements for somatic insertions at one close-range target and two long-range ta rgets. Eight close-range Ds insertions near the right border of the T- DNA were recovered. Sequence analysis showed a precise junction betwee n the transposon and the target for all insertions. Two insertions in separate plants occurred at the same site, but others appeared dispers ed in the region of the right T-DNA border with no target specificity. However, insertions showed a preference for one orientation of the tr ansposon. Use of plants with multiple Ac (HiAc) or Ds (HiDs) elements allowed detection of somatic insertions at two single-copy genes, PG ( polygalacturonase) and DFR (dihydro-flavonol 4-reductase). Certain HiD s plants showed much higher rates of insertion into PG than others. In sertions in PG and DFR were found throughout the gene regions monitore d and, with the exception of one insertion in PG, the junctions betwee n transposon and target were exact. SSI analysis of progeny from the H iDs parents revealed that in some cases the tendency to incur high lev els of somatic insertions in PG was inherited. Inheritance of this cha racter is an indication that SSI could be used to direct a search for germinal PG insertions in tomato.