Site-selected insertional mutagenesis of tomato with maize Ac and Ds elements.
Site-selected insertion (SSI) is a PCR-based technique which uses primers located within the transposon and a target gene for detection of transposon insertions into cloned genes. We screened tomato plants bearing single or multiple copies of maize Ac or Ds transposable elements for somatic insertions at one close-range target and two long-range targets. Eight close-range Ds insertions near the right border of the T-DNA were recovered. Sequence analysis showed a precise junction between the transposon and the target for all insertions. Two insertions in separate plants occurred at the same site, but others appeared dispersed in the region of the right T-DNA border with no target specificity. However, insertions showed a preference for one orientation of the transposon. 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 (dihydroflavonol 4-reductase). Certain HiDs plants showed much higher rates of insertion into PG than others. Insertions in PG and DFR were found throughout the gene regions monitored and, with the exception of one insertion in PG, the junctions between transposon and target were exact. SSI analysis of progeny from the HiDs parents revealed that in some cases the tendency to incur high levels of somatic insertions in PG was inherited. Inheritance of this character is an indication that SSI could be used to direct a search for germinal PG insertions in tomato.
Cooley
M B
MB
Department of Vegetable Crops, University of California, Davis 95616, USA.
Goldsbrough
A P
AP
Still
D W
DW
Yoder
J I
JI
D000429:Alcohol Oxidoreductases / Q000235:genetics; D001483:Base Sequence; D015139:Blotting, Southern; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D017931:DNA Primers; D004251:DNA Transposable Elements / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D017343:Genes, Plant / Q000235:genetics; D018551:Solanum lycopersicum / Q000235:genetics*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D011096:Polygalacturonase / Q000235:genetics; D003313:Zea mays / Q000235:genetics*
D017931:DNA Primers; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D010940:Plant Proteins; C036483:T-DNA; D000429:Alcohol Oxidoreductases; C056570:dihydroflavanol 4-reductase; D011096:Polygalacturonase