Mapping of transposable element Dissociation inserts in Brassica oleracea following plant regeneration from streptomycin selection of callus.
To investigate the potential of heterologous transposons as a gene-tagging system in broccoli (Brassica oleracea var. italica), we have introduced a Dissociation ( Ds)-based two-element transposon system. Ds has been cloned into a 35S-SPT excision-marker system, with transposition being driven by an independent 35S-transposase gene construct. In three successive selfed generations of plants, there was no evidence of germinal-excision events. In a previous study, we overcame this apparent inability to produce B. oleracea plants with germinal excisions by performing a novel tissue-culture technique to select for fully green shoots from seed with somatic excision events. The results showed a very high efficiency of regeneration of fully green plants (up to 65%), and molecular analysis showed that the plants contained the equivalent of a germinal-excision event. In this study, we followed the previous work by using inverse and nested PCR to generate probes of flanking genomic DNA adjacent to independently reinserted Ds elements, and these were hybridised to DNA from a double-haploid mapping population of B. oleracea. Seventeen Ds insertions and the original Ds T-DNA site have been localised, and these are spread over six (out of nine) linkage groups. Distribution of inserts show that 15 were found on a different linkage group to the original 'launch' site, and of these 11 were found to be clustered on two separate groups. Previous studies in other plant species have found that germinal excision of Ds predominantly moves to sites linked close to the donor site. However, this study shows a potential to produce plants with Ds insertion scattered over many unlinked sites.
McKenzie
Neil
N
John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK.
Dale
Philip J
PJ
D015139:Blotting, Southern; D001937:Brassica / Q000235:genetics* / Q000502:physiology; D002874:Chromosome Mapping; D017931:DNA Primers; D004251:DNA Transposable Elements / Q000235:genetics*; D015335:Molecular Probes / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D012038:Regeneration; D013307:Streptomycin; D014170:Transformation, Genetic
D017931:DNA Primers; D004251:DNA Transposable Elements; D015335:Molecular Probes; D013307:Streptomycin