Submitted to: Plant Sciences Institute Symposia
Publication Type: Proceedings
Publication Acceptance Date: 6/5/2003
Publication Date: 6/5/2003
Citation: Interpretive Summary:
Technical Abstract: Our goal is to establish a population of barley lines, each containing a single, stable and mapped Ds insertion to enable mutagenesis targeted to specific genomic locations in barley. We have produced lines derived from a transformed parent containing the maize Ds element that was induced to transpose by crossing with an Ac transposase-expressing line. The transposed Ds insertions, which include a selectable marker, have been stabilized by selecting plants lacking Ac transposase, mapped and characterized in the different lines. The population in its aggregate will include Ds elements dispersed throughout the diploid barley genome. Upon reactivation by crossing with the Ac transposase line, the Ds element is expected to transpose primarily to nearby locations for targeting known regions of interest. Here, we report the characteristics of single or low-copy Ds insertions after initial transposition. Genomic 5' and 3' sequences flanking the Ds insert were amplified via inverse PCR or TAIL-PCR and sequenced for mapping, BAC screening, and classification of insertion sites and Ds element integrity. Preliminary analyses show preferential insertion of Ds into distal chromosomal locations. Many transpositions mapped in a cluster near the original site of insertion, as expected, but others were found at unlinked locations on five of the seven barley chromosomes. BLAST searches indicate the transposed inserts are generally found within genes. In most lines, characteristic 8 base pair duplications were observed in genomic sequences adjacent to the Ds terminal inverted repeats. Approximately 20% of the lines contained transposed Ds elements with defective inverted repeats that were consistently associated with incomplete 8 base pair genomic duplications. Thus, the Ac/Ds system is seen to behave in barley as described for other species, and the initial transposition events tagged putative genes. We are employing the Ds flanking genomic sequences as probes to assign corresponding map locations to BAC clone inserts.