Submitted to: Molecular Genetics and Genomics
Publication Type: Peer reviewed journal
Publication Acceptance Date: 6/11/2004
Publication Date: 7/30/2004
Citation: Cooper, L.D., Marquez-Cedillo, L., Singh, J., Zhang, S., Carollo, V.L., Edwards, V.M., Johnson, K., Klienhofs, A., Rangel, S., Sturbaum, A.K. 2004. Mapping ds insertions in barley using a sequence-based approach. Molecular Genetics and Genomics. 271: 181-193. Interpretive Summary: A system to perform mutagenesis on barley is described. This method uses the transposon (jumping gene) system, called Ac/Ds, to mobilize into genes and disrupt their expression. The transposon can then be mapped and gene can be discovered.
Technical Abstract: A transposon tagging system, based upon maize Ac/Ds elements, was developed in barley (Hordeum vulgare). The long-term objective is a set of lines with Ds insertions dispersed throughout the genome as a comprehensive tool for gene discovery and reverse genetics. AcTPase and Ds elements were introduced into immature embryos of Golden Promise by biolistic transformation. Subsequent transposition and segregation of Ds away from AcTPase and the original site of integration resulted in a number of new lines, each containing a stabilized Ds element in a new location. Using a sequence-based mapping strategy, we determined the genome locations of the Ds insertions in 19 independent lines using primarily restriction digest-based assays of PCR amplified single nucleotide polymorphisms and PCR-based assays of insertion or deletions. The principal strategy was to clone the corresponding regions in the Wolfe Dominant and Recessive Multiple Marker stocks and to map the sequence polymo! rphisms in the Oregon Wolfe Barley mapping population. The mapping results obtained by the PCR-based approach were confirmed by RFLP analyses in four of the lines. In addition, the cloned DNA flanking sequences are being used to assign corresponding map locations to genomic BAC library inserts, thus integrating barley genetic and physical maps. BLAST search results indicate that the majority of the transposed Ds elements are found within non-repetitive, predicted coding sequences. Transposon tagging in barley using Ac/Ds promises to provide a useful tool for Triticeae functional genomics.