Plant & Animal Genomes XIV Conference

January 14-18, 2006
Town & Country Convention Center
San Diego, CA

Targeting Genes And Their Function In The "Genome Haystack": Transposon-Based Saturation Mutagenesis

Jaswinder Singh¹ , Shibo Zhang¹ , Calvin Chen¹ , Lol Cooper² , Anne Sturbaum³ , Michael Freudiger¹ , Phil Bregitzer³ , Pat Hayes² , Peggy Lemaux¹

¹  Department of Plant and Microbial Biology University of California, Berkeley CA, 94720 USA

²  Department of Crop and Soil Science, Oregon State University, Corvallis OR, 97331 USA

³  National Small Grains Germplasm Research Facility, USDA ARS, Aberdeen, ID, USA, 83210

In large-genome species, like barley, identifying genes and studying function presents significant challenges. The maize Ac/Ds transposon system for gene tagging in heterologous species offers an approach to this problem. An Ac/Ds gene tagging system developed in the true diploid barley, demonstrates the power of this system in the Triticae family. Extensive mapping efforts in barley provide a robust platform for the effective use of the transposon approach to characterizing genes. Locations of transposed Ds elements (TNPs) were mapped using single-copy Ds lines, generated by crossing Ds and AcTPase lines. This approach is useful in large-genomes species because Ds tends to transpose to genic regions. BLAST searches indicate ~90% of Ds flanking sequences were from known or putative genes, like MLA1, wall-associated kinases, ubiquitin-conjugating enzyme, ATP-binding transporter and cytochrome P450. Once mapped, TNPs near genes of interest or located in clustered gene regions can be reactivated to perform targeted mutagenesis, based on the propensity of Ds to transpose locally. For maximal utility of this approach, an understanding of the re-mobilization characteristics of TNPs is required. A detailed study of TNP re-activation over 3-4 consecutive generations of transposition was performed to provide the foundation for tagging and “transposon-walking” approaches. From 120 TNPs, flanking sequences adjacent to Ds in 50 lines provided insights into re-activation behavior of Ds, particularly the status of terminal inverted repeats (TIRs), 8 bp duplications and the nature of insertion sites. Re-activation frequencies in secondary, tertiary and quaternary populations were 13.7-16.9%; intactness of the TIRs impacts re-activation frequency.