Transposon-Mediated Functional Genomics in Barley.

Transposable elements have advantages over other approaches for determining gene function in large genome cereals. Different strategies have been used to exploit maize Ac/Ds for such studies in heterologous species. First, large numbers of independent Ds insertion lines (TNPs) are generated and screened phenotypically. Alternatively, smaller numbers of transposed elements are identified, mapped and then remobilized for localized gene targeting. A robust platform was developed to use transposon targeting approaches in barley to complement existing, extensive genomic resources. In our NSF Plant Genome Research Project we (i) generated 200 single-copy Ds TNPs, (ii) determined flanking sequences in >100 lines, (iii) mapped 43 DsT loci on the genetic linkage map, (iv) identified 250 BAC addresses using flanking sequence probes and (v) quantified remobilization frequencies of primary, secondary and tertiary TNPs for saturation mutagenesis schemes. Results from BLAST searches of flanking sequences indicated that ~86% are from known or putative genes; examples include MLA1, wall-associated kinases, ubiquitin-conjugating enzyme, ATP-binding transporter, terpene synthase, ankyrin1-like protein and cytochrome P450. Mapped insertion sites are valuable for tagging linked genes by reactivating Ds elements close to traits/genes of interest to achieve saturation mutagenesis. Because the marked tendency of Ds to transpose to linked locations is critical for this approach, we investigated characteristics important to TNP reactivation, i.e., status of terminal inverted repeats (TIRs) and 8 bp duplications and the nature of insertion sites. Remobilization frequencies of 12-17% over 3-4 generations of TNPs with intact TIRs provide the necessary foundation for tagging and “transposon-walking” (repeated localized transposition) strategies. The Bregitzer laboratory, which maintains and distributes these materials to interested researchers, has continued to generate additional tagging resources. Outreach efforts, which included the animated educational game, “Genetic Dartboard” (http://barleyworld.org/oregonwolfe.php), were aimed at increasing understanding of the Ac/Ds transposon tagging system.