Poster: Genomics Resources

Abs # 949: Ac/Ds Transposon-mediated Gene Tagging in Barley: Characteristics and Implications for a Heterologous System

Presenter:	Singh, Jaswinder , jsingh@nature.berkeley.edu
Authors	Singh, Jaswinder  (A)   Zhang, Shibo  (A)   Cooper, Lol  (B)   Rangel, Sergio  (A)   Hayes, Patrick M (B)   Lemaux, Peggy G (A)
Affiliations:	(A): University Of California (B): Oregon State University

The Ac/Ds transposon system, which causes heritable mutations in maize, is now used to identify genes in other plant species. Barley, a diploid and closely related to important cereal species, is an ideal platform in which to use Ac/Ds for functional genomics in cereals. Because Ac/Ds transposes into gene-rich regions and linked locations, this approach is being used to tag genes via saturation mutagenesis. Crossing transposase-expressing lines with Ds-containing lines activates Ds to move; however, the behavior and reactivation characteristics of Ds in the heterologous systems are not fully understood. In this study, we generated single or low-copy Ds insertion lines and reactivated Ds as described above. Detailed analyses of 18 transposed events were conducted using iPCR/TAIL-PCR to generate the 5’- and 3'- flanking sequences adjacent to Ds. In most lines, characteristic 8 base pair duplications were observed and confirmed by obtaining overlapping sequences from 5’- and 3’- flanks in wild-type DNA. Approximately 20% of lines had defective inverted repeats, always associated with incomplete 8 base pair duplications. 5’- and 3'- flanking sequences were also analyzed using blast-searches against DNA and protein databases and gene prediction programs. Almost 82% of flanking sequences were from either known or putative genes, which include a wall-associated kinase, cytochrome P450 and several ESTs from barley and wheat. Preliminary sequence analyses confirm the preferential insertion of Ds into gene-rich regions, providing an ideal tool for functional genomics. It also provides a platform for selective sequencing of gene-rich regions in barley and wheat, which have large genomes and a high percentage of repetitive DNA.