Rapid, large‐scale generation ofDstransposant lines and analysis of theDsinsertion sites in rice

Abstract

The ®rst six authors contributed equally to the paper. SummaryRapid, large-scale generation of a Ds transposant population was achieved using a regeneration procedure involving tissue culture of seed-derived calli carrying Ac and inactive Ds elements. In the F 2 progeny from genetic crosses between the same Ds and Ac starter lines, most of the crosses produced an independent germinal transposition frequency of 10±20%. Also, many Ds elements underwent immobilization even though Ac was expressed. By comparison, in a callus-derived regenerated population, over 70% of plants carried independent Ds insertions, indicating transposition early in callus formation. In the remaining population, the majority of plants carried only Ac. Most of the new Ds insertions were stably transmitted to a subsequent generation. An exceptionally high proportion of independent transposants in the regenerated population means that selection markers for transposed Ds and continual monitoring of Ac/Ds activities may not necessarily be required. By analyzing 1297 Ds-¯anking DNA sequences, a genetic map of 1072 Ds insertion sites was developed. The map showed that Ds elements were transposed onto all of the rice chromosomes, with preference not only near donor sites (36%) but also on certain physically unlinked arms. Populations from both genetic crossing and tissue culture showed the same distribution patterns of Ds insertion sites. The information of these mapped Ds insertion sites was deposited in GenBank. Among them, 55% of Ds elements were on predicted open-reading frame (ORF) regions. Thus, we propose an optimal strategy for the rapid generation of a large population of Ds transposants in rice.

Ac-Immobilized, a Stable Source of Activator Transposase That Mediates Sporophytic and Gametophytic Excision of Dissociation Elements in Maize

“…The tendency of Ds to transpose to linked sites has also been exploited to disrupt genes present in tandem arrays (Takken et al 1999;Tantikanjana et al 2004) and to perform localized saturation mutagenesis (Nishal et al 2005). Ac/Ds mutagenesis platforms in Arabidopsis (Sundaresan et al 1995;Ito et al 1999;Muskett et al 2003;Kuromori et al 2004), tomato (Healy et al 1993;Knapp et al 1994;Meissner et al 2000), tobacco (Scofield et al 1992), rice (Chin et al 1999;Zheng-Ge et al 2003;Kim et al 2004;Kolesnik et al 2004), and barley (Koprek et al 2000;Cooper et al 2004) have been developed. Yet, despite the demonstrated utility of Ds in gene tagging in maize (Hake et al 1989;Colasanti et al 1998), a two-component Ac/Ds genetagging program for this important crop plant has not yet been developed.…”

The Institute for Genomic Research Osa1 Rice Genome Annotation Database

“…Multiple efforts are under way to generate a large collection of tagged insertion mutants in rice using T-DNA tagging (Sallaud et al, 2004), Ac/Ds (Greco et al, 2001;Kim et al, 2004), and Tos17 (Miyao et al, 2003). We downloaded from the dbGSS division of GenBank a total of 26,725 sequences reported to be flanking sequence tags (FSTs) from various projects.…”

RAV-Like1Maintains Brassinosteroid Homeostasis via the Coordinated Activation ofBRI1and Biosynthetic Genes in Rice

“…RAVL1 was identified by examination of two mutant alleles identified in a Ds transposant rice population (Kim et al, 2004) as dark-green and semidwarf plants. The mutants were named ravl1-1 and ravl1-2 (see below), and the trap Ds elements were located in the N-terminal region of ravl1-1 and the C-terminal region of ravl1-2.…”