Establishing an efficient Ac/Ds tagging system in rice: large‐scale analysis of Ds flanking sequences

Abstract

A two-element Activator/Dissociation (Ac/Ds) gene trap system was successfully established in rice (Oryza sativa ssp. japonica cv. Nipponbare) to generate a collection of stable, unlinked and single-copy Ds transposants. The germinal transposition frequency of Ds was estimated as an average of 51% by analyzing 4413 families. Study of Ds transposition pattern in siblings revealed that 79% had at least two different insertions, suggesting late transposition during rice development. Analysis of 2057 Ds¯anking sequences showed that 88% of them were unique, whereas the rest within T-DNA. The insertions were distributed randomly throughout the genome; however, there was a bias toward chromosomes 4 and 7, which had two times as many insertions as that expected. A hot spot for Ds insertions was identi®ed on chromosome 7 within a 40-kbp region. One-third of Ds¯anking sequences was homologous to either proteins or rice expressed sequence tags (ESTs), con®rming a preference for Ds transposition into coding regions. Analysis of 200 Ds lines on chromosome 1 revealed that 72% insertions were found in genic region. Anchoring of more than 800 insertions to yeast arti®cial chromosome (YAC)-based EST map showed that Ds transposes preferentially into regions rich in expressed sequences. High germinal transposition frequency and independent transpositions among siblings show that the ef®ciency of this system is suitable for large-scale transposon mutagenesis in rice.

Genome Rearrangements in Maize Induced by Alternative Transposition of ReversedAc/DsTermini

“…In contrast, the orientation of insertion of the Ds termini appears to be random in several large collections produced for gene tagging (Kolesnik et al 2004;Nishal et al 2005;Pan et al 2005). If the insertion orientation of reversed-ends transposition is also random, then one would expect that inversions and deletions would be generated at a similar frequency.…”

Trans-Kingdom Transposition of the Maize Dissociation Element

“…There have been multiple reports of successful transposition of the hAT members in heterologous species, reflecting remarkable adaptation and flexibility of their transposition mechanism (Baker et al 1986;Weil and Kunze 2000;Ishikawa et al 2002;Kawakami and Noda 2004;Kolesnik et al 2004). On the basis of sequence conservation between distant hAT members it was previously hypothesized that horizontal transmission between distinct kingdoms occurred during their evolution (Calvi et al 1991).…”

“…Several features, including accurate cut-and-paste mechanism of transposition, small size of cis-required sequences ($600 bp of minimal Ds), large cargo-insert capacity, reasonably high transposition frequency, moderate copy number, and preferential insertion in transcribed regions and in 59 regions of genes have made the Ac/Ds elements particularly amenable for genetic studies (Parinov et al 1999;Kolesnik et al 2004;Kuromori et al 2004). Moreover, Ac/Ds elements have been successfully utilized in many heterologous plant species, although the activity in different plant hosts varied considerably.…”

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.…”