Transpositional behaviour of an Ac/Ds system for reverse genetics in rice

Abstract

A collection of transposon Ac/ Ds enhancer trap lines is being developed in rice that will contribute to the development of a rice mutation machine for the functional analysis of rice genes. Molecular analyses revealed high transpositional activity in early generations, with 62% of the T0 primary transformants and more than 90% of their T1 progeny lines showing ongoing active transposition. About 10% of the lines displayed amplification of the Ds copy number. However, inactivation of Ds seemed to occur in about 70% of the T2 families and in the T3 generation. Southern blot analyses revealed a high frequency of germinal insertions inherited in the T1 progeny plants, and transmitted preferentially over the many other somatic inserts to later generations. The sequencing of Ds flanking sites in subsets of T1 plants indicated the independence of insertions in different T1 families originating from the same T0 line. Almost 80% of the insertion sites isolated showing homology to the sequenced genome, resided in genes or within a range at which neighbouring genes could be revealed by enhancer trapping. A strategy involving the propagation of a large number of T0 and T1 independent lines is being pursued to ensure the recovery of a maximum number of independent insertions in later generations. The inactive T2 and T3 lines produced will then provide a collection of stable insertions to be used in reverse genetics experiments. The preferential insertion of Ds in gene-rich regions and the use of lines containing multiple Ds transposons will enable the production of a large population of inserts in a smaller number of plants. Additional features provided by the presence of lox sites for site-specific recombination, or the use of different transposase sources and selectable markers, are discussed.

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

“…Ac/Ds tagging systems have been utilized to isolate a number of genes (Bancroft et al 1993;Jones et al 1994;James et al 1995;Bhatt et al 1996;Meissner et al 1999), to define promoter and enhancer elements (Sundaresan et al 1995;Chin et al 1999;Greco et al 2003;Wu et al 2003;Jin et al 2004), and to create genetic mosaics for clonal analysis (Peng and Harberd 1997;Jenik and Irish 2001). 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).…”

Generating Novel Allelic Variation ThroughActivatorInsertional Mutagenesis in Maize

“…Dissociation (Ds) are powerful insertional mutagens in plants and have been used to isolate genes (Kunze and Weil 2002), to define promoter and enhancer elements (Athma et al 1992;Moreno et al 1992;Sundaresan et al 1995;Chin et al 1999;Greco et al 2003;Wu et al 2003;Jin et al 2004), and to create genetic mosaics for clonal analysis (Emerson 1917;Dawe and Freeling 1990;Peng and Harberd 1997;Jenik and Irish 2001). An important feature of both Ac and the nonautonomous derivative Ds is the tendency for short-range transposition.…”

Mutant Resources in Rice for Functional Genomics of the Grasses

“…In rice, the two-component maize transposon Ac-Ds (Chin et al, 1999;Upadhyaya et al, 2002;Greco et al, 2003;Kolesnik et al, 2004) and En/Spm-dSpm (Greco et al, 2004;Kumar et al, 2008) systems have been well characterized, and good genetic selection schemes have been developed to select for transpositional activity useful for large-scale mutagenesis (Hirochika et al, 2004;Zhu et al, 2007). In addition, the endogenous rice Tos17 retrotransposon is active in specific genotypes and conditions and is an effective insertion mutagen in the rice genome .…”