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

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 T-0 primary transformants and more than 90% of their T-1 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 T-2 families and in the T-3 generation. Southern blot analyses revealed a high frequency of germinal insertions inherited in the T-1 progeny plants, and transmitted preferentially over the many other somatic inserts to later generations. The sequencing of Ds flanking sites in subsets of T-1 plants indicated the independence of insertions in different T-1 families originating from the same T-0 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 T-0 and T-1 independent lines is being pursued to ensure the recovery of a maximum number of independent insertions in later generations. The inactive T-2 and T-3 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.