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

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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.

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Acknowledgements

We thank Gert van Arkel and Wim Dirkse for assistance in the isolation of flanking sequences, Hans Sandbrink and Joost de Groot for assistance in Blast analyses and Dick Geurtjes for taking care of the plants in the greenhouse. This research was funded in the participating labs by a European Union project BIO4 CT 972132 on "Transposon mutagenesis in rice". The work has been carried out in compliance with the current laws governing genetic experimentation in The Netherlands.

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  1. Plant Research International, PO Box 16, 6700 AA, Wageningen, The Netherlands

    R. Greco & A. Pereira

  2. Institute of Biology Leiden, Clusius Laboratory, Leiden University, Wassenaarseweg 64, 2333 AL , Leiden, The Netherlands

    P. B. F. Ouwerkerk, R. J. de Kam, A. H. Meijer & J. H. C. Hoge†

  3. CIRAD-BIOTROP, TA 40/0, Avenue Agropolis, 34398 cedex 5, Montpellier, France

    C. Sallaud & E. Guiderdoni

  4. Department of Biology, University of Milan, Via Celoria 26, 20133, Milano, Italy

    C. Favalli & L. Colombo

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Correspondence to A. Pereira.

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Communicated by F. Salamini

This report is dedicated to the loving memory of our colleague Dr J. Harry C. Hoge

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Greco, R., Ouwerkerk, P.B.F., de Kam, R.J. et al. Transpositional behaviour of an Ac/Ds system for reverse genetics in rice. Theor Appl Genet 108, 10–24 (2003). https://doi.org/10.1007/s00122-003-1416-8

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