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Plant Cell, Vol. 11, 1853-1866, October 1999, Copyright © 1999, American Society of Plant Physiologists

GENOMICS ARTICLE

A Two-Component Enhancer-Inhibitor Transposon Mutagenesis System for Functional Analysis of the Arabidopsis Genome

Elly Speulmana, Peter L. J. Metza, Gert van Arkela, Bas te Lintel Hekkerta, Willem J. Stiekemaa, and Andy Pereiraa
a Centre for Plant Breeding and Reproduction Research, Department of Molecular Biology, P.O. Box 16, NL-6700 AA Wageningen, The Netherlands

Correspondence to: Andy Pereira, A.Pereira{at}cpro.dlo.nl (E-mail), 31-317-418094 (fax)

A modified Enhancer-Inhibitor transposon system was used to generate a series of mutant lines by single-seed descent such that multiple I insertions occurred per plant. The distribution of original insertions in the population was assessed by isolating transposon-flanking DNA, and a database of insertion sites was created. Approximately three-quarters of the identified insertion sites show similarity to sequences stored in public databases, which demonstrates the power of this regimen of insertional mutagenesis. To isolate insertions in specific genes, we developed three-dimensional pooling and polymerase chain reaction strategies that we then validated by identifying mutants for the regulator genes APETALA1 and SHOOT MERISTEMLESS. The system then was used to identify inserts in a class of uncharacterized genes involved in lipid biosynthesis; one such insertion conferred a fiddlehead mutant phenotype.




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