The genom e of modern sugarcane cultivars is highly polyploid (~12x), aneuploid, of interspecific origin, and contains 10 Gb of DNA. Its size and complexity represent a major challenge for the isolation of agronomically important genes. We have undertaken the first attempt to isolate a gene from sugarcane by map-based cloning, targeting a durable major rust resistance gene (Bru1).To overcome constraints associated with high polyploidy, we developed stategies including diploid/polyploid syntenic shuttle mapping with model diploid species (sorghum and rice) and haplotype-specific chromosome walking. These strategies allowed us to develop a high-resolution genetic map including 17 markers in an interval of 0.42 cM comprising Bru1 and to build a physical map of the target haplotype that still includes two gaps at this stage due to the discovery of an insertion specific to this haplotype. BAC clones representing seven differents hom(oe)ologous haplotypes have been sequenced. These sequence s are being used to complete the physical map of the target haplotype. (Texte intégral)