Bananas are the developing world's fourth most important food crop. Many cultivated clones are hybrids between M. acuminata (A genome) and M. balbisiana (B genome). Recently, banana streak disease infection has been causing serious yield loses. The disease is caused by banana streak badnavirus (BSV) and the infections seem to result from the activation of retroviral sequences integrated into the B genome. In order to facilitate positional cloning, physical mapping and genome sequencing, we have constructed a BAC library from M. balbisiana. We have isolated high-molecular-weight genomic DNA using a standard and novel protocols, the latter involving flow cytometry to purify nuclei from tissue homogenates. The library was constructed by partial digestion of genomic DNA with HindIII restriction enzyme and cloning into the pIndigoBAC-5 vector. The average insert size of the library is 135 kb with more than 70% of inserts larger than 120 kb. The entire library consists of 36,864 clones and represents 9x genome equivalents. Screening with chromosome-specific markers proved representation of the whole B genome and confirmed estimated genomic coverage. On average, the library is contaminated with 3% chloroplast clones and 0.004% mitochondrial clones. However, only 0.09% clones obtained from flow-sorted nuclei contain chloroplast DNA, indicating the advantage of the novel procedure. This library provides an essential resource for the analysis of BSV activation mechanisms and isolation of genes responsible for resistance to biotic and abiotic stress. The project was supported by CIRAD, Academy of Sciences of the Czech Republic (IAA6038201) and French Ministry of Foreign Affairs (COCOP 17/01). (Texte intégral)