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<p>Abstract</p> <p>Background</p> <p>Many commercial banana varieties lack sources of resistance to pests and diseases, as a consequence of sterility and narrow genetic background. Fertile wild relatives, by contrast, possess greater variability and represent potential sources of disease resistance genes (R-genes). The largest known family of plant R-genes encode proteins with nucleotide-binding site (NBS) and C-terminal leucine-rich repeat (LRR) domains. Conserved motifs in such genes in diverse plant species offer a means for isolation of candidate genes in banana which may be involved in plant defence.</p> <p>Results</p> <p>A computational strategy was developed for unbiased conserved motif discovery in NBS and LRR domains in R-genes and homologues in monocotyledonous plant species. Degenerate PCR primers targeting conserved motifs were tested on the wild cultivar <it>Musa acuminata </it>subsp. <it>burmannicoides</it>, var. Calcutta 4, which is resistant to a number of fungal pathogens and nematodes. One hundred and seventy four resistance gene analogs (RGAs) were amplified and assembled into 52 contiguous sequences. Motifs present were typical of the non-TIR NBS-LRR RGA subfamily. A phylogenetic analysis of deduced amino-acid sequences for 33 RGAs with contiguous open reading frames (ORFs), together with RGAs from <it>Arabidopsis thaliana </it>and <it>Oryza sativa</it>, grouped most <it>Musa </it>RGAs within monocotyledon-specific clades. RFLP-RGA markers were developed, with 12 displaying distinct polymorphisms in parentals and F1 progeny of a diploid <it>M. acuminata </it>mapping population. Eighty eight BAC clones were identified in <it>M. acuminata </it>Calcutta 4, <it>M. acuminata </it>Grande Naine, and <it>M. balbisiana </it>Pisang Klutuk Wulung BAC libraries when hybridized to two RGA probes. Multiple copy RGAs were common within BAC clones, potentially representing variation reservoirs for evolution of new R-gene specificities.</p> <p>Conclusion</p> <p>This is the first large scale analysis of NBS-LRR RGAs in <it>M. acuminata </it>Calcutta 4. Contig sequences were deposited in GenBank and assigned numbers <ext-link ext-link-type="gen" ext-link-id="ER935972">ER935972</ext-link> – <ext-link ext-link-type="gen" ext-link-id="ER936023">ER936023</ext-link>. RGA sequences and isolated BACs are a valuable resource for R-gene discovery, and in future applications will provide insight into the organization and evolution of NBS-LRR R-genes in the <it>Musa </it>A and B genome. The developed RFLP-RGA markers are applicable for genetic map development and marker assisted selection for defined traits such as pest and disease resistance.</p>