Plant & Animal Genomes XIV Conference Plant & Animal Genomes XIV ConferenceJanuary 14-18, 2006
Town & Country Convention Center
San Diego, CA
Robert N.G. Miller , Georgios J. Pappas Jr. , Manoel T. Souza Jr. , David J. Bertioli
Most commercial banana varieties (Musa acuminata Colla) show high sterility and little genetic diversity, with sources of resistance to important pests and diseases frequently lacking. The wild diploid genotype Musa acuminata subsp. burmannicoides, var. Calcutta 4 shows considerable resistance to fungal pathogens and represents a potential source of resistance related genes. Amongst these, the NBS-LRR family is the most abundant class, and is characterized by the presence of conserved nucleotide binding site (NBS) and leucine rich repeat (LRR) domains. To identify resistance gene analogs (RGAs) we devised a computational strategy for degenerate primer design based on analysis of available monocotyledon sequences. Genomic DNA from Calcutta 4 was PCR amplified with 15 degenerate primer pairs targeting NBS or both NBS-LRR domains, with products cloned and randomly sequenced. Out of a total of 672 sequences with NBS targeting primers, approximately 10% (68 sequences) showed significant similarity with RGAs. When using NBS-LRR spanning primers, 257 out of 901 (29%) sequences showed significant hits with RGAs, a 3-fold improvement in selectivity. Assembly of all 325 RGA sequences generated 54 contigs, with 28 full length sequences. Phylogenetic analyses including an A. thaliana and O. sativa RGA sequence dataset revealed the expected prevalence of Musa and monocotyledon-specific clades, as well as paraphyletic clades, indicating amplification of a wide spectrum of RGA sequences. Representative RGAs will be used to screen a Calcutta 4 BAC library for shotgun sequencing and genetic marker development.