Computational Identification and Characterization of Novel Genes from Legumes

The Fabaceae, the third largest family of plants and the source of many crops, has been the target of many genomic studies. Currently, only the grasses surpass the legumes for the number of publicly available expressed sequence tags (ESTs). The quantity of sequences from diverse plants enables the use of computational approaches to identify novel genes in specific taxa. We used BLAST algorithms to compare unigene sets from Medicago truncatula, Lotus japonicus, and soybean (Glycine max and Glycine soja) to nonlegume unigene sets, to GenBank's nonredundant and EST databases, and to the genomic sequences of rice (Oryza sativa) and Arabidopsis. As a working definition, putatively legume-specific genes had no sequence homology, below a specified threshold, to publicly available sequences of nonlegumes. Using this approach, 2,525 legume-specific EST contigs were identified, of which less than three percent had clear homology to previously characterized legume genes. As a first step toward predicting function, related sequences were clustered to build motifs that could be searched against protein databases. Three families of interest were more deeply characterized: F-box related proteins, Pro-rich proteins, and Cys cluster proteins (CCPs). Of particular interest were the >300 CCPs, primarily from nodules or seeds, with predicted similarity to defensins. Motif searching also identified several previously unknown CCP-like open reading frames in Arabidopsis. Evolutionary analyses of the genomic sequences of several CCPs in M. truncatula suggest that this family has evolved by local duplications and divergent selection.