Lignin is a major recalcitrant factor in the production of bioethanol from lignocellulosic biomass. The object of this project is to isolate and clone genes involved in the biosynthesis of lignin in grasses to facilitate reduction of recalcitrance of temperate grasses. We present the cloning and characterization of five cinnamyl alcohol dehydrogenase (CAD) genes from the Brachypodium distachyon genotype Bd21-3. One of these genes BdCAD1 exhibits 86% identity to CAD2 genes from maize and sorghum, respectively. Phylogenetic analyses of known CAD genes from different species places BdCAD1 together with CAD2 genes from rice, maize and sorghum and CAD4 and CAD5 from dicot Arabidopsis. This branch of CAD genes has been implicated in the lignification of vascular tissues. Mutations in maize and sorghum CAD2 genes have been linked to the brown midrib phenotype which is associated with reduced lignin content and increased digestibility. We are currently generating a mutant population of the model grass Brachypodium distachyon using sodium azide and γ-ray. We are screening the mutant population for mutations in the lignin biosynthesis pathway using the TILLING approach. As an alternative strategy we also employ a saccharification assay in an attempt to identify mutant lines with a higher saccharification potential.

Keywords: biofuel; lignin; cellulosic; cinnamyl alcohol dehydrogenase; CAD; Brachypodium distachyon