Identification of MAPK and MAPKK Genes in Brachypodium distachyon

The B. distachyon genome assembly version 1.2 was downloaded from the website ftp://ftp.brachypodium.org/to construct a local protein database. Method used to identify the MAPK and MAPKK genes in B. distachyon was similar to that described in rice and poplar [19] (link). For the MAPKK gene family, the predicted proteins derived from B. distachyon pseudo-molecules were queried using a profile Hidden Markov Model-based search (HMMER: http://hmmer.wustl.edu/) with an HMM built from the ten Arabidopsis MAPKKs [32] (link). MAPKK gene models were only accepted if they displayed the consensus sequences for dual-specificity protein kinases, including the conserved aspartate and lysine residues within the active site motif (–D(L/I/V)K-), and the plant-specific phosphorylation target site motif (–S/TxxxxxS/T-) within the activation loop. Similarly, the predicted B. distachyon proteins were queried using a profile Hidden Markov Model-based search with an HMM built from the twenty Arabidopsis MAPKs for the MAPK gene family. MAPK gene models were only accepted if they contained the canonical consensus sequences for serine/threonine protein kinases, as well as an appropriately positioned activation loop (-TXY-motif). And then the predictions of MAPK and MAPKK coding sequences were verified with available EST. Finally, multiple alignments of the identified B. distachyon amino acid sequences of these two gene families with that of Arabidopsis and rice were performed by Clustal W [33] (link) with default options and the phylogenetic trees were constructed based on the bootstrap neighbor-joining (NJ) method with a Kimura two-parameter model by MEGA [34] (link).