Peptide sequences for each gene were aligned individually with the Muscle software (Edgar 2004); conserved alignment blocks were identified and extracted using Gblocks (Castresana 2000) and concatenated using a custom Perl script.
A second folder named "Rejected – no primers" holds those which contain at least one hot spot region but no conserved alignment blocks suitable for primer development.
We therefore used only conserved alignment blocks, located using Gblocks version 0.91 b [ 72].
Because of the e-value threshold in the homology search, poorly conserved alignments are filtered off.
The upstream regions of Tetraodon and Fugu also displayed highly conserved alignments, in particular one, positioned very close to the miRNA, with 88% identity along 126 bp.
These results indicate that the iterative bi-directional mapping strategy is not only useful to identify one-to-one mappings, but also helpful to reveal more functional conserved alignments.
Alignment of homologue protein sequences of different species with ClustalW (http://www.ebi.ac.uk/clustalw/) showed that the serine (position 95), valine (position 140) and asparagine (position 317) residues are highly conserved (alignments, see Supplementary data), supporting that these mutations are most likely pathogenic.
Since NB-LRR loci can comprise up to seven different domain types connected by partially conserved linkers, the RBH approach can result in false positives due to short but highly conserved alignments of highest-scoring sequence pairs (HSPs) in functionally non-relevant (i.e. structural) parts of the protein.
Hence, to facilitate the identification of regions of the model expected to be of high quality (i.e. highly conserved sequence alignment with no or few insertions and deletions), the structurally derived target-template alignment is shown in colors, highlighting regions of conservation between the modeled protein and the structure template (41).
Although the sequences had reduced overall similarity, there were blocks of highly conserved sequence (alignment supplementary material S3b, Supplementary Material online).
The conformation of this loop is also stabilized by another strongly differentially conserved site: alignment position 35 (D35 in eEF1A and P35 in EFL, Figure 2).
