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The water temperature was monitored with a thermocouple placed in the water and manually adjusted using a coil-type water heater submerged in the water bath.
The sequences were aligned and manually adjusted using ClustalW.
Sequences were aligned using Clustal X [50] and manually adjusted using MacClade v. 4.06 [60].
Sequences of functional genes were aligned using Clustal X [65] and manually adjusted using BioEdit [66].
The initial model was refined by alternating automatic minimization protocols performed with CNS inspecting visual electron density map and manually adjusted using the program O [37].
In order to identify and trim sequences to the anchor regions, nrITS and psbA-trnH regions were aligned using CLUSTAL W [39] and then manually adjusted using MacClade (Sinauer Associates, Inc., Sunderland, MA).
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Since the algorithm is designed to 'over cluster', clusters were then manually verified and adjusted using Klusters (Hazan et al., 2006).
AFF4 was built manually and the model was adjusted using Coot (Emsley and Cowtan, 2004).
Nucleotide sequences were aligned with MAYV sequences available in GenBank by using Clustal X (http://bips.u-strasbg.fr/fr/Documentation/ClustalX/), and manually adjusted by using Se-Al (http://tree.bio.ed.ac.uk/software/seal/).ac.uk/software/seal/
To validate the putative uORFs, the first 100 amino acids of the Arabidopsis mORF were aligned to Genbank plant ESTs using tBLASTn (E-value = 1e-10, limit: Viridiplantae [orgn] NOT Arabidopsis [orgn], complexity filter off), and all retrieved plant uORF sequences were aligned to rice and Arabidopsis uORFs using ClustalW [ 74], manually adjusted, and visualized using Jalview [ 75].
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