Megalign tool

Nucleotide sequences were edited and assembled using the SeqManII tool of the Lasergene DNAStar program 7.1 (DNAStar) (Madison, WI, USA, 2006). The Megalign tool (DNAStar) was used for pairwise and multiple alignments of DNA sequences and for deducing amino acid sequences. Shannon entropy was calculated using BioEdit software [27 ]. Diversity and distance were calculated using the Molecular Evolutionary Genetics Analysis (MEGA) program 5 and a pairwise deletion option. Phylogenetic and molecular evolutionary analyses were conducted using MEGA5 (Center for Evolutionary Functional Genomics Biodesign Institute, Arizona State University, Tempe, AZ, USA, 2011) by the neighbor joining method [28 (link)]. Bootstrap values (based on 1000 replicates) for each node were provided if they were at least 70 %. N-glycosylation sites were predicted using N-GlycoSite (http://www.hiv.lanl.gov/content/sequence/GLYCOSITE/glycosite.html). The stable mutation rates (marginal frequencies at each position of the amino acid sequence) were calculated using the MargFreq software (http://sray.med.som.jhmi.edu/SCRoftware/MargFreq/). The statistical analysis of the sequence variations was performed using SAS (Statistical Analysis System) (version 9.2, SAS Institute Inc, SAS Campus Drive, Cary, NC, USA, 2008).

For multiple sequence analysis, all Arabidopsis CrRLK1L gene and protein sequences were aligned with the help of the MegAlign tool of the DNASTAR (Lasergene) sequence analysis software. GeneDoc software was used for editing multiple sequence alignments⁷³ . Definition and sizing of the CrRLK1L protein domains were done with Interpro 65.0 (http://www.ebi.ac.uk/interpro/)^{74 (link)}. Multiple alignment for phylogenetic analysis of the extracellular domains was calculated by ClustalW. The tree was constructed using the Maximum Likelihood method with 1000 bootstrap repeats in MEGA6. Public transcriptomics data were consulted using the eFP browser^{31 (link),75 (link)}, Genevestigator^{34 (link),35 (link)} and Araport in the ThaleMine data warehouse^{36 (link),37 (link)}.

The MegAlign tool (DNASTAR, USA) was used to calculate nucleotide sequence similarity. Clones showing ≥90% identity in their nucleotide sequence were categorized as part of the same genetic group. Sequence similarity searches were conducted using BLASTX 2.2.30 against the GenBank non-redundant protein sequences database [56 (link)]. The ExPASy translation tool [57 (link)] was used to translate and predict the correct reading frames for the nucleotide sequences. Partial PhaC sequences were aligned using the MUSCLE algorithm [58 (link)] of the BioEdit program [59 ]. Protein sequences were also checked for the presence of the conserved catalytic lipase-box (G-X-[S/C]-X-G) [9 (link)]. Protein neighbor-joining (NJ) phylogenetic trees were constructed using MEGA5 [60 (link)] with MUSCLE alignment and 1000 bootstrap tests.