Computational protocol: Delineation of plant caleosin residues critical for functional divergence, positive selection and coevolution

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Protocol publication

[…] Caleosin gene families were identified from 15 completely sequenced genomes representing the plant lineage from unicellular green algae to multicellular plants. The search was performed using "caleosin" as a keyword in the Phytozome ( database; eight Arabidopsis thaliana caleosin genes were first retrieved and then used as a query sequence in BLAST searches (BLASTP and TBLASTN) and the sequences retrieved from corresponding plant-genome annotation resources were analyzed. Partial and redundant sequences were excluded. Sequences were obtained from the following groups and species: the unicellular green algae Chlamydomonas reinhardtii and Volvox carteri (; the moss Physcomitrella patens (; the lycophyte Selaginella moellendorffii (; the monocotyledonous angiosperms Brachypodium distachyon (, Oryza sativa (, Setaria italica (, Zea mays (, and Sorghum bicolor (; and the dicotyledonous angiosperms Arabidopsis thaliana (, Citrus sinensis (, Cucumis sativus (, Brassica rapa (, Glycine max (, and Populus trichocarpa ( In addition, the Pfam ( and SMART ( databases were employed to detect conserved domains with caleosin or caleosin-like protein candidates. Finally, we manually refined the search results based on the Pfam (PF05042) and SMART analyses to further reduce hits with partially conserved functional domains and other false positives. [...] Multiple sequence alignment was the first step in the phylogenetic analysis; alignment quality may have a significant impact on the final phylogenetic tree [,]. Amino acid sequences of caleosin genes were aligned using Clustal X [,] with the default parameters, excluding poorly aligned positions, gap positions, and divergent regions. Phylogenetic analyses were performed with a neighbor-joining (N-J) method using MEGA5 [] and the reliability of interior branches was assessed with 1000-bootstrap resampling. To confirm the tree topologies, maximum likelihood (ML) and Bayesian trees were constructed using PhyML 3.0 [] and MrBayes 3 [,], respectively, the results of which showed similar topology with only minor modifications at deep nodes (data not shown). Finally, the N-J phylogenetic tree was determined with sub V as the root (Figure  A). […]

Pipeline specifications

Software tools BLASTP, TBLASTN, Clustal W, MEGA, PhyML, MrBayes
Databases Pfam Phytozome
Applications Phylogenetics, Amino acid sequence alignment
Organisms Arabidopsis thaliana
Chemicals Calcium