Computational protocol: Comparative analysis of the RTFL peptide family on the control of plant organogenesis

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Protocol publication

[…] The database used for searching sequences of RTFL members are listed in TableĀ . To select RTFL members, HMMER was used to search the Pfam database and BLASTP was used to search SALAD, Pytozome v8.0, NCBI, and Sol genomics network (TableĀ ). Whole-sequence comparison of RTFLs was obtained from the Surveyed Conserved Motif Alignment Diagram and the Associating Dendrogram (SALAD) database (Mihara et al. ). SALAD is commonly used for genome-wide comparative analysis of annotated protein sequences in plants. The evolutionarily conserved short amino acid sequences in homologous proteins were identified as motifs using MEME software (http://meme.sdsc.edu/meme/intro.html) in SALAD. The motif significance is reported as the E-value upon MEME analysis, and the motif number in SALAD depends on this E-value. However, this numbering system sometimes results in dispersive motif numbers among the paralogs. In order to keep the motif numbers consecutive in the text, six motif numbers (Motif 5, 11, 12, 14, 16, 24) were slightly modified and re-numbered by considering a generality in distribution of each motif among various plant taxa (see the modification in Table S2). Annotations with similar motif patterns were grouped into the same clades according to the value of approximately unbiased (AU) or bootstrap probability (BP) (Shimodaira , ). Clustering was calculated using pvclust in R software (http://www.r-project.org/).Phylogenetic relationships between RTFLs in O. sativa and Arabidopsis were evaluated using MEGA version 6 (Tamura et al. ). A total of 43 RTF sequences of O. sativa and Arabidopsis were used for the analysis. Molecular Phylogenetic analysis was inferred using the maximum likelihood method based on the JTT matrix-based model (Jones et al. ). The bootstrap consensus tree inferred from 1000 replicates (Felsenstein ) was used to represent the evolutionary history of the taxa analyzed. Initial tree(s) for the heuristic search were obtained by applying the Neighbor-Joining method to a matrix of pairwise distances estimated using a JTT model. […]

Pipeline specifications

Software tools HMMER, BLASTP, Pvclust, MEGA
Databases Pfam SGN SALAD
Application Phylogenetics
Organisms Arabidopsis thaliana, Oryza sativa