Computational protocol: Characterization of cytokinin signaling and homeostasis gene families in two hardwood tree species: Populus trichocarpa and Prunus persica

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Protocol publication

[…] Populus and Prunus homologues of Arabidopsis genes were identified by searching the Populus trichocarpa genome database versions 1.1 and 3.0 using various bioinformatic tools and databases available via the Populus genome portal (http://genome.jgi-psf.org/Poptr1_1/Poptr1_1.home.html; http://www.phytozome.net/search.php), and the Prunus persica genome version 1 [], using databases available via The Genome Portal of the Department of Energy Joint Genome Institute [] (http://genome.jgi-psf.org/Poptr1_1/Poptr1_1.home.html), and Phytozome portal [] (http://www.phytozome.net/search.php?org=Org_Ptrichocarpa_v3.0; http://www.phytozome.net/search.php?method=Org_Ppersica). The bioinformatics tools included BLAST searches, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), EuKaryotic Orthologous Groups Database (KOG) and ortholog finder. Arabidopsis sequences were identified using The Arabidopsis Information Resource (TAIR). Amino acid sequences were aligned using ClustalW followed by manual adjustments when needed []. The best bidirectional hit (BBH) method was used as the first approach to determine orthologous pairs of the cytokinin signaling and response genes in Prunus as described by []. Protein sequences were aligned using Jalviewand ClustalW2 [] followed by manual adjustments where needed. The resulting alignment was precisely back-translated to yield a data matrix of the corresponding nucleotide sequences. Gene models, transcript IDs and physical loci of Populus genes used in construction of phylogenetic trees can be found in Additional file : Table S1. Gene models and EST support for Prunus genes can be found in Additional file : Table S2 (physical loci of the genes are not available in the current 1.0 version of the genome), and TAIR gene numbers of Arabidopsis genes in Additional file : Table S3. [...] We used a maximum likelihood search strategy on amino acid alignments to investigate orthologs and paralogs in the cytokinin signaling and homeostasis gene families. Sequences were aligned using MUSCLE with default settings []. A single most optimal tree for each data set was computed using the RaxML BlackBox web server (http://embnet.vital-it.ch/raxml-bb/) running RaxML version 7.2.8 []. Default settings were used with the WAG model of molecular evolution including a gamma parameter. One hundred bootstrap samples were generated to assess support for the inferred relationships. Local bootstrap values (in percentages) are indicated for branches with ≥50% support. […]

Pipeline specifications

Software tools Ortholog-Finder, Clustal W, MUSCLE, RAxML
Databases TAIR Phytozome KEGG KOG
Applications Genome annotation, Phylogenetics, Nucleotide sequence alignment
Organisms Populus trichocarpa, Prunus persica, Arabidopsis thaliana