Computational protocol: Miiuy Croaker Transferrin Gene and Evidence for Positive Selection Events Reveal Different Evolutionary Patterns

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Protocol publication

[…] Healthy miiuy croakers (800±25 g) were collected from Zhoushan Fisheries Research Institute (Zhejiang, China). The fishes were acclimatized in a recirculating seawater system at ambient temperature and under a natural photoperiod for at least one week. The fishes were fed twice daily with commercial pellets until satiety.Miiuy croaker challenge experiments with V. anguillarum were performed as previously described , the miiuy croaker was injected with 1 mL bacteria V. anguillarum suspension, which was made after V. anguillarum centrifuge to approximately 3×107 CFU/ml in phosphate-buffered saline. And uninfected fish were maintained in separate tanks as control group. Infected fish were respectively killed at 6, 12, 24, 36, 48 and 72 h after injection treatment. Tissues (liver, spleen and kidney) were removed and then stored at −70°C until use. To determine the TF gene expression in the healthy miiuy croakers, ten tissue samples from the heart, muscle, kidney, eye, gill, intestine, brain, spleen, fin and liver were collected from uninfected miiuy croakers and placed in tubes. The samples were then immediately frozen in liquid nitrogen after dissection and then separately stored at −80°C prior to RNA extraction. [...] All the TF cDNA sequences used in this study were obtained from the GenBank ( and Ensemble ( databases: eight TF sequences from eight mammals and twenty-eight sequences from twenty-three teleosts, together with two TF from two invertebrates as outgroups. Taxonomic information and accession numbers are provided in . The SignalP 4.0 Server was adopted for signal peptide prediction . The MUSCLE software was used for the alignment of the putative amino acid sequences of miiuy croaker and other known vertebrates using default alignment parameters . The sequence identities were determined using the MEGALIGN program of DNASTAR .The jModeltest software was used to select the optimal substitution model for our collected nucleotides data . The Akaike information criterion (AIC) was used to choose the optimal model. A phylogenetic tree was constructed via the Bayesian approach using MrBayes v3.1.2 ; the program was run using 10,000,000 generations under GTR+I+G model, with 25% of trees burned. The resulting phylogenetic tree was then visualized and edited using TreeView . [...] The phylogenetic tree () was used to determine the possible effects of diverse environments on the selective pressures on the ancestors of aquatic or terrestrial organisms. The selective pressures at the ancestral branches of vertebrates, mammals and teleosts were analyzed using the maximum likelihood (ML) methods in the CODEML program of PAML v4 . First, the one-ratio model which assumed that all branches have only one ratio, was used to identify the selective pressures in all TF genes. Second, the free ratio that allowed varied ω ratios in every branch was used to determine via the likelihood ratio test (LRT) whether this model suitably fitted the data compared with the one-ratio model. The branch-site model was then used to identify the ancestral foreground lineages of interest of vertebrates, mammals and teleosts.The subsets of mammalian and teleost TF sequences, both of which used the TF of the two invertebrates as the out group, were used to account for the different functional and structural constraints experienced by individual site–domains in the site model . Six site models were used on the teleost and mammalian TF sequences subsets to investigate the possible positively selected sites among those lineages. In all cases, the two-fold differences of log-likelihood values (2ΔlnL) between each of the two nested models were calculated following a chi-square distribution, with the degrees of freedom equal to difference in the number of parameters between the nested models . The Bayes empirical Bayes (BEB) were used in the M2a and M8 models to calculate the Bayesian posterior probability (BPP) of the codon sites under a positive selection . […]

Pipeline specifications

Software tools MUSCLE, SignalP, jModelTest, MrBayes, TreeViewX, PAML
Applications Phylogenetics, Nucleotide sequence alignment
Organisms Miichthys miiuy, Acanthamoeba polyphaga mimivirus, Vibrio anguillarum
Diseases Bacterial Infections
Chemicals Iron