Computational protocol: Miiuy Croaker Hepcidin Gene and Comparative Analyses Reveal Evidence for Positive Selection

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Protocol publication

[…] The mRNA expression patterns of hepcidin gene in different tissues (liver, spleen, kidney, intestines, heart, muscle, stomach, brain, swim bladder, and fin) of healthy miiuy croakers and in four tissues (liver, spleen, kidney, and intestines) of infected and health miiuy croakers were determined using real-time RT-PCR. Tissue samples from three individuals were mixed for RNA preparation. Primers HAMP-RT-F (5′-TCGTGCTCGCCTTTATTTG-3′) and HAMP-RT-R (5′-AACCGCAGCCTTTGTTCTT-3′) were used for amplifying gene fragment. Real-time quantitative PCR was conducted on a 7500 Real-time PCR system (Applied Biosystems, USA). Amplifications were carried out at a final volume of 20 µl, containing 1 µl cDNA sample, 10 µl SYBR Green Real-time PCR master mixtures (Takara), 0.4 µl ROX II, 1 µl of each primer and 6.6 µl ddH2O. The reaction carried out without the template was used as blank control. PCR amplification was performed in triplicate wells, using the following conditions: 10 sec at 95°C, followed by 40 cycles consisting of 5 sec at 95°C and 34 sec at 60°C, dissociation curve analysis was performed after each assay to determine target specificity. Expression of β-actin was used as internal control in gene expression analysis. The primers β-actin-RT-F and β-actin-RT-R were used for RT-PCR of β-actin expression. Comparisons between groups were made by one-way analysis of variance followed by a Duncan test for identification of the statistically distinct groups. [...] Multiple alignments of the HAMP sequences from different species were performed using the Muscle . The appropriate model of sequences evolution was determined with jModeltest under Bayesian Information Criterion (BIC). The GTR+I+G model of sequence evolution for phylogenetic analysis. Phylogenetic tree was reconstrucred using Bayesian inference in MrBayes3.1 . The program was run with 5,000,000 generations with a burn-in of 25%. [...] Phylogenetic analysis showed that HAMP of fish can be divided into two paralogus lineages (HAMP1 and HAMP2), therein, HAMP1 is an orthologue of the mammalian sequences; paralogs gene (HAMP2) is generated in 3R genome replication [16.28]. Closely related genes show rapid functional diversification immediately after a duplication event until they have adapted to their new function . A total of twenty-seven unique sequences representing twelve HAMP1 sequences and fifteen HAMP2 sequences, respectively, were used for evolutionary analyses.In order to estimate the selective constraints on the HAMP gene, evolutionary analysis were performed with PAML 4.5 program suite , . The hypothesis of positive selection was tested using site-specific model in the CODEML program. We employed the random-sites models assuming several heterogeneous sites with different ω parameters without a priori knowledge of which class (neutral, purifying, or positive selection) a given codon belongs to. To account whether positive selection has been operating on any codon sites, we estimated parameters under six different codon substitution models (M0, M1a, M2a, M3, M7, and M8 model) . The likelihood ratio tests (LRT) were performed to compare the corresponding models with and without selection (ie, M0 vs M3, M1a vs M2a, and M7 vs M8). When the alternative models M2a and M8 suggest the presence of codons with ω>1, this can be considered as evidence of positive selection . Posterior probabilities for site classes have been calculated by Bayes empirical Bayes (BEB) in the case of models M2a and M8 . Statistical significance is determined by comparing twice the log-likelihood difference (2ΔLnL) to a χ2 distribution with degrees equal to the difference in the number of parameters between the models to be compared . The posterior means of ω for some sites classes are great than one (calculated are the average of ω over all sites classes weighted by the posterior probabilities), those sites are likely to be under positive selection . […]

Pipeline specifications

Software tools MUSCLE, jModelTest, MrBayes, PAML
Applications Phylogenetics, Nucleotide sequence alignment
Organisms Miichthys miiuy, Vibrio anguillarum, Homo sapiens
Diseases Bacterial Infections
Chemicals Cysteine