Computational protocol: Comparative Analysis of Mitochondrial Genomes in Distinct Nuclear Ploidy Loach Misgurnus anguillicaudatus and Its Implications for Polyploidy Evolution

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Protocol publication

[…] The sequence fragments obtained were edited in the Seqmen program (DNAstar, Madison, WI, USA) for contig assembly to obtain the complete mitogenome sequences. Annotation of protein-coding and ribosomal RNA genes, and definition of their respective gene boundaries were carried out with DOGMA software . The tRNAs and their secondary structures were identified by tRNAscan-SE 1.21 software. Putative origin of light strand replication (OriL), control region (CR), and conserved motifs were identified via sequence homology.Sequences were aligned using the Clustal W . Pairwise distances of the nucleotide sequences of the mitogenomes were estimated using Kimura's two-parameter method. The protein-coding gene alignment at the nucleotide level was based on the information provided by the protein alignment. Base composition and pattern (models of substitution, transition-to-transvertion [ts/tv] ratios, base composition distances) of evolution for each gene or each protein-coding gene, as appropriate, were determined with MEGA 5.0 . The numbers of variable sites, nucleotide diversity, synonymous and non-synonymous distance among the five-level ploidy M. anguillicaudatus mitogenomes were estimated with DnaSP v5.0 . The nucleotide variations in each genes/regions of the five-level ploidy M. anguillicaudatus were calculated by percent of variable sites. [...] As protein-coding genes are informative in inferring species phylogeny , the nucleotide sequences of mitochondrial proteins from five ploidy M. anguillicaudatus mitogenomes were sequenced in this study. The previously reported mitogenome sequences of 28 cobitidae species and two out-group species (Danio rerio and Cyprinus carpio) were downloaded from GenBank (). The nucleotide sequences of 13 mitochondrial protein-coding genes were separately aligned using Clustal X , the gaps and ambiguous areas were excluded manually. The phylogenetic analysis were performed based on 13 concatenated mitochondrial protein-coding genes by the maximum likelihood (ML) and Bayesian inference (BI) methods, using MEGA version 5.0 and MrBayes 3.1.2 , respectively.The jModeltest program was used to determine the best fitting models of nucleotide substitution. The Akanke's Information Criterion (AIC) indicated that the GTR+I+G model are the most appropriate for the dataset. In the BI analyses, the following settings were applied: number of Markov chain Monte Carlo (MCMC) generations  =  three million, sampling frequency  =  1000, burn-in  =  250. The robustness of the resultant ML tree was evaluated using bootstrap probabilities calculated from nonparametric bootstrap analyses with 500 pseudo-replications. […]

Pipeline specifications

Software tools DOGMA, tRNAscan-SE, Clustal W, MEGA, DnaSP, MrBayes, jModelTest
Applications Genome annotation, Phylogenetics
Organisms Misgurnus anguillicaudatus