Computational protocol: Mitochondrial Genetic Differentiation of Spirlin (Actinopterigii: Cyprinidae) in the South Caspian Sea basin of Iran

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Protocol publication

[…] In the laboratory, muscle tissues were dissected immediately and stored at −20 °C prior to total DNA extraction. DNA was extracted from the tissue using the cetyl-trimethylammonium bromide method. A fragment of cytochrome b was sequenced using two primer combinations that amplify a region of 774 base pairs starting from the 5′ terminus of the mitochondrial cytochrome b primers in the flanking (GluDG.L TGACTTGAARAACCAYCGTT) and threonine (cb6b.HGGAATTCACCTCTCC) regions. Polymerase chain reaction products were cleaned for sequencing and aligned using Mega 4. Alignment of cytochrome b was based on the inferred amino acid sequence. Our data set was complemented by cytochrome b sequences of four A. bipunctatus (GenBank accession AF090740, HM560059, Y10445, and AF090741). The sequences generated in this study for each gene have been submitted to GenBank under accession numbers (HQ658865–HQ658893, ). [...] The nucleotide sequences obtained were subjected to phylogenetic analysis by the neighbor-joining, maximum parsimony, and maximum likelihood methods using the PAUP 4.0b10 program. The aligned sequences were also estimated by Bayesian inference using MrBayes 3.1.2. The choice of model for the Bayesian inference and maximum likelihood method was determined based on the Akaike information criterion as implemented in the Model test by Aliabadian et al. For maximum likelihood purposes, the estimated models were used in a subsequent maximum likelihood heuristic tree search with 10 random addition sequence replicates, using tree bisection and reconnection branch swapping. To assess the nodal support for each branch, 100 and 1000 bootstrap replicates were run under maximum likelihood and maximum parsimony, respectively, with parameters estimated automatically. The choice of model for the Bayesian analyses was determined based on an Akaike information criterion as implemented in the Model test. With four Metropolis-coupled MCMC chains, incremental heating temperatures of 0.1 °C were run for 20 million generations and sampled every 100 generations. The first 10 million generations of each run, well after the chain reached apparent stationarity, were discarded as burn-in. The resulting 50% majority rule consensus tree was considered for the hypothesis.The Kimura-2 parameter distance was used to calculate the genetic distances for cytochrome b to quantify sequence divergences between individuals. We compared alternative phylogenetic hypotheses using the Shimodaira-Hasegawa test, as implemented in RAxML version 7.0.3. The tested topologies were obtained, enforcing the monophyly of 10 selected populations in the maximum likelihood searches for RAxML. […]

Pipeline specifications

Software tools MUSCLE, MEGA, PAUP*, MrBayes, RAxML
Applications Phylogenetics, Nucleotide sequence alignment
Chemicals Nucleotides