Computational protocol: Phylogenetic analysis of the Dactylogyroides longicirrus (Monogenea: Dactylogyridae) based on the 18S and ITS 1 ribosomal genes

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[…] The ribosomal DNA of parasite was extracted using DNeasy Tissue Kit (Qiagen). The purified DNA obtained was suspended in buffer and stored -200C. The PCR amplification of 18S and ITS 1 ribosomal RNA gene was carried out by specifically designed primer, (forward, 5'- CGGTTGCAATTTTTATGTGG-3') and (reverse, 5'- GAGTGATCCACCACTTGCAG-3'). Reaction was performed in final 25 µl volume containing 3 µl of lysate, 10 X polymerase chain reaction (PCR) buffer, 1 unit of Taq polymerase (Biotools, Madrid, Spain), 0.4mM dNTP and 10 pM of each primer pair. PCR products were examined on 1.5% agarose–TBE (Trisborate- EDTA) gels, stained with ethidium bromide and visualized under ultraviolet light. Amplification products were purified by a Chromous PCR clean up kit (#PCR 10, Chromous Biotech, Bangalore, India). Gel-purified PCR products were sequenced using a Big Dye Terminator version 3.1 cycle sequencing kit in ABI 3130 genetic analyser (Applied Biosystems) with the same primers. The closely related homologous sequences were identified by comparing the 18S and ITS 1 rRNA gene sequence of D. longicirrus with the monogenean sequences available at NCBI. ClustalW2 [] was used to align all sequences with default settings. Phylogenetic trees were reconstructed using MEGA version 5 []. Phylogenetic analysis was performed based on neighbourjoining (NJ) and maximum-parsimony (MP) methods. In reconstructing the NJ tree, the Kimura two-parameter model [] was used to estimate the distances. […]

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