Phylogenetic Analysis of hsp70-234 Gene

PCR-hsp70-234 products were purified with the EXOSAP-ITTM Express PCR Product Cleanup (AppliedBioystems^®, University Park, IL, USA) and prepared for sequencing using the BigDye Terminator Kit v3.1 (Thermo Fisher Scientific^®, Waltham, MA, USA) according to the manufacturer’s instructions. The forward and reverse sequences were obtained using the ABI3500XL automatic DNA analyzer. The sequences obtained were mounted in CAP3 software (Iowa State University, Ames, IA, USA), aligned using MAFFT v.7.221 (Universitu of Osaka, Osaka, Japan) [22 (link)], edited in Geneious v.8.1.7 software (Biomatters Ltd., Auckland, New Zealand) [23 (link)], and compared with sequences available in GenBank using the BLAST tool. Phylogenetic analyses were performed in three stages: (i) determination of the evolutionary model and likelihood analysis using the IQ-TREE v.1.3.2; (ii) phylogenetic reconstruction in IQ-TREE v.1.3.2 software (Center for Integrative Bioinformatics Vienna, Wien, Austria) using a non-parametric reliability test of 1000 replicates and bootstraps; and (iii) editing of the phylogenetic tree using FigTree v.1.4.2 software (University of Edinburgh, Edinburgh, UK) [24 (link)].