The ligated product was introduced into the E. coli strain JM109 by chemical transformation. One colony from each cloning reaction was selected. The recombinant plasmids were purified using Wizard® Plus SV Minipreps DNA purification system (Promega, Madison, USA) and bidirectional sequenced using universal primer T7 and SP6. DNA sequencing was conducted by 1st Base Pte. Ltd., Singapore.
The chromatograms were validated and assembled in BioEdit version 7.0.1. Phylogenetic analysis The sequences were multiple-aligned with a set of Leishmania strains retrieved from the GenBank using ClustalX, version 2.0.12 [23]. The pairwise genetic distances among isolates were estimated using program MEGA (Molecular Evolutionary Genetics Selleck Sepantronium Analysis), version 4.0 [24]. To investigate the relationships among L. siamensis isolates and other Leishmania species, Leishmania sequences of each locus examined in this study from GenBank were included in the dataset. The evolutionary history was inferred by phylogenetic tree construction using three methods, i.e., Neighbor Joining (NJ), Maximum Parsimony (MP) and Bayesian inference. The NJ and MP trees were constructed using program MEGA, version 4.0 [24]. Reliability of the inferred trees was tested by 1000 Linsitinib bootstrap replications.
For the Bayesian method, starting trees were random: four simultaneous Markov chains were run for 500,000 generations, burn-in values were set at 30,000 XMU-MP-1 generations and trees were sampled every 100 generations. Bayesian posterior probabilities were calculated using a Markov Chain Monte Carlo sampling approach implemented in MrBAYES, version 3.1.2 [25]. The Akaike information criterion in Modeltest, version 3.06, was used to select a DNA substitution model of all phylogenetic analyses [26]. The following models were selected for the dataset of each gene: K2P (SSU-rRNA), TrN+Γ (ITS1 and hsp70), and GTR+Γ (cyt b). The nucleotide sequences generated in this study have been deposited in GenBank under accession
no. JX195633-JX195637, JX195639-JX195640, and KC202880-KC202883. Results Sequence analysis PCR amplification of each target locus resulted in amplicons of the expected sizes as follows: SSU-rRNA (540 bp), nearly ITS1 (340–348 bp), hsp70 (1422 bp), and cyt b (865 bp). Due to the limited amount of DNA samples, studied loci of some samples were not successfully amplified. Twelve amplicons were successfully amplified and bidirectionally sequenced. As a result, a total of 15L. siamensis sequences were analyzed in this study. These consisted of four isolates from SSU-rRNA (CU1, PCM1, PCM2, and PCM4; sequences of PCM1 and PCM2 were reported by Bualert et al. [8]), four isolates from ITS1 (CU1, PCM1, PCM2, and sequences of PCM4; PCM1 were reported by Sukmee et al. [7]), four isolates from hsp70 (CU1, PCM2, PCM4, and PCM5), and three isolates from cyt b (CU1, PCM1, and PCM2).