2.5.1 Sequence Analysis
Chromatogram signals of all sequencing were examined with chromas 2.2.6. Sequences without ambiguous signals were selected. Vector sequence from the MHC class I gene was removed using seqMan in the DNAStar7.1 package. Sequence editing and organization were done with BioEdit [28]. Sequences were aligned individually and then altogether four sampled species using CLUSTAL X [29]. The unique alleles were named according to the nomenclature for MHC in non-human species [30]. NCBI BLAST [31] was used for sequences confirmation representing close identity to passerine species previously published MHC class I exon 3 sequences. Sequences having at least one stop codon (shift in the reading frame due to indels or nonsense sequences) were classified as pseudogenes. Based upon sequences found to be translatable, a minimum number of functional loci MHC class I was estimated using a conservational approach that all Loci from samples species’ individual were in heterozygote state.
The average pairwise nucleotide distances (Kimura 2-parameter model - K2P), and the Poisson-corrected amino acid distances were calculated using MEGA7.0. Standard errors were obtained through 1000 bootstrap replicates. Haplotypes identification (Na) , the average number of nucleotide differences (K), polymorphic sites (S) ) and nucleotide diversity (π) were measured by DnaSP 5.10 [32].