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].