DNA barcoding is a precise technique that uses molecular genetics tools for accurate identification, categorizing, relating and separating the phylogenies of species. Being a small sized genome and agile enough to show rapid mutation, mtDNA has been used as a pertinent marker of molecular biodiversity.The aim of this study was to develop DNA barcode for genetic characterization and classification of Sub-family Phasianinaeof Pakistani bird species. Theyhave not been genetically identified yet in Pakistan. It includes birds like domestic chicken(Gallus gallusdomesticus), aseel chicken(Gallus gallusdomesticus strain),blue peafowl(Pavo cristatus), green peafowl (Pavo muticus), white peafowl (Pavo cristatus leuticus), Kalij pheasant (Lophura leucomelanos),monal pheasant (Lophophorus impejanus),koklass pheasant(Purcrasia macrolopha), ring necked pheasant (Phasianus colchicus), Tragopan (Tragopan melanocepals) andred junglefowl (Gallus gallus). These birds are considered an important part of an ecosystembecause they play a significant role in seed dissemination, pollination of plants and disease spread which are the basic constituents of an ecosystem. They are used for food, hunting and entertainment purposes.
Mitochondrial geneCytochrome c oxidase subunit 1 (CO1)of 500bps was used as a marker for identification at specie level.Genomic DNA was extracted by each blood and tissue sample of eleven bird species (33 samples). Amplification of CO1 gene was a done by using a universal set of primers (BIRDF1 and BIRDR1)containing region of almost 750 bps (Hebert et al. 2003).Amplicons were purified and sequenced Sanger sequencing method (Sanger et al. 1977). Forward and reverse sequences were analyzed using softwaresEMBOSS merger,ClustalW, BioEdit and nBLAST. Phylogenetic analysis of selected bird species was done. Each sequence was aligned
with its reference sequences of CO1 gene present on NCBI. Every nucleotide position which did not align with the reference sequence was studied to identify SNPs. Fixation index (FST) were used to measure species diversity within a same sub population relative to that found in the entire population. Consensus sequences (500bps) generated was used to construct their phylogenetic tree to see their evolutionary relationship with other bird species. All species showed their closest linkage with their respective species. Pakistani population of peafowl and chicken species showed the close relation with same sequences generated in China. Tranopans showed its closest linkage with T. temminckii.
In conclusion, seven species ofPhasianinaesub-family of Pakistani bird species was genetically characterized first time in Pakistan by using CO1 as a barcode. It proves that DNA barcoding is an efficient and accurate molecular tool for species identifica¬tion and phylogenetic implication. This study leads to establish a DNA Data Bank that helped scientists to investigate the biodiversity, taxonomic classification, specie identification, in forensic purposes and to study the genetic and phenotypic evolution of these species. DNA barcoding through CO1 gene works as a functional tool for detectingmeat mislabeling and preventing illegitimate trade. This study has established foundations for molecular biologists to study taxonomic uncertainties at sub species level using SNP based identifying marker. It helps in preservation and identification of endangered species by generating their barcodes from even minimal evidence available.