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Molecular Classification of Pakistani Rose-Ringed Parakeet using Mitochondrial ND2 Gene.

Byline: Sehrish Firyal Tahir Yaqub Aftab Ahmad Anjum Muddasar Saeed Khan Muhammad Mansha Muhammad Tayyab Muhammad Wasim and Ali Raza Awan


The present study aimed to genetically identify the indigenous Rose-ringed parakeet of Pakistan using NADH dehydrogenase subunit 2 (ND2) gene polymorphism. Blood samples of 24 unrelated Pakistani rose-ringed parakeets were utilized for isolation of genomic DNA followed by amplification and sequencing of ND2 gene. The analysis of genetic phylogeny of the ND2 gene indicated that the Pakistani rose-ringed parakeet was cladded with Psittacula krameri with DNA and amino acid sequence similarity of 97.6 and 98.27% respectively. Further comparative analysis indicated 25 changes in nucleotide and six changes in amino acid sequences in ND2 gene of Pakistani rose-ringed parakeet suggestive of an independent evolution of this avian species. On the basis of unique genotype and distinguishing phenotypic characteristics Pakistani rose-ringed Parakeet should be classified as Psittacula krameri.

The present report is the first documentation on molecular classification of Pakistani Roseringed parakeet on the basis of ND2 gene polymorphism. Copyright 2014 Friends Science Publishers

Keywords: Pakistani rose-ringed parakeet; ND2 gene polymorphism; Mitochondrial DNA; Psittacula krameri; Phylogenetic analysis


Pakistan is endowed with a majestic array of wild avian fauna. Rose-ringed parakeet commonly known as Kathy parrot belongs to family Psittacidae. On the basis of geographical distribution Rose-ringed parakeets (Psittacula krameri) have been classified into four subspecies viz; P. k. krameri P. k. parvirostris P. k. manillensis and P. k. borealis but they have not been genetically characterized as yet (Ahmad et al. 2012a b). Genetic based investigations are needed for identification of reproducible molecular markers which could be employed for accurate species identification and differentiation. Among the genetic approaches mitochondrial DNA (mtDNA) markers are potentially authentic tools for species characterization and molecular classification (Lane 2005; Awan et al. 2013).

Mitochondrial genome is considered very important for evolutionary analysis and has potential for identification and delineation of species (Galtier et al. 2009). Furthermore the mutations in mtDNA occur in a chronological manner that enable researchers to investigate these changes and can be associated with geographical distribution and origin of populations. Mitochondrial DNA is an excellent milestone to predict the ancestry of the individuals and leads to accurate conclusions regarding taxonomic relationships (Barrowclough 1983; Zink 1991).

It harbors greater importance than nuclear DNA and phenotypic traits in order to determine the evolutionary pattern (Irwin et al. 1991). Mitochondrial ND2 (mtND2) gene evolves comparatively at a high rate (Otto et al. 1996) and has reportedly been an effective mean in discerning the relationships among closely related species (Macey et al. 1998). In the present study we utilized ND2 gene sequences analysis to define the phylography of Pakistani rose-ringed parakeet.

Materials and Methods

A total of 24 Pakistani Rose-ringed parakeets were selected on the basis of their unique morphological characteristics. Blood samples (200 L) were collected and utilized for DNA isolation using organic DNA extraction method followed by ND2 gene amplification. Amplicons were visualized on 1.2% agarose gel and were purified using DNA extraction kit (GeneAll General Biotechnology Seoul Korea). Then the amplicons were sequenced using BigDye terminator cycle sequencing kit (Applied Biosystems USA) on ABI 3130 Genetic Analyzer (Applied Biosystems Foster City USA). Chromatograms were analyzed using Chromas Ver. 1.45 ( The sequence alignments were done using BioEdit version 5.0.9 (Hall 1999). Aligned

ND2 sequences were imported to MEGA 5.1 (Kumar et al. 2004) and Expasy Bioinformatics Resource Portal ( for phylogenetic and amino acid sequence analysis respectively.


We amplified and analyzed full length ND2 gene of Pakistani rose-ringed parakeets to determine their molecular phylogeny. The nucleotide sequences of ND2 gene from 24 birds were submitted to NCBI GenBank (Accession No. KC823233 to KC823256).

ND2 gene based comparative analysis indicated that Pakistani Rose-ringed parakeets contain 97.6% similarity with Asian and African rose-ringed parakeet (P. krameri)

92.22% with Indian Blue-winged parakeet (P. columboides) 91.83% with Indian malabar parakeet (Psittinus cyanurus) 91.45% with Blue-naped parrot (Tanygnathus lucionensis) 90.20% with Song parrot (Geoffroyus heteroclitus) 86.45% with Australian and Indonesian Rainbow Lorikeet (Trichoglossus haematodus) and 86.16% with Indonesian Red-and-blue lory (Eos histrio).

Comparison of ND2 gene sequence of Pakistani roseringed parakeet with its closest homologue P. krameri indicated 25 variations in nucleotide sequence (Fig. 1A). Deduced amino acid sequences were also utilized for homology analysis (Fig. 1B). Amino acid sequence based comparative analysis revealed similarity of 98.27% with P. krameri 93.64% with P. columboides 93.35% with P.

cyanurus 92.77% with T. lucionensis and G. heteroclitus 85.83% with T. haematodus and 85.83% with T. haematodus. The phylogenetic analysis demonstrated that Pakistani rose-ringed parakeet clustered in Clade B with P. krameri P. columboides P. roseate G. heteroclitus P. cyanurus T. lucionensis Eclectus roratus and Prioniturus montanus (Fig. 2). Members of Clade B (P. krameri P. columboides P. roseate and P. cyanurus) belonging to Asia shared an identity of 90 to 97.97% with Pakistani rose-ringed parakeets. Clade A consisting of T. haematodus P. elegans M. undulates P. fulgidus and E. histrio members and belonging to Australia and Indonesia shared a homology of 80 to 92% with Pakistani parakeets. Clade C consisted of two members P. picta belong to Indonesia while Anodorhynchus leari was from Brazil.


In the present study mtND2 gene was utilized for characterization of Pakistani rose-ringed parakeet. Previously the same gene was utilized for characterization of various avian species like Otus megalotis and Mimizuku gurneyi (Zink et al. 1991; Gonzalez et al. 2009; Bradman et al. 2011). Comparative analysis revealed that the use of ND2 gene was an alternative of cytochrome b (Cyt b) gene

Comparison of ND2 gene sequence from Pakistani rose-ringed parakeet (Pakistani parakeet) with its closest homologue Psittacula krameri. Fig. 1A and 1B shows the comparison of nucleotide and amino acid sequences respectively. Asterisks at the bottom of sequence indicate sequence identity while the highlighted nucleotides are the variation among the two species for the identification of various avian species of Phasianidae Anatidae Gruidae Scolopacidae Accipitridae Falconidae Struthionidae and Psittacidae families (Boonseub et al. 2009). We have also studied the Cytb gene of Pakistani rose-ringed parakeet (Unpublished data) and the results confirmed the findings of present study.

ND2 gene based homology analysis showed 22 transitions and 1 transversion but not a single indel. The other two nucleotides could not be declared as transition or transversion due to ambiguity in the reference sequence. Quantification of the transitions/ transversions is primarily important for coding genes based characterization and phylogenetic analysis. Transitions are mostly downweighted as compared to the nucleotide transversion in phylogenetic investigations. Reconstruction of accurate phylogenetic relationship is fairly data dependent because it plays integral role in numerous genetic indices (Broughton et al. 2000). Previous studies of Fitch (1967) and Li et al. (1984) also were in favor of high transitional frequency in nuclear and mitochondrial genomes. The elevated rate of nucleotide transition has been reported to show multiple vibrational events in specific nucleotide sites and increase the frequency of homoplasmy (Meyer 1994; Simons et al.

1994). Furthermore the nucleotide transitional accumulation exhibits the plateau" effect. That means beyond a specific level increase in nucleotide transitions becomes neutral even with the increase of overall divergence (Irwin et al. 1991). mtDNA variations are pivotal tools in evolutionary molecular ecology and population genetics. They paved the way for modeling the population history of individuals. Coalescent events in mitochondrial genome (Schierup and Hein 2000) including ways of population expansion and divergence time (Kuhner et al. 1998; Beerli and Felsenstein 1999) greatly impact on biogeographical distributions of individuals.

It was observed that most of the changes in nucleotide sequence were at third base of codon so the coded amino acids were found same-sense. The comparison of amino acids sequences from Pakistani rose-ringed parakeet with P. krameri indicated 6 variations; P92S T156A T191A X193I T239M and A277T (Fig. 1B). The replacement of P92S and A277T were conversion of non-polar to polar amino acids whereas the change of T156A T191A and T239M were from polar to nonpolar amino acids. The presence of X at position 193 in the reference sequence of P. krameri indicates the presence of I or replacement with other amino acid. X-ray crystallographic studies are required to explain the structural and functional relationship due to these replacements.

Phylogenetic analysis further suggested that the Pakistani Rose-ringed parakeet is monophyletic with Asian P. krameri P. columboides P. roseate G. heteroclitus P. cyanurus T. lucionensis Eclectus roratus and Prioniturus montanus. In view of the preliminary nature of the present study similar additional studies on the Rose-ringed

Phylogenetic tree constructed using the deduced amino acid sequence. The DNA sequences were taken from present study or from NCBI databank; Phylogram is based on ND2 gene sequences of Psittaciformes indicating the molecular classification of the Pakistani rose-ringed parakeet parakeets (including those endemic in other provinces of Pakistan) are clearly warranted.

This is the first study on molecular classification of Pakistani rose-ringed parakeet at sub-species level using nucleotide and amino acid sequences of ND2 gene. These novel polymorphisms might act as marker for the molecular classification of Pakistani rose-ringed parakeet.


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Author:Firyal, Sehrish; Yaqub, Tahir; Anjum, Aftab Ahmad; Khan, Muddasar Saeed; Mansha, Muhammad; Tayyab, M
Publication:International Journal of Agriculture and Biology
Article Type:Report
Geographic Code:9PAKI
Date:Dec 31, 2014
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