Molecular Identification of Pakistani Lathy Rock Pigeon using Cytochrome B Gene

Sehrish Firyal1,*, Ali Raza Awan1, Muhammad Umair Latif1, Muhammad Tayyab1, Muhammad Wasim1, Shagufta Saeed1 and Imaad Rashid2

1Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Civil Lines, Outfall Road, Lahore

2Department of Clinical Medicine and Surgery, University of Agriculture, Faisalabad

ABSTRACT

Single nucleotide polymorphism (SNPs) analysis is a powerful tool for taxonomic and phylogenetic analysis. These are bi-allelic molecular markers, easy to interpret and uniformly distributed within genomes. In this study cytochrome b (Cytb) gene based SNPs analysis was employed to genetically characterize the Pakistani domestic breed Lathy pigeon. Cytb haplotype for local domestic breed was developed. Homology analysis of Cytb gene revealed Columbia livia as closest homologue of Pakistani Lathy pigeon with five novel SNPs. Phylogenetic analysis of domestic lathy pigeon indicated the monophyletic relation with C. livia. We are reporting for the first time, the novel SNPs in the Cytb gene of local domestic pigeon breed that might act as panel of molecular marker for the identification of Pakistani lathy pigeon.

Article Information

Received 25 October 2016

Revised 27 June 2018

Accepted 31 December 2018

Available online 09 October 2020

Authors’ Contributions

SF, MUL and ARA designed the study, analysed the data and wrote the manuscript. MT, SS and MW edited and revised the manuscript. IR collected the samples.

Key words

Domestic lathy pigeon, Mitochondrial DNA, Cytochrome B, SNPs, Phylogenetic analysis.

DOI: https://dx.doi.org/10.17582/journal.pjz/20161025081051

* Corresponding author: [email protected]

0030-9923/2020/0006-2409 $ 9.00/0

Copyright 2020 Zoological Society of Pakistan

Pigeon have played significant role throughout the human history, interacted with human for a variety of purposes and are being used as pet for racing competitions as a source of amusement and sports in many countries of the world. Some varieties of pigeon used in racing competitions are very costly and are of high economic value, due to which stealing, smuggling and mugging issues are common for these pigeons. Apart from these issues, the specie and genetic lineage of these pigeons is also questioned very often. So, it is very important to develop panel of genetic markers to identify unambiguity of the breeds (Lee et al., 2008). Pakistan is bestowed by a diversified avian fauna (Awan et al., 2013). Among them pigeons are distinctive birds which have been bred for centuries. Lathy Rock is a unique Pakistani domestic pigeon; colloquially is Salara in Punjab. The Lathy Rock is semi-solid in color having light and dense gray feather pattern throughout the body. They have orange colored iris with golden paler inner ring (http://faisalabad.olx.com.pk/). Traditionally, they have been identifying on the basis of morphological and anatomical characteristics (Danish et al., 2008). No molecular data is available to authentically identify this pigeon breed.

DNA based molecular markers provide new scaffold for critique breed identification (Tautz et al., 2003). Among these molecular markers, mitochondrial Cytb gene has proven to be an efficient tool for species identification and characterization (Firyal et al., 2013; Saif et al., 2012). Cytb gene has been extensively employed to resolve relationships between closely related taxa and to build higher level phylogenies of vertebrates (Johns and Avise, 1998). Phylogenetic analysis is an imperative molecular tool which can underpins the taxonomic affinities, molecular diversities and lineage assignment of avian species (Allan and Max, 2010). As no documentation is available for Pakistani pigeon breeds herein we describe through DNA sequencing, the gene specific novel polymorphisms in Cytb gene of the Pakistani domestic pigeon; Lathy Rock and its molecular classification within the genetic spectrum of other Columbiformes.

Materials and methods

A total of 23 unrelated domestic lathy pigeons were selected on the basis of yellowish iris color and dense gray feather pattern throughout the body, from 3 cities (Lahore, Bahawalpur and Sialkot) of Punjab province of Pakistan. The samples were named as PKDRP1 to PKDRP23. Blood samples (200 µl) were collected from each bird and utilized for the isolation of genomic DNA (Sambrook and Russel, 2001). Primers were designed using the Cytb gene sequence of Columbia livia (Accession No. GQ240309) as reference sequence. Full length Cytb gene was amplified with the help of specific primers. Amplicons were purified and subjected for sequencing using an ABI Prism 3130 genetic analyzer (Applied Biosystems, Foster City, USA). The obtained DNA sequences were analyzed using various Bioinformatic tools. ClustalW (http://www.ebi.ac.uk) was used to perform the multiple sequence alignment. On the basis of pairwise alignments, phylogenetic tree was constructed with the help of NCBI distance tree matrix and Blast Tree View; in which the Cytb gene sequences of the Lathy Rock pigeon was compared with all the available Cytb gene sequences of Columbiformes (pigeons and doves). The nucleotide sequences of Cytb gene of Pakistani Lathy Rock pigeons were submitted to GenBank using Sequin Submission Portal.

Results and discussion

From all the selected birds, PCR resulted in amplification of 1.143kb Cytb gene. The Cytb gene sequences of 23 domestic Lathy Rock pigeons were submitted to GenBank under the Accession No. KC811442 to KC811463. The sequence similarity analysis indicated Columba livia as closest homologue of domestic Lathy Rock pigeons with 99.61% homology. Table I demonstrated the percentage homology of domestic Lathy Rock pigeons with the various members of Columbiformes. Comparative analysis of Cytb gene from domestic Lathy Rock pigeons and C. livia revealed the presence of 5 SNPs in the Cytb gene (T174A, C232T, G261A, G703A and G816A). Phylogenetic tree constructed based on Cytb gene polymorphisms of Lathy Rock with all available Cytb gene sequences of the Columbiformes indicated that domestic Lathy pigeon is monophyletic with respect to C. livia and C. rupestris (Fig. 1).

Table I.- Percentage homology of Pakistani domestic Lathy Rock pigeon with other members of Columbiformes.

Present study describes the molecular characterization and phylogeny of Pakistani domestic Lathy Rock pigeons using Cytb gene polymorphism. Mitochondrial Cytb gene is extensively used to underpin the taxonomic classification and systematic resolution of an individual (Heidrich and Wink, 1998; Saif et al., 2012; Tautz et al., 2003; Firyal et al., 2013) and has been considered one of the most useful genes for evolutionary studies (Esposti et al., 1993). It has been extensively employed to address the diversity ambiguities as it contains both conserved and rapidly evolving codon positions (Irwin et al., 1991).

Comparative analysis of Cytb gene from domestic Lathy Rock pigeons and C. livia divulged the presence of 5 SNPs in the Cytb gene. These 5 SNPs in the Cytb gene would prove to be novel for identification of Pakistani Lathy Rock pigeon breed.

Further, Cytb gene polymorphisms based evolutionary analysis of lathy Rock explored the continuum of shallower to deeper genetic divergences. This unique continuum evolution defines the biological relationships of individuals. Phylogenetic tree constructed based on Cytb gene polymorphisms of lathy Rock with all available Cytb gene sequences of the Columbiformes indicated that domestic lathy pigeon is monophyletic with respect to C. livia and C. rupestris (Fig. 1). In addition to this, analyses of present study significantly revealed that rock pigeon (C. livia) is the ancestor of Pakistani domestic pigeons (Gonzalez et al., 2009; Levi, 1996; Stringham et al., 2012). Rock Pigeon and Pakistani domestic pigeons diverged from Hill pigeon (C. rupestris): likely to be allopatric. Branch lengths separating them are very short and believe to be a recent radiation. No empirical data is available about this radiation because little work has been undertaken to address this issue. These findings are unswerving even with the whole genomic studies (Shapiro et al., 2013). The present study provides the basis for further genetic investigations of avian species. This is the first report of novel SNP identification of Cytb gene of the Pakistani Lathy Rock pigeon and its phylogeny.

Statement of conflict of interest

The authors have no conflict of interest and are agreed to submit this piece of information to Pakistan Journal of Zoology for publication.

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