Assessment of Mitochondrial ATPase 8/6 Genes Based Genetic Diversity in Sindh Ibex (Capra aegagrus Blythi)

Javeria Zafar1, Asif Nadeem1*, Maryam Javed1, Fehmeeda Fatima1, Wasim Shehzad1, Ghulam Abbas1, Rajput Zahid Iqbal2 and Muhammad Muddassir Ali1

1Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences Lahore

2Shaheed Benazir Bhutto University of Veterinary and Animal Sciences, Sakrand

JZ and AN made equal contributions to this article.

ABSTRACT

The current study was designed to characterize mitochondrial ATPase8/6 genes in Sindh ibex and to determin its phylogenetic relationship with other capra species. Blood samples were collected from Kirthar National Park, Sindh, Pakistan. PCR product was sequenced bi-directionally using dideoxy chain termination method after mitochondrial ATPase8/6 genes amplification. Polymorphism, Genetic diversity and Phylogenetic analysis were carried out by the MUSCLE, DnaSP and MEGA6 tools. Total of 20 variations at different positions were found in aligned sequence results. Sequence conservation was observed for the ibex population. The overall results showed a close evolutionary relationship among Capra aegagrus, Capra nubiana, Capra falconeri, Capra hircus and Capra caucasica, demonstrating that they all were evolved from same ancestor.

Article Information

Received 22 August 2019

Revised 01 September 2019

Accepted 07 September 2019

Available online 16 January 2020

Authors’ Contribution

AN designed the study. JZ and AN carried out the genomic work. GA and RZI collected the samples. MJ, FF and MMA analyzed the data and wrote the manuscript. AN and WS revised the manuscript.

Key words

ATP synthase, Phylogenetic relationship, Parsimony analysis, Genetic diversity

DOI: https://dx.doi.org/10.17582/journal.pjz/20190822100843

* Corresponding author: [email protected]

0030-9923/2020/0002-0441 $ 9.00/0

Copyright 2020 Zoological Society of Pakistan

INTRODUCTION

Wild goat species live at mountains and at higher altitudes, may face different environmental stress according to their habitat. Moreover, domestication of these species led to the decrease in genetic variety due to increase inbreeding and hence might causes the extinction of many novel and diverse breeds.

Pakistan is fairly rich in biological diversity range and has been conventionally known for its abundance in wildlife, mainly game animals such as Bradford urial (Ovisorientalis), Sindh ibex or Sara (Capra aegagrus blythi) and Chinkara (Gazella benneti) (Jacobson et al., 2003). The estimated Sindh ibex population is almost 13,155 and densely present on the Kirthar Range, with lesser concentration on Khambu and Dumber and small numbers somewhere else. Higher altitude appear critical to this species (Sultana and Mannen, 2003). Till now many studies have been done on goat using mitochondrial DNA (mt-DNA) and of nuclear DNA (Kibegwa et al., 2016; Kadowaki et al., 2016) but no data has been published on phylogenetic diversity and genetic characterization of Sindh ibex except Heat Shock Protein 70-1 as reported by Fatima et al. (2019).

Mitochondrial ATPase-8 and ATPase-6 genes (mtATPase 8/6) are considered as one of the most potential genes for the whole mitochondrial genome due to its structure and function, and it is consisted of both conserved and rapidly evolving regions which are considered as more reliable regions for evolutionary studies (Farias et al., 2001). It is considered as one of the most valuable genetic marker to identify the relation within families and genera. (Parson et al., 2000). Any mutation in ATPase 6 gene causes the abrasion in single structure, tissue or organ (e.g., myopathies, encephlopathies and cardiopathies) to multisystem syndromes (Stewart et al., 2009). So, in the present study, the sequencing of the whole mtATPase 8/6 genes of Sindh ibex has been done. The variants were identified in mtATPase 8/6 genes in Sindh ibex and phylogenetic tree was constructed to understand the genetic diversity of Sindh ibex.

 

MATERIALS AND METHODS

Sample collection and storage

Blood samples (n=15) of wild goat (Capra aegagrus blythi) were collected from Kirthar National Park, Sindh, Pakistan in ethylenediamine tetra acetic acid (EDTA) coated vacutainers. Samples were stored at 200C before the extraction of DNA.

Genomic DNA extraction

The blood samples were thawed and subjected to DNA extraction through organic extraction method for whole genome. Quantification of DNA was done through gel electrophoresis (0.8% agarose gel) and spectrophotometric analysis (Thermo scientific 2000 nano spectrophotometer). The gel was run at 110 volts for 30 min. BIO-RAD Gel Documentation system was used to visualize DNA bands.

PCR optimization

The PCR conditions for the amplification of mtATPase8/6 genes were optimized by using different concentration of MgCl2, dNTPs (deoxynucleotide triphosphate) and Taq polymerase to get the maximum amplification of the required product.

Touchdown PCR (Bio-Rad thermo cycler) was used to optimize the primers and PCR was performed.

Sequencing of PCR amplicon

PCR amplicons were precipitated with 100µl of 80% ethanol and then sequenced in both directions using dideoxy chain termination method by Big DyeTM Terminator on ABI 3130XL Genetic analyzer.

Bioinformatics analysis

Sequences were analyzed manually by using BioEdit software V.7.0. Sequencing homology search in public databases was carried out through Nucleotide blast program (Altschul et al., 1990). Polymorphism was studies by the multiple sequence alignment using ClustalW2 and DnaSP, v.5 (Librado and Rozas, 2009). Any change in the DNA sequence was confirmed by sequencing both sense and antisense strands. Molecular Evolutionary Genetics Analysis V.6.0 (Tamura et al., 2013) was used to construct he phylogenetic trees. Percentage similarity was calculated by using the MUSCLE tool.

RESULTS AND DISCUSSION

Wild life is a vital part of ecosystem and important assets of a country. However, very few studies have been carried out on genomic and proteomic parameters of Pakistan’s wild animals.

Genetic characterization of Sindh ibex was done on the basis of mtATPase8/6 gene. For his, the amplicon of 990bp size were amplified using the PCR. Afterwords, the products of PCR were sequenced and Chromatogram of all sequenced samples were analysed.

The results showed 20 variations at different positions. All variations were found to be homozygous Transition mutations. Results of polymorphic sites, Haplotype Diversity, G+C and Tajima’s test ATPase 8/6 gene in Sindh Ibex are shown in Tables I, II, III, IV. Twenty transitional mutations were observed, in which two were singleton variable sites with two variants positioned (Table I), while rest of eighteen variations were parsimony variable sites with two variants (Table II). Average number of nucleotide differences was 7.408, whereas stochastic variance of k (no recombination), Vst (k) was calculated as 11.695 and G+C contents in whole population of Sindh ibex were 0.361 (519.00 sites).

 

Table I. Polymorphic sites of ATPase 8/6 gene in Sindh ibex.

Number of sequences	15
Number of sequences used	15
Selected region	1-973
Number of sites	973
Total number of sites (excluding sites with gaps / missing data	519
Sites with alignment gaps or missing data	454
Invariable (monomorphic) sites	501
Variable (polymorphic) sites	20
Total number of mutations	20
Singleton variable sites	2
Parsimony informative sites	18
Singleton variable sites (two variants)	2
Site positions	193, 505
Parsimony informative sites (two variants)	18
Site positions	120, 133, 162, 183,185, 187, 201, 209, 220, 223, 291, 391, 418, 441, 448, 468, 509, 517

 

Conserved region identification showed the sequence conservation for the ibex population, 0.967 and the conservation threshold for population was 1. Conserved region for ibex specie was from 518-653 bp, comprising 135 nucleotides as shown in Table V.

Furthermpre, the results of homology analysis indicated that partial sequence of ATPase8/6 genes of Sindh ibex showed maximum similarity (99%) with that of domestic goat and sheep.

 

Table II. Haplotype diversity of ATPase 8/6 gene in Sindh ibex.

Number of Haplotype, h	5
Haplotype (gene) diversity, Hd	0.0808
Variance of Haplotype diversity	0.00277
Standard Deviation of Haplotype diversity	0.053
Nucleotide diversity, Pi	0.01427
Theta (per site) from Eta	0.01045
Theta (per site) from S, Theta-W	0.01045
Variance of theta (no recombination)	0.0000190
Standard deviation of theta (no recombination)	0.00436
Variance of theta (free recombination)	0.0000061
Standard deviation of theta (free recombination)	0.00246
Average number of nucleotide differences, k	7.408
Stochastic variance of k (no recombination), Vst(k)	11.695
Sampling variance of k (no recombination), Vs(k)	1.675
Total variance of k (no recombination), V(k)	13.369
Stochastic variance of k (free recombination), Vst(k)	2.469
Sampling variance of k (free recombination), Vs(k)	0.329
Total variance of k (free recombination), V(k)	2.799
Variance of theta (no recombination)	5.123
Variance of theta (free recombination)	1.635

 

Table III. G+C Contents of ATPase 8/6 gene in Sindh ibex.

G+C content,
G+C	0.361 (519.00 sites)

 

Table IV. Tajima’s test and Tajima’s test of ATPase 8/6 gene in Sindh ibex.

Tajima's D	1.45787
Statistical significance	Not significant, P > 0.10
Fu and Li's D* test statistic	1.01041
Statistical significance	Not significant P > 0.10

 

Phylogenetic analysis showed two distinct clades and ATPase8/6 gene of Sindh ibex was highly homologous to that of other animals as shown in Figues 1 and 2.

The overall results showed a close evolutionary relationship with other animals, demonstrating that they all were evolved from same ancestor. Phylogenetic analysis showed that Capra aegagrus, Capra nubiana, Capra falconeri, Capra hircus and Capra caucasica were sharing a common ancesters. Previous studies revealed that the bezoar, markhor and other wild goats are distantly related to the domestic goat, based on mtDNA analysis (Pidancier et al., 2006; Sultana et al., 2003).

 

Table V. Conserved region identification of ATPase 8/6 gene in Sindh ibex.

Net number of analyzed sites, L	613
Number of variable/polymorphic sites S	20
Sequence conservation, C	0.967
Net number of analyzed sites, L	613
Number of variable/polymorphic sites, S	20
Conserved Region
Conservation threshold, CT	1
[Region Start-End Conservation Homozigosity P-value] Region_1, 518-653, 1.000, 1.000,0.0061
ATCCTATTTATTGGATCTACAAACCTATTAGGCCTTCTACCCCACTCATT TACACCAACTACACAACTATCAATAAATCTAGGCATGGCTATTCCCTTAT GAGCAGGGGCTGTAATTACAGGTTTTCGCAACAAAA

 

 

CONCLUSION

The results of the current study will help in understanding the life and evolutionary patterns of Sind Ibex. However, further molecular studies are needed by using the domestic goats from other countries, for better understanding and planning the conservation strategies of wildlife.

 

Statement of conflict of interests

The authors declare that they have no competing interests.

 

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