Phylogeny of Pakistani Cattle Breeds using Mitochondrial Cytochrome b Gene

Tanveer Hussain1,*, Masroor Ellahi Babar1, Marcos De Donato2, Abdul Wajid1, Asif Nadeem3, Zahoor Ahmad3, Waqas Ahmad Khan4, Sunday O. Peters5 and Ikhide G. Imumorin6

1Department of Molecular Biology, Virtual University of Pakistan, Lahore

2Tecnologico de Monterrey, Campus Queretaro, Department of Bioengineering,

Queretaro, Mexico

3Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Lahore

4Department of Biotechnology, University of Sargodha, Sargodha

5Department of Animal Sciences, Berry College, Mount Berry, Georgia, USA

6School of Biological Sciences, Georgia Institute of Technology, Atlanta, Georgia, USA

ABSTRACT

Zooarchaeological data indicates that the Near East and the Indus Valley were the centers of domestication and diffusion of modern day cattle. To better understand the origin and genetic diversity of native cattle breeds (Bos indicus) in Pakistan, partial sequencing of the mitochondrial gene cytochrome b (339 bp) was done in 136 individuals from 10 different breeds. Analysis of the Cyt b gene showed high conservation in Pakistani cattle as expected, with only 26 individuals showing nucleotide changes. Only 5 point mutations were present in multiple individuals (SNP), but one was specific for indicine cattle. Two Lohani and 5 Nari Master cattle showed nucleotide changes specific to taurine cattle. Of the changes found, only three produced amino acid changes in the protein sequence. The UPGMA tree showed a clear differentiation between taurine and indicine cattle, except mitochondrial taurine sequences in Lohani and Nari Master breeds. The within-breed estimates of divergence were very low in all breeds except for Nari Master (mixed-bred). The estimates of divergence among breeds were also low for most breed pairs, except for Nari Master and Dhanni. While the overall genetic divergence within the B. indicus or within B. taurus were also very low (0.002 and 0.003, respectively), however the genetic difference between B. indicus and B. taurus was significantly higher (0.014 ).

Article Information

Received 12 August 2015

Revised 24 July 2017

Accepted 18 November 2017

Available online 31 August 2018

Authors’ Contribution

TH and MEB designed and planned the study. TH, MDD, AW, AN, ZA and WAK performed the sampling and lab work. TH, MEB, MDD, SOP and IGI contributed to data analysis and manuscript writing.

Key words

Pakistani Cattle, Cytochrome b, Genetic diversity, Phylogenetics.

DOI: http://dx.doi.org/10.17582/journal.pjz/2018.50.6.2029.2035

* Corresponding author: [email protected]

0030-9923/2018/0006-2029 $ 9.00/0

Copyright 2018 Zoological Society of Pakistan

Introduction

Pakistan had 44.4 million cattle in 2016-17 (GoP, 2017) with 15 distinct breeds for which very little information on genetic architecture is available. Present day domestic cattle were domesticated from the extinct aurochs, Bos primigenius (Payne, 1991; Troy et al., 2001). After the last glacial event of 15,000 years ago, B. primigenius was found throughout almost the whole of Eurasia and North Africa. The zooarchaeological data indicates the Near East and the Indus Valley (Pakistan, northwest India, and eastern Afghanistan) as domestication and diffusion centers of modern cattle (Helmer et al., 2005). The mitochondrial genome (mtDNA) of vertebrates has become a common tool for resolving phylogenetic relationships, domestication and to estimate degrees of relatedness and divergence times. The use of mtDNA relies on calibrated molecular clocks at different evolutionary depths due to its peculiar properties of maternal inheritance and high mutation rate which allows for the accumulation of molecular diversity within the time frame of domestic history (Carmela et al., 2000; Simon et al., 2006).

The phylogenetic information contained in cytochrome b (Cyt b) gene has been widely used for intra- and interspecies comparisons and it is considered to be a good marker to study the genetic differentiation and phylogenetic relationships among species within the same genus or the same family (Browers et al., 1994; Zardoya and Meyer, 1996), and it has been also widely used to study the origin, taxonomy and phylogeny of the Bovinae subfamily (Birungi and Arctander, 2001; Hassanin and Ropiquet, 2004). Some studies have been carried out to determine the genetic relationships and structure of some of the cattle populations in this region (Metta et al., 2004; Mukesh et al., 2004; Karthickeyan et al., 2006, 2009; Sodhi et al., 2006; Rehman and Khan, 2009; Azam et al., 2012). This study was carried out to assess the genetic differentiation and phylogeny of 10 major cattle breeds in Pakistan using the mtDNA cytochrome b region.

 

Table I.- The nucleotide changes in CYTB sequences of Pakistani cattle and the amino acid changes, where appropriate. Shaded changes are found typically in taurine cattle.

Position	93	196	161	280	312	318	331	345	384
Consensus indicine	G	G	T	T	T	T	G	C	C
Consensus taurine	A	G	T	T	C	T	G	T	T
Amino acid change		V→I					E→K
Lohani 8	A				C			T	T
Lohani 11	A				C			T	T
Nari Master 3	A
Nari Master 4	A				C			T
Nari Master 6	A				C			T	T
Nari Master 9	A				C			T	T
Nari Master 11	A				C			T	T
Nari Master 22	A		C		C			T	T
Dajal 20		A
Dahnni 1				C
Dahnni 2				C
Achai 7						C
Achai 10						C
Dajal 16						C
Nari Master 14						C
Red Sindhi 2023						C
Red Sindhi 2038						C
Tharparker 1909						C
Sahiwal 1						C
Sahiwal 2						C
Sahiwal 3						C
Dahnni 1						C
Dahnni 2						C
Cholistani 3							A
Cholistani 4							A
Achai 3									G*

*Change C→G in Achai 3 produces a change F→L in the amino acid sequence.

 

Materials and methods

Whole blood (10 mL) was collected from unrelated representative individuals of selected cattle breeds from several breeding areas. The sampling from sibs was deliberately avoided by restricting the number of samples from a given herd (Table I). DNA was extracted from frozen blood after thawing using inorganic extraction method (Babar et al., 2012) and each sample was brought to a concentration of 50 ng/µL. Specific primers mtCytbF 5’-TCCATAAATACAAAGAGCCTTATCAGT-3’and mtCytbR 5’-GCGGCATGGTAATTAAGCTC-3’ were designed for cattle Cytochrome b gene (GeneBank Accession No JN817351) using the Primer3 software (v. 0.4.0 ); Rozen and Skaletsky, 2000). The 367 bp of Cyto b was amplified in all samples using 25 µL reaction mixture with 2mM MgCl2, 100µM of dNTPs, 0.2µM of each forward and reverse primer and 1 U of Taq DNA polymerase (Fermentas, Thermo Fisher Scientific Inc., USA) was used. The amplification was carried out at an initial denaturation at 94ºC for 4 min, 35 cycles of denaturation at 94ºC for 30s, annealing at 56°C for 30s and extension at 72°C for 45s followed by final extension at 72 ºC for 10 min and cooling at 4ºC for 1 h. All products were sequenced using an automated sequencer ABI prism 3130 XL Genetic Analyzer (Applied Biosystems, Inc., Foster City, CA). MEGA software (V 5.05) (Tamura et al., 2011) was used for sequence alignments, translations and phylogenetic analysis.

The numbers of base substitutions per site, averaged over all sequence pairs within and between each group were calculated using the Maximum Composite Likelihood model (Tamura et al., 2004). The analysis involved 152 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 339 positions in the final dataset. Evolutionary analyses were conducted in MEGA5 software.

 

Table II.- List of cattle breeds, number (total = 130), species, type and sampling area in Pakistan used in this study.

Breed	n	Species	Type	Sampling area
Sahiwal	3	B. p. indicus	Dairy	Punjab
Cholistani	13	B. p. indicus	Dairy	Punjab
Red Sindhi	17	B. p. indicus	Dairy	Sindh
Tharparker	23	B. p. indicus	Dual purpose	Sindh
Dhanni	2	B. p. indicus	Draught	Punjab
Lohani	16	B. p. indicus	Draught	Punjab, KPK
Dajal	10	B. p. indicus	Draught	Punjab
Bhangnari	21	B. p. indicus	Draught	Balochistan
Achai	12	B. p. indicus	Draught	KPK
Nari Master	13	B. p. indicus x B. p. taurus	Beef	Balochistan

 

Results

Mitochondrial cytochrome b gene sequences (GenBank accession numbers: JN117611 - JN117615 and JX668003 - JX668127) were highly conserved in all the Pakistani cattle (Fig. 1), evidenced by the fact that only 26 of the 130 individuals studied showed nucleotide changes from the consensus sequence (Table II). Nine nucleotide changes were detected in the sequences analyzed, but only changes at 5 positions were present in multiple individuals: one polymorphic site (SNP) was found in nucleotide 318 (T→C) in several breeds of indicine cattle, but not in any of the taurine cattle from GenBank (Fig. 1). The other four changes found in multiple animals of Pakistani cattle, are typical of the taurine cattle haplotype, but some were found in two Lohani and five Nari Master Cattle from Pakistan. However, the one animal, Nari Master 3 had only one of the changes found in taurine haplotype, while Nari Master 4 had 3 of the 4 changes. In addition, three changes were present in only one animal and two changes in two animals (Table II). Of all the changes found, only one was a transversion and three (including this transversion) produced amino acid changes in the protein sequence.

The phylogenetic tree built using Unweighted Pair Group Method with Arithmetic Mean (UPGMA) showed a clear differentiation between taurine and indicine cattle (Fig. 1), except for those Pakistani cattle showing mitochondrial taurine sequences. All the indicine cattle showed almost identical sequences. In addition, cattle sequences were significantly different from Bison bonasus, Bos javanicus, Bos grunniens and Bubalus bubalis, as well as the sequences of Ovis aries and Capra hircus. The within breed estimates of divergence were very low in all breeds except for Nari Master (Table III). The estimates of divergence among breeds were also low for most breed pairs, except for Nari Master and Dhanni. However the overall genetic divergence within the B. indicus as well as within B. taurus were also very low i.e. 0.002 and 0.003, respectively, the genetic difference between B. indicus and B. taurus was significantly higher i.e. 0.014.

 

Discussion

Domestic cattle are generally considered to be derived from two major subspecies, B. t. taurus and B. t. indicus. It is suggested that these two subspecies contributed independently to modern domestic cattle and were domesticated separately 8,000-10,000 years ago (Payne 1991; Loftus et al. 1994). It is also possible that the origin of the Asian cattle is much more complicated; namely, the population may have genes introgressed from other Bovinae species such as B. (Bibos) javanicus, B. (Bibos) gaurus or B. (Poephagus) grunniens. Therefore, these species have been suggested as a third source of Asian cattle (Namikawa et al., 1984, 1995; Kawamoto et al., 1992; Kikkawa et al., 1995; Yu et al., 1999; Tu et al., 2000).

It has been previously demonstrated that mitochondrial DNA (mtDNA) haplotypes specific for each subspecies exist in native populations of Asian cattle (Watanabe et al., 1989; Bhat et al., 1990; Kikkawa et al., 1995, 2003; Cai et al., 2007), which agree with our results. Gene flow from taurine cattle into Asian zebu populations has been suggested as an unusual pattern of introgression (Namikawa 1981; Namikawa et al., 1984, 1995; Amano et al., 1994; Kawamoto et al., 1992; Tu et al., 2000; Kikkawa et al., 2003; Cai et al., 2007, 2010). Currently, there is more evidence to show that the origin of the Asian cattle populations include genes introgressed from B. t. taurus and other Bos species (Yu et al., 1999; Tu et al., 2000; Kikkawa et al., 2003; Cai et al., 2010) such as the Bangladesh and Nepali cattle which have mixed breed taurus/indicus mtDNA and SRY genotypes, as well as Indonesian and Nepali cattle showing mixedbred Bali/indicus and yak/indicus, respectively (Kikkawa et al., 2003).

 

Table III.- Estimates of evolutionary divergence over sequence pairs between groups. The number of base substitutions per site from averaging over all sequence pairs within each group are shown. The diagonal bold faced numbers show the mean interpopulational evolutionary diversity estimates.

	Other B. indicus	Achai	Bhag nari	Cholis tani	Dajal	Loh ani	Nari Master	Tharp arker	Sahi wal	Dha nni	Other B. taurus
Other B. indicus	0.001
Achai	0.001	0.001
Bhagnari	0.000	0.001	0.000
Cholistani	0.001	0.001	0.000	0.001
Dajal	0.001	0.001	0.001	0.001	0.001
Lohani	0.002	0.002	0.001	0.002	0.002	0.003
Nari Master	0.005	0.006	0.005	0.006	0.006	0.005	0.007
Red Sindhi	0.001	0.001	0.000	0.001	0.001	0.002	0.005
Tharparker	0.000	0.001	0.000	0.001	0.001	0.002	0.005	0.000
Sahiwal	0.003	0.003	0.003	0.003	0.003	0.004	0.008	0.003	0.000
Dhanni	0.006	0.006	0.006	0.006	0.006	0.007	0.011	0.006	0.003	0.00
Other B. taurus	0.014	0.014	0.013	0.014	0.014	0.012	0.009	0.014	0.017	0.02	0.003

 

Here we have shown a very distinctive pattern of Cytb gene for each of B. t. taurus and B. t. indicus in the Pakistani breeds. This gene was used to study the genetic differentiation among 18 breeds of cattle from China, successfully identifying taurine, indicine or hybrid origins, and showing a declining south-to-north gradient of female zebu introgression and a geographical hybrid zone of Bos taurus and Bos indicus in China (Cai et al., 2007, 2010). Higher nucleotide diversity in the taurine cattle than in the indicine cattle was found in the Chinese breeds. In addition, Ripamonte et al. (2012) found that the Zebu (Nellore) cattle in Brazil showed large influence of taurine mtDNA. We also found a low degree of introgression from taurine mtDNA into indicine but only in the Lohani breed. This could be caused by the crossbreeding of this breed with taurine cattle, since there is no specific breeding program established for most cattle producers in Pakistan.

It is interesting to see that even though Bison belongs to a different genus, this species and B. javanicus were the species phylogenetically closest to taurine/ indicine cattle. This has been reported previously and introgression produced by cattle/bison crossbreeding is suggested as one of the explanation for this result (Cai et al., 2011; Hassanin et al., 2012). In addition, a GenBank sequence reported for indicine cattle from Malaysia (Brakmas breed) was not observed among the indicine cattle analyzed here. This could be due to the reported introgression found in several breeds of cattle from Southeast Asia (Yu et al., 1999; Tu et al., 2000; Kikkawa et al., 2003; Cai et al., 2010). Because native breeds of cattle have been selected for many generations for specific traits and for the resistance to environmental conditions and tolerance to various diseases, they represent a valuable resource for modern cattle production in diverse, non-optimal environments. In order to better use these breeds, this study represent a contribution for their genetic characterization and the cytb gene could be used to detect gene introgression from taurine into indicine cattle, and vice versa.

 

ACKNOWLEDGEMENTS

Special thanks to Livestock and Dairy Development Department of Punjab, Khyber Pakhtunkhwa, Balochistan and Livestock and Fisheries Department of Sindh for helping in collection of blood samples from different cattle breeds. The Higher Education Commission of Pakistan is acknowledged for funding (Project No. 20-1703).

 

Statement of conflict of interest

Authors have declared no conflict of interest.

 

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