Mitochondrial
Phylogeny and Population Structure of Pakistani Dromedary Camel (Camelus dromedarius)
Abdul Fatih1, Abdul Wajid2,
Muhammad Masood Tariq1*, Masroor Ahmad Bajwa1, Masroor
Ellahi Babar3, Pamela Anna Burger4 and Tanveer Hussain5*
1Center for Advanced Studies in Vaccinology and
Biotechnology, University of Balochistan, Pakistan
2Department of Biotechnology, Virtual University of
Pakistan
3University of Agriculture, D.I. Khan,
Khyber-Pakhtunkhwa, Pakistan
4Research Institute of Wildlife Ecology, University of
Veterinary Medicine Vienna, Austria
5Department of Molecular Biology, Virtual University of
Pakistan
*For correspondence: tanveer.hussain@vu.edu.pk
Received 23 November 2020;
Accepted 22 January 2021; Published 16 April 2021
Abstract
Dromedary camels (Camelus
dromedarius) are one of the most important livestock species mainly used
for milk and meat production in semi-arid and hot-desert expanses of the
Arabian-Peninsula, Africa, and Southwest Asia. This study investigated the
genetic diversity and population structure within and between eight dromedary
camel breeds (n = 210) inhabiting
Balochistan province, Pakistan, by mitochondrial cytochrome b (Cyt b). Sequences (1140 bp) analysis
showed a total of 18 variable sites resulting in 16 haplotypes. The average
haplotype and nucleotide diversities were Hd = 0.484 ± 0.051 and π =
0.00272 respectively. Phylogenetic analysis showed different clusters for
camelids. The neutrality tests did not support the population demographic
expansion for these camel breeds. Based on these results, we suggest that an
imperative genetic management and breeding strategies are required for the
effective conservation of this species. © 2021 Friends Science Publishers
Keywords: Cyt
b; Haplotypes; Phylogeny; Dromedary camels; Balochistan
Introduction
The genus Camelus
is an interesting species inhabiting cold and hot deserts and as well as high
and low altitude worldwide. The family Camelidae is divided into two groups,
New-World (Lamini) and Old-World (Camelini) camels. Within Camelini, two
domesticated species are described including the single-humped dromedary or
Arabian camel, (Camelus dromedaries)
found in Middle-East, Horn of Africa and Southeast of Asia (Bahbahani et al. 2019) and the two-humped Bactrian
camel (Camelus bacterianus) found in
Central and Eastern-Asia, as well as the critically endangered two-humped wild
camel (Camelus ferus) distributed in
remote areas of the Gobi and Taklamakan deserts in Mongolia and Northwest China
(Yadamsuren et al. 2019; Fitak et al. 2020).
The
world population of the camel is estimated at around 35 million with the
majority (90%) are the dromedaries (FAO 2019). In Pakistan, the population of
the camel is estimated to be around 1.0 million distributed in arid, semi-arid,
and desert areas of the country with 20 different recognized breeds. They have
been providing rich organic milk, meat, hides, wool, dancing/racing animal, and
transport facility provider. Camels are an important part of the country's
economy and have a significant role in the socio-economic uplift of the local
community. The hardy nature of camel with wonderful physiological
characteristics has made it the best-suited animal for draft purposes in the
climatically harsh and inaccessible regions (Chen et al. 2019).
Dromedaries are one of the major pastoral domestic
species in Pakistan along with few herds of Bactrian camels found in Northern
areas. Balochistan shares a total of 41% of dromedary camels in Pakistan,
followed by Punjab (22%), Sindh (30%) and Khyber Pakhtunkhawa (7%)
respectively. Balochistan is situated in the southwestern part of Pakistan in a
desert belt between 25ºN to 32ºN latitude and 60ºE to 72ºE longitude.
Balochistan contains mostly arid and semi-arid areas, deserts and mountainous
regions. Camels are an important animal in the province's livestock herds and
play a vital role in the economy of rural pastoral communities. Overall, the
camel is still a neglected species in Pakistan and has not given proper
attention by scientists and researchers. Comprehensive information on the
population structure and diversity found in dromedaries from Balochistan
province is still missing. To fill this gap and to understand the phylogenetic
relationship between populations, we investigated the genetic diversity among
eight camel breeds using mitochondrial Cyt
b gene sequences. With this study we aim to provide first genetic data for
genetic-based breed management in dromedaries of Balochistan province.
Materials and
Methods
In total, we sampled 210 animals from eight dromedary
camel breeds in geographically different districts of Balochistan province (Fig.
1), including the Brahvi, Kharani, Kachhi, Mukrani, Roadbari, Pishin, Lassi and
Kohi well adapted to hot, arid and cold, dry environments of Balochistan. The blood samples were collected in
EDTA-containing vacutainer-tubes and were carried on ice-box to Animal Genomic
Laboratories, Virtual University of Pakistan. The DNA was isolated from the
blood using revised phenol/chloroform procedure previously described by Wajid et
al. (2014). The extracted DNA samples were quantified using the Nanodrop
spectrophotometer (Thermo-scientific, U.S.A.) and stored at -20ºC until further
use. The complete Cyt b gene was
amplified and sequenced using three-overlapping primers previously reported by
Babar et al. (2015). PCR amplification was carried out in a final volume
of 30 µL mixture contained genomic DNA (20 ng) 2 µL, forward and
reverse primers (10 pmol) 1.5 µL, dNTPs (2.5 mM) 2 µL,
reaction-buffer (10X) 3.5 µL, MgCl2 (1.5 mM) 3.5 µL,
Taq DNA-polymerase (5Unit) 0.5 µL (Thermo-scientific) and H2O
17 µL. The amplification was carried out in BioRad Thermos-cycler using
initial denaturation of 95ºC for 5 min followed by 33 cycles of denaturation at
95ºC for the 30s, annealing at 60ºC for 30s and extension at 72ºC for 1 min and
then with a final extension at 72ºC for 7 min. The purified Cyt b gene PCR products were sequenced
by ABI3130 automated-sequencer (ABI, Inc., Foster City, C.A.). A Maximum
Likelihood method phylogenetic tree was constructed to investigate the
evolutionary relationship of the studied camel breeds with other dromedary, and
Bactrian camel breeds using MEGAv6 software. DnaSP v. 5 program
was used for conducting neutrality tests, i.e.,
Tajima’s D and Fu and Li’s F to detect a deviation from neutrality, and to
calculate within species diversity parameters, e.g., number of haplotypes, nucleotide and haplotype diversities.
The complete Cyt b haplotypes (n = 16) obtained in this study were
submitted to GenBank and are available under accession number MT578032 to
MT578047.
Results
The mtDNA sequence analysis of 1140 bp in 210 animals
showed a total of 18 variable sites resulting in 16 different haplotypes that
demonstrated a moderate mtDNA genetic diversity in the Pakistani camel
populations. The detail of the number of variations in each breed, haplotype,
haplotype diversities, and nucleotide diversities are shown in Table 1. Among
the studied camel breeds, the average haplotype and nucleotide diversities were
Hd= 0.484 ± 0.051 and π = 0.00272 respectively. Highest number of
haplotypes (n = 3) was detected in Pishin and Kharani dromedaries while only a
single haplotype was identified in Kachhi and Rodbari breeds, respectively. The
haplotype diversity ranged from Hd = 0.500 ± 0.048 in the three populations
Brahvi, Kohi, and Lassi to Hd = 0.690 ± 0.023 in Pishin and Kharani (Table 1).
Nucleotide diversity ranged from π = 0.00044 (Lassi) to π = 0.00242
(Kharani). The haplotypes identified in camel populations under study were unique
to each population, except Markrani and Rodbari shared one haplotype; this is a
likely crossbreeding as geographically nearest neighbors.
The Rodbari camel breeds showed highest genetic
relatedness (0.1%) with other two Makrani and Lassi camel breeds. Among all
studied breeds, Kachhi was divergent camel exhibited 1.3% genetic distance from
Makrani and Kharani camel breeds. A Maximum Likelihood method phylogenetic tree
was constructed to investigate the evolutionary history of the studied camel
breeds with other dromedary and Bactrian camels from Arabian Peninsula, Iran,
Ethopia, Kenya, Russia, China and Mongolia. The result clearly exhibited two
distinct clades, dromedary and Bactrian. The Bactrian camels further divided
into two different lineages, the domestic and wild animals (Fig. 2). Two
classical tests were employed to detect deviation from neutrality. We obtained
positive values of Tajima’s D and Fu and Li’s F tests in all studied camel
breeds, indicative of balancing selection or a population subdivision event,
but the result was statistically significant (P < 0.05) only in three camel breeds.
Discussion
In this study, we investigated the genetic diversity and
phylogenetic relationship among 210 dromedaries belonging to eight camel breeds
reared in Baluchistan province using complete mitochondrial Cyto b amplification. Retrieving this
genetic information is a first indispensable step to facilitate the breed
conservation program in an effective and reminiscent way. Camel ecotype is not
well defined in Pakistan and very little information is available on
genetic-studies in camels found all over the country. Hence, the molecular
studies on genetic diversity in this specie can facilitate the development of
national camel-breeding program to establish an effective strategy for the
conservation of these important genetic resources.
Cyt
b is a highly conserved region of mitochondrial DNA (mtDNA) genome used
for studying species classification and detection of phylogenetic relations
among diverse mammalian species.
Table 1: The genetic diversity analysis of studied camel
populations
Species
|
Samples |
Detected
variations |
H |
Haplotype
diversity (Hd) |
Nucleotide
diversity (Pi) |
K |
Tajima's
D |
P value |
Fu
and Li's F |
Pishin |
30 |
2 |
3 |
0.690
± 0.023 |
0.00081 |
0.91954 |
1.68264 |
P
> 0.10 |
1.21673
P > 0.10 |
Brahvi |
25 |
3 |
2 |
0.500
± 0.048 |
0.00132 |
1.50000 |
2.20530 |
P
< 0.05* |
1.52520
P < 0.05* |
Kachhi |
25 |
0 |
1 |
0.000 |
0.00000 |
0.00000 |
0.00000 |
0.00 |
0.00000 |
Kharani |
30 |
6 |
3 |
0.690
± 0.023 |
0.00242 |
2.75900 |
2.34410 |
P
< 0.05* |
1.80694
P < 0.05* |
Kohi |
25 |
2 |
2 |
0.500
± 0.048 |
0.00088 |
1.00000 |
1.92637 |
P
> 0.10 |
1.30700
P > 0.10 |
Lassi |
25 |
1 |
2 |
0.500
± 0.048 |
0.00044 |
0.50000 |
1.47274 |
P
> 0.10 |
0.97500
P > 0.10 |
Makrani |
25 |
4 |
2 |
0.513
± 0.037 |
0.00180 |
2.05300 |
2.53549 |
P
< 0.01* |
1.73014
P < 0.05* |
Rodbari |
25 |
0 |
1 |
0.000 |
0.00000 |
0.00000 |
0.00000 |
0.00 |
0.00000 |
H: Haplotype
K: the average number of nucleotide differences
Fig.
1: Geographical locations of the samples used in this
study
Fig.
2: Maximum-Likelihood
method phylogenetic tree inferred from Cyto b gene
sequences
The partial and complete Cyt b has been used for detection of genetic conservation and
phylogenetic relationship among Bactrian camel (Ming et al. 2016), dromedaries (Babar et al. 2015; Legesse et al.
2018) and other livestock species (Hussain et
al. 2018). In this study, complete Cyto
b gene has been used for comparing genetic diversity between eight camel
breeds rearing in different geographical regions of Balochistan. The breeds
showed low differences in their genetic diversity. The haplotypes identified
were unique to each breed except the two camel breeds except Markrani and
Rodbari shared one haplotype. The uniqueness of the haplotypes identified in
the studied camel populations generally suggests the haplotypes specific to a
geographic region and low level of crossbreeding.
Two classical tests were employed to detect deviation
from neutrality. The positive D values indicate an excess of intermediate
frequency alleles and may suggest balancing selection or population
contraction. Neutrality tests were not performed on Kachhi and Rodbari camel
breeds, because these species had no genetic variation in the Cyt b gene. The neutrality tests showed
no population demographic expansion for the eight studied camel populations.
Phylogenetic analysis was performed based on the complete Cyt b gene and showed different clusters for camelids. The
phylogeny of the Cyt b gene was
consistent with known phylogeny for the ruminant classification based on other
genes (Iqbal et al. 2020).
Conclusion
This study provides initial information on the genetic
diversity of camel populations reared in Balochistan province. The studied
camel populations exhibited low levels of haplotype and nucleotide diversities,
which may be due to the small effective population size or many years
inbreeding that may further diminish the genetic diversity in these camel
populations. Indeed, the camels are well adaptable domestic animals to the
harsh ecosystems where these animals are essential for the economy and food
security of local communities. We suggest based on these results, an imperative
genetic management and breeding strategies are required for effective
conservation of this species.
Acknowlegment
We would like to thank to the farmers and the staffs of the
Livestock & Dairy Development Department (L&DD) of Balochistan
province, helped us in samples collection. Laboratory work was performed under
the HEC grant (NRPU-9336) provided to Dr. Tanveer Hussain
Author Contributions
AF, AW and MTM designed and perceived the experiment,
AF, MEB and AW execute the experiment, AF and TH collected the samples and AF
and TH helped in data analysis, AW wrote the manuscript.
Conflicts
of Interest
All authors declare no
conflicts of interest.
Data
Availability
Data presented in this study
are available on fair request to the corresponding author.
Ethics
Approval
The experiments were carried
out in accordance with the guidelines issued by the Ethical Committee of
University of Balochistan, Quetta, Pakistan.
References
Babar ME, T Hussain, A Wajid, A Nawaz, A Nadeem, SA
Shah, MA Shahid, N Ahmad, K Javed, M Abdullah (2015). Mitochondrial
cytochrome-b and D-loop sequence-based genetic diversity in Mareecha and
Bareela camel breeds of Pakistan. J Anim
Plant Sci 25:591‒594
Bahbahani H, HH Musa, D Wragg, ES Shuiep, F Almathen, O
Hanotte (2019). Genome diversity and signatures of selection for production and
performance traits in dromedary camels. Front
Genet 10; Article 893
Chen SG, J Lic, F Zhang, B Xiao, JM Hu, YQ Cui, M
Hofreiter, XD Hou, GL Sheng, XL Lai, JX Yuan (2019). Different maternal
lineages revealed by ancient mitochondrial genome of Camelus bactrianus from China. Mitochondrial
DNA A DNA Mapp Seq Anal 30:786–793
FAO (2019). The Food and Agriculture Organization of the
United Nations. Italy: FAOSTAT
Fitak
RR, E Mohandesan, J Corander, A Yadamsuren, B Chuluunbat, O Abdelhadi, A Raziq,
P Nagy, C Walzer, B Faye, PA Burger (2020). Genomic signatures of domestication
in Old World camels. Commun Biol 3:316-325
Hussain T, ME Babar, MD Donato, A Wajid, A Nadeem, Z
Ahmad, WA Khan, SO Peters, IG Imumorin (2018). Phylogeny of Pakistani Cattle
Breeds using Mitochondrial Cytochrom-b Gene. Pak J Zool 50:2029‒2035
Iqbal LN, Q Ain, A Wajid, A Mohiuddin, TM Yousaf, N Hussain,
S Sherzada, F Manzoor, T Hussain, ME Babar (2020). Detection of genetic
variations in pattern recognition receptors (PRRs) gene of Tharparkar cattle
breed. Pak Vet J 40:400–402
Legesse YW, CD Dunn, MR Mauldin, N Ordonez-Garza, GR
Rowden, YM Gebre, MY Kurtu, SM Ali, WD Whibesilassie, M Ballou, M Tefera, G
Perry, RD Bradley (2018). Morphometric and genetic variation in 8 breeds of
Ethiopian camels (Camelus dromedarius).
J Anim Sci 96:4925‒4934
Ming L, L Yi, FC Guo, G Siriguleng, J Jirimutu (2016).
Molecular phylogeny of the Bactrian camel based on mitochondrial Cytochrome-b
gene sequences. Genet Mol Res 15:1–8
Wajid A, M Wasim, T Yaqub, S Firyal, M Tayyab, S
Siddique, T Hussain (2014). Assessment of genetic diversity in Balochi and
Rakhshani sheep Breeds of Balochistan using microsatellite DNA-markers. J Anim Plant Sci 24:1348‒1354
Yadamsuren A, O Daria, S Liu (2019). The seasonal
distribution of wild-camels (Camelus ferus) in relation to changes of the environmental
conditions in Mongolia. Open J Ecol 9:293‒314