Molecular Phylogeny and Characterization of Mundri Sheep (Ovis aries) of Pakistan through Sequencing of Mitochondrial Cytochrome b and Cytochrome Oxidase Subunit I
T. Noreen#, T. Hussain#, M. Mansha#, Z. Mansoor, A. Wajid, M. R. Ashraf, G. Ayub, M. M. M Musthafa and F. M. M. T. Marikar*
#Contributed equally
Abstract
The main focus of this research is to determine the molecular phylogeny and characterization of Mundri Sheep (Ovis aries) through sequencing of mitochondrial Cytochrome b and Cytochrome Oxidase Subunit I (COI). This sheep breed appears morphologically different from other local sheep breeds of Pakistan. The current research is carried out to appraise the status of Mundri sheep whether it is a different breed from other breeds or not. Blood samples of Mundri sheep were collected from Livestock Experiment Station, (LES) Fazilpur in district Rajanpur (Punjab). DNA was isolated and subjected to Polymerase Chain Reaction (PCR) for amplification of Cytochrome b and COI genes using appropriate primers. PCR products were sequenced and analyzed by MEGA X software. The phylogeny analysis categorized Ovis aries including Mundri sheep into three and two groups for Cytochrome b and COI genes respectively. It showed Mundri sheep as a separate group and thus as a separate breed from all other local sheep breeds. Hence the study validates based on Cytochrome b and COI that Mundri sheep is a distinctive breed from the rest of the local sheep breed.
Key words: Mundri Sheep; Ovis aries; Molecular Phylogeny; Characterization; Cytochrome b; Cytochrome Oxidase Subunit I; COI; Pakistan
Introduction
Although there exists controversy and uncertainty regarding the origin of sheep (Ovis aries) evidence shows that, sheep were probably first domesticated in the Fertile Crescent region of the Near East about 11,000 years ago. Sheep (Ovis aries), is a well-known quadrupedal livestock animal. The ancestor of household sheep is the Asian mouflon (O. orientalis).
Urial (Ovis vignei) is one of the closest related species of all modern sheep, found primarily in the mountainous area of Central Asia. Mouflon, Urial, and Argali are wild sheep and thought to be ancestors of domestic sheep (Aljumaah et al., 2014; Atavliyeva and Tarlykov, 2018). Human beings domesticated sheep around 10,000 BC (Taberlet et al., 2011). The classification of sheep is Domain: Eukaryota (Whittaker and Margulis, 1978) Kingdom: Animalia (Cuvier, 1812) Phylum: Chordata (Bateson, 1885) Subphylum: Vertebrata (Cuvier, 1812) Class: Mammalia (Cuvier, 1812) Subclass: Theria (Bateson, 1885) Order: Artiodactyla (Owen, 1848) Suborder: Ruminantia (Owen, 1848) Family: Bovidae (Owen, 1848) Subfamily: Caprinae (Owen, 1848) Genus: Ovis (Owen, 1848) Species: Aries (Owen, 1848) (Özgen, 2008).
Countries that have large areas of grassland are the major producers of sheep.
Most of the population of sheep lives in areas where the source of feeding includes the residues of crops, natural shrubs, and different types of grasses. New Zealand, Australia, India, China, South Africa, the United States, Argentina, and Turkey are the major national producers of sheep. Pakistan ranks 10th in the world in terms of sheep population (Khan et al., 2008). Pakistan is at 3rd position in Asia as far as the population of small ruminants is concerned, with a yearly growth rate of 4%, the most elevated in Asia (Khan and Ashfaq, 2010). The current population of farm animals in Pakistan consists of 31.2 million sheep, 78.2 million goats, 41.2 million buffaloes, 1.1 million camels, and 49.6 million cattle (Khan et al., 2007). Sheep cultivation or sheep farming is the raising and rearing of household sheep. Commonly people from everywhere throughout the world, keep sheep for an agricultural reason so as to acquire their meat, milk, and wool.
They also yield sheepskin and parchment.
There are approximately 200 sheep breeds in Asia. Local breeds of sheep in Pakistan are 30 (Afzal and Naqvi, 2004).
The classification has been made on the basis of tail morphology. These breeds are classified into thin tail and fat tail sheep. In the irrigated areas the thin tail sheep breeds are generally found whereas fat tail sheep breeds are found in arid lands and mountainous areas of Sindh, KPK, and Azad Kashmir. Thin tail sheep consist of Buchi, Baltistani, Damani, Cholistani, Kaghani, Kail, Kajli, Kooka, Hissardale, Kali, Lohi, Kachhi, Pahari, Poonchi, Sipli, and Thalli. Fat tail sheep consist of Balochi, Bibrik, Balkhi, Gojal, Dumbi, Hashtnagri, Harnai, Khijloo, Kohai-Ghizar, Michni, Latti/Salt Range, Rakhshani, Tirahi, and Waziri (Khan et al., 2007). Sheep breeds Buchi, Lohi, Thalli, Latti/Salt Range, Cholistani, Sipli, Khijloo, Hissardale, and Kajli are found in Punjab; Dumbi, Kachhi, and Kooka are found in Sindh; Balkhi, Damani, Kaghani, Hashtnagri, Michni, Tirahi, and Waziri are found in KPK; Balochi, Bibrik, Harnai, and Rakhshani are found in Balochistan; Baltistani, Gojal, Kail, Kali, Kohai-Ghizar, Pahari, and Poonchi are found in the Northern area, and Azad Jammu and Kashmir AJK (Khan and Ashfaq, 2010). There may be still other sheep breeds available in the country which awaits documentation. In the Rajanpur district of Punjab province, a breed of sheep exists which is not still recognized as a separate breed, called the Mundri sheep by the local people. The sheep is found in other parts of the country too but exists in large numbers in this region. Mundri sheep appear morphologically different from other local breeds of sheep. This sheep has different physical features from other local Pakistani breeds therefore, there is also a need to study genetically that this sheep is different from other breeds or part of some already existing breed.
Mundri sheep are fairly large, a thin tail breed found in district Rajanpur province of Punjab with white color extremely short ears and sharp roman nose. Other characteristic of Mundi sheep is listed in the Table 1.
Source: Livestock Experiment Station, (LES) Fazilpur, Rajanpur
Mundri sheep breed habitats the region between the bed of Koh-e-Suleman range to the delta region of river Indus in District Rajanpur and also few flocks were reported in Koh-e-Suleman and across the river bed in Muzaffargarh especially in Alipur and Zahir Pir.
Genetic characterization helps in the detection of variations as a result of differences either in modifying factors, DNA sequences, or in specific genes. For phylogenetic examination, mitochondrial DNA is used (Rubinoff and Holland, 2005). Phylogenetic can be defined as the process in which the relationships that are related to evolution can be estimated and investigated effectively. In order to find out the evolutionary relationships between different organisms, the nucleic acid sequences are explored and then compared with the sequences on nucleic acid of the other organism. The results of the study may be interpreted in such a way that those organisms that will be having fewer differences in these components will be considered to be having a close relationship with the other organism while the organisms in which there will be many differences in the sequence of the components of nucleic acids will be having a less close relationship between them (Chenna et al., 2003).
In the past, various old methods were used for the similar purpose of finding out the evolutionary relationships between the organisms, and these included the approaches based on the morphology or the traits reading life cycle. However, in this era, with the advancement of technology, more advanced ways are used in order to find out the evolutionary relationships between the organisms. One of the most important advantages of the recent methods or approaches is that the results or the differences among the sequences of different organisms can be quantified effectively. This quantification is based on the number of differences that are observed in the sequences of the components of nucleic acids of different organisms. The results that are obtained by phylogeny are generally represented in the form of a ‘tree’.
This tree is having various branches that are actually the indications of the relationships of that organism with other organisms (Saitou and Imanishi, 1989).
According to the past studies, in order to determine the phylogeny of the organisms and to find out the level at which those organisms are related to each other in one way or the other, the most commonly used and effective tool is the mitochondrial DNA (mtDNA) (Brown et al., 1979). The reason behind the use of mtDNA in order to find out the phylogeny of different organisms is that there are various molecular markers at different levels of the evolutionary history of those organisms. These molecular markers are actually based on the distinct and special properties and characteristics that have been inherited from the ancestors of those organisms (Galtier et al., 2009).
There is no certain information regarding the origin of the domestic sheep also called Ovis aries. In this regard, studies have shown that some wild species of sheep are the actual source or origin of the domestic sheep and the various breeds of domestic sheep have been the results of breeding of those wild species (Mason and Mason, 1984). Some of the examples of wild sheep breeds that have been discussed in the literature include Urial, Mouflon, and Argali (Zeuner, 1963). All these species or breeds are supposed to have a contribution in the domestic breeds of sheep.
Cytochrome c oxidase I (COI), a mitochondrial gene, could fill in as the center of a worldwide bio identification framework for animals. Sometimes, the success rate is 100% in the accurate identification of cases. Hence, the COI is a dependable accessible solution and cost-effective identification system for the existing issue of identification of species (Jiang et al., 2015).
Abbas et al. (2017) investigated the usefulness of mitochondrial cytochrome b, cytochrome c, and d-loop region to find out the molecular phylogeny and diversity of hog deer. PCR method was employed and only nucleotide polymorphisms were found. A phylogenetic tree was constructed by using bioinformatics apparatus. The results revealed a minor genetic variation.
Savar Sofla et al. (2017) examined genetic relationships and differences between two Iranian breeds of sheep by exploiting the cytochrome b (cyt-b) gene sequence. The findings of the study revealed a separate group of Iranian sheep.
From this study, future researchers can have help and information for the determination of the genetic structure of different breeds.
Genetic characterization of Mundri sheep and its comparison with other sheep breeds is still an unexplored area within Pakistan. Therefore, this current study has been conducted to fill this niche through exploratory research. A large number of Mundri sheep are present in Rajanpur District. No study has so far been conducted to establish whether Mundri sheep is a separate breed or it is a part of some existing breed. The purpose of this study is to investigate the genetic characteristics of Mundri sheep in order to determine the molecular phylogeny of this sheep. As a pioneer work, this study will contribute significantly to determine the evolutionary relationship of Mundri sheep among various breeds of sheep based on the similarities and differences in its genetic characteristics. The scope of this study is to understand the genetic diversity of Mundri sheep of District Rajanpur and to evaluate the genetic structure/make-up of this breed, using mitochondrial Cytochrome b and Cytochrome Oxidase Subunit I (COI) gene sequences.
Materials and methods
Blood collection and Ethical clearance
The research work was performed at the Animal Genomics Laboratory of the Department of Molecular Biology of the Virtual University of Pakistan located at its 1-Davis Road, Lahore campus. Blood samples of the Mundri sheep breed were collected from Livestock Experiment Station, (LES) Fazilpur, Rajanpur. By using venipuncture procedure 10 mL blood samples were collected from 30 sheep (male and female) and transferred into ethylenediaminetetraacetic acid (EDTA) containing tubes/Vacutainer immediately (Sambrook and Russell, 2001). The tubes were shaken to ensure mixing of blood with EDTA, to avoid coagulation.
The blood samples were placed in an Ice chilled container and transferred to a lab and stored at -20 °C until further use. Sample collection was performed according to standard procedures without any stress or harm to animals (European Union, 2010). All laboratory works were conducted at the Virtual University of Pakistan. Institutional Animal Care and Use Committee (IACUC) from Virtual University of Pakistan was obtained the Ethical Clearance before the experiment.
DNA Extraction
DNA was extracted from the blood samples by using this protocol (Sambrook and Russell, 2006). Blood sample of 200 μL of Mundri Sheep was taken in an Eppendorf tube. Lysis buffer 1000 μL was added to it. It was vortexed for a few seconds. It was centrifuged at 10,000 rpm for 10 minutes. Pellet formation was checked and the supernatant was discarded. Lysis buffer 1000 μL was added and centrifuged at 10,000 rpm for 10 minutes and this step was repeated 3 times. 250 μL buffer A1, 80 μL 10% SDS, and 20 μl proteinase K were added. It was incubated at 58 °C overnight for degradation of the protein. On the next day, 300 μL PCI was added to each sample. It was vortexed for a few seconds. It was centrifuged at 13,000 rpm for 15 minutes. Three layers were formed. The upper aqueous layer was carefully transferred into a separate Eppendorf tube. Isopropanol 600 μL was added and it was mixed gently with a pipette. It was centrifuged at 13,000 rpm for 15 minutes. The upper layer was discarded. Ethanol 1000 μL was added.
It was centrifuged at 13,000 rpm for 10 minutes. The upper layer was discarded and the pellet was dried. Injection water 150 μL was added and the pellet was dissolved in it. DNA was stored at -20 °C for further use. Inorganic method (Sambrook and Russell, 2001) was used for genomic DNA extraction. The final concentration of DNA was brought to 50 ng/uL and stored at -80 °C before further use.
Mitochondrial Genome analysis
To amplify the complete mitochondrial Cyt b gene (1609 bp), three pairs were of primers was designed from Bos indicus (NCBI accession number AF492350) using software Primer3 (Steve and Skaletsky, 2000). Primer set : MtC-CF1 (5’-GT-CATCATCATTCTCACATGGAATC-3’) and MtCCR1 (5’-CTCCTTCTCTGGTTTACAAGACCAG-3’). A fragment of the mitochondrial gene cox1 was amplified using a set of forward primer set: MtC-CF2 (5’-GCAGAGTTTGAAGCTGCT-3’) and MtCCR2 (5’-AGCTGACGTGAAGTAAGC-3’). PCR was performed in a 25 µL reaction mixture containing 1 µL of template (the genomic DNA of each sample was used as a template for PCR), 1 µL of each primer (10 pmol/µl), 12.5 µL of 2× Taq PCR MasterMix and 9.5 µL of ddH2O. Negative controls were always included in PCR reactions to assess possible contamination.
Polymerase Chain Reaction
Polymerase Chain Reaction (PCR) is a technique to amplify the target DNA segment, also for the detection of bacteria, viruses, and other scientific researches. In this research, we have used the touchdown PCR method for the amplification of Cytochrome b and Cytochrome Oxidase Subunit I (COI) genes. In the touchdown method, the temperature is reduced in every cycle by the specified value. The touchdown value for Cytochrome b is 50-48 °C (-0.3 per cycle) 30 seconds and for COI is 54-50 °C (-0.5 per cycle) 1 minute. The standard PCR conditions for Cyt b were followed: initial denaturation temperature 4 min at 95 °C, 35 cycles of denaturation at 94 °C for 30 s, annealing at 50 °C for primer sets 1, for 30 s and extension at 72 °C for 45 s followed by final extension at 72 °C for 10 min. PCR was performed in 25 µL reaction mixture using about 50 ng DNA as template with 2 units Taq DNA polymerase (Fermentas, Thermo Fisher Scientific Inc. USA). For COI, PCR was performed using initial denaturation at 95 °C for 4 min, and then 35 cycles of denaturation at 94 °C of for 30 s, annealing at 54 °C for 30 sand extension at 72 °C for 45 s following 10 min of final extension at 72 °C. 25 µL reaction mixtures were used for PCR using 50ng DNA as template and 1 unit Taq DNA polymerase. PCR product was purified by ethanol precipitation and sequenced using an automated 300 DNA sequencer ABI PRISM® 3130Xl Genetic Analyzer (Applied Biosystem Inc, Foster City, CA).
Gel Electrophoresis, DNA Sequencing and Analysis
The PCR product of both Cytochrome b and COI genes were run on 1.2% Agarose gel for 45 minutes at 120 volts. 5:3 composition of the sample is loaded on the gel i.e. 5 µL DNA is mixed with 3 µL Bromophenol dye. The target product size of the Cytochrome b gene is 1140 bp and the product size of the Cytochrome Oxidase Subunit I (COI) is 1035 bp. Thermo Scientific 1Kb ladder is used for this purpose. 3 µL of the ladder is loaded on the gel. The results were observed on the gel doc system. PCR products were purified with 80% ethanol and sent for sequencing. 19 PCR products of Cytochrome b gene and 19 PCR products of Cytochrome Oxidase Subunit I (COI) were sequenced from the Lab Genetix G-3 AL-Hafeez Business Center, 89, Block B 3 Gulberg III, Lahore, Pakistan. 35 µL of PCR product of each sample with 100 µL of Cytochrome b-F and COI-F primer were sent for sequencing. The products from Sanger sequencing were analyzed by using Bioedit 7.2.5 (alignment editor for biological sequences) software. For phylogenetic analysis, various bioinformatics tools will be used, such as MEGA 6 software (Tamura et al., 2013). The resultant sequences were analyzed for the nucleotide analysis by using the software MEGA X (Kumar et al., 2018). These alignments were used to make the phylogenetic tree of Cytochrome b and COI.
Multiple Sequence Analysis
The newly determined complete mitochondrial Cyt b gene and COI sequences from Pakistani Mundri Sheep were submitted in GenBank for Cyt b gene and for COI. 667 nucleotides of Cytochrome b were used for the molecular characterization of Mundri sheep (Ovis aries).
Sequences from our samples of Mundri sheep were aligned with other sheep sequences through MEGA X software by using CLUSTAL O (1.2.4). To discover the evolutionary relationships between genes and to find out shared patterns amongst functionally or a structurally related gene, multiple sequence alignment is a tool used in research to find out about closely related genes or proteins.
The multiple alignments were saved in FASTA format for further analysis. These Mundri sheep sequences were aligned with the downloaded FASTA files of other Ovis aries sequences on MEGA X along which showed the highest similarity ratio with our query sequences from GenBank. All the accession numbers (when a biological polymer sequence (DNA, protein) submitted to a sequence database a unique identifier assigned to it) of the sequences used in this analysis are given below (Table 2 and Table 3).
Phylogenetic analysis
Phylogenetic trees were constructed through MEGA X software by using multiple Cytochromes b and COI sequences alignments of Mundri sheep, and other sheep breeds. The Maximum likelihood (ML) statistical method was used for the construction of a Phylogenetic tree using bootstrap methods. Further, we used the Jukes-Cantor model for this phylogenetic analysis. The bootstrap test method was used by adjusting 500 replicates values.
In this study, goat (Capra hircus) genes (Cytochrome b and COI) were used as an out-group. The length of the branches shows genetic distance. Scale 0.02 (2%) shown at the bottom of phylogenetic tree is the amount of genetic change/variation. The Bootstrap value of >0.6 shows strong support for tree topology.
Results
Cytochrome b and Cytochrome Oxidase Subunit I (COI) are the main players for the determination of the molecular Phylogeny of animals. To evaluate the genetic phylogeny of Mundri sheep, DNA isolation was done by extraction protocol as described by Sambrook and Russell (2006). The quantity of DNA was measured by NanoDrop (Thermo Fisher Scientific).
Cytochrome b and COI genes were amplified by touchdown Polymerase chain reaction (PCR) was done. PCR products were confirmed by Agarose gel (1.2%) electrophoresis with 1Kb DNA ladder (Thermo Fisher Scientific). For the sequencing of Cytochrome b and COI, PCR products were sent to Lab Genetix G-3 AL-Hafeez Business Center, 89, Block B 3 Gulberg III, Lahore, Pakistan.
The sequencing of the Cytochrome b gene was analyzed by using Bioedit 7.2.5 (alignment editor for biological sequences) software. We trimmed some sequences from the 5` end and 3` ends of all our sequence samples because the quality of the sequencing was not so good. 09 query sequences of Cytochrome b whose sequence result was very clear were chosen for further analysis.
Figure 1 shows genetic distance (P-distance) of Mundri sheep with other closely related local sheep breeds and from the rest of the world taken from the NCBI Genbank.
Analyses were carried out using the Maximum Composite Likelihood model. 60 nucleotide sequences were involved in the analysis. There were a total of 667 positions in the final dataset. Maximum evolutionary divergence between sequences shown by the Cytochrome b gene was 0.06; which implies that there is a little variation amongst Mundri Sheep and other sheep breeds compared with it.
Figure 2 is a phylogenetic tree for Cytochrome b dividing the sheep breeds into 3 clades. The phylogenetic tree shows Mundri sheep (Ovis aries) from Pakistan as a separate group thus indicating as a different breed. In this clade, the tree shows that Mundri Sheep P11 and P 14 shared evolutionary history with Mundri sheep P1 which further shared history with Mundri sheep, P2, and P 15 but this is away from P16, P7, P 20, and P9. This also shows that the parents of Mundri P11, P14, P1, P2, and P15 are the same and all Mundri sheep having a common ancestor. The divergence between goat (Capra hircus) which has been used as out-group and Mundri sheep breeds query sequence varies up to 0.06 whereas divergence between other local, and other sheep breeds varies up to 0.04. This study shows that more morphological and genetic data can produce more resolution in the relationship of sheep breeds.
The analysis of the Cytochrome Oxidase Subunit I (COI) gene was made through Bioedit 7.2.5 software. Some sequences were terminated from the 5` end and 3` ends of all our sequence samples having not good quality. Very clear 14 query sequences of COI results were chosen for further analysis. 747 nucleotides of Cytochrome Oxidase Subunit I (COI) were used for the molecular characterization of Mundri sheep (Ovis aries). Sequences from our samples of Mundri sheep were aligned with other sheep sequences through MEGA X software by using CLUSTAL O (1.2.4). To discover the evolutionary relationships between genes and to find out shared patterns amongst functionally or a structurally related gene, multiple sequence alignment is a tool used in research to find out about closely related genes or proteins. The multiple alignments were saved in FASTA format for further analysis.
The analysis of the Cytochrome Oxidase Subunit I (COI) gene was made through Bioedit 7.2.5 software. Some sequences were terminated from the 5` end and 3` ends of all our sequence samples having not good quality. Very clear 14 query sequences of COI results were chosen for further analysis. 747 nucleotides of Cytochrome Oxidase Subunit I (COI) were used for the molecular characterization of Mundri sheep (Ovis aries). Sequences from our samples of Mundri sheep were aligned with other sheep sequences through MEGA X software by using CLUSTAL O (1.2.4). To discover the evolutionary relationships between genes and to find out shared patterns amongst functionally or a structurally related gene, multiple sequence alignment is a tool used in research to find out about closely related genes or proteins. The multiple alignments were saved in FASTA format for further analysis.
Figure 3 shows genetic distance (P-distance) of Mundri sheep with other closely related local sheep breeds and from rest of the world taken from the NCBI Genbank. Analyses were carried out using the Maximum Composite Likelihood model. The analysis involved 32 nucleotide sequences. There were a total of 655 positions in the final dataset. The maximum evolutionary divergence between sequences shown by Cytochrome oxidase subunit I (COI) gene was 0.03; which implies that there is a little variation amongst Mundri Sheep and other sheep breeds compared with it.
Figure 4 is the second maximum likelihood phylogenetic tree dividing the sheep breeds into two clades. Goat (Capra hircus) COI was used as an out-group.
Clade first shows Mundri sheep from Pakistan showed a very close relationship with Ovis aries from India and China (Jialuo sheep). Clade 2 of the tree shows that sheep breeds from China are closely related to sheep breeds from Turkey, Scotland, Italy, Poland, Canada, Kazakhstan, and Kenya.
Genetic distance graphs (P-distance) and Phylogenetic trees of both genes indicate that mitochondrial Cytochrome b gene is the best gene for Phylogenetic studies rather than mitochondrial Cytochrome Oxidase Subunit I (COI) gene.
Discussion
This study on molecular phylogeny and characterization through sequencing of mitochondrial Cytochrome b and Cytochrome Oxidase Subunit I (COI) genes of Mundri sheep was conducted. Mundri sheep breed is fairly large, thin tailed with white color extremely short ears and sharp roman nose, habitats in the Rajanpur district of Punjab province, appears physiologically different from other local sheep breeds present in the country.
The exploration of the phylogenetic relationship between species is one of the major areas of interest since the conception of the theory of evolution. It is found that variations occur between species, whether in physiology, morphology, ecology, in ways of behavior, or in geographical distribution, during the course of phylogenesis. With the advancement in such fields of study Linnaean system has stepped forward to the modern system of biological classification such as systematics, cladistics, and phylogenetics based upon the evolutionary relationships between organisms. Genetic characterization of different breeds of particular species has been made possible with advances in science. For phylogenetic examination in animals, mitochondrial DNA is one of the most commonly used molecular markers. In the past, various studies have been conducted to determine the genetic characteristics and evolutionary relationship of various sheep breeds as well as goats, buffalo camel, etc. by using mitochondrial Cytochrome b and Cytochrome Oxidase Subunit I (COI). However, this study is conducted to explore the genetic characterization of Mundri sheep and its comparison with other sheep breeds which is still an unexplored area within Pakistan.
Phylogenetic analysis was conducted to find out the relationship of Mundri sheep with other breeds of sheep using mitochondrial Cytochrome b and Cytochrome Oxidase Subunit I (COI) gene sequencing. These genes were selected due to their unique properties of specie’s identification and for determining a phylogenetic association between organisms.
The evolutionary relationship of Mundri sheep with local sheep breeds as well as other sheep breeds was studied by constructing phylogenetic trees.
Cytochrome b and COI genes of Mundri sheep were amplified by PCR and sequenced to confirm the separate breed of Mundri sheep A total of 1,414 number of nucleotides (Cytochrome b=667 and Cytochrome Oxidase Subunit I (COI)=747) were used for the molecular characterization of Mundri sheep (Ovis aries). We aligned our query sequences (Mundri sheep) along with the FASTA files of other Ovis aries sequences on MEGA X. Our findings showed high similarity of Mundri sheep sequences from Pakistan and other parts of the world whose genetic characterization and evolutionary relations were determined using either Cytochrome b gene sequence or Cytochrome Oxidase Subunit I (COI) genes sequence. The alignments were saved for further analysis.
The phylogenetic tree of Cytochrome b was constructed by using our query sequences and other Ovis aries sequences of local breeds and from different countries which showed the highest similarity.
This analysis groups the Mundri sheep in a separate clad due to evolutionary divergence. Moreover, our results divide the sheep into three groups. Most of the sheep breeds from Pakistan, India, Iran, and China showed close relationship with Australian, Georgian and Russian sheep breeds. Some of the sheep breeds from Pakistan China, Iran also showed close relationship with sheep breeds from Poland, Italy, China, Finland, Russia, Iraq, Ukraine, and Turkey. Mundri sheep totally grouped separately in the phylogenetic analysis of the Cytochrome b gene thus appearing as a different breed from all other sheep breeds. The separate breed of Mundri sheep is also validated by the genetic distance (P-distance) table deduced from the Cytochrome b gene.
The 0.06 evolutionary divergence value of the P-distance table with other sheep breeds is ample proof of its separate clad.
These results are also supported by earlier studies conducted by various researchers on sheep phylogenetic relationship and taxonomic status.
Although “Thalli” and “Lohi” sheep breed are very close to Indian sheep breed but these findings were derived by using the cytochrome b gene and other conserved markers. It was reported that “Thalli” and “Lohi” sheep were very close to the Indian breed as we inferred that Mundri sheep showed its close resemblance with other sheep breeds of the world. Genetic relationship and differences between two Iranian breeds of sheep by exploiting Cytochrome b (cyt-b) gene sequence were examined.
The findings of the study revealed a separate group of Iranian sheep (Savar Sofla et al., 2017) as we reported about Mundri sheep.
The second maximum likelihood phylogenetic tree was constructed using Cytochrome Oxidase Subunit I gene (COI) gene sequences. In the case of the COI gene, the phylogenetic analysis was done between query sequences of Mundri sheep with other Ovis aries local sheep breeds and from different countries.
All sheep were divided into 2 groups.
Mundri sheep from Pakistan showed a very close relationship with Ovis aries from India and China (Jialuo sheep).
The findings of the study revealed that some sheep breeds from China showed a close relationship with sheep breeds from Scotland, Italy, Poland, Canada, Kazakhstan, and Kenya. P-distance table with COI gene showed a maximum evolutionary divergence of 0.03.
Earlier Sharifi et al. (2017) differentiated the genetic features of various breeds of goats in the Iranian context through sequence analysis and Polymerase Chain Reaction (PCR). The finding of the study revealed that Iranian goats possessed 1286 (bp) cytochrome Oxidase Subunit I (COI) gene in partial sequence and had four sites of variables and three haplotypes. The analysis showed that Iranian goats were clustered in a separate lineage in reference to the combination of GenBank.
In conclusion, the current study determined the molecular phylogeny of Mundri sheep in comparison to other local sheep breeds of Pakistan as well as the rest of the world using two mitochondrial molecular markers i.e. Cytochrome b and Cytochrome Oxidase Subunit I (COI). Based on Cytochrome b sequencing, Mundri sheep (Ovis aries) appeared as a separate group whereas this study also revealed a close association of Mundri sheep with Ovis aries from India and China (Jialuo sheep).
Phylogenetic relationship and characterization of Mundri sheep and its comparison with other breeds is an unexplored area within Pakistan. No study has so far been conducted to establish whether Mundri sheep is a separate breed or it is a part of some existing breed. The purpose of this study was to analyze the genetic characteristics of Mundri sheep in order to determine the molecular phylogeny of this sheep by Mitochondrial Cytochrome b and Cytochrome Oxidase Subunit I (COI) genes. Blood samples of Mundri sheep were collected from Livestock Experiment Station, (LES) Fazilpur district Rajanpur for this study. DNA was isolated from these blood samples using the inorganic method and subsequently quantified. Mitochondrial Cytochrome b and COI were amplified by using designed primers through PCR. PCR products were sequenced and analyzed through MEGA X software. For phylogenetic analysis, the maximum likelihood (ML) statistical method was used with the bootstrapping of 500 replications. Evolutionary divergence of Mundri sheep was observed by genetic distance (P- distance) tables of Cytochrome b and COI genes.
Two different Maximum Likelihood phylogenies were obtained from Cytochrome b and COI gene. Goat (Capra hircus) was used as an out-group.
The phylogenetic analysis categorized Ovis aries including Mundri sheep into three and two groups for Cytochrome b and COI genes respectively. Further, it showed that that Mundri sheep (Ovis aries) from Pakistan appeared as a separate group and thus grouped as a distinctive breed from local and rest of the world breed. Furthermost of the sheep breeds from Pakistan, India, Iran, and China showed a close relationship with Australian, Georgian and Russian sheep breeds. Some of the breeds from Pakistan China, Iran also showed a close relationship with sheep breeds from Poland, Italy, China, Finland, Russia, Iraq, Ukraine, and Turkey in this phylogenetic analysis.
There found a close relationship of Mundri sheep with the Ovis aries from India and China (Jialuo sheep). Moreover, the potential of Cytochrome b and COI for the identification of species and subspecies can be explored. The present study will help to evaluate the diversity of mammals, birds, reptiles, fish, and insects.
Conclusion
In the past, the classification of organisms was based upon morphological characters. Mundri sheep from Pakistan is already accepted as a separate breed because the physical appearance of this sheep is different from other local breeds of sheep. This study expressed the potential of mitochondrial Cytochrome b and Cytochrome Oxidase Subunit I (COI) genes to help in determining the molecular phylogeny and characterization of this sheep. Mundri sheep from Pakistan appeared as a different breed from all other local sheep breeds as derived from phylogenetic tree. This study will also be helpful in the genetic conservation of Mundri Sheep.
Recommendations
This study recommended that mitochondrial Cytochrome b and Cytochrome Oxidase Subunit I (COI) genes are effective tools in determining genetic characteristics and also are helpful for species identification and phylogeny. A substantial number of local sheep (n=30) breeds have so far been recognized in Pakistan.
Most of the work on sheep breeds awaits documentation even there may still be other sheep breeds available in country which are not recognized till now. So by using these genes other sheep breeds and diversity of other fauna (birds, mammals, reptiles, fishes, and insects) should be studied. Further, the present molecular techniques should be applied on already recognized species/subspecies to get more insight about them.
Acknowledgements
We acknowledge Dr. Hafiz Khalid Ijaz Qaisrani, Veterinary Officer, Livestock Experiment Station Fazilpur, District Rajanpur and the Livestock and Dairy Development Department (L&DD) Punjab, Pakistan for the kind facilitation to conduct this study. The HEC project NRPU-4485 is also acknowledged for lab facilities at the Virtual University of Pakistan.
References [… show]
Molekularna filogenija i karakterizacija mundri ovce (Ovis aries) u Pakistanu sekvenciranjem mitohondrijskog citokroma b i podjedinice I citokrom oksidaze
Tuba NOREEN, Department of Zoology, University of Education, Lahore, Pakistan; Tanveer HUSSAIN, Department of Molecular Biology, Virtual University of Pakistan, Rawalpindi, Pakistan; Muhammad MANSHA, Department of Zoology, University of Education, Lahore, Pakistan; Zinnia MANSOOR, Department of Biotechnology, Virtual University of Pakistan, Lahore, Pakistan; Abdul WAJID, Department of Biotechnology, Balochistan University of Information Technology, Engineering and Management Sciences, Quetta, Balochistan, Pakistan; Muhammad Rizwan ASHRAF, Goher AYUB, Department of Biotechnology, Virtual University of Pakistan, Lahore, Pakistan; Muneeb M. M. MUSTHAFA, Department of Biosystems Technology, Faculty of Technology, Southeastern University of Sri Lanka, Sri Lanka; Faiz M. M. T. MARIKAR, General Sir John Kotelawala Defense University, Ratmalana, Sri Lanka
Glavni je cilj ovog istraživanja bio odrediti molekularnu filogeniju i karakterizaciju mundri ovce (Ovis aries) sekvenciranjem mitohondrijskog citokroma b i podjedinice I citokrom oksidaze (COI). Ova se pasmina ovaca morfološki čini drugačijom od ostalih lokalnih pasmina ovaca u Pakistanu. Ovo je istraživanje provedeno da bi se procijenio status mundri ovce, da bismo mogli odrediti radi li se o pasmini drugačijoj od ostalih pasmina. Uzorci krvi mundri ovce prikupljeni su iz Stanice za eksperimente na stoci (engl. Livestock Experiment Station – LES) Fazilpur u okrugu Rajanpur (Punjab). DNK je izolirana i podvrgnuta lančanoj reakciji polimerazom (PCR) zbog pojačanja citokrom B i COI gena uporabom prikladnih primera. PCR proizvodi su sekvencirani i analizirani pomoću MEGA X softvera. Filogenetska analiza kategorizirala je Ovis aries uključujući mundri ovcu, u tri i dvije skupine za citokrom b, odnosno COI gene. Istraživanje je pokazalo da je mundri ovca posebna skupina i time zasebna pasmina ovaca u odnosu na ostale lokalne pasmine. Na temelju citokroma b i COI, naša je studija potvrdila da je mundri ovca zasebna pasmina i da se razlikuje od ostalih lokalne pasmine ovaca.
Ključne riječi: mundri ovca, Ovis aries, molekularna filogenija, karakterizacija, citokrom b, podjedinica I citokrom oksidaze, COI, Pakistan
