1.
Phylogenetic Analysis Of Major Fresh Water Carps Of Pakistan Through DNA Barcoding
by Madeeha Awan (2012-VA-650) | Dr. Ali Raza Awan | Dr.Sehrish Firyal | Dr. Muhammad Tayyab.
Material type: ; Literary form:
not fiction
Publisher: 2014Dissertation note: Pakistan is bestowed with the land of geological and topographic diversity. The ecological variation is uniformly reflected in all water lands of the country. Pakistan has significantly huge natural inland water resources in the form of ocean, rivers, networks of canals and lakes (Mirza and Rafique 1994). The country is blessed with one of the largest freshwater resources in the world correspondingly large number of freshwater living vertebrates is available from which fishes are quite significant considering the ecological balance and its consumption as food. It is one of the food sources which solely provide all the essential nutrients, minerals and high quality protein which is not common from other food items (Muhammad Rafique 2007). Out of 33,100 fish species identified worldwide as per Fish Base organization report published in April 2015 (http://www.fishbase.org). Out of 233 (indigenous and exotic) freshwater fish species, 78 economically important indigenous fish species are available in the water bodies of the Pakistan. According to this report fishes are the largest vertebrate group, constituting about 50% of all vertebrate species. Systematically fishes are widely spread in nature, ranging from prehistoric jawless fishes to cartilaginous fishes and also from old to current day bony fishes. The taxonomic placement of these fishes shows their belonging to class Actinopterygii, sub-class Teleostei, 3 cohorts, 6 superorders, 13 orders, 30 families and 86 genera (Rafique 2007; Rafique and Khan 2012).
Demand of fish is increasing day by day not only being the naturally available source of food rather the health benefits associated with its consumption. This necessitates to develop a more efficient and sustainable system to increase their growth. DNA based technologies are being competently employed in aquaculture production fields for pedigree information.
Introduction
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Moreover, tagging each fish individually is not an easy task so these DNA based methods help in avoiding intrusion of environmental factors which may result from raising fish families in separate reservoirs (Martinez 2007). Fish identification has been traditionally based on phenotypic features. However, due to high multiplicity and morphological similarity, in many cases, fish at its different developmental stages are hard to be identified by relying only on morphological characteristics (Victor et al. 2009).
For phylogenetic studies of the animals the use of mt-DNA is very common and reliable compared to nuclear DNA due to its high evaluation capabilities, which results in gathering of differences even between closely related species (Moore 1995; Mindell et al. 1997).“Bar-coding gap" is the name given to the property that is inter-specific variation in this region is markedly higher than intra-specific variability (Hebert et al. 2003).
Approximately each and every animal contain 13 protein-coding genes (PCGs) as an essential component of their mt-genome (mitochondrial genome), which helps in encoding of several proteins responsible for the oxidative phosphorylation machinery (Richly A et al. 2004, Song H et al. 2008). Being maternally inherited, mt-DNA is better as compared to genomic DNA such as quick evolution, less exposure to recombination, high copy number, high conservation, little duplications and negligible intergenic regions (Waugh J 2007, Xu J 2005). Clonal inheritance is the main property which makes it more worthy and suitable marker in comparison with other available molecular bio-diversity tools (Galtier et al. 2009).
DNA barcoding is one of the taxonomic tools. It is being used to distinguish animal species based on the small segment of their genome such as mitochondrial DNA, designated as an identification tag or barcode of particular species (Herbert et al. 2003). Identification of species using DNA barcoding is quite debatable. Still many researchers consider it as a reliable
Introduction
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basic tool to ascertain the genetic characterization of diverse eukaryotic species, especially after establishment of the Consortium for the Barcode of Life (CBOL) in 2004 [http: //www. barcodeoflife.org/].
Ideally DNA barcoding should provide quick, reliable and cost effective species identification, even to those user who has little or negligible knowledge of taxonomy (Herbert et al. 2003, Hajibabaei M et al. 2005, Herbert et al. 2005). Identification of unknown source is possible by using distance based tree which can be created by comparing unidentified sequences against retrieved known sequences of different species (Hebert et al. 2003, 2004a, 2004b). DNA barcoding identification system has been recognized universally as standardized method to recognize species and unveil their genetic diversity (Herbert et al. 2003; Herbert et al. 2004). The ideal DNA barcoding is robust, with conserved, universal primer binding sites, reliable DNA amplification and sequencing.
From whole mitochondrial genome, Cytb (Cytochrome b) is considered as one of the most promising gene due to its function and structure, even it is composed of both conserved and rapidly evolving regions which are more related to evolutionary studies (Farias et al. 2001). To identify unknown or ambiguous species it is considered more reliable as it contain sequences which provide the specific information about particular species (Parson W et al. 2000a, 2000b). It is also one of the most useful genetic marker to identify the linkage within families and genera (Meyer 1994; Teletchea 2009). Cytb gene is involved in comparative studies which results in development of new classification schemes and been used to assign a genus to a newly described species as well as improve the understanding of evolutionary relationships of genra (Castresana 2001).
Introduction
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One of the core objectives of this study is to identify and classify four freshwater indigenous fish species of Pakistan, which includes Labeo rohita (Rohu), Labeo calbasu (Calbans), Catla catla (Thalla) and Cirrhinus mrigala (Mori) using Cytb gene. Morphologically, Labeo rohita shows compressed body with convex dorsal profile while mouth bears a pair of barbells and fins are gray and orange in color. Catla catla shows compressed body with broad head. Mouth is wide with thick lower lip. Labeo Calbasu`s dorsal profile is more convex than that of abdomen and two pairs of barbells are present on fringed upper lip. Cirrhinus mrigala has elongated and streamlined body shape which is grayish and silver in color (Bhuiyan AL 1964; Rahman AK 1989). All of these species are found in freshwater bodies mostly lakes, rivers and ponds except Labeo calbasu which is a bottom dweller. These fishes are harvested by using rod and line or by using nets (Talwar PK and Jhingran AG 1991). These fishes are known as major carps and economically very important for the country due to their high consumption as food. These fishes are also used for fish farming due to their greater muscle mass thus also possess very high commercial value for fish farming business.
Another objective of this study is to resolve the taxonomic anomalies related to above mentioned species. Selling of fish meat in mislabeled packaging is a serious issue now days. Most commonly Hypophthalmichthys molitrix (silver carp) and Ctenopharyngodon idella (grass carp) are sold under the label of Labeo rohita. DNA barcoding is also helpful in detecting such fraudulent mislabeling.
It would be the first study in Pakistan to genetically characterize commercially important fish species. It would help scientists to know about their phylogenetic and taxonomic status and also assist fish fanciers to genuinely identify their species of interest. Identification of fish species is also important for conservation of biodiversity as it helps in preservation and
Introduction
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identification of endangered species by generating their barcodes from even minimal evidence available. This study has paved the way for molecular biologists to study taxonomic ambiguities at sub species level using SNP (Single nucleotide polymorphism) based identifying marker. Availability: Items available for loan: UVAS Library [Call number: 2207-T] (1).
2.
Application Of Microsatellite Markers For Genetic Diversity Analysis Of Endangered Punjab Urial (Ovis Orientalis Punjabiensis) In Pakistan
by Anam Aftab (2012-VA-534) | Dr.Tanveer Hussian | Dr. Wasim Shehzad | Dr. Muhammad Tayyab .
Material type: ; Literary form:
not fiction
Publisher: 2014Dissertation note: Biological diversity is now recognized as common concern of mankind and genetic diversity is the major driver of variation within and across breeds, which helps populations to adapt to environmental changes. There is very little importance is given to conserve wild sheep in previous years and its genetic diversity is decreasing day by day. Every now and then breeds are being haunted for crossing to some other imported breed without attempting to see if such efforts will be sustainable. For any breed development efforts thus, available genetic resources need to be characterized both at phenotypic and genetic levels (Khan et al. 2007).
Among the three levels of Biodiversity, one is Genetic variation which is suggested bythe International Union forNatureconservation (IUCN) for preservation (Mc Neely et al. 1990). The reason for it is that firstly; genetic diversity favors the changes as the environment changes and secondly; it prevents inbreeding depression (Reed and Frankham 2003). In this way genetic diversity increase the survival status and increase fitness of individuals.
Among many other wild animals present in Pakistan, there are 6 to 9 species of wild sheep (Ovis orientalis) are present which have different color and size of their winter neck ruff of males, saddle patches and horns color. Urial is a picture of Marco Polo in texture and hue. In Pakistan, ladakh urial, Blanford urialand Punjab urial are found in Gilget, Baluchistan and Punjab respectively. The discrepancy lies in the color of ruff among these 3 sub species (Roberts, 1977). Urial is among those precious wild animals that were hunted severely for trophy and other purposes in the past, that’s why included in red list of IUCN in vulnerable category (IUCN 2000).
Punjab Urial (A type of wild sheep – Ovis vignei punjabiensis) belongs to family bovidae which is the large family consisting of 140 species (Glazko et al. 2011) is facing serious threat of extinction in Pakistan is a medium-sized wild sheep which is included in IUCN red list of endangered animals (IUCN 2002). Urial is inhabitant in Western Central Asianregion stretching from northeast side of Iran and west side of Kazakhstan to Balochistan (Pakistan) and Ladakh regions of North India. The local name of Urial is Shapo, Arkar and Gad. Reddish-brown outstretched pelt that achromatizes during the winter is one of the distinct traits of urial (Aleem 1977; Schaller 1977). Urial is gregarious and sexually dimorphic as males are called rams and females as eves (Awan 2001). Male have weightof 40 kgand have large spiralled horns having height of 80 to 100 cm and females have comparatively less weight and height of 25 kg and 12 cm respectively and have uncurled horns.Females give birth to 1 or 2lambs in recent days of April (Awan, 2001). Males have a black ruff expanded from the neck to the trunk and notably longer horns. Table 1.1:Some physical features of the Punjab Urial (Awan et al. 2001)
Features Urial
Body weight 40 kg male; 25 kg female
Shoulder size 31-35 inches or 80-90 cm
Horn size 39 inches or 80-100 cm long male; 12 cm long female
Urial are found in moderate to very arid habitats, especially grasslands including agricultural fields and woodland areas (Valdez, 1982). Urial is herbivorous and eats grasses, shrubs and grains. The patch of salt range of Pakistan which fall in the area of Pind Dadan Khan, Choa Saidan Shah and Kallar Kahar is considered like a paradise on earth for the wild fauna. The fascinating hills of these areas are covered with thick trees and different wild plants are also the sanctuary of urial.
Table 1.2: The details of these sub species in IUCN list of endangered mammals (IUCN 2002).
Subspecies Citation in IUCN list
Ovis vegnei blanfordi VUC 1 Appendix ll
Ovis vegnei punjabienses ENA1cde,c1+2a Appendix ll
Ovis vegnei vegnei VUC 1 Appendix ll
In Pakistan, Punjab Urial dispersed throughout the Kala Chitta and Salt Range in a very little number(Hess et al, 1997). The Afghan Urial inhabits Baluchistan, Khyber Pakhtunkhwa, and Sindh Provinces. In Chitral District, little segregated populations of Ladakh urial are stillextensively distributed near the west bank of the Kunar river from Chitral southwards to Drosh. Ladakh Urial existence at the east bank of Kunar river and north of Chitral are not proved (Malik 1987). Total population estimate of urial is recorded by conducting several surveys. Reasonably 2,500 to 3,000 urial existsin Baluchistan (Hess et al. 1997). In 1993 the overall population assessment of Northern Areas was four hundred to five hundred Urial (Rasool 1999). In Pakistan, there were seemingly less than 600 Ladakh urial (Hess et al. 1997;NWFP 1992; Schaller 1971, 1977) but the number of urial decreased to 200 to 300 urial in all over northern areas (Rasool 1999). Based on the facts mentioned above there is dire need to conserve the Urial population in the country. The urial is one the precious fauna of Pakistan and provides us with wool and meat (fat, flesh or any eatable part). It is also important in economic way and in maintaining of ecosystem balance. In the beginning, sheep were reared for meat, milk and skin (Ensminger and parker, 1986). After 3500 B.C. men learnt to spin wool and so used wool in textile industry (Smith et al. 1997). So, because of increasing world population, there is great demand of these products and increasing day by day. That’s why we have to conserve is natural resource of Pakistan.Habitat fragmentationleads to the risk of exaggerated genetic drift and inbreeding in isolated population. So, there is need to save urial from these threats by enforcement of law and conserving it for future.
In all over the euchromatic genome Microsatellite markers are present. These markers are highly polymorphic (Ellegren 2000; Schlotterer 2000).A lots of polymorphic microsatellites have been analyzed in ruminants like domesticsheep, cattle etc. (de Gortari et al. 1997,1998 ;Hayes et al.1996; Jenkings et al. 1997) aiding the use of these in parentage testing.
Microsatellite markers are among the most reliable molecular markers for genetic characterization studies in animal species (Sunnucks, 2001) and are simple sequence repeats (SSRs) of 1-6 base pairs, repeated tandemly in coding as well as noncoding portion of DNA in prokaryotes and eukaryotes (Weber and May, 1989; Toth et al., 2000). Microsatellite markers have often used for genetic diversity studies because they areabundant, unbiased, widely distribution all over the DNA, highly polymorphic, easy in assessment like genotyping of these markers (Canon etal. 2001).Microsatellite markers aid in genetic differentiation and conservation studies (Peter et al. 2007;Rendo et al. 2004; Arranz et al. 2001).These are considered very useful markers for assessment of genetic diversity, parentage confirmation, genome mapping, disease research population genetic studies and conservation genetics. These are also reported to be efficient enough to identify within and among breed differentiation and population sub structuring in cattle (Glowatzki-Mullis et al. 1995; Ciampolini et al. 1995; Garcia-Moreno et al. 1996; Jarne and Lagoda, 1996; MacHugh et al. 1998).Therefore the conservation activities are very important to save Punjab Urial from extinction and the study is designed to explore its genetic diversity.
Availability: Items available for loan: UVAS Library [Call number: 2261-T] (1).
3.
Genetic Identification And Molecular Classification Of Sub-Family Phasianinae Of Pakistani Bird Species Through Dna Barcoding
by Maryem Hussain (2008-VA-349) | Dr. Ali Raza Awan | Dr. Sehrish Firyal | Dr. Muhammad Tayyab.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: DNA barcoding is a precise technique that uses molecular genetics tools for accurate identification, categorizing, relating and separating the phylogenies of species. Being a small sized genome and agile enough to show rapid mutation, mtDNA has been used as a pertinent marker of molecular biodiversity.The aim of this study was to develop DNA barcode for genetic characterization and classification of Sub-family Phasianinaeof Pakistani bird species. Theyhave not been genetically identified yet in Pakistan. It includes birds like domestic chicken(Gallus gallusdomesticus), aseel chicken(Gallus gallusdomesticus strain),blue peafowl(Pavo cristatus), green peafowl (Pavo muticus), white peafowl (Pavo cristatus leuticus), Kalij pheasant (Lophura leucomelanos),monal pheasant (Lophophorus impejanus),koklass pheasant(Purcrasia macrolopha), ring necked pheasant (Phasianus colchicus), Tragopan (Tragopan melanocepals) andred junglefowl (Gallus gallus). These birds are considered an important part of an ecosystembecause they play a significant role in seed dissemination, pollination of plants and disease spread which are the basic constituents of an ecosystem. They are used for food, hunting and entertainment purposes.
Mitochondrial geneCytochrome c oxidase subunit 1 (CO1)of 500bps was used as a marker for identification at specie level.Genomic DNA was extracted by each blood and tissue sample of eleven bird species (33 samples). Amplification of CO1 gene was a done by using a universal set of primers (BIRDF1 and BIRDR1)containing region of almost 750 bps (Hebert et al. 2003).Amplicons were purified and sequenced Sanger sequencing method (Sanger et al. 1977). Forward and reverse sequences were analyzed using softwaresEMBOSS merger,ClustalW, BioEdit and nBLAST. Phylogenetic analysis of selected bird species was done. Each sequence was aligned
with its reference sequences of CO1 gene present on NCBI. Every nucleotide position which did not align with the reference sequence was studied to identify SNPs. Fixation index (FST) were used to measure species diversity within a same sub population relative to that found in the entire population. Consensus sequences (500bps) generated was used to construct their phylogenetic tree to see their evolutionary relationship with other bird species. All species showed their closest linkage with their respective species. Pakistani population of peafowl and chicken species showed the close relation with same sequences generated in China. Tranopans showed its closest linkage with T. temminckii.
In conclusion, seven species ofPhasianinaesub-family of Pakistani bird species was genetically characterized first time in Pakistan by using CO1 as a barcode. It proves that DNA barcoding is an efficient and accurate molecular tool for species identifica¬tion and phylogenetic implication. This study leads to establish a DNA Data Bank that helped scientists to investigate the biodiversity, taxonomic classification, specie identification, in forensic purposes and to study the genetic and phenotypic evolution of these species. DNA barcoding through CO1 gene works as a functional tool for detectingmeat mislabeling and preventing illegitimate trade. This study has established foundations for molecular biologists to study taxonomic uncertainties at sub species level using SNP based identifying marker. It helps in preservation and identification of endangered species by generating their barcodes from even minimal evidence available.
Availability: Items available for loan: UVAS Library [Call number: 2376-T] (1).
4.
Molecular Characterization of Pakistani Common Leopard
by Muhammad Usman Ijaz (2012-VA-908) | Dr. Sehrish Firyal | Dr. Ali Raza Awan | Dr. Muhammad Tayyab.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: CD not available. Availability: Items available for loan: UVAS Library [Call number: 2379-T] (1).
