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Study of Genetic Diversity in the Threatened Ornamental Fishes, Badis Badis (Hamilton- Buchanan, 1822) and Amblyceps Mangois (H

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Study of Genetic Diversity in the Threatened Ornamental Fishes, Badis Badis (Hamilton- Buchanan, 1822) and Amblyceps Mangois (H Study of Genetic Diversity in the Threatened Ornamental Fishes, Badis badis (Hamilton- Buchanan, 1822) and Amblyceps mangois (Hamilton-Buchanan, 1822) from Terai and the Dooars region of North Bengal, India A THESIS SUBMITTED TO THE UNIVERSITY OF NORTH BENGAL FOR THE AWARD OF DOCTOR OF PHILOSOPHY (Ph.D.) IN ZOOLOGY BY TANMAY MUKHOPADHYAY SUPERVISOR PROF. SOUMEN BHATTACHARJEE DEPARTMENT OF ZOOLOGY UNIVERSITY OF NORTH BENGAL November, 2018 DEDICATED TO MY PARENTS -: PREFACE :- The research work has been started in 2013 and documented in the dissertation entitled “Study of Genetic Diversity in the Threatened Ornamental Fishes, Badis badis (Hamilton-Buchanan, 1822) and Amblyceps mangois (Hamilton-Buchanan, 1822) from Terai and the Dooars region of North Bengal, India.” under the supervision of Prof. (Dr.) Soumen Bhattacharjee, Cell and Molecular Biology Laboratory, Department of Zoology, University of North Bengal. The studied region is located in the sub-Himalayan Terai and Dooars region of North Bengal, India. The important feature of this region is rich diversity in the faunal and floran resource. The snow capped mountain peaks, the dense foothill, the luxuriant forests, the frothing rivers, the lush paddy fields, vibrant green tea gardens makes this region of North Bengal a hotbed of bio-diversity or biodiversity hotspot. This region is intersected by numerous streams, rivers and rivulets. All their sources are the Darjeeling Himalaya in the north and are mainly snow fed and rain fed. Most of the rivers drain considerable amount of water during rainy season due to their large catchment area in the hills. Over last few years, wild populations two ornamental fishes viz., Badis badis and Amblyceps mangois have declined steadily mainly due of habitat loss, overexploitation, pollution, and destructive fishing procedures; the information is confirmed by the NBFGR (National Bureau of Fish Genetic Resources), India country’s apex body for the purpose. There exists scanty information on the status of genetic diversity of Badis badis and Amblyceps mangois in India. Absolutely no information exists from Terai-Dooars region of West Bengal, India under the Eastern Himalayan biodiversity hotspot. Therefore, to the research work has been targeted to ascertain the present status of genetic diversity of this fishes that have potential ornamental and export values in a region of India. Moreover, proper exploitation and management of such ichthyofauna with regard to sustainable aquaculture can substantially improve human livelihood and also to conserve the germplasms in the wild. Page | I Genetic variability of an organism is of great importance for its sustenance and loss of this curtails its adaptability and increases the risk of its extinction. However, maintaining the genetic variation in a threatened species is essential for survivability and its adaptation. Molecular markers are realistic and useful tool to investigate the genetic structure of populations of different species. Three major river systems have been studied from Terai-Dooars region of West Bengal, India and total seventeen populations (for Badis badis) and fourteen (for Amblyceps mangois) were selected from different geographic locations for collection of fish samples. The catch of the fishes from each location was very low these indicate the threatened population status of this species in this region. Therefore, a non-invasive DNA isolation technique was developed and used for extraction of genomic DNA from the fishes. The genomic DNA was extracted from the minute quantities of fins without sacrificing the organisms. In the present study RAPD and ISSR techniques are used for genetic diversity study because they are easy, cost effective and fast methods, especially when other sophisticated methods are not yet developed. Moreover, mitochondrial cytochrome oxidase sub-unit 1 (mtCOI) gene is also analyzed to observe the genetic diversity and the phylogenetic relationship with other populations from geographically isolated regions; and to substantiate the findings based on RAPD and ISSR markers A correlative study based on several anthropogenic factors causing the dwindling population genetic structure with that of the available genetic diversity in the studied species can add a newer dimension to find out the cause of this low level of genetic variability as well as a possible conservational strategy. Information on intra and inter-specific genetic variation from the present study should form a baseline on which further studies can be undertaken. As the species is commercially important and has an ornamental value; and the region being located in the sub- Himalayan hotspot region, the management and proper rehabilitation of this ichthyofauna in the wild is essential from the standpoint of livelihood of rural fishermen and their economic restoration. Page | II ABSTRACT Badis badis or Chameleon Fish (Hamilton-Buchanan, 1822) (Actinopterygii, Perciformes, Badidae) and Amblyceps mangois or Indian Torrent Catfish (Hamilton- Buchanan, 1822) (Actinopterygii, Siluriformes, Amblycipidae) are two a tropical, freshwater species that has ornamental importance and thus possesses commercial value. The species has been categorized as vulnerably susceptible in the list of threatened freshwater fishes of India by NBFGR, Lucknow, India- country’s apex body for the purpose. However, studies with regard to genetic diversity of fish species especially in the eastern sub-Himalayan region of West Bengal, India have been found to be very inadequate. On the standpoint of validating its threatened status, assessment of its genetic diversity from this particular region with the efforts to conserve its wild population seem to be of great significance. Genetic variability of an organism is of paramount importance from the standpoint of survival and reproductive fitness as well as for sustenance of the whole population; therefore, the erosion of genetic diversity of a population curtails its capability for adaptation and increases the risk of its extinction. The vulnerable organisms having small/minute population size may experience a perpetual reduction in the genetic variation. However, maintaining the genetic variation in a threatened imperilled species is essential to ascertain its adaptation, expansion and opportune reestablishment in natural populations. The study of intra and inter - population genetic variability within and between local populations is profoundly utilizable to gain information on individual identity, breeding patterns, genetic degree of relatedness and genetic diversity/variability within as well as between them. Genetic variations can be assessed by means of DNA polymorphisms. Molecular markers are realistic and useful tools to for the investigation and monitoring the genetic structure of populations both in native and captive lots condition. RAPD and ISSR techniques are of prime importance for widely used for genetic diversity study because they are easy, cost effective and fast methods, especially when other sophisticated methods are not yet developed. Moreover, the RAPD and ISSR amplifications do not require prior knowledge of flanking sequence of the genome of the concerned species. RAPD-PCR technique has been extensively used to characterize genetic structure as well as to study genetic diversity of different fishes. ISSR primers usually amplify DNA segments present at an amplifiable distance between two oppositely oriented identical microsatellite repeat regions. ISSR markers are generated via PCR reactions amplification with a single Page | III microsatellite primer designed from repetitions of two or three nucleotides anchored (either 5´ or 3´) with in a sequence of one to three nucleotides that aim to eliminate slippage-related artefacts. Some advantages related to these markers are rapidness, requirement of small quantities of template DNA, a fewer/less number of PCR reactions, high PCR reactions annealing temperatures that reduce the quantity of artifacts and multi-locus highly polymorphic marker in nature. Thus, ISSR-PCR technique was preferred many times over to other dominant markers by several groups of researchers in studying genetic diversity at the interspecific and intraspecific levels of species. Therefore, due to the unavailability of microsatellite or SNP markers in Badis badis and Amblyceps mangois till date, we resorted to the time-tested RAPD and ISSR primers to ascertain and compare the available genetic variations in this ichthyofauna. Moreover, I have analyzed mtCOI gene to observe the genetic and phylogenetic relationship with other geographically isolated population across worldwide; to corroborate the findings based on the RAPD and ISSR markers with that of mtCOI gene Three major river systems (Mahananda, Teesta and Jaldhaka) have been studied and total seventeen populations (for Badis badis) and fourteen (for Amblyceps mangois) were selected from different geographic locations for the collection of the fish samples. The catch of the fishes from each location was very low and as the species are belong to the threatened category a non-invasive DNA isolation technique was developed and used for extraction of genomic DNA from the fishes. The genomic DNA was extracted from the minute quantities of fins without sacrificing the organisms. Different diversity indices like total number of polymorphic loci, percentage of polymorphic loci, Nei’s genetic diversity, Shannon’s information index, and measure of evenness are used to study the genetic architecture
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