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1(L 01 - 0 DETERMINATION OF THE BOTANICAL COMPOSITION OF BLACK RHINOCEROS (Diceros bicornis) DUNG USING THE rbcL GENE AS A MOLECULAR MARKER, AND ANALYSIS OF ANTIOXIDANT AND PHENOLIC CONTENT OF ITS BROWSE 1(L 01 - 0"1 A thesis submitted in fulfillment of the .. lon~q I..{I TJ. requirements for the degree of MASTER OF SCIENCE of RHODES UNIVERSITY by SlYA VUYA ISHMAEL BULANI January 2007 DECLARATION This work is the original work produced by Siyavuya 1. Bulani, submitted to Rhodes University in January 2007, for a Master of Science degree in Biochemistry. Siyavuya I. Bulani Supervisor: Dr. Brendan S. Wilhelmi Co-supervisor: Prof. John M. Brand 11 ACKNOWLEDGEMENTS .:. First I would like to thank the Almighty God for giving me strength throughout this project. .:. I extend my sincere gratitude to my supervisor, Dr. B.S. Wilhelmi for providing a good research environment that encouraged me to work and think independently, and for his support and understanding . •:. My co-supervisor, Prof. J .M. Brand for his invaluable and critical input in this study . •:. Mr. B. Fike, manager of the GFRR, for allowing us to obtain samples from the reserve . •:. Mr. T. Dold for identification and verification of the plant species . •:. Mr. N. Ragubeer for his assistance on cyclic voltammetric analyses . •:. To all the friends I have made in the department, thank you for your support, God bless you all. .:. To my Mother, family and friends thank you for your prayers and supporting my ambitions and goals . •:. Finally, I would like to thank the National Research Fund and Mellon Foundation for financial assistance. 111 ABSTRACT The black rhinoceros remains one of the world's extremely endangered species despite a variety of policies to protect it. The black rhinoceros population at the Great Fish River Reserve (GFRR) in the Eastern Cape in South Africa has increased steadily since their re-introduction in 1986. This megaherbivore is a browser, with a diet obtained largely from the short and medium succulent thicket of the GFRR. Knowledge of the preferential diet of the black rhinoceros on the reserve is an important factor for the effective management of the land and the herbivores that compete for its resources. The dietary preferences of the black rhinoceros at the reserve have been established using backtracking methods. In this study the rbcL gene was used to establish an rbcL gene database of the plants from the GFRR and determine the botanical composition of the black rhinoceros dung from the GFRR. Due to the limited number of rbcL gene plant sequences from the GFRR deposited in the GenBank database, 18 plant species from the GFRR were sequenced. Sequence analyses between the partial rbcL gene sequences generated were able to distinguish between plants down to species level. Plant species from the family Euphorbiaceae and Fabaceae showed sequence variation at intra-specific level compared to those of Tiliaceae which were more conserved. The generated rbcL gene sequences from seasonal dung samples were compared to the rbcL gene sequenced from 18 plant species obtained from the GFRR and those from the GenBank database. A wide range of plant species were identified from the dung samples. There were no major differences in botanical composition between the dung samples, except that Grewia spp. were found to dominate in almost all seasons. The results obtained on the free radical scavenging activity of the extracts against 2,2-Diphenyl-l­ picrylhydrazyl (DPPH) increased in the order of methanol > ethyl acetate > chloroform. The DPPH free radical scavenging activity of the methanol plant extracts increased in the order Brachylaena elliptica > Plumbago auriculata > Grewia robusta > Azima tetracantha. Methanol extracts on the TLC plate sprayed with Fe3+-2,4,6-Tri-2-pyridyl-s-triazine (TPTZ) showed that the compounds present in the extracts react differently to ferric ion, with most compounds unable to reduce fen·ic ion. Furthermore the methanol extracts were able to exhibit reduction potentials vs. Ag/ AgCl at low concentrations. The compounds in the extracts were shown to be phenolic acids and flavonoid glycosides. IV 1 .. no. ulghi;J:J.t I (. h jO~ _or (tthcI r ,,,(1,,n. T ABLE OF CONTENTS Page DECLARATION ........... .............................. .......... ........... .......... ................. ....... ..... ......................... ii ACK.!'<OWLEDGEMENTS .......................................... ... ... ..................... ......... ....... .. .... ..... ........... iii ABSTRACT .................................... ..................... .. ........................................ .... .............. ..... .. .... .... iv TABLE OF CONTENTS .............. ... ............................. .... .. ........ ....... .. ..... ... ....................... ............ v LIST OF APPENDIXES .... .. ... ...... .. ... ........................................................................................... vii LIST OF FIGURES ....................... ................ ................ .. ...... ........................................... ... ......... viii LIST OF TABLES .................................................... ................. ....... .. .... ..... ............ ..... ................... x LIST OF ABBREVIATIONS ............................................................... ........................................ xii CHAPTER ONE LITERATURE REVIEW ........................................................ ...... ........................... .. ............. ....... I 1.1 Introduction ............ .............................. .. ........ ........ ...... ........ .......... ........................................ I 1.2 The black rhinoceros ... ..... ............... .................... ....... ......... ..... ............................................. 3 1.2.1 The black rhinoceros subspecies ........................................ ............................................... 3 1.2.2 Decline of black the rhinoceros population .... .................................................................. 4 1.2.3 Black rhinoceros and predators ...................................... ................................................... 6 1.2.4 Free-ranging black rhinoceros ... .................................................. ..... ........ ........................ 6 1.2.5 Captive black rhinoceros ............. ......................................................................... .. ........... 7 1.3 Great Fish River Reserve ........ .......................................................... .... ........... .. .. .... ............. 8 1.3.1 Diet of the Black rhinoceros in the GFRR ... ................ ... .. .............................................. 10 1.4 Methods used to study the diet composition of herbivores ... ... ........................................ 13 1.4.1 Direct observation ............................................................................... .. .... .... .................. 14 1.4.2 Fistula methods ....... .. ..... .... .. .. ... .. .... ... ... ......................... .... ..... ...... ..................... .. .. ..... .... 14 1.4.3 Fecal analysis using micro histology ............................................................................... IS 1.5 Project objectives .... ........... ......................... .. .... ... ... ........ .. .... .. ......... .................................... 16 CHAPTER TWO VARIATION OF THE rbcL GENE BETWEEN SELECTED PLANT SPECIES ................. 17 2.1 Introduction .... .......... ............... .. ............................ ........... .. .................................................. 17 2.1.1 Size, base composition and genomic structure ... .............................................. .............. 18 2.1.2 Genes and coding capacity ...................... ................................... ....................... ............ .. 19 2.2 Ribulose-l,S-bisphosphate carboxylase/oxygenase (rhcL) gene ...................................... 19 2.3 Materials and methods ................... .. ........................ .................. ... .. ... ................................. 20 2.3.1 Sample collection ............................................ .. ................. .. ..... ....................... ............... 21 2.4 Genomic DNA extraction from plants .... .......................... .. ................... ... ......................... 22 2.5 Polymerase Chain Reaction (PCR) ......... ... ......... .......... .. .... .. ............................................. 22 v 2.5.1 Primers .. ..... ......................... .... ...... .. ........................................... "" ..... " ........ " " .............. 22 2.5.2 Amplification of the rbcL gene from plants ...... " .. .... .. " .... "" ................ " ........ " ...... .... ... 23 2.6 Cloning ..................................................... .............. ........... ..... ............................................... 24 2.6.1 Plasmid iso lation ............. "" .............. .. ................ " ... .. " " .. .. "" ..... "" ....... , ......... " ............. 24 2.7 Cycle seq ueneing ................... .. ........ "" .. .. .......... " .... " ...... "" ...... " ..... "" .... "." ........ " ........... .. 25 2.8 Results ........... ...... ... ............ ... ... .. ... ............................ " ....... " .... ." ........ " ..... "" ..... ." .. ... .... ...... 26 2.8.1 DNA recovery ............................................ " ..... "" ...... " ..... " ...... "" ... .... " .. .. ... ""
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