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Rabia Arif Department of Botany University of the Punjab Lahore, Pakistan 2018

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Rabia Arif Department of Botany University of the Punjab Lahore, Pakistan 2018 DNA Profiling of Coprophilous Fungus Sordaria fimicola by PCR Based Molecular Markers RABIA ARIF DEPARTMENT OF BOTANY UNIVERSITY OF THE PUNJAB LAHORE, PAKISTAN 2018 DNA Profiling of Coprophilous Fungus Sordaria fimicola by PCR Based Molecular Markers A Thesis Submitted to the University of the Punjab in Partial Fulfilment to the Requirements for the Degree of Doctor of Philosophy in Botany By Rabia Arif Research Supervisor Prof. Dr. Muhammad Saleem DEPARTMENT OF BOTANY UNIVERSITY OF THE PUNJAB LAHORE, PAKISTAN. January, 2018 ABSTRACT The present study is restricted to the isolation, morphological and molecular characterization of Sordaria fimicola by applying PCR based molecular markers coupled with high resolution melt analysis to obtain adequate data for measuring genetic variations. All the collected samples isolated from different herbivore dung samples were plated; incubated and S. fimicola colonies were identified on solid PDA media. A total of 61 fungal isolates of S. fimicola were isolated by moist chamber and single plate isolation techniques. The effect of different physical parameters such as pH (4.5-8), temperature (20-45 ºC), carbon (cellulose, glucose and maltose), nitrogen sources (sodium nitrate, ammonium sulphate and casein hydrolysate) and UV light on the mycelial and perithecial growth was also determined on different strains of S. fimicola. Present attempt was made to gauge molecular differences on Sordaria fimicola‟s genome present on site of Evolution Canyon. All strains and isolates were authenticated by targeting internal transcribed spacer and hypervariable regions (ITS1, ITS2, full ITS, V3, V4 and V9). No sequence variations were observed in case of ITS and V9 regions while two polymorphic sites on position 212 A (C); 213 G (A) were found in V3 and V4 region in strains isolated from the south slope of Evolution Canyon. The PCR-based technique of randomly amplified polymorphic DNA (RAPD) was used to fingerprint and assess the genetic relatedness among eight isolates of the fungus S. fimicola isolated from various areas of Lahore, Pakistan. Each DNA sample was amplified with each of eight RAPD primers during the initial screening and the products were resolved on 1.5 % agarose gel, stained with ethidium bromide and snapped under gel documentation system. i Four primers failed to show any amplification but the remaining four (50 %) primers generated a total of 22 bands (5.5 bands per primer) among the eight isolates of S. fimicola. Of these 22 resolved bands, 13 (3.5 per primer) were polymorphic and the remaining 9, bands were common (monomorphic) among these isolates. The least efficient primer was R8 (1.79 %), while the most efficient one was R3 (8.93 %). Primer R3 had the highest PIC value (0.5) and identified all eight isolates through unique patterns of banding. Jaccard similarity coefficient ranged between 0.2-0.6. Sequence products of 485 bp and 811 bp long nucleotides were obtained from two RAPD loci (RL-7 and RL4) and converted to sequence characterized amplified region (SCAR400 and SCAR500) markers for S. fimicola. Another aim of the experiment was to look for natural genetic variation in different natural strains of Sordaria fimicola isolated from natural and stressed ecological conditions of Evolution Canyon. It was challenging because there was no sequence information at the time, and I was required to design a set of PCR primers that would reliably amplify in S. fimicola and it was hoped that the amplified products were polymorphic. Hence the decision was made to target randomly selected genes and regions containing short sequence repeats, putting primers in the exonic parts to amplify the intron(s) they flank and used S. macrospora sequence information to strategically place the "exon-primed, intron-crossing" primers to determine nucleotide variations in fifty strains of Sordaria fimicola by sequencing PCR amplicons. As compared to the strains isolated from the north slope, strains isolated from the south slope exhibited point mutations on various positions and enrichment of short sequence repeats was observed more in strains isolated from the south slope of EC. Frequency clock proteins are significant factor of circadian clocks which regulate gene expression that react to various environmental conditions in many organisms and are ii principally involved in rhythmic movements displayed by numerous filamentous fungi. Mating type a-1 proteins, encoded by mat-a-1 genes, control the sexual compatibility and vegetative incompatibility with A mating types in many ascomycetes. In this study, I have also explored the phosphorylation and glycosylation status of frequency clock and mating type a1 proteins on serine/threonine/tyrosine residues using NetPhos3.1 and YinOYang 2.1 server. iii ACKNOWLEDGEMENTS Although feelings are deep but unfortunately the words are too shallow, that cannot express the depth of my feelings. The names may be mentioned but the extent and the significance of their help is impossible to capture. All praises for Almighty Allah, the creator of this universe, who guides us in the ocean of darkness and enables us to overcome the difficulties in crucial situations and all respect and love for Hazarat Muhammad (PBUH) who enables us to recognize our creator and to understand the philosophy of life. No words can adequately express the depth of my gratitude to my supervisor Professor Dr. Muhammad Saleem, University of the Punjab, Lahore whose admirable guidance; stimulation, devotion and affectionate behaviour helped me to complete the present manuscript. His marvellous guidance and supervision throughout the progress of this research and in the preparation of this manuscript is highly commendable. I have the honour to express my profound sense of gratitude and indebtedness to Siu Fai Lee, my advisor at Department of Genetics, University of Melbourne, Australia who helped me a lot to perform my all major experiments and to complete this manuscript. I am really thank full to him who always showed cooperative behaviour, encouragement, patience and moral support during research work and my stay at Melbourne University, Australia. With profound gratitude and deep sense of devotion and obligation, I wish to especially recognize the guidance and encouragement extended by Ary Hoffman and Dr. Nancy who facilitate me with all chemicals and apparatuses at Hoffman Laboratory, Bio 21 Institute, Melbourne Australia to perform my all experiments. I am also thankful to Professor Dr. B.C. Lamb, Imperial College London, who read my thesis for English grammar corrections and suggestions that improved my research work. iv No words of acknowledgements can be adequate to sublime love and benevolent cooperation of my Parents (Mr and Mrs Arif) and Mother –in- law whose love, prayers and sympathies are with me in every moment of life. So far whatever I have achieved is due to Allah‟s blessings and my parent‟s prayers. I am nothing without family members. A big hug goes to my beloved husband Atef Nawaz; who stands by me no matter what. I would like to express my appreciation for his nice cooperation, encouragement, patience, loving attitude and moral support during research work. I am also thankful to my Sisters (Humera Arif, Nowida Nawaz and Amna Arif) and my sweet and precious Brothers who are so nice to me in every moment of my Life that sometimes I literally feel proud of myself. I am highly obligated to and cannot be precise for their consummate guidance, kind cooperation and valuable suggestions during the progress of my studies and research. Last but not the least I am really thankful to my friend and lab fellow Tazeen Jamil who provided valuable help, guidance during the preparation, production and composition of this thesis; I am also intended to Dr. Muhammad Ishfaq, Miss Faiza Akram, Miss Asifa Irshad Kiyyani, Mrs Uzma Naureen, Mr Muhammad Zahid, Mr Jabbar and Mr Atif Khan Lodi who helped me a lot during my stay at Molecular Genetics Research Lab. Botany, PU. RABIA ARIF v LIST OF ABBREVIATIONS µg Micro gram µM Micro molar AS African slope AFLP Amplified fragment length polymorphism AP-PCR Arbitrarily primed PCR ATP Adenosine triphosphate Bp Base pair c-DNA Complementary DNA CTAB Cetyl trimethyle ammonium bromide DAF DNA amplification fingerprinting DNA Deoxyribose nucleic acid dNTPs Di nucleotide triphosphate dsDNA Double stranded DNA EC Evolution canyon EF1α Elongation factor 1 alpha EPIC Exon prime intron crossing markers ES European slope EtBr Ethidium bromide F Forward FRET Fluorescence resonance energy transfer Frq Frequency g-DNA Genomic DNA HRMA High resolution melt analysis ISSR Inter simple sequence repeats ITS Internal transcribed spacer region Kb Kilobase KOH Potassium hydro-oxide LCR Ligase chain reaction MAAP Multiple arbitrary amplicon profiling Mat Mating type MEA Malt extract agar MgCl2 Magnesium chloride Min Minutes vi mL Milli litter MMS Methyl methanosulfonate MW Molecular weight NFS North facing slope Ng Nano gram NGS Next generation sequencing Nm Nano meter ºC Degree centigrade OD Optical density PB Polymorphic bands PCR Polymerase Chain Reaction PDA Potato dextrose agar pH Power of hydrogen PIC Polymorphic information content PTMS Post translational modifications R Reverse RAPD Randomly amplified polymorphic DNA READNA Revolutionary Approaches and Devices for Nucleic Acid Analysis RFLP Restriction fragment length polymorphism RNA Ribose nucleic acid Rps Ribosomal proteins RT-PCR Real time Polymerase Chain Reaction SCAR Sequence characterized amplified region Ser Serine
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