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Phytosociology and Genetic PHYTOSOCIOLOGY AND GENETIC DIVERSITY OF PRUNUS SPECIES (WILD AND CULTIVATED) GROWING IN DIFFERENT AGRO-ECOLOGICAL ZONES OF MUZAFFARABAD DIVISION, AZAD JAMMU AND KASHMIR, PAKISTAN Muhammad Nasir (Regd. No. 2002-Gmdb-3652) Session 2011-2014 Department of Botany Faculty of Sciences University of Azad Jammu and Kashmir, Muzaffarabad, Pakistan. 1 2 PHYTOSOCIOLOGY AND GENETIC DIVERSITY OF PRUNUS SPECIES (WILD AND CULTIVATED) GROWING IN DIFFERENT AGRO-ECOLOGICAL ZONES OF MUZAFFARABAD DIVISION, AZAD JAMMU AND KASHMIR, PAKISTAN By Muhammad Nasir (Regd. No. 2002-Gmdb-3652) A Thesis Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Botany Session 2011-2014 Department of Botany Faculty of Sciences University of Azad Jammu and Kashmir, Muzaffarabad, Pakistan. 3 CERTIFICATION Certified that contents and form of Thesis entitled “Phytosociology and genetic diversity of Prunus species (wild and cultivated) growing in different agro- ecological zones of Muzaffarabad division, Azad Jammu and Kashmir, Pakistan” submitted by Muhammad Nasir have been satisfactory for the requirements of the degree. Supervisor: ______________________________ (Prof. Dr. Muhammad Qayyum Khan) Member: ________________________________ (Prof. Dr. Ghulam Murtaza) Member: ________________________________ (Prof. Dr. Syed Abdul Majid) External Examiner: ________________________ Chairman Department of Botany Dean Director Faculty of Sciences Advanced Studies & Research 4 DEDICATED To My beloved and great father Muhammad Fareed (late) Whose love and inspiration encourage at each step of my life and Also My dearest mother Whose love and care potentiate me For every Nobel venture CONTENTS 5 Pages Acknowledgments xiii Abstract xv 1. INTRODUCTION 1 1.1 NATURAL VEGETATION 1 1.2 ASSESSMENT OF VEGETATION 5 1.2.1 Phytosociology 5 1.2.2 Phytodiversity 7 1.2.3 Life-Form and Leaf Size Spectra 11 1.3 DISTRIBUTION AND TAXONOMY OF GENUS PRUNUS 13 1.4 SOCIO-ECONOMIC IMPORTANCE OF GENUS PRUNUS 16 1.4.1 Ethno- Medicinal Uses 16 1.4.2 Nutritional Values 17 1.4.3 Economic Importance 17 1.5 CULTIVATION AND PRODUCTION OF GENUS PRUNUS 19 1.6 GERMPLASM CONSERVATION AND CHARACTERIZATION IN GENUS PRUNUS. 19 1.7 USE OF MARKERS FOR GENETIC CHARACTERIZATION IN PLANTS 24 1.7.1 Morphological Markers 24 6 1.7.2 Biochemical Markers 25 1.7.3 Molecular Markers 25 1.7.4 General Objectives 39 1.7.5 Specific Objectives 39 2. MATERIALS AND METHODS 41 2.1 Phytosociological studies 41 2.1.1 Qualitative Characters 41 2.1.1.1 Floristic composition 41 2.1.1.2 Biological Spectrum 42 2.1.1.2.1 Phenarophytes 45 2.1.1.2.2 Chamaephytes 45 2.1.1.2.3 Hemicryptophytes 45 2.1.1.2.4 Therophytes 45 2.1.1.2.5 Geophytes 45 2.1.1.2.6 Lianas 45 2.1.1.3 Leaf spectra 46 2.1.1.3.1 Leptophyll (L) 46 2.1.1.3.2 Nanophyll (N) 46 2.1.1.3.3 Microphyll (Mi) 46 2.1.1.3.4 Mesophyll (Me) 46 2.1.1.3.5 Macrophyll (Ma) 46 2.1.1.3.6 Megaphyll (Mg) 46 2.1.2 Quantitative Characters 47 7 2.1.2.1 Community structure 47 2.1.2.2 Determination of suitable size 47 2.1.2.3 Number of quadrats 47 2.1.2.4 Distribution of quadrats 47 2.1.2.5 Components of Phytosociological Diversity 48 2.1.2.5.1 Density 48 2.1.2.5.2 Relative Density 48 2.1.2.5.3 Frequency 48 2.1.2.5.4 Relative frequency 48 2.1.2.5.5 Canopy cover 48 2.1.2.5.6 Relative Canopy Cover 49 2.1.2.5.7 Importance Value 49 2.1.3 Components of Diversity 50 2.1.3.1 Index of diversity 50 2.1.3.2 Species Richness 50 2.1.3.3 Equitability 50 2.2 MOLECULAR CHARACTERIZATION OF GENUS PRUNUS 53 2.2.1 Collection of Plant Materials 53 2.2.2 DNA extraction 53 2.2.2.1 Chemicals and Reagents 53 2.2.2.2 DNA Extraction Protocol 54 2.2.3 DNA Quantification 56 2.2.3.1 PCR Amplification and Gel Electrophoresis 56 8 2.2.3.2 Allele sizing 57 2.2.3.3 SSR Data analysis 57 3. RESULTS AND DISCUSSION 64 3.1 Floristic Composition of the Study Area 64 3.2 Cluster Analysis of Plant Communities 68 3.3 Principle Component Analysis of the Species Data 72 3.4 Analysis of Diversity and its Components in Plant communities 74 3.5 Life Form and leaf size spectra of plant communities in Study Area 77 3.5.1 Life form spectrum 77 3.5.1.1 Prunus-Dichanthium-Cynodon Community 77 3.5.1.2 Prunus-Zanthoxylum-Seteria Community 77 3.5.1.3 Prunus-Phragmitis-Capsella Community 78 3.5.1.4 Prunus-Geranium-Arundo Community 78 3.5.1.5 Prunus community 78 3.5.1.6 Prunus-Indigofera-prunus Community 78 3.5.1.7 Prunus-Impatiens-Cynodon Community 79 3.5.1.8 Cynodon-Caltha - Sambucus community 79 3.5.1.9 Heteropogon-Cynodon - Rumax community 79 3.5.1.10 Achelia-Mantha- Parrotiopsis community 79 3.5.1.11 Phalaris-Trifolium- Prunus community 79 3.5.1.12 Oxalis-Viburnum-Juglans community 80 3.5.1.13 Heteropogon-Cynodon-Poa community 80 9 3.5.1.14 Cedrus-Avena-Prunus community 80 3.5.1.15 Prunus-Pinus-Prunus community 80 3.5.1.16 Prunus armeniaca community 81 3.5.1.17 Prunus community 81 3.5.1.18 Prunus-Prunus community 81 3.5.1.19 Prunus community 81 3.5.1.20 Picea-Cedrus community 81 3.5.1.21 Prunus-Juglans community 82 3.5.2 Leaf size Spectra 86 3.5.2.1 Prunus-Dichanthium-Cynodon Community 86 3.5.2.2 Prunus-Zanthoxylum-Seteria Community 86 3.5.2.3 Prunus-Phragmitis-Capsella Community 86 3.5.2.4 Prunus-Geranium-Arundo Community 86 3.5.2.5 Prunus community 87 3.5.2.6 Prunus-Indigofera-prunus community 87 3.5.2.7 Pinus-Impatiens-Cynodon Community 87 3.5.2.8 Cynodon-Caltha - Sambucus community 87 3.5.2.9 Heteropogon-Cynodon - Rumax community 87 3.5.2.10 Cedrus-Avena-Prunus community 88 3.6 Analysis of Genetic Diversity of genus prunus using SSR markers 93 3.7 Phylogenetic Analysis Using SSR Markers 101 3.7.1 Interspecific diversity 104 3.8 DISCUSSION 107 3.8.1 Phytosociology 107 10 3.8.2 Genetic diversity 122 SUMMARY 130 LITERATURE CITED 132 APPENDICES 185 11 LIST OF TABLES Tables Page 1. Selected sites from the study area along with coordinates and altitude42 2. Coverage classes for the coverage of herbs and shrubs 48 3. List of genus Prunus accessions collected from different ecological zones of Muzaffarabad division 58 4. List of 19 SSR primers used to assess the genetic diversity of 96 Prunus accessions collected from study area 60 5. Phytosociological attributes of the investigated plant communities 75 6. Life form classes recorded for the investigated plant communities 83 7. Leaf size spectra of the investigated plant communities from the study area 89 8. Genetic variability parameters of 12 microsatellite loci in 96 accessions of genus Prunus 95 9. Characteristics of 12 microsatellite loci in 96 accessions of Prunus species collected from different ecological zones of Muzaffarabad division 96 12 LIST OF FIGURES Figures Page 1. Map of study area along with selected sites 43 2. Raunkierian’s diagramme for leaf size spectra 51 3. Predominant Plant families in term of species number recorded from the study area 65 4. Percentage of Life Form Spectrum Recorded from Study area, Western Himalayas Muzaffarabad Division 66 5. Percentage of Leaf Spectra of plant species recorded in the selected study area of Western Himalayas. 66 6. Cluster analysis Dendrogram representing species assemblage based on species correlation matrix 70 7. Principle component analysis by ordination biplot based on correlation between species and sites 72 8. Graphical presentation of diversity and its components of plant communities recorded from Muzaffarabad Division, Azad Kashmir 76 9. Percentage of the life form classes recorded from the studied plant communities 83 10. Percentage of the leaf spectra recorded from the investigated plant communities 92 11. : Phylogenetic tree of the 96 accessions and cultivars of prunus species based on UPGMA method and Nei (1983) genetic distances 105 13 ACKNOWLEDGEMENTS All praises and prayers to Almighty Allah, the merciful, the beneficent, whose blessings we enjoy throughout our lives. This is due to His blessings and mercy that I have successfully completed this research work and crossed another milestone of my academic career. Countless Darood-o-Salam upon the Holy Prophet Hazrat Muhammad (P.B.H.H.), who enabled me to recognize my creator and declared it to be an obligatory of every Muslim man and woman to acquire Knowledge. In the first place I would like to record my gratitude to my supervisor Prof. Dr. Muhammad Qayyum khan, Department of Botany, University of Azad Jammu and Kashmir Muzaffarabad for his Supervision, advice, and guidance. Above all and the most needed, he provided me unflinching encouragement and support in various ways. His truly scientist intuition has made him as a constant oasis of ideas and passions in science, which exceptionally inspire and enrich my growth as a student, a researcher and a scientist want to be. I am thankful to Prof. Dr. Syed Abdul Majid, Chairman, Department of Botany, UAJ&K, for his encouragement, help, support and valuable suggestions during the entire period of my PhD. Study. I gratefully acknowledge Prof. Dr.
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