Dissertation vt yen full 10 5 21

Faculty of Environmental Sciences, Institute of Forest Botany and Forest Zoology Distribution, genetic diversity and eurycomanone content of Eurycoma longifolia Jack in the province of Thua Thien Hue, Vietnam Dissertation in fulfillment of the requirements for the Degree of Doctor of Forest Science (Dr. Yen Van Thi Born on 02.1983 in Thua Thien Hue, Vietnam Supervisor: Prof. Doris Krabel, Dresden University of Technology Co-supervisor 1: Prof. Andreas Roloff, Dresden University of Technology Co-supervisor 2: Prof.

Nguyen Hoang Loc, Hue University of Science Date of defense: Declaration of conformity I hereby confirm that this copy conforms to the original dissertation on the topic: “Distribution, genetic diversity and eurycomanone content of Eurycoma longifolia Jack in the province of Thua Thien Hue, Vietnam” …………………………………… Location, date …………………………………… PhD candidate’s signature Acknowlegement ACKNOWLEDGEMENT First, I would like to express my gratitude and deepest thanks to my first supervisor, Prof. Doris Krabel, for her guidance, teaching, useful advice, support and encouragement for the entire doctoral study at the Dresden University of Technology. Without her support, I would not have enough strength to complete my thesis. I am so grateful to be her student.

I want to thank to my co-supervisors, Prof. Andreas Roloff and Prof. Nguyen Hoang Loc, for the willingness to give valuable comments and remarkable advice on the dissertation. Doris Krabel and Prof.

Nguyen Hoang Loc have been strongly supported the valuable research budgets and the laboratory facilities. I also would like to acknowledge the scholarship program from the Vietnamese Government (VIED) and the financial supports from the Graduate Academy (GA) and Association of Friends and Sponsors of TUD, e. (GFF) of TU Dresden, the family foundation of Nyamjav Ayur in Mongolia; my bosses, Ass. Dang Thai Duong and Dr.

Hoang Huy Tuan - Faculty of Forestry, Hue University of Agriculture and Forestry (HUAF) for their support, encouragement and facilitation of my application for the VIED scholarship and WWF (World Wide Fund) for their support in the next steps of this dissertation. I respectfully offer my sincerest thanks to my colleagues at the Chair of Forest Botany, Dresden University of Technology for their help and encouragement during my stay in Dresden. Special thanks go to Ms. Kristin Morgenstern, Mr.

Jens-Ulrich Polster and the working group “Molecular Physiology of Woody Plants” for their guidance and support in lab work and data analysis. Besides, I would like to express my thank to Prof. Karsten Wesche and Dr. Veit Herklotz from Senckenberg Museum of Natural History (Görlitz) and Dr.

Cao Thi Thu Hien from University of Vietnam National Forestry, for their valuable teaching, support and advise in data analysis. I also want to thank to Mr. Alexander Solger, Dr. Britt Kniesel, Dr.

Matthias Mayer, Ms. Sri Astutik, Ming, Khiem, Bayartaa, Nicole, Anne and Birgit for the great support and valuable discussions. I am thankful to all my colleagues Hue University of Agriculture and Forestry for providing useful information of my research in Vietnam, especially Dr. Tran Minh Duc, M.

Nguyen Hoi, Ass. Nguyen Van Loi and to my students from HUAF who helped me between 2016 and 2018 of my field work: Dien, Mot, Huy, Nhat, Dang, Quang, Hau, Dieu, Cuong, Minh, Thuy, Tien and Ngoc Hoa. I Acknowlegement Special thanks to the research group of Prof. Nguyen Hoang Loc, especially Dr.

Hoang Tan Quang, Mr. Trinh Huu Tan, Dr. Nguyen Ngoc Luong, Dr. Le Thi Ha Thanh and all his students from Hue University of Science: Tuan, Tue and Uyen for the valuable support of my laboratory work in Vietnam.

I also respect and appreciate the kind support of the managers of the nursery garden in Huong Thuy town, Bach Ma National Park, forest rangers in Nam Dong, A Luoi Saola Nature Reserve and the People’s Committee of Phong Dien district for my field work. Most importantly, to my husband, my little son, my parents and the family members who always support me by their love, beliefs and encouragement. Thank you very much again. Vielen Dank! Chân thành cảm ơn! Van Thi Yen II Table of Contents TABLE OF CONTENTS ACKNOWLEDGEMENT………………………………………………………………………I TABLE OF CONTENTS…………………………………………………………………….III LIST OF FIGURES………………………………………………………………………….VI LIST OF TABLES………………………………………………………………………….VIII LIST OF APPENDICES……………………………………………………………………….XI 1 General introduction .1 Status of medical plant resources in Vietnam.

longifolia tree and its importance .2 Scope of study .2 Ecology and distribution.2 Importance and use .3 Cultivation and propagation of the species .2 Propagation and germination of seeds .4 Pests and diseases.1 Assessment of genetic variation .2 Parameters for comparing the utility of markers .3 Genetic variation – Selected Examples. 20 3 Material and research methods .2 Nam Dong district .3 Bach Ma National Park.2 Field and lab design. 29 III Table of Contents 3.4 RAPD analysis and primer screening .5 SCoT and BPS analysis and primer screening .1 Data of distribution, phenotype, propagation and eurycomanone component .2 Analysis of genetic variation and population structure .1 Population distribution status of E.1 Terrain and soil .1 Leaf morphology and anatomy of mature trees .2 Leaf characteristics of seedlings and seedling’s growth performances .1 RAPD, SCoT and BPS amplification .2 Genetic diversity in E.4 Principle Coordinate Analysis (PCoA) .6 Genetic cluster analysis .7 Genetic variation across different generations .1 Biological traits and root water content .2 Eurycomanone content of E. longifolia root tissues .3 Correlation between the eurycomanone content and plant traits and geographical factors .1 Ecological distribution of E.2 Morphological and anatomical adaptability of E.

longifolia to different distribution areas .1 Leaf morphology and anatomy of mature trees under different site conditions .2 Leaf characteristics of seedlings and mature trees of E. 111 IV Table of Contents 5.3 Genetic diversity of E.1 Comparative utility of different marker systems .2 Genetic diversity between different distribution areas .3 Genetic structure of different distribution areas .4 Comparison of the genetic variation between mature trees and seedlings .4 The correlation between eurycomanone content and the distribution areas. 122 6 Conclusions and recommendations.2 Suggestions for further research .… 156 V List of Figures LIST OF FIGURES Figure 2.1 Natural occurrence of E. longifolia in a mountainous area in Vietnam (A) and typical flowers (B) and fruits (C) .2 Distribution map of E.

longifolia in Asia .3 Geographic distribution of six E. longifolia populations selected in Peninsular, Malaysia .1 Map of the province of Thua Thien Hue located in central Vietnam .2 Average temperatures in Nam Dong, A Luoi, Bach Ma and Phong Dien between 2013 and 2017 .3 Average precipitation in different areas between 2013 and 2017 .4 Diagram of baseline and plot design .5 Five different positions of leaflets from one leaf to estimate the number of stomata through microscope and microscope slides in the paper-clip boxes .6 Measurement of the size of the leaves and the number of leaflets in the field .7 a) Before taking the root sample and b) after digging the hole and taking the root sample .8 Geographical distribution map of DNA samples, mother trees and root samples of four (wild) E. longifolia populations in the province of Thua Thien Hue .9 Research Design Framework .1 Topographic distribution of E. longifolia based on elevation (m) .2 Distribution map of E.

longifolia Jack in the province of Thua Thien Hue. longifolia occurencies along the observation baselines .4 Distribution of trees and saplings in diameter (a) and height classes (b) .5 Differences of sapling diameter and height between sandy and mountainous areas .6 The increase of tree height and diameter with elevation, slope steepness and the positive correlation of trees and saplings between height and diameter. longifolia in mountainous area (A) and sandy area (B) .8 Comparison of seedling density and height between mountains (N = 1,752) and sandy areas (N = 99) .9 Distribution of seedling number along the height classes (cm) .10 Leaf area of E. longifolia mature trees .11 Leaf morphology in different areas: A Luoi (1), Nam Dong (2), Bach Ma (3) and Phong Dien (4) .12 Leaf area differences in moist (mountain) and dry (sand) sites .13 Stomatal density differences of mature trees at the four distribution areas .14 Stomatal density differences of mature trees in moist and dry sites .15 Different distribution of stomatal density in dry (sandy) site (a) and moist (mountainous) site (b).

65 VI List of Figures Figure 4.16 Cross section of the leaves: a) pit-type stomata in dry site and b) flat-type stomata in moist site .17 Negative correlation of leaf area with stomatal density of mature trees .18 Positive correlation of leaf length (cm) with leaf width (cm) .19 Differences between mature trees and seedlings in aspects of a) leaf area and b) stomatal density in different sites .20 Typical examples of fruit from a) sandy area (dry site) and b) mountainous area (moist site).21 Germination response of the ripe seeds of E. longifolia from different provenances in the period between 15 and 55 days .22 The growth pattern of E. longifolia seedlings in terms of height, collar diameter and number of leaves .23 Correlation between seedling height and seedling collar diameter, from different sites .24 Images of seedlings from different stages .25 A gel image generated by SCoT21 primer (12 samples) and one sample investigated with five different primers .26 Principle Coordinate Analysis (PCoA) based on genetic relationship of four subpopulations of 276 E.28 STRUCTURE Q plots generated utilizing the maximum value of ΔK indicate E.29 Dendrogram agglomerative clustering using Ward’s method and Euclidean distances among individuals. longifolia mother tree samples and 269 seedlings analysed from four different sites .31 The weight of all the root samples from four sites.32 a) Difference of water content in moist and dry areas and b) positive correlation between root water content and elevation factor .33 Standard curve of eurycomanone .34 HPLC chromatogram of eurycomanone standard at 300 µg/µL .35 HPLC chromatogram of eurycomanone extract from root .36 a) Eurycomanone content of the root of E.

longifolia in four different areas and b) eurycomanone content in moist and dry sites .37 Positive correlation between eurycomanone and root water content .38 Root diameter dependence on eurycomanone content. 100 VII List of Tables LIST OF TABLES Table 2.1 Summary of several main bioactive compounds and pharmacological effects from E.2 Summary of the previous studies of genetic diversity in E.3 Main indices of genetic diversity in E. longifolia populations studied by Osman et al.1 Forest status in the province of Thua Thien Hue .2 Population name, geographical location and sample size of DNA samples .3 Number of different provenances, propagated and natural seedlings and seedlings, for estimating germination, growth pattern and total DNA isolation .4 Variables measured of baselines, plots and tools for these measurements .5 PCR pipetting instructions and cycling protocol for GoTaq® Green Master Mix used for SCoT21, SCoT36, OPC02, OPB05 primers and TopTaq Master Mix used for LA2a primer .6 List of RAPD, SCoT and BPS primer sequences tested .7 Summary of the methodologies of data collection .1 Soil factors and forest status .2 Tree density (including saplings), tree size and the frequency of individuals based on elevation in four different areas .3 The diameter and height classes of trees and saplings .4 Stomatal density (stomata/mm2) and the number of stomata per leaf for moist- and dry-grown E.5 Average ± standard deviation, range of leaf length, leaf width and number of leaflets of E. longifolia in four different sites and statistical differences .6 Average ± standard deviation, range of leaf length, leaf width and number of leaflets of E.

longifolia in moist and dry sites .7 Spearman’s correlation coefficient (r2) and p of correlation among leaf characteristics (N = 505) .8 Average ± standard deviation of seedling characteristics from four different provenances .9 Average ± standard deviation, range of fruit morphology of moist and dry sites .10 Seedling’s provenances and seed maturity from the seed germination (days) within a 55-day period and survival rate >22 months .11 Details of banding pattern revealed through RAPD, SCoT and BPS markers.12 Summary of the genetic variation as revealed through RAPD, SCoT and BPS markers among four populations of E.