Genetic diversity analysis of 12 cyclocodon spp accessions by rapd and issr molecular markets

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY - - GRADUATION THESIS TITLE: GENETIC DIVERSITY ANALYSIS OF 12 CYCLOCODON SPP ACCESSIONS BY RAPD AND ISSR MOLECULAR MARKERS HA NOI - 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY - - GRADUATION THESIS TITLE: GENETIC DIVERSITY ANALYSIS OF 12 CYCLOCODON SPP ACCESSIONS BY RAPD AND ISSR MOLECULAR MARKERS Student Name : Vu Tien Dung Class : K62CNSHE Student Code : 620571 Instructor : Dinh Truong Son, Ph.D HA NOI – 2022 COMMITMENT I assure that the entire study procedure was carried out by myself under the scientific supervision of PhD Dinh Truong Son I assure that all research contents, conclusions, and information in my graduation thesis are entirely honest and unpublished Hanoi, February 09th 2022 Student Vu Tien Dung i ACKNOWLEDGEMENTS First of all, I would like to express deeply my gratitude to my supervisor’s Ph.D Dinh Truong Son has supported me complete my thesis with their patience and knowledge Moreover, their guidance helped me in the period of the writing of this thesis Secondly, I would like to thank all my teachers in the Faculty of Biotechnology, especially the Department of Plant Biotechnology They provided useful scientific knowledge as well as technique for me This is one of the most important things to help me to successful my thesis I would like to thank the Department of Plant Biotechnology where supporting equipment and finances for my thesis Then, I would like to thank my lab companion for his significant assistance I will cherish the time we spent working together Last but not least, my heartfelt gratitude goes to my loved ones for their unending and unequalled love, as well as their emotional support through times of adversity in our lives My graduation thesis would not have been finished if it had not been for them, and I dedicate my achievement to them Hanoi, February 09th 2022 Student Vu Tien Dung ii CONTENTS COMMITMENT i ACKNOWLEDGEMENTS ii CONTENTS iii ABBREVIATION v LIST OF TABLES vi LIST OF FIGURES vii ABSTRACT viii PART I INTRODUCTION 1.1 Preface 1.2 Purposes and requirements 1.2.1 Purposes 1.2.2 Requirements PART II LITERATURE OVERVIEW 2.1 Introduction of Cyclocodon 2.1.1 Taxonomy 2.1.2 Morphology characteristics and distributions 2.1.3 Researches on Cyclocodon 2.2 Molecular markers and applications of RAPD and ISSR markers in genetic diversity analysis 2.2.1 Classical markers 2.2.2 Molecular markers 10 2.2.2.1 RAPD molecular markers 11 2.2.2.2 ISSR molecular markers 12 2.2.3 Applications of RAPD and ISSR markers in genetic diversity analysis 13 2.3 Researches on Cyclocodon using RAPD and ISSR molecular markers 14 iii PART III MATERIALS AND METHODS 15 3.1 Materials 15 3.2 Time and Place of the Study 15 3.3 Research equipment 15 3.4 Chemical substances 16 3.5 Contents and methods 18 3.5.1 Contents 18 3.5.2 Methods 18 3.5.2.1 DNA extraction 18 3.5.2.2 PCR reaction 20 3.5.2.3 Gel electrophoresis 20 3.5.2.4 Data analysis 21 PART IV RESULTS AND DISCUSSION 23 4.1 Total DNA extraction results 23 4.2 Genetic diversity analysis using RAPD molecular markers 24 4.3 Genetic diversity analysis using ISSR molecular markers 30 4.4 Genetic diversity analysis combined RAPD and ISSR molecular markers 34 PART V CONCLUSION AND SUGGESTION 40 Conclusion 40 Suggestion 40 REFERENCES 41 APPENDIX 43 Supplemental table: 43 Supplemental table Electrophoresis results of the PCR products (RAPD Primers) 43 Supplemental table Electrophoresis results of the PCR products (ISSR Primers) 46 iv ABBREVIATION CTAB Cetyl trimethylammonium bromide DNA Deoxyribonucleic Acid dNTPs Deoxynucleotide Solution Mix EDTA Etylen Diamine Tetra Acetic Acid ISSR Inter-Simple Sequence Repeats RAPD Random Amplified Phlymorphic DNA PCR Polymerase chain reaction TAE Tris-base, acetic acid and EDTA PVPP Poly Vinyl Poly Pyrrolidone v LIST OF TABLES Table List of Cyclocodon accessions 15 Table List of chemical substances used in research 16 Table 3 List of RAPD primers used in research 16 Table List of ISSR primers used in research 17 Table Components of PCR reaction 20 Table OD260/280 index results and concentration of 12 accessions 23 Table Polymorphism of 12 Cyclocodon spp accessions revealed by RAPD markers 25 Table Matrix of genetic similarity among 12 Cyclocodon spp accessions revealed by RAPD markers calculated by similarity coefficient of SokalMichener 27 Table 4 Polymorphism of 12 Cyclocodon spp accessions indicated by ISSR markers 30 Table Matrix of genetic similarity among 12 Cyclocodon spp accessions revealed by ISSR markers calculated by similarity coefficient of SokalMichener 32 Table Matrix of genetic similarity among 12 Cyclocodon spp accessions revealed by RAPD and ISSR markers calculated by similarity coefficient of Sokal-Michener 36 Table Matrix comparisons of Mantel test/two-tailed test between markers38 vi LIST OF FIGURES Figure Cyclocodon Celebicus (Blume) D.Y.Hong Figure 2 Cyclocodon Lancifolius (Roxb.) Kurz Figure Distribution’s map of Cyclocodon in Vietnam .9 Figure Results of electrophoresis product with primer OPB-01 24 Figure UPGMA cluster analysis of 12 Cyclocodon spp accessions with a similarity coefficient of RAPD marker 28 Figure Principal component analysis (PCA) of 12 Cyclocodon spp accessions based on RAPD marker .29 Figure 4 UPGMA cluster analysis of 12 Cyclocodon spp accessions with a similarity coefficient of ISSR marker 33 Figure Principal component analysis (PCA) of 12 Cyclocodon spp accessions based on ISSR marker 34 Figure UPGMA cluster analysis of 12 Cyclocodon spp accessions with a similarity coefficient combined RAPD and ISSR marker 37 Figure Principal component analysis (PCA) of 12 Cyclocodon spp accessions based on pooled RAPD and ISSR marker 38 vii ABSTRACT Cyclocodon (Campanulaceae) is a dicotyledonous herbaceous perennial plant found mostly in Central, East, and South Asia This plant has long been used in traditional medicine to treat illnesses and symptoms such as bronchitis, coughs, spasms, psychoneurosis, cancer, obesity, hyperlipidemia, edema, hepatitis, colitis, and lung damage Many physiologically active chemicals, including as polyphenols, saponins, tannins, triterpenes, alkaloids, and steroids, are found in Cyclocodon and contribute to its various pharmacological actions As a result, the focus of this graduation thesis is on assessing the genetic diversity of 12 Cyclocodon spp accessions using ISSR and RAPD markers The RAPD marker's average polymorphism level (73.81%) was greater than the ISSR marker's (67.16%) The results of this study contribute to the provision of data for research and assessment, as well as the enrichment of genetic resources for medical plant development and germ plant viii conservation in Vietnam indicated that, at 71.9% similarity level, 12 accessions were split into three distinct major cluster (I,II and III) which consisted of 3, and accessions, respectively A combination of RAPD and ISSR analysis has been widely employed since both markers can give important information for germ-plant resources, which can be used for categorization and breeding Both RAPD and ISSR markers were employed to assess genetic connections and variation, because pooled data from several types of markers can offer a more meaningful categorization than a single technique alone These approaches provide additional information for determining genetic similarity 35 Table Matrix of genetic similarity among 12 Cyclocodon spp accessions revealed by RAPD and ISSR markers calculated by similarity coefficient of Sokal-Michener C.TL07 C.TL08 C.BT01 C.PXL01 C.PXL02 C.PXL03 C.PXL04 C.HRS01 C.HRS02 C.SP01 C.TP01 C.QB01 C.TL07 1.000 C.TL08 0.911 1.000 C.BT01 0.606 0.585 1.000 C.PXL01 0.579 0.558 0.699 1.000 C.PXL02 0.588 0.576 0.699 0.904 1.000 C.PXL03 0.581 0.579 0.692 0.87 0.938 1.000 C.PXL04 0.579 0.572 0.695 0.845 0.891 0.916 1.000 C.HRS01 0.588 0.563 0.759 0.686 0.704 0.706 0.759 1.000 C.HRS02 0.576 0.565 0.729 0.656 0.67 0.677 0.724 0.938 1.000 C.SP01 0.583 0.558 0.745 0.681 0.695 0.702 0.727 0.932 0.943 1.000 C.TP01 0.59 0.574 0.779 0.674 0.697 0.704 0.706 0.856 0.872 0.902 1.000 C.QB01 0.601 0.595 0.781 0.681 0.704 0.706 0.731 0.886 0.879 0.909 0.925 36 1.000 Figure UPGMA cluster analysis of 12 Cyclocodon spp accessions with a similarity coefficient combined RAPD and ISSR marker Clusters II had accessions, among them, C.HRS02 and C.SP01 had a maximum similarity coefficient of 0.94 (Figure 4.6) Although dendrograms generated by ISSR, RAPD and combined ISSR with RAPD indicate some variations, all 12 accessions were always assigned in the same cluster More ever, accessions C.HRS02 and C.SP01 reached the highest similarity level when analyzed by ISSR and combined ISSR and RAPD (Figure 4.4 and 4.6) 37 Figure Principal component analysis (PCA) of 12 Cyclocodon spp accessions based on pooled RAPD and ISSR marker In addition to UPGMA clustering analysis, the combined RAPD and ISSR markers were also analyzed with PCA scatter plots which show the first two principal components accounted for 52.46% and 37.33% of the total genetic variation of the total variation, respectively Moreover, the PCA also indicates the much distance in genetic diversity between accessions in Figure 4.7 These results again indicate the high genetic diversity of the collected samples, which might be used in cross-breeding projects to improve this valuable medicinal plant in Vietnam Table Matrix comparisons of Mantel test/two-tailed test between markers p-value Sokal-Michener matrix Matrix correlation (r) RAPD vs ISSR 0.985