Assessment of the genetic diversity of 15 lotus accessions by rapd and issr markers

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -  - GRADUATION THESIS TITLE: ASSESSMENT OF THE GENETIC DIVERSITY OF 15 LOTUS ACCESSIONS BY RAPD AND ISSR MARKERS HANOI- 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -  - GRADUATION THESIS ASSESSMENT OF THE GENETIC DIVERSITY OF 15 LOTUS ACCESSIONS BY RAPD AND ISSR MARKERS Student : HA THI LOI Student’s code : 637245 Class : K63CNSHE Faculty : BIOTECHNOLOGY Instructor : Dinh Truong Son, PhD HANOI- 2022 COMMITMENT I hereby declare that this thesis’s data and research results are true and have never been used in any publication I hereby declare that any assistance in the presentation of this thesis has been received and that the sources cited have been acknowledged Hanoi, December 2022 Student Ha Thi Loi i ACKNOWLEDGEMENT While completing my graduation thesis, I received a lot of encouragement and support, along with the enthusiastic and thoughtful guidance of teachers in the Department of Plant Biotechnology, Biotechnology Faculty, Vietnam National University of Agriculture I would also like to express my deep gratitude to Dr Dinh Truong Son, Head of the Department of Plant Biotechnology, Biotechnology Faculty, Vietnam the National University of Agriculture was wholeheartedly guided and helped me throughout the process of doing my graduation thesis I also sincerely thank friends at the Department of Microbial Biotechnology for creating favorable conditions for me to complete the report best Finally, I would like to thank my family and friends, who have always helped and encouraged me throughout the process Sincere thanks! Hanoi, December 2022 Student Ha Thi Loi ii INDEX COMMITMENT i ACKNOWLEDGEMENT i INDEX ii LIST OF TABLES vi LIST OF FIGURES vii ABSTRACT vii I INTRODUCTION 1.1 Introduction 1.2 Purpose and requirements 1.2.1 Purpose 1.2.2 Requirements II LITERATURE REVIEW 2.1 An overview of lotus 2.1.1 Classification and taxonomy of lotus 2.1.2 Distributions of lotus 2.1.3 Characteristics of lotus 2.1.4 The Use of lotus 2.1.5 Lotus are being and studied in Vietnam across the world 2.2 Random Amplified Polymorphic DNA (RAPD) 10 2.2.1 Definitions 10 2.2.2 Principles 10 2.2.3 Applications of RAPD Analysis 11 2.3 Inter-Simple Sequence Repeats (ISSR) 13 2.3.1 Definition 13 2.3.2 Principles 14 2.3.3 Applications 15 iii III MATERIAL AND METHODS 17 3.1 Materials, chemicals, and equipment 17 3.1.1 Materials 17 3.1.2 Chemicals and equipment 18 3.2 Methods 21 3.2.1 DNA extraction 21 3.2.2 PCR 23 3.2.3 Gel electrophoresis 24 3.2.4 Data Analysis 25 IV RESULTS AND DISCUSSION 27 4.1 DNA extraction 27 4.2 Genetic diversity analysis of 15 lotus accessions using RAPD molecular 28 4.3 Genetic diversity analysis of 15 lotus accessions using ISSR molecular 35 4.4 Genetic diversity analysis of 15 lotus accessions using combined RAPD and ISSR molecular markers 41 4.5 Comparison primers 44 V CONCLUSION AND PROPOSAL 47 5.1 Conclusion 47 5.2 Proposal 47 REFERENCES 48 APPENDIX 53 iv LIST OF ABBREVIATIONS B Blank CTAB Cetyltrimethylammonium bromide DNA Deoxyribonucleic acid RNA Ribonucleic acid EDTA Ethylenediamine tetraacetic acid et al et alii (Latin), and others L Ladder ISSR Inter-Simple Sequence Repeats ml milliliter PCR Polymerase chain reaction PVPP Polyvinylpyrrolidone RAPD Random Amplified Polymorphic DNA TAE Tris-acetate EDTA TE Tris- EDTA Tm Annealing temperature ℃ Degree Celsius % Percent spp Species v LIST OF TABLES Table 3.1 Shows the 15 lotus samples used in the research 17 Table 3.2 List of chemicals used in the study 18 Table 3.3 List of RAPD primers 19 Table 3.4 List of ISSR primers 20 Table 3.5 Components of each PCR reaction 23 Table 3.6 Thermal cycler of PCR 24 Table 4.1 Spectrophotometric results in determining DNA quality and quantity of extracted samples 27 Tables 4.2 Performance of RAPD primers in the genetic diversity analysis of 15 lotus accessions 30 Table 4.3 The matrix of genetic similarity among 15 lotus accessions revealed RAPD calculated by the similarity coefficient of Sokal & Michener 32 Tables 4.4 Performance of ISSR primers in the genetic diversity analysis of 15 lotus accessions 36 Table 4.5 Matrix of genetic similarity among 15 lotus accessions revealed by ISSR markers calculated by similarity coefficient of Sokal & Michener 38 Table 4.6 Matrix of genetic similarity among 15 lotus accessions revealed by combined RAPD and ISSR markers calculated by similarity coefficient of Sokal & Michener 42 Table 4.7 Comparison of the discriminating capacity of RAPD and ISSR of 15 lotus accessions 45 Table 4.8 Matrix comparisons of Mantel test / Two-tailed test between markers 46 vi LIST OF FIGURES Figure 2.1 Map the showing disjunctive distribution of the genus Nelumbo (Li et al., 2014) Figure 2.2 Anatomy of lotus (Watson, 1992) Figure 2.3 The principle of RAPD-PCR technique Arrows indicate primer annealing sites (Arif et al., 2010) 11 Figure 2.4 Process flow chart for ISSR genotyping experiments (Ng et al, 2015) 14 Figure 4.1 Electrophoresis results of the PCR products with OPN01 Primer 28 Figure 4.2 UPGMA cluster analysis of 15 lotus accessions with similarity coefficient of RAPD marker Dash line indicates the mean similarity (0.759) The dendrogram the 15 lotus accessions in indicated group (I, II, III and IV) 34 Figure 4.3 PCA analysis of 15 lotus accessions based on RAPD markers 35 Figure 4.4 UPGMA cluster analysis of 15 lotus accessions with similarity coefficient of ISSR marker Dash line indicates the mean similarity (0.795) The dendrogram the 15 lotus accessions in indicated group (I, II, III and IV) 40 Figure 4.5 PCA analysis of 15 lotus accessions based on ISSR markers 40 Figure 4.6 UPGMA cluster analysis of 15 lotus accessions with similarity coefficient of ISSR marker Dash line indicates the mean similarity (0.785) The dendrogram the 15 lotus accessions in indicated group (I, II, III and IV) 43 Figure 4.7 PCA analysis of 15 lotus accessions based on both markers: RAPD and ISSR 44 ABSTRACT vii Lotus is a common plant in the world that lives in lakes and rivers with many uses such as food, medicine, decoration, and water environment protection In this study, inter-simple sequence repeats (ISSR) and Random Amplified Polymorphic DNA (RAPD) markers were applied to assess genetic diversity of 15 lotus accessions in Vietnam Our results showed that 15 lotus accessions exhibited a high level of genetic diversity (the percentage of polymorphic bands ranged from 58.72 to 69.93%) 113 loci were produced by 12 RAPD primers, 81 of which were polymorphic and accounted for 71.68% of the total ISSR primers generated 1717 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