Genetic diversity analysis of 15 cyclocodon spp accessions by rapd markers

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FALCULTY OF BIOTECHNOLOGY GRADUATION THESIS SUBJECT: “GENETIC DIVERSITY ANALYSIS OF 15 CYCLOCODON SPP ACCESSIONS BY RAPD MARKERS” Hanoi – 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FALCULTY OF BIOTECHNOLOGY GRADUATION THESIS SUBJECT: “GENETIC DIVERSITY ANALYSIS OF 15 CYCLOCODON SPP ACCESSIONS BY RAPD MARKERS” Student: Dao Ngoc Hai Student Code: 620485 Class: K62CNSHE Faculty: Biotechnology Supervisor: Dinh Truong Son , PhD Hanoi – 2022 COMMITMENT I guarantee that the graduate thesis: “Genetic diversity analysis of Cyclocodon spp accessions by RAPD markers” is my own research wich is done with the instruction of Mr Dinh Truong Son, PhD The parts where I use references in the thesis have been mentioned in the references section The data and results presented in the thesis are completely honest If wrong, I will take responsibility and all the discipline that the faculty and the academy offer Hanoi, / /2022 Student Dao Ngoc Hai i ACKNOWLEDGEMENT In order to complete this graduation thesis, in addition to my own efforts, i would like to express my sincere thanks to: Thanks to the Vietnam National University of Agriculture for supporting me about facilities with modern library system, diverse types of documents that are convenient for my research I would like to express my deepest gratitude to my instructor, Mr Dinh Truong Son, whose sincerity and encouragement I will never forget This thesis would not have been possible without Mr Dinh Truong Son, whose guidance from the initial step in research enabled me to develop an understanding of the subject I am thankful for the extraordinary experiences he arranged for me It is an honor to learn from Mr Dinh Truong Son I would also like to thank some students at the Department of Plant Biotechnology, Faculty of Biotechnology, Vietnam National University of Agriculture for creating favorable conditions for me to complete this thesis Finally, i look forward to receiving comments and suggestions from the teachers to make my graduation thesis more complete Hanoi, / /2022 Student Dao Ngoc Hai ii CONTENT COMMITMENT i ACKNOWLEDGEMENT ii CONTENT iii LIST OF ABBREVIATIONS v LIST OF TABLES vi LIST OF FIGURES vii PART I INTRODUCTION 1.1 Rationale of the Study 1.2 Research Objective 1.3 Research Requirements PART II LITERATURE REVIEW 2.1 Research Review 2.1.1 Origin 2.1.2 Distributions 2.1.3 Characteristics 2.1.4 Researches on Cyclocodon spp 2.1.5 Genetic diversity 2.2 RAPD and the applications of RAPD in genetic diversity 2.2.1 Classical markers 2.2.2 Molecular markers/DNA markers 2.2.3 Randomly amplified polymorphic DNA (RAPD) 2.2.4 Some Researches on genertic diversity using RAPD 10 PART III MATERIALS AND METHODS 12 3.1 Materials, Chemicals, tools and equipments used in the research 12 3.1.1 Materials 12 3.1.2 Chemicals, tools and equipment used in the research 13 3.2 Contents and Methods 14 3.2.1 Contents 14 3.2.2 Methods 15 PART IV RESULTS AND DISCUSSION 20 4.1 DNA extraction results 20 4.2 Results of using RAPD molecular markers in genetic diversity analysis 22 iii 4.3 Discussion 33 PART V CONCLUSIONS AND RECOMMENDATIONS 35 5.1 Conclusions 35 5.2 Recommendations 35 REFERENCES 36 APPENDIX 37 iv LIST OF ABBREVIATIONS CTAB Cetyltrimethylammonium bromide DNA Deoxyribonucleic acid RNA Ribonucleic acid EDTA Ethylendiamin Tetraacetic Acid et al et alii (Latin), and others ml Milliliter PCR Polymerase chain reaction PVPP Polyvinylpyrrolidone RAPD Random Amplified Polymorphic DNA TAE Tris-acetate EDTA °C Degree Celsius % Percent spp Species v LIST OF TABLES Table 3.1 15 accessions of Cyclocodon spp 12 Table 3.2 List of chemicals used in the study 13 Table 3.3 List of RAPD primers used in the research 13 Table 3.4 Components of each PCR reaction 17 Table 3.5 PCR Procedure 18 Table 4.1 Spectrophotometric results of extracted samples 20 Table 4.2 Polymorphism among 15 Cyclocodon spp accessions revealed by RAPD markers 22 Table 4.3 PIC values and Rp values of RAPD Primers 23 Table 4.4 Matrix of genetic similarity among 15 Cyclocodon spp accessions revealed by RAPD markers calculated by similarity coefficient of Sokal-Michener 31 vi LIST OF FIGURES Figure 2.1 Cyclocodon spp characteristics and distributions Figure 2.2 Cyclocodon Lancifolius Figure 2.3 Cyclocodon Celebicus Figure 2.4 RAPD Principle 10 Figure 4.1 Results of electrophoresis with primer OPB-02 21 Figure 4.2 UPGMA cluster analysis of 15 Cyclocodon spp accessions with a similarity coefficient of RAPD marker 32 Figure 4.3 PCA analysis of 15 Cyclocodon spp accessions based on RAPD markers 32 vii PART I INTRODUCTION 1.1 Rationale of the Study From ancient times to the present, medicinal plants have always played an important role in maintaining the health and well-being of human communities around the world Vietnam is fortunate to be located in the tropical monsoon belt with ¾ of the area being mountainous, stretching from north to south Such natural conditions give our country a rich and diverse forest ecosystem According to statistics, our country has nearly 12,000 species of vascular plants belonging to more than 2,256 genera, 305 families Not only playing the role of a green lung to regulate the climate, the forest also brings a great potential for medicinal plant resources Medicinal plants are widely distributed throughout Vietnam with key regions: Northwest, Northeast, Red River Delta, North Central, East Truong Son, South Central Coast, Central Highlands, Southeast and Mekong River Delta Among the announced species, there are many species classified as rare such as: Panax vietnamensis, Tam That Hoang, Bach Hop, Cyclocodon spp , etc Cyclocodon spp are relatively rare genetic resource in Vietnam The root is used as a tonic and wine yeast Young leaves are edible vegetables In areas where they are distributed, fewer and fewer individuals have been found Although they are only recently exploited to a limited extent, deforestation for cultivation has directly damaged their habitat In Vietnam, although studies on medicinal plants have been conducted early, the genetic diversity of Cyclocodon spp is not yet analyzed Therefore, I decided to this research Table 4.4 Matrix of genetic similarity among 15 Cyclocodon spp accessions revealed by RAPD markers calculated by similarity coefficient of Sokal-Michener L.KL 01 L.TL 01 L.TL 01 1.000 L.KL 02 L.KL 03 L.KL 04 C.KL 01 C.KL 02 C.KL 03 C.KL 04 C.TL 01 C.TL 02 C.TL 03 C.TL 04 C.TL 05 L.KL 01 0.910 1.000 L.KL 02 0.854 0.887 1.000 L.KL 03 0.906 0.939 0.929 1.000 L.KL 04 0.906 0.929 0.920 0.991 1.000 C.KL 01 0.519 0.495 0.533 0.519 0.519 1.000 C.KL 02 0.547 0.514 0.561 0.547 0.557 0.962 1.000 C.KL 03 0.528 0.514 0.561 0.547 0.547 0.943 0.953 1.000 C.KL 04 0.509 0.495 0.552 0.528 0.528 0.943 0.953 0.943 1.000 C.TL 01 0.500 0.495 0.552 0.528 0.528 0.915 0.925 0.962 0.943 1.000 C.TL 02 0.519 0.495 0.552 0.528 0.538 0.896 0.915 0.934 0.925 0.953 1.000 C.TL 03 0.528 0.505 0.552 0.538 0.547 0.906 0.934 0.925 0.915 0.925 0.962 1.000 C.TL 04 0.505 0.472 0.519 0.486 0.495 0.854 0.873 0.892 0.873 0.882 0.882 0.854 1.000 C.TL 05 0.505 0.481 0.528 0.495 0.495 0.882 0.873 0.901 0.882 0.901 0.892 0.873 0.925 1.000 C.TL 06 0.519 0.495 0.524 0.500 0.491 0.906 0.887 0.887 0.906 0.887 0.868 0.868 0.892 0.948 C.TL 06 1.000 31 Figure 4.2 UPGMA cluster analysis of 15 Cyclocodon spp accessions with a similarity coefficient of RAPD marker Dash line indicates the mean similarity (0.725) The dendrogram distributed the 15 Cyclocodon spp accessions in indicated groups (I and II) Observations (axes F1 and F2: 98.78 %) 0.6 C.KL02 C.TL03 C.KL03 0.4 C.KL04 C.TL02 C.TL01 C.KL01 0.2 L.KL04 L.KL03 L.KL02 F2 (1.28 %) -0.2 L.KL01 L.TL01 -0.4 -0.6 C.TL06 -0.8 C.TL05 C.TL04 -1 -4 -3 -2 -1 F1 (97.51 %) Active observations Figure 4.3 PCA analysis of 15 Cyclocodon spp accessions based on RAPD markers 32 15 Cyclocodon spp accessions assessed by RAPD marker displayed high genetic diversity, with a mean similarity level of 0.725, varying from 0.472 (L.KL01 vs C.TL04) to 0.991 (L.KL03 vs L.KL04) in Table 4.4 Using the UPGMA hierarchical clustering method, the obtained pairwise similarity matrix was used to create a dendrogram At the mean of 72.5% similarity level, 15 accessions were grouped into main clusters in Figure 4.2 Clusters I comprises accessions including L.TL01, L.KL01, L.KL03, L.KL04, L.KL02 Clusters II comprise 10 accessions including C.KL01, C.KL02, C.KL04, C.KL03, C.TL01, C.TL02, C.TL03, C.TL04, C.TL05, C.TL06 (Figure 4.2) In addition to UPGMA clustering analysis, the RAPD markers were also analyzed with PCA scatter plots which show the first two principal components accounted for 97.51% and 1.28% 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 4.3 The RAPD analysis detected 212 polymorphic loci (of which 66.54 % were polymorphisms) that unambiguously classified 15 accessions into main clusters (Figure 4.2) In addition to UPGMA clustering analysis, the relationship between 15 accessions was also analyzed with PCA scatter plots which show the first two principal components accounted for 97.51% and 1.28% of the total genetic variation of the total variation, respectively Moreover, the PCA also indicates the much distance in genetic diversity between accessions (Figure 4.3) 4.3 Discussion In this study, the genetic variations among 15 Cyclocodon spp accessions were revealed by RAPD The pairwise similarity values obtained with the RAPD marker ranged between 0.472 and 0.991 (Table 4.4) The result has shown a high genetic diversity among 15 accessions To add another layer of data analysis, the relationship among 15 accessions was established using Principal 33 Component Analysis (PCA), which was performed using the XLSTAT 2018 software In agreement with the dendrogram result (Figure 4.2), when 15 accessions were evaluated using PCA, they were divided into two primary groups that were comparable to dendrogram analysis (Figure 4.3) Furthermore, the PCA illustrates how widely apart the accessions are from one another These results again indicate the high genetic diversity of the collected samples which might be used in the crossing programs to enhance these valuable medicinal plant in Vietnam 34 PART V CONCLUSIONS AND RECOMMENDATIONS 5.1 Conclusions The genetic diversity of 15 Cyclocodon spp accessions collected in Vietnam revealed that the RAPD marker is a useful tool At the mean of 72.5% similarity level, 15 accessions were grouped into main clusters in Figure 4.2 Clusters I comprises accessions including L.TL01, L.KL01, L.KL03, L.KL04, L.KL02 Clusters II comprise 10 accessions including C.KL01, C.KL02, C.KL04, C.KL03, C.TL01, C.TL02, C.TL03, C.TL04, C.TL05, C.TL06 The high genetic variability among 15 Cyclocodon spp accessions could be used in genetic improvement, sustainable management and germplasm conservation of Cyclocodon spp in Vietnam 5.2 Recommendations For a better evaluation of the genetic diversity of 15 Cyclocodon spp accessions, the research should be expanded with other types of molecular markers, such as ISSR 35 REFERENCES Thomas G Lammers (2011) Revision of the Infrageneric Classification of Lobelia L (Campanulaceae: Lobelioideae) Tzu-chao lin, Chang-chi hsieh, Dinesh Chandra Agrawal, Chao-lin kuo, Fu-shin Chueh and Hsin-sheng Tsay (2007) ITS Sequence Based Phylogenetic Relationship of Dangshen Radix Đinh Đoàn Long (2012) Đánh giá đa dạng di truyền số loài dược liệu Việt Nam thuộc Đảng Sâm (Codonopsis sp) kỹ thuật AND mã vạch, Luận văn Thạc sỹ, Đại học Khoa học tự nhiên - Đại học Quốc gia Hà Nội De Padula, Bunyapraphatsara LS, Lemmens RHMJ (1999) Plant Resources of South East Asia PROSEA, Bogor, Indonesia 21: Griffith, W (1858) Journal of the Proceedings of the Linnean Society 2: 17 Deyuan Hong, Thomas G Lammers (2011) Cyclocodon Griffith ex J D Hooker & Thomson, J Proc Linn Soc., Bot 2: 17 1857 Rohlf, F.J (1987) NTSYS-pc: Microcomputer programs for numerical taxonomy and multivariate analysis Am Stat., 41: 330-330 XLSTAT Addinsoft (2020) XLSTAT statistical and data analysis solution New York, USA 36 APPENDIX ELECTROPHORESIS RESULTS OF THE PCR PRODUCTS Electrophoresis results of the PCR products with APH18 Primer Electrophoresis results of the PCR products with OPA01 Primer 37 Electrophoresis results of the PCR products with OPA03 Primer Electrophoresis results of the PCR products with OPB01 Primer Electrophoresis results of the PCR products with OPB02 Primer 38 Electrophoresis results of the PCR products with OPB03 Primer Electrophoresis results of the PCR products with OPB04 Primer Electrophoresis results of the PCR products with OPB05 Primer 39 Electrophoresis results of the PCR products with OPB17 Primer Electrophoresis results of the PCR products with OPC01 Primer Electrophoresis results of the PCR products with OPC03 Primer 40 Electrophoresis results of the PCR products with OPC04 Primer Electrophoresis results of the PCR products with OPC08 Primer Electrophoresis results of the PCR products with OPC13 Primer 41 Electrophoresis results of the PCR products with OPC15 Primer Electrophoresis results of the PCR products with OPD01 Primer Electrophoresis results of the PCR products with OPD02 Primer 42 Electrophoresis results of the PCR products with OPN01 Primer Electrophoresis results of the PCR products with OPR09 Primer Electrophoresis results of the PCR products with OPR12 Primer 43 Electrophoresis results of the PCR products with OPS05 Primer Electrophoresis results of the PCR products with OPS08 Primer Electrophoresis results of the PCR products with OPS10 Primer 44 Electrophoresis results of the PCR products with OPV03 Primer 45