Analysis of genetic diversity of 15 cyclocodon spp accessions by issr markers

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY  GRADUATION THESIS SUBJECT: “ANALYSIS OF GENETIC DIVERSITY OF 15 CYCLOCODON spp ACCESSIONS BY ISSR MARKERS” Hanoi, 3/2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY  GRADUATION THESIS SUBJECT: “ANALYSIS OF GENETIC DIVERSITY OF 15 CYCLOCODON spp ACCESSIONS BY ISSR MARKERS” Student : Nguyen Thi Hong Phuc Student Code : 620619 Class : K62-CNSHE Instructor : Dinh Truong Son, Ph.D Hanoi, 3/ 2022 COMMITMENT I guarantee that the graduation thesis "Analysis of genetic diversity of Codonosis varieties using ISSR markers" is the result of my own research, conducted under the scientific guidance of Ph.D Dinh Truong Son Data and research results in this thesis are completely honest, comply with the regulations on citation, annotation and have never been used or published in any other works I take full responsibility for this commitment In the process of researching and implementing this thesis, I have inherited the achievements of scientists with respect and gratitude Hanoi, February 9th 2022 Student Nguyen Thi Hong Phuc i ACKNOWLEDGEMENTS First and foremost, I would like to express deeply my gratitude to my supervisors Ph.D Dinh Truong Son who have supported me complete my thesis with their patience and knowledge Moreover, their guidance helped me in the period of writing of this thesis I would like to thank all my teachers in Faculty of Biotechnology, especially 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 Department of Plant Biotechnology where supported financial for my thesis I thank my lab-mate for her meaning contribution I will appreciate the time when we were working together Last but not least, I would specifically like to thank my family, especially my parents who have loved me, cared for me, trusted me and given the best study condition Hanoi, February 9th 2021 Student Nguyen Thi Hong Phuc ii CONTENTS COMMITMENT i ACKNOWLEDGEMENTS ii CONTENTS iii ABBREVIATION v LIST OF TABLES vi LIST OF FIGURES vii ABSTRACT viii PART I INTRODUCTION viii 1.1 Rationale of the study 1.2 Research purposes 1.3 Requirements PART II DOCUMENTARY OVERVIEW 2.1 Study overview 2.1.1 Origin 2.1.2 Taxonomy 2.1.3 Characteristics 2.1.4 The value of Cyclocodon 2.1.5 Medicinal value of Cyclocodon 55 2.1.6 Research and development situation of Cyclocodon in the world and in Vietnam 10 2.1.6.1 Research and production situation of Cyclocodon in the world 10 2.1.6.2 Research on Cyclocodon in Vietnam 11 2.1.7 Genetic diversity 12 2.2 Molecular markers and their applications in genetic diversity analysis 2.2.1 Molecular directive concept 13 2.2.2 The ISSR primer 10 2.2.3 Some studies on the development of the bank are based on the ISSR primer 18 iii PART III MATERIALS AND METHODS 21 3.1 Materials 21 3.1.2 Place and time of study 22 3.1.3 Research equipment 22 3.1.4 Chemistry 22 3.2 Research content and methods 24 3.2.1 Research content 24 3.2.2 Research Methods 24 3.2.2.1 Total DNA extraction method 24 3.2.2.2 PCR method 26 3.2.2.3 Gel Electrophoresis 25 3.2.2.4 Methods of evaluating and processing data 27 PART IV RESULTS AND DISCUSSION 30 4.1 Total DNA extraction results 30 4.2 Results of using ISSR molecular markers in genetic diversity analysis 31 4.2.1 Evaluation results of genetic diversity of 15 Cyclocodon spp accessionsError! Book 4.2.2 Analysis of ISSR data using NTSYS 2.1 software 42 PART V CONCLUSION AND SUGGESTION 46 Conclusion 46 Suggestion 46 REFERENCES 47 APPENDUM 54 iv ABBREVIATION AFLP CTAB DNA DPPH EDTA ISSR PCR RAPD RFLP TAE Amplified fragment length polymorphism Cetyltrimethylammonium bromide Deoxyribonucleic acid 2,2-diphenyl-1-picrylhydrazyl Ethylendiamin Tetraacetic Acid Inter Simple Sequence Repeats Polymerase chain reaction Random Amplified Polymorphic DNA Restriction fragment length polymorphism Tris-acetate EDTA v LIST OF TABLES Table 3.1 Lists the accessions and sampling locations used in the study Table 3.2 Chemicals used in the study 22 Table 3.3 List of ISSR primers used in the study 23 Table 3.4 Table of ingredients for PCR reaction – ISSRError! Bookmark not defined Table 3.5 PCR reaction cycle Error! Bookmark not defined Table 4.1 OD260/280 index results and concentration of research accessions 30 Table 4.2 Polymorphism of 15 Cyclocodon spp accessions based on ISSR primer Error! Bookmark not defined Table 4.3 Details of PIC and Rp value of ISSR primers.Error! Bookmark not defined Table 4.4 Similarity coefficient of 15 Cyclocodon spp accessions 4Error! Bookmark no vi LIST OF FIGURES Figure 2.1 Cyclocodon spp Error! Bookmark not defined Figure 2.2 PCR amplification using ISSR primer 18 Figure 4.1 Results of electrophoresis of PCR product primer UBC835 31 Figure 4.2 Results of electrophoresis of PCR product primer UBC858Error! Bookmark n Figure 4.3 Tree diagram showing the genetic relationships of 15 varieties of Cyclocodon Error! Bookmark not defined Figure 4.4 PCA analysis of 15 Cyclocodon spp accessions based on pooled ISSR markers Error! Bookmark not defined vii ABSTRACT Study to evaluate genetic diversity of 15 Cyclocodon spp accessions collected at different locations in Vietnam using ISSR primer In this study, 37 ISSR markers were used to analyze genetic diversity of 15 Cyclocodon accessions and detected a total of 239 loci with 2703 bands The mean PIC of the 15 primers was 0.18 and the mean Rp difference was 9.53 The similarity coefficient of 15 accessions ranged from 0.640 to 0.966 At genetic similarity coefficient of 0.791, 15 Cyclocodon accessions studied were classified into groups viii 51.Fu PP, Hong T, Yang Z (2008) Effect of polysaccharides fromRadix Cyclocodon on insulin resistance in diabetic mice Li-shizhen Med Mater Med Res 19:2414–2416 52.He K, Li XG, Chen X, Ye XL, Huang J, Jin YN, Li PP, DengYF, Jin Q, Shi Q, Shu HJ (2011) Evaluation of antidiabeticpotential of selected traditional Chinese medicines in STZ-induced diabetic mice J Ethnopharmacol 137:1135–1142 53.Xu AX, Zhang ZM, Ge B, Pu JF (2006) Study effect and itsmechanism on resisting senility of Cyclocodon pilosula Nannf.Chin J Mod Appl Pharm 23:729–731 54.Liu L, Wang JH, Hou N (1989) Studies on the pharmacologicaleffects and mechanisms of Cyclocodon pilosula (CP) and itsefficacious chemical ingredients on preventing the gastricmucosal damage of rats I The effects of the decoction of CP.Pharmacol Clinics Chin Mater Med 5:11–14 55.Liu L, Wang JH, Hu Y, Hou N, Chen JW (1990) Studies on thepharmacological effects and mechanisms of Cyclocodon pilo-sula (CP) and its efficacious chemical ingredients on preventingthe gastric mucosal damage of rats III The effects of factionsextracted from the section VII of CP Pharmacol Clinics ChinMater Med 6:20–23 56.Song D, Wang ZT, Li LY, Zhong GY (2008) Protective effect ofLobetyolin on gastric mucosa of experimental gastric ulcer inrats J Emerg Tradit Chin Med 17:963–964 57.Ng TB, Liu F, Wang HX (2004) The antioxidant effects ofaqueous and organic extracts of Panax quinquefolium, Panaxnotoginseng, Cyclocodon pilosula, Pseudostellaria heterophyllaand Ethnopharmacol 93:285–288 52 Glehnia littoralis J 58.He B, Zhang YT, Yuan XG, Sun JS, Wei GH, Lin T (2011)Protective effects of Radix Cyclocodon on ischemia–reperfusioninjury in rats after kidney transplantation Pediatr Surg Int27:1203–1212 59.Zhang RX, Wang FL (1992) Regulatory effect of CPPS on cell-mediated immunity in mice J Lanzhou Med Coll 18:161–16 60.Zhang RX, Wang FL (1993) Effect of CPPS on humoralimmunity and IL-2 production J Lanzhou Med Coll 19:14–17 61.Yoo CS, Kim SJ (2013) Methanol extract of Cyclocodon pilo-sula inhibits inducible nitric oxide synthase and protein oxida-tion in lipopolysaccharidestimulated raw cells Tropical JPharm Res 12:705–710 62.Liu JH, Bao YM, Song JJ, An LJ (2003) Cyclocodon pilosula(Franch.) Nannf total alkaloids potentiate neurite outgrowthinduced by nerve growth factor in PC12 cells Acta PharmacolSin 24:913–917 63.Pan SY, Zhang YF, Liu Y, Xu QP, Jiang MY, Guo YJ (1992)Effects of alkaloids of Dangshen (Cyclocodon pilosula)onamnesia induced by scopolamine and the acetylcholine and choline acetyltransferase of brain in mice Pharmacol ClinicsChin Mater Med 8:22–24 53 APPENDUM Figure Gel electrophoresis pattern of ISSR1 primer Figure Gel electrophoresis pattern of ISSR3 primer 54 Figure Gel electrophoresis pattern of ISSR4 primer Figure Gel electrophoresis pattern of ISSR6 primer Figure Gel electrophoresis pattern of UBC807 primer 55 Figure Gel electrophoresis pattern of UBC808 primer Figure Gel electrophoresis pattern of UBC809 primer Figure Gel electrophoresis pattern of UBC811 primer 56 Figure Gel electrophoresis pattern of UBC811 primer Figure 10 Gel electrophoresis pattern of UBC812 primer Figure 11 Gel electrophoresis pattern of UBC813 primer 57 Figure 12 Gel electrophoresis pattern of UBC814 primer Figure 13 Gel electrophoresis pattern of UBC815 primer Figure 14 Gel electrophoresis pattern of UBC817 primer 58 Figure 15 Gel electrophoresis pattern of UBC819 primer Figure 16 Gel electrophoresis pattern of UBC820 primer Figure 17 Gel electrophoresis pattern of UBC823 primer 59 Figure 18 Gel electrophoresis pattern of UBC824 primer Figure 19 Gel electrophoresis pattern of UBC826 primer Figure 20 Gel electrophoresis pattern of UBC827 primer 60 Figure 21 Gel electrophoresis pattern of UBC834 primer Figure 22 Gel electrophoresis pattern of UBC835 primer Figure 23 Gel electrophoresis pattern of UBC836 primer 61 Figure 24 Gel electrophoresis pattern of UBC841 primer Figure 25 Gel electrophoresis pattern of UBC843 primer Figure 26 Gel electrophoresis pattern of UBC845 primer 62 Figure 27 Gel electrophoresis pattern of UBC848 primer Figure 28 Gel electrophoresis pattern of UBC855 primer Figure 29 Gel electrophoresis pattern of UBC856 primer 63 Figure 30 Gel electrophoresis pattern of UBC858 primer Figure 31 Gel electrophoresis pattern of UBC860 primer Figure 32 Gel electrophoresis pattern of UBC864 primer 64 Figure 33 Gel electrophoresis pattern of UBC873 primer Figure 34 Gel electrophoresis pattern of UBC888 primer Figure 35 Gel electrophoresis pattern of UBC889 primer 65 Figure 36 Gel electrophoresis pattern of UBC890 primer Figure 37 Gel electrophoresis pattern of UBC891 primer 66