Genetic diversity of stephania rotunda lour accessions using issr molecular markers

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY - - GRADUATION THESIS PROJECT: GENETIC DIVERSITY OF STEPHANIA ROTUNDA LOUR ACCESSIONS USING ISSR MOLECULAR MARKERS HA NOI - 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY - - GRADUATION THESIS PROJECT: GENETIC DIVERSITY OF STEPHANIA ROTUNDA LOUR ACCESSIONS USING ISSR MOLECULAR MARKERS Student Name : Nguyen Tien Phat Class : K62CNSHE Student Code : 620407 Instructor : Ninh Thi Thao, Ph.D HA NOI – 2022 COMMITMENT I assure that the entire study procedure was carried out by myself under the scientific supervision of Ph.D Ninh Thi Thao I assure that all research contents, conclusions, and information in my graduation thesis are entirely honest and unpublished Hanoi, August 09th 2022 Student Nguyen Tien Phat i ACKNOWLEDGEMENTS First of all, I would like to express deeply my gratitude to my supervisor’s Ph.D Ninh Thi Thao 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, August 09th 2022 Student Nguyen Tien Phat ii CONTENTS COMMITMENT i ACKNOWLEDGEMENTS ii CONTENTS iii ABBREVIATION v LIST OF TABLES vi LIST OF FIGURES vii PART I INTRODUCTION viii 1.1 Preface 1.2 Purposes and requirements .2 1.2.1 Purposes 1.2.2 Requirements PART II DOCUMENTARY OVERVIEW 2.1 Introduction of Stephania rotunda Lour .3 2.1.1 Taxanomy 2.1.2 Morphology characteristics and distributions .3 2.1.3 Medicinal value of Stephania rotunda Lour 2.2 ISSR molecular marker and its applications in medicinally plants 2.2.1 Definition of molecular marker .6 2.2.2 ISSR molecular markers 2.2.3 Applications of ISSR marker in genetic diverstiy of medicinal plants PART III MATERIALS AND METHODS 10 3.1 Materials 10 3.2 Time and place of the Study 11 3.3 Research equipment 11 3.4 Chemical substances 11 3.5 Contents and methods 12 3.5.1 Contents .12 3.5.2 Methods .13 3.5.2.1 DNA extraction 13 3.5.2.2 PCR reaction .14 iii 3.5.2.3 Agarose gel electrophoresis 14 3.5.2.4 Data analysis .15 PART IV RESULTS AND DISCUSSION 17 4.1 Quantity and quality of total genomic DNA 17 4.2 Amplification of S rotunda Lour accessions with ISSR-PCR 20 4.3 Genetic diversity analysis based on ISSR-PCR results 25 PART V CONCLUSION AND SUGGESTION 31 Conclusion .31 Suggestion .32 REFERENCES 33 APPENDIX 35 Banding profile obtained with ISSR primers 35 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 3.1 List of chemical substances used in research 11 Table 3.2 Components of PCR reaction 14 Table 3.3 Cycles of PCR reaction 14 Table 4.1 DNA concentration and purity index of genomic DNA extracted from leaves of 32 S rotunda Lour accessions 17 Table 4.2 Polymorphism of 14 ISSR markers 22 Table 4.3 The respective marker performance indexes of 14 ISSR primers used with 32 Stephania rotunda Lour accessions 23 Table 4.4 Matrix of genetic similarity among 32 Stephania rotunda Lour accessions using ISSR markers calculated by Jaccard’s similarity coefficient 27 vi LIST OF FIGURES Figure 1 Stephania rotunda Lour plant (source: internet) Figure 4.1 Agarose gel electrophoresis of total genomic DNA extracted from 32 Stephania rotunda Lour 19 Figure 4.2 ISSR banding profile obtained with primer UBC-824 20 Figure 4.3 ISSR banding profile obtained with primer UBC-889 21 Figure 4.4 Dendrogram obtained with UPGMA from the Jaccard’s similarity coefficients of 32 Stephania rotunda Lour accessions based on ISSR data analysis 29 vii ABSTRACT In several Asian countries, Stephania rotunda Lour (Vietnamese name: Bình vơi) has long been used to treat asthma, tuberculosis, diarrhea, hyperglycemia, cancer, fever, digestive problems, sleep difficulties, and inflammation It mostly contains alkaloids, and over 30 alkaloids have been isolated from its tuber, including bisbenzylisoquinolines, hasubanalactams, berberines, and aporphines The total genomic DNA extracted from 32 Stephania rotunda Lour accessions using the CTAB method was suitable for the purpose of using as a template for ISSR - PCR reaction with the DNA purity index (OD260/OD280) ranging from 71 – 2.07; DNA concentrations ranged from 117 to 1054 ng/µl 14 ISSR primers on 32 Stephania rotunda Lour accessions produced with a total of 74 loci with average of 5,14 loci per primer The average PIC index was 0.2 and the index of difference between primer pairs Rp averaged of 1.31 At the mean of 0.79 similarity level, 32 accessions were grouped into main clusters viii PART V CONCLUSION AND SUGGESTION Conclusion The total genomic DNA extracted from 32 Stephania rotunda Lour accessions using the CTAB method was suitable for the purpose of using as a template for ISSR - PCR reaction with the DNA purity index (OD260/OD280) ranging from 71 – 2.07; DNA concentrations ranged from 117 to 1054 ng/µl 14 ISSR primers on 32 Stephania rotunda Lour accessions produced with a total of 74 loci with average of 5,14 loci per primer The polymorphism ranged from 40% for primer UBC-889 to 100% for primer UBC-864 and UBC-827 with an average of 69.33% A total of 1701 bands with an average of 121.5 bands per primer and 53.16 bands per accession was generated The average PIC index was 0.2 and the index of difference between primer pairs Rp averaged of 1.31 The genetic similarity coefficient of 32 Stephania rotunda Lour accessions based on the ISSR molecular markers ranged from 0.583 to 0.972 with the lowest similarity coefficient are between accession No 12 ( collected from Hoa Binh) and accession No (collected from Ha Giang The highest similarity coefficient are between accession No 28 and accession No 29 ( both collected in Da Bac, Hoa Binh) At the mean of 0.79 similarity level, 32 accessions were grouped into main clusters Cluster I has only accession collected from Kim Boi, Hoa Binh Cluster II comprises 21 accessions including: 2, 3, 4, 5, 16, 17, 18, 19, 20 (collected from Kim Boi, Hoa Binh); 31, 28, 30, 29 (collected from Da Bac, Hoa Binh); 13, 14, 15 (collected from Luong Son, Hoa Binh); 21, 22, 23, (collected from Lac Thuy, Hoa Binh); 24 (collected from Muong Nhe, Dien Bien); 27 (collected from Lai Chau) Cluster III comprises accessions including: 6, 7, 8, 9, 10 ( all collected from Ha Giang) Cluster IV has only accession 32 (collected from Thanh Hoa) Clusters V comprises accessions including 25 and 26 (collected from Muong Thin, Dien Bien) Clusters VI 31 comprises accessions including 11 and 12 ( collected from Cao Son, Luong Son, Hoa Binh) Suggestion It is necessary to collect more accession of Stephania rotunda Lour for research and evaluation, enriching genetic resources to medicinal plant improvement and germ plant conservation in the future 32 REFERENCES 10 11 12 Semwal DK, Semwal RB Efficacy and safety of Stephania glabra: An alkaloid-rich traditional medicinal plant Vol 29, Natural Product Research Taylor and Francis Ltd.; 2015 p 396–410 Vaid P, K Saini A, v Saini R, v Saini R, v Saini R, v Saini R EVALUATION OF ANTIOXIDANT, ANTI-CANCER AND ANTIMICROBIAL ACTIVITY OF DIFFERENT EXTRACTS AND FRACTIONS OF STEPHANIA GLABRA Plant Arch 2021 Apr 20;21(no 1) K Semwal D, Badoni Semwal R, Semwal R, Jacob V, Singh G Analgesic and Antipyretic Activities of Gindarudine, a Morphine Alkaloid from Stephania glabra Curr Bioact Compd 2011 Aug 15;7(3):214–7 Chombe D, Bekele E Genetic diversity analysis of cultivated Korarima [Aframomum corrorima (Braun) P.C.M Jansen] populations from southwestern Ethiopia using inter simple sequence repeats (ISSR) marker Mil Med Res 2018;5(1) Abdel-Latif A, Osman G Comparison of three genomic DNA extraction methods to obtain high DNA quality from maize Plant Methods 2017 Jan 3;13(1) Hieu HD, Thu Ha CT, Ngoc PB, Nhan LD, Thuy Huong NT, Ha CH Study on genetic diversity in Morinda officinalis F C How populations in Quang Ninh using issr marker Tap Chi Sinh Hoc 2016 Jan 14;38(1) Ninh TT, Doan HT, Nguyen MT, Tran TT, Dang PDD, Dinh ST, et al Genetic diversity of avocado (Persea americana Mill.) germplasm in Vietnam using RAPD and ISSR molecular markers Aust J Crop Sci 2022;16(6):856–62 Sen NT, Vinh T, Thị M, Hoa P, Công K, Học S, et al Nhân giống Bình vơi (stephania glabra (roxb.) miers) in vitro Kylin M Genetic diversity of Roseroot (Rhodiola rosea L.) from Sweden, Greenland and Faroe Islands Lam Tuang K, Myoe Min H, Sein MM EXTRACTION, ISOLATION, ANTIMICROBIAL STUDY AND STRUCTURAL ELUCIDATION OF A PURE COMPOUND ISOLATED FROM THE TUBER OF STEPHANIA GLABRA (ROXB.) MIERS Vol 1, J Myanmar Acad Arts Sci 2020 Gilmer R, Heitmann RC ON Pic(R[X]) FOR R SEMINORMAL Vol 16, Journal of Pure and Applied Algebra North-Holland Publishing Company; 1980 Marakli S A Brief Review of Molecular Markers to Analyse Medicinally Important Plants Vol 1, International Journal of Life Sciences and Biotechnology 2018 33 13 14 15 16 17 18 19 20 21 22 23 Pourmohammad A Application of molecular markers in medicinal plant studies Acta Universitatis Sapientiae, Agriculture and Environment 2013 Dec 1;5(1):80–90 Lộc Hiền N, Thị Nhựt T, Kỳ H, Huỳnh V, Tùng T SỰ ĐA DẠNG DI TRUYỀN CỦA QUẦN THỂ CÂY NGHỆ (CURCUMA SP.) Ở TỈNH BÌNH DƯƠNG Title: Evaluating genetic diversity of turmeric population in Binh Duong province Silva Júnior AL, Souza LC, Pereira AG, Caldeira MVW, Miranda FD Genetic diversity of schizolobium parahyba var Amazonicum (Huber ex Ducke) barneby, in a forest area in Brazil Genetics and Molecular Research 2017 Sep 21;16(3) Serrote CML, Reiniger LRS, Silva KB, Rabaiolli SM dos S, Stefanel CM Determining the Polymorphism Information Content of a molecular marker Gene 2020 Feb 5;726:144175 Bhakuni DS, Gupta S, Jain S The biosynthesis of the alkaloids of stephania glabra (ROXB.) miers Tetrahedron 1983 Jan 1;39(23):4003– 10 Nagy E, Marton LC The use of morphological traits and genetic markers to estimate genetic relationships in maize Cereal Res Commun 2006;34(2–3):887–94 Botstein D, White RL, Skolnick M, Davis4 RW Construction of a Genetic Linkage Map in Man Using Restriction Fragment Length Polymorphisms Vol 32, Am JHum Genet 1980 Hashemifar Z, Horticulturae MRS, 2018 undefined Genetic structure and variation in Perovskia abrotanoides Karel and P atriplicifolia as revealed by molecular and morphological markers Elsevier [Internet] [cited 2022 Sep 13 Govindaraj V, medicine ARS biology in reproductive, 2015 undefined Comparative proteomic analysis of primordial follicles from ovaries of immature and aged rats Taylor & Francis [Internet] 2015 Nov [cited 2022 Sep 13];61(6):367–75 Agarwal M, Shrivastava N, reports HPP cell, 2008 undefined Advances in molecular marker techniques and their applications in plant sciences Springer [Internet] 2008 Apr [cited 2022 Sep 13];27(4):617–31 Nguyen MC, Vo TT (1999) Avocado production in Vietnam In: Papademetriou MK (ed) Avocado production in Asia and the Pacific 34 APPENDIX Banding profile obtained with ISSR primers UBC-808 35 UBC-848 36 UBC-813 37 UBC-811 38 UBC-812 39 UBC-823 40 UBC-873 41 UBC-807 42 UBC-888 43 UBC-889 44 UBC-827 45