VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY - - GRADUATION THESIS PROJECT: EVALUATION OF GENETIC DIVERSITY AND PRESENCE OF LATE BLIGHT (PHYTOPHTHORA INFESTANS) RESISTANCE GENES IN SOME PROMISING HYBRID POTATO LINES BY MOLECULAR MARKERS HA NOI - 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY - - GRADUATION THESIS PROJECT: EVALUATION OF GENETIC DIVERSITY AND PRESENCE OF LATE BLIGHT (PHYTOPHTHORA INFESTANS) RESISTANCE GENES IN SOME PROMISING HYBRID POTATO LINES BY MOLECULAR MARKERS Student Name : Le Van Ngoc Class : K63CNSHE Student Code : 637424 Instructor : Nguyen Xuan Truong, Ph.D HA NOI – 2022 COMMITMENT I assure that the whole research process was performed by myself under the scientific guidance of Ph.D Nguyen Xuan Truong I assure that all research contents, results, and information in my thesis is completely honest and unpublished Hanoi, December 05th 2022 Student Le Van Ngoc i ACKNOWLEDGEMENTS In order to complete the graduation thesis, besides my own efforts, I have received a lot of enthusiastic support from teachers, friends and relatives First, I would like to start by expressing my sincere gratitude to Nguyen Xuan Truong, a PhD supervisor for helping me finish my thesis with their guidance and expertise Additionally, their advice was helpful to me as I was preparing this thesis I want to thank PhD Dinh Truong Son and 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 and where supporting equipment and finances for my thesis One of the most crucial elements that will enable me to successfully complete my thesis is this I appreciate the passionate assistance from the Institute of Biotechnology and everyone who works there so that I can have the finest thesis I thank my lab-mate for his meaningful 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 me the best study condition Hanoi, December 05th 2022 Student Le Van Ngoc ii CONTENTS COMMITMENT i ACKNOWLEDGEMENTS ii CONTENTS iii ABBREVIATION v LIST OF TABLES vi PART INTRODUCTION 1.1 Preface 1.2 Purposes and requirements .1 1.2.1 Purposes 1.2.2 Requirements PART LITERATURE OVERVIEW 2.1 Introduction of potato .3 2.1.1 Origin .3 2.1.2 Genetics 2.1.3 Botanical characteristics 2.1.4 Nutrient content in potatoes 2.1.5 Potato production in the world 2.1.6 Potato production in Vietnam 2.2 Phytophthora infestans in potato 2.2.1 Discovery history 2.2.2 Distribution range 10 2.2.3 Symptoms 10 2.2.4 The cause to the illness 11 2.3 Molecular markers and applications of RAPD and ISSR markers in genetic diversity analysis 13 2.3.1 Definition .13 2.3.2 RAPD molecular marker .13 2.3.3 ISSR molecular marker .14 iii 2.4 Researches on potato (Solanum tuberosum L.) using RAPD and ISSR molecular markers 15 PART MATERIALS AND METHODS 17 3.1 Materials 17 3.2 Time and Place of the Study 17 3.3 Research equipment 17 3.4 Chemical substances .18 3.5 Methods 20 3.5.1 DNA extraction 20 3.5.2 PCR reaction 21 3.5.3 Gel electrophoresis 22 3.5.4 Data analysis 23 PART RESULTS AND DISCUSSION 24 4.1 DNA extraction 24 4.2 Genetic diversity analysis of 28 potato lines using RAPD molecular 28 4.3 Genetic diversity analysis using ISSR molecular markers .33 4.4 Genetic diversity analysis combined RAPD and ISSR molecular markers 37 PART CONCLUSION AND SUGGESTION 42 5.1 Conclusion 42 5.2 Suggestion 42 REFERENCES 43 APPENDIX 44 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 2.1 Potato yield of the top countries in the world in 2012 and 2013 Table 3.1: 48 potato lines and three commercial varieties used for the study 17 Table 3.2 List of chemical substances used in research 18 Table 3.3 List of RAPD primers used in research 18 Table 3.4 List of ISSR primers used in research 18 Table 3.5 List of molecular markers associated with late blight resistance genes in potato 19 Table 3.6 Components of each PCR reaction 22 Table 3.7 Thermal cycler of PCR 22 Table 4.1 Concentration and quality of DNA isolated from 48 potato line and varieties 24 Table 4.2 The present of resistant gene in studied potato lines, symbol “+” for presence the gene, empty cells represent no detection 26 Table 4.3 Polymorphism of 28 lines of potato revealed by RAPD markers 28 Table 4.4 Matrix of genetic similarity among 28 accessions of potato revealed by RAPD markers calculated by similarity coefficient of Sokal-Michener 30Error! Bookmark not defined Table 4.5 Polymorphism of 28 potato lines accessions revealed by ISSR markers 33 Table 4.6 Matrix of genetic similarity among 28 potato accessions revealed by ISSR markers calculated by similarity coefficient of Sokal-Michener .35 Table 4.7 Matrix of genetic similarity among 28 potato accessions revealed by combined RAPD and ISSR markers calculated by similarity coefficient of Sokal-Michener 38 Table 4.8 Matrix comparisons of Mantel test / Two-tailed test between markers 41 vi LIST OF FIGURES Figure 2.1 Botanical characteristics of potato Figure 4.1 Results of electrophoresis product with primer APG-05 27 Figure UPGMA cluster analysis of 28 accessions of potato lines with a similarity coefficient of RAPD marker .32 Figure Principal component analysis (PCA) of 28 potato accessions based on RAPD marker 33 Figure 4 UPGMA cluster analysis of 28 potato accessions with a similarity coefficient of ISSR marker 36 Figure 4.5 PCA analysis of 28 potato accessions based on ISSR markers 37 Figure 4.6 UPGMA cluster analysis of 28 potato accessions with a similarity coefficient combined RAPD and ISSR marker 39 Figure Principal component analysis (PCA) of 28 potato accessions based on pooled RAPD and ISSR marker 40 vii PART INTRODUCTION 1.1 Preface Potato (Solanum tuberosum L.) is a short-term crop that plays a significant role in human food crops It is a member of the Solanaceae family and is native to Bolivia and Peru’s Andes mountains In terms of food production, potatoes are currently the fourth most widely planted crop after rice, wheat and maize They are the most widely grown tuber in the world They can adapt to a variety of environment situations, are simple to grow and produce a speedy harvest (International Year of the Potato 2008; Jeff Chapman et al 2011) The productivity and quality of tubers are, however, greatly impacted by a number of illnesses that are frequently transmitted to potatoes Diseases on potatoes are very diverse, rich in composition and cause of disease such as late blight, yellow wilt, viral leaf curl… Late blight caused by Phytophthora infestans is considered the most harmful in the world, resulting approximately 16% output losses globally (Haverkort A et al 2009) With the strong development of molecular biology, the breeding technology of molecular markers ensure accuracy, and be able to gather many targeted genes into one variety Traditional breeding methods are difficult or impossible to To create a new potato variety, the first thing is to have a source of resistance genes and molecular markers associated with those resistance genes, then evaluate and determine the resistance to late blight of the variety to use for future breeding Therefore, I conduct this work: “Evaluation of genetic diversity and presence of late blight (Phytophthora infestans) resistance genes in some promising hybrid potato lines by molecular markers” 1.2 Purposes and requirements 1.2.1 Purposes - Finding promising potato lines containing late blight resistance genes - Besides, Analysis the genetic diversity and relationships between 28 potato samples by RAPD and ISSR molecular markers Table 4.7 Matrix of genetic similarity among 28 potato accessions revealed by combined RAPD and ISSR markers calculated by similarity coefficient of Sokal-Michener 1906-03 1906-44 1944-37 1944-11 1944-30 1944-31 1944-29 2001-26 2001-05 2001-12 2001-13 2001-28 2001-36 2001-40 Bliss Alantic No-10 Marabel 2043-35 2043-18 2043-50 2043-26 2056-13 2061-22 2061-17 2076-36 2081-22 2081-17 1906-03 1906-44 1944-37 1944-111944-30 1944-31 1944-29 2001-26 2001-05 2001-12 2001-13 2001-28 2001-36 2001-40 Bliss Alantic No-10 Marabel 2043-35 2043-18 2043-50 2043-26 2056-13 2061-22 2061-17 2076-36 2081-22 2081-17 1.000 0.760 1.000 0.705 0.705 1.000 0.680 0.700 0.795 1.000 0.640 0.740 0.773 0.740 1.000 0.540 0.500 0.636 0.580 0.680 1.000 0.619 0.714 0.750 0.738 0.810 0.667 1.000 0.667 0.729 0.738 0.688 0.729 0.604 0.825 1.000 0.620 0.660 0.591 0.640 0.740 0.580 0.690 0.833 1.000 0.640 0.600 0.682 0.640 0.700 0.580 0.643 0.667 0.680 1.000 0.740 0.620 0.636 0.600 0.700 0.580 0.738 0.833 0.840 0.800 1.000 0.760 0.700 0.682 0.660 0.800 0.640 0.738 0.813 0.740 0.780 0.860 1.000 0.700 0.640 0.682 0.740 0.720 0.640 0.738 0.771 0.620 0.700 0.700 0.760 1.000 0.643 0.619 0.667 0.786 0.714 0.714 0.810 0.775 0.786 0.643 0.690 0.690 0.690 1.000 0.640 0.600 0.591 0.580 0.720 0.720 0.738 0.750 0.700 0.580 0.700 0.760 0.680 0.786 1.000 0.580 0.580 0.568 0.520 0.660 0.620 0.714 0.688 0.680 0.560 0.640 0.660 0.660 0.762 0.820 1.000 0.556 0.644 0.718 0.733 0.644 0.556 0.676 0.744 0.644 0.644 0.600 0.644 0.689 0.622 0.667 0.644 1.000 0.500 0.600 0.659 0.600 0.740 0.580 0.643 0.708 0.640 0.720 0.640 0.700 0.740 0.548 0.660 0.680 0.844 1.000 0.526 0.553 0.563 0.553 0.579 0.658 0.526 0.611 0.500 0.605 0.553 0.658 0.605 0.579 0.632 0.579 0.576 0.605 1.000 0.540 0.540 0.636 0.600 0.660 0.660 0.667 0.750 0.640 0.600 0.680 0.700 0.660 0.619 0.780 0.640 0.778 0.720 0.711 1.000 0.614 0.636 0.636 0.614 0.682 0.773 0.806 0.738 0.591 0.591 0.636 0.636 0.682 0.722 0.818 0.750 0.769 0.705 0.781 0.818 1.000 0.660 0.680 0.705 0.700 0.720 0.560 0.762 0.729 0.620 0.700 0.660 0.720 0.800 0.667 0.680 0.660 0.844 0.820 0.632 0.740 0.750 1.000 0.600 0.620 0.659 0.680 0.660 0.620 0.738 0.708 0.640 0.680 0.640 0.620 0.820 0.690 0.700 0.720 0.756 0.760 0.711 0.720 0.795 0.860 1.000 0.620 0.640 0.636 0.660 0.680 0.560 0.690 0.771 0.700 0.620 0.660 0.600 0.720 0.738 0.680 0.660 0.644 0.700 0.711 0.660 0.773 0.760 0.820 1.000 0.520 0.620 0.568 0.600 0.660 0.660 0.738 0.729 0.600 0.600 0.600 0.580 0.780 0.690 0.660 0.680 0.622 0.720 0.711 0.720 0.795 0.740 0.840 0.820 1.000 0.560 0.500 0.636 0.680 0.540 0.580 0.667 0.604 0.600 0.600 0.640 0.580 0.660 0.667 0.660 0.680 0.778 0.680 0.605 0.680 0.727 0.740 0.760 0.660 0.640 1.000 0.600 0.600 0.727 0.700 0.640 0.520 0.738 0.771 0.660 0.660 0.700 0.640 0.720 0.690 0.600 0.580 0.756 0.740 0.658 0.700 0.682 0.760 0.780 0.800 0.740 0.740 1.000 0.580 0.620 0.614 0.560 0.580 0.580 0.714 0.688 0.600 0.560 0.640 0.620 0.660 0.571 0.740 0.680 0.711 0.680 0.658 0.680 0.750 0.740 0.760 0.700 0.680 0.760 0.660 1.000 38 Figure 4.6 UPGMA cluster analysis of 28 potato accessions with a similarity coefficient combined RAPD and ISSR marker 39 Figure Principal component analysis (PCA) of 28 potato accessions based on pooled RAPD and ISSR marker PCA scatter plots were used to assess the genetic diversity of 28 potato lines analyzed by combined RAPD and ISSR markers PCA analysis revealed that the top two principal components accounted for 16.88% and 26.69%, respectively, of the total genetic variance Additionally, the PCA shows in Figure 4.7 how far apart the lines are genetically diverse The genetic diversity of 28 potato lines were examined using inter simple sequence repeat (ISSR) or random amplified polymorphic DNA (RAPD) techniques or a combination of RAPD and ISSR markers (Figs 4.2, 4.4 and 4.6) The correlation between two matrices was tested using the Mantel test It uses permutations of the rows and columns of one of the input distance matrices to calculate the significance of the correlation in a non-parametric manner The Pearson productmoment correlation coefficient (r), which ranges from -1 to +1, is the test statistic A 40 value near to -1 denotes a strong negative correlation, while a value close to +1 denotes a strong positive correlation A correlational value of means there is none For the Mantel test, we employed similarity coefficient matrices, and the results showed a RAPD and ISSR correlation coefficient (r) of -0.022 (p 0.677) This result showed that there was no correlation between the two markers (Table 4.8) There was correlation between RAPD and the combined RAPD-ISSR data (r = 0.816, p