Analysis of genetic diversity among accessions of lonicera japonica

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY - - GRADUATION THESIS TITLE: ANALYSIS OF GENETIC DIVERSITY AMONG ACCESSIONS OF Lonicera japonica HA NOI - 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY - - GRADUATION THESIS TITLE: ANALYSIS OF GENETIC DIVERSITY AMONG ACCESSIONS OF Lonicera japonica Student name : NGUYEN HUU DUC THINH Student code : 637076 Class : K63CNSHE Supervisors : Ph.D NGUYEN VAN KHIEM Ph.D NGUYEN THI THUY HANH Departerment : Biotechnology HA NOI – 2022 COMMITMENT I hereby commit that the thesis is completely done by me under the guidance of PhD Nguyen Van Khiem, PhD Nguyen Thi Thuy Hanh All the data and results that I have provided in this study are true, accurate, and not used in any other reports I also assure that the literatures cited in the thesis indicated the origin and all help were thankful Hanoi, December 2022 Student Nguyen Huu Duc Thinh i ACKNOWLEDGEMENTS In order to complete the graduation after 4,5 years of training, learning and improving knowledge, I would like to sincerely thank teachers for their dedicated guidance during the study, research and training process at the Vietnam National University of Agriculture First of all, I want to express my sincere thanks to the National Research Center for Medicinal plant germplasm and breeding, Biotechnology Department and Molecular Technology Department, because this thesis would not have been possible without the involvement and support of the assistance and guidance of dedicated teachers at the laboratories of the Department First and foremost, I pay my deep sense of gratitude to my principal supervisors PhD Nguyen Van Khiem- National Research Center for Medicinal Plant Germplasm and Breeding, PhD Nguyen Thi Thuy Hanh- Vietnam National University of Agriculture, who encouraged me to complete my thesis and gave me lots of advice I would like to thank all staffs, working at the Laboratory of the Department of Biotechnology - National Research Center for Medicinal Plant Germplasm and Breeding, especially MSc Dinh Thanh Giang, for helping and creating favorable conditions for me during doing thesis They were all very friendly and helpful to me, I was lucky to study and work in a great environment Despite trying to complete the Thesis in the most complete way However, due to some limitations in terms of knowledgment and practical experiences, it is inevitable that certain short comings that you have not seen I look forward to the suggestions of teachers to make the thesis more complete Last but not the least, my family is also an important inspiration for me So, with due regards, I express my gratitude to them Thank you sincerely! Hanoi, December 2022 Student Nguyen Huu Duc Thinh ii INDEX COMMITMENT i ACKNOWLEDGEMENTS ii INDEX iii LIST OF TABLES v LIST OF FIGURES vi LIST OF ABBREVIATIONS vii ABSTRACT viii PART I INTRODUCTION 1.1 Introduction 1.2 Objectives and requirements of the topic 1.2.1 Objectives 1.2.2Scientific significance PART II LITERATURE REVIEW 2.1 Overview of Honeysuckle 2.1.1 Taxonomic position of Honeysuckle genus 2.2 Distribution and feature of Lonicera in Vietnam 2.2.1 Distribution 2.2.2 Diversity and plant characteristics of Lonicera japonica in Vietnam 2.2.3 Chemical composition 2.3 Biological effects and uses 2.3.1 Biological effects 2.3.2 Usage value 2.4 World research on Lonicera 2.4.1 Research on Lonicera taxonomy 2.4.2 Research on the therapeutic ability of Honeysuckle 11 2.4.3Research on plant classification, propagation, planting, and processing of Honeysuckle 11 2.5 The situation of domestic research on honeysuckle 12 iii 2.5.1 Taxonomic study of honeysuckle in Vietnam 12 2.5.2 The therapeutic effect 13 2.5.3 About planting techniques, care, collection, processing and product specifications 14 2.6 Markers in the assessment of genetic diversity 14 2.6.1 Morphological marker 14 2.6.2 Biological markers 15 2.6.3 Molecular marker 16 PART III MATERIALS AND STUDY METHODS 21 3.1 Materials 21 3.1.1 Materials 21 3.1.2 Chemicals, instruments and equipments 22 3.2 Experiment location and time 22 3.3 Study Methods 22 3.3.1 Collecting and describing and identification of accessions 22 3.3.2 DNA extraction 22 3.3.3 PCR using ISSR primers and PCR product analysis 23 3.3.4 Analysis of PCR products 24 PART IV RESULTS AND DISCUSIONS 26 4.1 Collect samples, describe plant characteristics, identify scientific names 26 4.2 Total DNA extration 27 4.3 Genetic diversity analysis among 20 accessions using the ISSR markers 29 4.4 Analysis of genetic relationships among 20 accessions 37 PART V CONCLUSIONS AND PROPOSALS 39 5.1 Conclusions 39 5.2 Proposals 39 REFERENCES 40 iv LIST OF TABLES Table List of aceesions collected used in the study 21 Table 3.2 List of ISSR primers used in the study 24 Table 3.3 Composition and concentration of substances for PCRs 24 Table Total DNA concentration of samples 29 Table Genetic diversity among 20 accessions using 10 ISSR primers 35 Table Genetic similarity coefficients among 20 accessions of Lorencea japonica 36 v LIST OF FIGURES Figure Photos of Lonicera japonica accoarding to classification system of A Takhtajan Figure 2.2 Chemical structures of some key compounds in honeysuckle (Nguyen Thanh Cong, 2015) Figure 4.1 Several photos of Lonicera japonica were collected from provinces/city 27 Figure Electrophoresis photo of total DNA on gel 0,8% isolated from 20 samples (KN 1-20: wells: 1-20) 28 Figure PCR products of 20 accessions 1kb DNA marker (KN 1-20: wells: 1-20) 33 Figure 4 Genetic relationships diagram among 20 accessions using 10 ISSR primers 37 vi LIST OF ABBREVIATIONS Abbreviations Meaning µg Microgram µl Microliter DNA Deoxyribonucleic acid AFLP Amplified fragment length polymorphism bp Base pair CAPs Cleaved amplified polymorphic sequence CTAB Cetyl trimethylammonium bromide dNTPs Deoxyribonucleotide triphosphates ISSR Inter simple sequence repeat kb Kilobase RAPD Random amplified polymorphic DNA SSR Simple sequence repeat SNP Single-nucleotide polymorphisms Na2EDTA Ethylene Diamine Tetra Acetate Sodium OD Optical Density PCR Polymerase chain reaction RNA Ribonucleic acid TAE Tris acetate EDTA TE Tris EDTA EDTA Etylendiamintetra axetic vii ABSTRACT According to traditional medicine, Honeysuckle (Lonicera japonica Thumb.) is used to treat many different diseases that have been prescribed in the Vietnam Pharmacopoeia and are listed in the List of essential traditional medicines issued by the Ministry of Health (Circular 19/2018/TT-BYT) In Vietnam, there are 11 species of naturally distributed from the North to the Central Highlands and most species of this genus in Vietnam are used as medicines Around the world there have been many studies assessing the genetic diversity of some species in Lonicera using DNA molecular markers In the present study, a total of 20 accessions of Lonicera japonica were identified, collected from provinces and cities throughout the country, grown in the field of National Institute of Medicinal Materials in Thanh Tri, Ha Noi Total DNA was extracted, quantified, and qualified to perform PCRs A total of 10 ISSR primers were used to amplify 20 accessions Analysis of genetic diversity based on ISSR markers using NTSYS 2.1 software (Rohlf, 1998) In this study, 20 accessions were collected from provinces/cities with different ecological conditions Results showed that a total of 85 loci were amplified, 74 of which were polymorphic, and the polymorphic rate was 88.03% A PIC index in the range of 0.29 - 0.43 indicated that the levels of polymorphism was moderate The cladogram analysis results based on the UPGMA method showed that the accessions of Lonicerae japonica had high genotype diversity, the similarity coefficient was between 0.45 and 1.00, and the average value was 0.64 The study revealed significant genetic variation among accessions 20 accessions were divided into main groups: The first group consists of KN1, KN2, KN3, KN6, KN7, KN8, KN9, KN10, KN13, KN14; the second group having samples including KN4 and KN5; the third group having samples inclunding KN11, KN12, KN15, KN16, KN17, KN18, KN19, KN20 The research results of the project contributed to conservation and breeding in Vietnam in the future viii M M C M M D M M E 31 M M F M M G M M H 32 M M I M M J Figure PCR products of 20 accessions 1kb DNA marker (KN 1-20: wells: 1-20) List of Primiers: A: UBC835; B: UBC842; C: UBC807, D: UBC810; E: UBC815; F: UBC 824; G: UBC827; H: UBC836; I: UBC848; J: UBC857 Sun et al (2013) used 12 ISSR primers to study the polymorphism of 36 Lonicera japonica samples in China Results showed a polymorphism rate of 88.37% Choi et al (2015) studied the genetic diversity of 161 individuals belonging to natural populations of Lonicera caerulea in Korea using the ISSR molecular marker showing a relatively low polymorphic locus rate of about 64.56% The highest difference between Rp of 6.4 belonged to the U824 primer, 33 the lowest was 2.5 of the U815 primer, and the average Rp index was 4.52 According to Prevost and Wilkinson (1999), the higher the Rp index indicated the correlation between genotypes and DNA molecular markers, the higher the Rp, the more effective the molecular marker was in genotype groups From the above results, UBC824 molecular markers were very effective in using to divide among accessions According to Botstein et al (1980), if the PIC > 0.5 the primer was used for high polymorphic results, whereas a PIC between 0.25 ≤ a PIC ≤ 0.5 gived a mean polymorphic result, and with a PIC < 0.25, the polymorphism result was low Table 4.3 showed that the lowest PIC is 0.29 (UBC807, UBC835, UBC842) and the highest PIC was 0.43 (UBC857) Overall, all PIC readings of the 10 ISSR primers were greater than 0.25 and lower than 0.5, and this showed that the 10 ISSR primers used in the study produced average polymorphisms Genetic similarity coefficient ranged from 0.45 (KN2 and KN12) to 1.00 (KN13 and KN14) It could be KN13 and KN14 was same (Table 4.4) 34 Table Genetic diversity among 20 accessions using 10 ISSR primers Indices of Primer Number Primer Melting Temperature difference Loci Polymorphic Polymorphic bands (%) Total between bands primer Tm (ºC) PIC pairs (Rp) UBC807 53 85.71 68 2.8 0.29 UBC810 53 10 80 126 4.4 0.3 UBC815 53 5 100 57 2.5 0.36 UBC824 53 11 11 100 98 6.4 0.38 UBC827 53 8 100 104 0.37 UBC835 53 11 63.64 143 5.3 0.29 UBC836 53 85.71 69 3.7 0.34 UBC842 53 88.89 99 3.3 0.29 UBC848 53 88.89 92 5.6 0.37 10 UBC857 53 87.50 94 6.2 0.43 4.52 0.34 Total 85 Average/Primer 8.5 Average/Sample 4.25 950 7.4 35 88.03 95 44.02 47.5 Table Genetic similarity coefficients among 20 accessions of Lorencea japonica KN1 KN2 KN3 KN4 KN5 KN6 KN7 KN8 KN9 KN10 KN11 KN12 KN13 KN14 KN15 KN16 KN17 KN18 KN19 KN1 1.00 KN2 0.91 1.00 KN3 0.73 0.68 1.00 KN4 0.72 0.69 0.73 1.00 KN5 0.53 0.58 0.56 0.72 1.00 KN6 0.61 0.56 0.79 0.68 0.61 1.00 KN7 0.84 0.84 0.66 0.62 0.51 0.54 1.00 KN8 0.87 0.87 0.69 0.68 0.56 0.60 0.92 1.00 KN9 0.86 0.86 0.68 0.67 0.55 0.59 0.93 0.99 1.00 KN10 0.69 0.69 0.85 0.60 0.55 0.66 0.76 0.75 0.76 1.00 KN11 0.52 0.49 0.55 0.52 0.66 0.58 0.52 0.51 0.49 0.54 1.00 KN12 0.47 0.45 0.55 0.54 0.64 0.58 0.52 0.46 0.47 0.59 0.88 1.00 KN13 0.69 0.72 0.59 0.65 0.60 0.52 0.74 0.80 0.81 0.65 0.52 0.54 1.00 KN14 0.69 0.72 0.59 0.65 0.60 0.52 0.74 0.80 0.81 0.65 0.52 0.54 1.00 1.00 KN15 0.55 0.51 0.61 0.51 0.62 0.61 0.55 0.54 0.55 0.58 0.87 0.80 0.48 0.48 1.00 KN16 0.53 0.53 0.61 0.51 0.62 0.56 0.55 0.56 0.58 0.62 0.78 0.73 0.48 0.48 0.91 1.00 KN17 0.55 0.53 0.64 0.53 0.60 0.59 0.58 0.56 0.58 0.62 0.78 0.73 0.48 0.48 0.91 0.98 1.00 KN18 0.62 0.62 0.68 0.65 0.67 0.71 0.51 0.56 0.55 0.58 0.68 0.66 0.53 0.53 0.69 0.67 0.67 1.00 KN19 0.64 0.61 0.65 0.64 0.64 0.69 0.52 0.55 0.54 0.54 0.74 0.69 0.52 0.52 0.73 0.68 0.71 0.94 1.00 KN20 0.65 0.62 0.73 0.72 0.69 0.75 0.53 0.56 0.55 0.60 0.66 0.68 0.58 0.58 0.67 0.62 0.65 0.93 0.89 36 KN20 1.00 4.4 Analysis of genetic relationships among 20 accessions Using the Sokal and Michener homology coefficients (1958) and the UPGMA method in NTSYS 2.1 software to assess the level of genetic diversity, we obtained the results shown in Table 4.4 The coefficient of genetic similarity reflected the genetic relationships among 20 accessions The closer the two genetic germplasms are in terms of genetic information, the greater their genetic coefficient and vice versa The results showed that the genetic coefficient ranged from 0.45 to 1.00, indicating that the fluctuations of these 20 accessions were relatively high and had a fairly long range of fluctuations Figure 4 Genetic relationships diagram among 20 accessions using 10 ISSR primers According to the results in Figure 4.12, it was possible to divide the 20 accessions into main groups based on an average similarity coefficient of 0.64 Group I including 10 samples: KN1, KN2, KN3, KN6, KN7, KN8, KN9, KN10, KN13, KN14 Group II consists of sampes; KN4, KN5 closely related to the similarity coefficient of 0.72 Group III consists of samples: KN11, KN12, KN15, KN16, KN17, KN18, KN19, KN20 Similarities between plants when observed over time, space, or other factors could occur due to spatial distribution and genetics, trees that live close to 37 each other are more likely to be similar than distant trees, and trees of the same species often have more common traits between them than other plants of different species (Fortin, 2005) 38 PART V CONCLUSIONS AND PROPOSALS 5.1 Conclusions A total of 20 accessions were collected from provinces/cities, described morphological traits and identified scientific name They belonged to Lorencera japonica Thumb Total DNAs of 20 accessions were isolated DNAs was qualified anf good quality for PCRs Genetic diversity of 20 accessions were analysed using the ISSR 10 primers The total number of loci produced was 85, the number of detected loci per primer ranged from to 11 loci, an average of 8.5 loci per primer with an average polymorphic locus of 7.4 loci per primer The proportion of polymorphisms of loci was from 63.64 to 100%, averaging 88.03% The PIC of the 10 ISSR primers averaged 0.34 Total of 20 accessions of Lorencea japonica were divided into main groups based on an average similarity coefficient of 0.64 Group I including 10 samples: KN1, KN2, KN3, KN6, KN7, KN8, KN9, KN10, KN13, KN14 Group II consists of samples: KN4, KN5 closely related to the similarity coefficient of 0.72 Group III consists of samples: KN11, KN12, KN15, KN16, KN17, KN18, KN19, KN20 5.2 Proposals Additional markers such as RAPD, SSR can be used to assess the genetic diversity of 20 Lonicera japonica Useful information from this study could be used for 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Japonica Flos, World Science and Technology-Modernization of Traditional Chinese Medicine, (9):1890-1895 29.Zhong-Hu Li (2018) Evolutionary Analysis of Plastid Genomes of Seven Lonicera L Species: Implications for Sequence Divergence and Phylogenetic Relationships, Int J Mol Sci 2018, 19, 4039; doi:10.3390/ijms19124039 42 Appendix: Binary data matrix UBC807 UBC810 UBC815 UBC824 KN1 KN2 KN3 KN4 KN5 KN6 KN7 KN8 KN9 KN10 KN11 KN12 KN13 KN14 KN15 KN16 KN17 KN18 KN19 KN20 0 0 0 1 0 1 0 0 0 1 0 1 1 1 1 1 1 1 0 0 0 0 1 0 1 0 0 0 0 0 0 1 0 1 1 1 1 1 1 1 1 1 1 1 1 0 1 0 0 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 0 0 1 0 1 1 0 0 0 0 0 1 0 1 1 0 0 0 1 0 1 1 0 0 1 1 0 1 0 0 0 0 1 0 0 0 1 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 0 1 1 1 0 1 0 0 0 0 0 1 0 0 1 1 0 1 0 1 1 1 1 0 0 1 0 1 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 1 1 0 1 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 1 0 0 0 0 1 0 1 1 1 1 1 1 0 0 0 1 1 1 1 1 1 0 0 0 0 0 1 1 1 1 0 1 0 1 0 0 1 1 1 43 UBC827 UBC835 UBC836 UBC842 KN1 KN2 KN3 KN4 KN5 KN6 KN7 KN8 KN9 KN10 KN11 KN12 KN13 KN14 KN15 KN16 KN17 KN18 KN19 KN20 1 1 1 1 1 1 1 1 0 1 0 0 1 0 1 0 0 0 1 0 0 1 1 1 1 1 1 0 0 1 1 0 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 1 1 1 1 1 1 1 0 1 1 0 1 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 0 1 1 1 1 1 1 1 1 1 1 0 0 0 1 1 1 1 1 1 0 0 1 1 0 1 1 0 0 0 0 1 0 1 1 1 1 0 1 0 1 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 1 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 1 1 1 0 0 0 1 0 1 0 1 0 1 1 0 1 1 1 1 1 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0 0 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 0 0 0 0 0 1 0 0 0 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 0 0 0 1 1 1 1 1 1 1 44 KN1 KN2 KN3 KN4 KN5 KN6 KN7 KN8 KN9 KN10 KN11 KN12 KN13 KN14 KN15 KN16 KN17 KN18 KN19 KN20 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 1 0 1 0 1 0 1 1 0 1 0 0 0 0 1 0 0 1 0 1 1 0 1 1 0 0 0 0 1 1 1 1 1 1 1 1 0 1 1 1 0 1 0 0 0 1 0 0 0 UBC848 0 1 1 1 1 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 0 1 0 0 0 1 0 1 1 1 1 1 0 0 0 0 0 1 0 1 1 1 0 1 0 0 1 0 1 1 1 0 1 0 0 1 1 0 1 UBC857 1 0 1 1 0 0 1 0 0 1 0 0 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 0 45