Phân lập, tuyển chọn chủng vi khuẩn lactic có khả năng kháng khuẩn và định hướng ứng dụng làm giống khởi động cho lên men

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -  - GRADUATION THESIS TITLE: GENETIC DIVERSITY ANALYSIS OF 16 LOTUS VARIETIES BY DNA MOLECULAR MARKERS Hanoi - 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -  - GRADUATION THESIS GENETIC DIVERSITY ANALYSIS OF 16 LOTUS VARIETIES BY DNA MOLECULAR MARKERS Student : NGUYEN THI HUE Student’s code : 637412 Class : K63CNSHE Faculty : Biotechnology Supervisor : Dinh Truong Son, PhD Hanoi - 2022 COMMITMENT I hereby declare that the data and research results in this thesis are true and have never been used in any publication I hereby declare that any assistance in the presentation of this thesis has been received and that the sources cited have been acknowledged Hanoi, December 2022 Student Nguyen Thi Hue i ACKNOWLEDGEMENT First of all, I would like to thank Vietnam National University of Agriculture and the Faculty of Biotechnology for giving me the opportunity to join the graduation thesis Due to the education policy of the institute, I got such a useful module Even so, I am also very grateful to my supervisor, Dr Dinh Truong Son, Head of Department of Plant Biotechnology, Biotechnology Faculty, Vietnam National University of Agriculture, and others at the Department of Plant Biotechnology accompanying, helping and motivating me during my graduation thesis The teachers always give me advice and suggestions Finally, I would like to thank the K63CNSHE class members for being with me, studying, helping and being with me in the meaningful moments of this graduation thesis And also to my family for always supporting me unconditionally with everything Sincerely thank! Hanoi, December 2022 Student Nguyen Thi Hue ii INDEX COMMITMENT i ACKNOWLEDGEMENT .ii INDEX iii LIST OF ABBREVIATIONS vi LIST OF TABLES vii LIST OF FIGURES viii ABSTRACT ix I INTRODUCTION 1.1 Introduction 1.2 Purpose and requirements 1.2.1 Purpose 1.2.2 Requirements II LITERATURE REVIEWS 2.1 Research Review 2.1.1 Origin of Nelumbo spp 2.1.2 Distributions of Nelumbo spp 2.1.3 Characteristics of Nelumbo spp 2.1.4 Research on Nelumbo spp 2.1.5 Genetic diversity 2.2 RAPD, ISSR and the applications of RAPD and ISSR in genetic diversity 2.2.1 Classical markers 2.2.2 Molecular markers/DNA markers iii 2.2.3 Randomly amplified polymorphic DNA (RAPD) 2.2.4 Inter-simple sequence repeat (ISSR) .11 2.2.5 Some research on genetic diversity using RAPD and ISSR 13 III MATERIAL AND METHODS 15 3.1 Materials, chemicals, and equipment .15 3.1.1 Materials 15 3.1.2 Chemicals and equipment 16 3.1.2.1 Chemicals 16 3.1.2.2.Tools and equipment 17 3.2 Methods 18 3.2.1 DNA extraction (Masoomi-Aladizgeh et al., 2016) 18 3.2.2 The PCR 20 3.2.3 Gel electrophoresis (Sambrook et al., 1989) 20 3.2.4 Data analysis 21 IV RESULTS AND DISCUSSION 23 4.1 DNA extraction results .23 4.2 Genetic diversity of 16 lotus varieties analyzed by RAPD molecular markers 24 4.3 Genetic diversity of 16 lotus varieties analyzed by ISSR molecular markers 31 4.4 Genetic diversity of 16 lotus varieties analyzed by combined ISSR and RAPD molecular markers 37 4.5 Comparative study of RAPD- and ISSR-based results 41 iv V CONCLUSION AND PROPOSAL 44 5.1 Conclusion 44 5.2 Proposal 44 REFERENCES 45 APPENDIX 49 v LIST OF ABBREVIATIONS CTAB Cetyltrimethylammonnium bromide % Percent °C Degree Celsius DNA Deoxyribonucleic acid EDTA Ethylenediaminetetraacetic acid et al et alii (Latin), and others ISSR Inter Simple Sequence Repeat ml NTSYScp Milliliter Numerical Taxonomy and Multivariate Analysis System for personal computer PCR Polymerase Chain Reaction PVPP Polyvinylpolypyrrolidone RAPD Random Amplified Polymorphic DNA RNA Ribonucleic aicd SDS Sodium Dodecyl Sulfate spp Species TAE Tris-acetate-EDTA TE USDA Tris + Ethylenediaminetetraacetic acid United States Department of Agriculture vi LIST OF TABLES Table 3.1 16 varieties of Nelumbo spp 15 Table 3.2 List of chemicals used in the research 16 Table 3.3 List of RAPD primers used in the research 16 Table 3.4 List of ISSR primers used in the research 17 Table 3.5 Components of each PCR reaction 20 Table 3.6 PCR Procedure 20 Table 4.1 Spectrophotometric results of extracted samples 23 Table 4.2 Performance of RAPD primers in the genetic diversity analysis of 16 lotus varieties 26 Table 4.3 Matrix of genetic similarity among 16 lotus varieties revealed by RAPD markers calculated by similarity coefficient of Sokal-Michener 28 Table 4.4 Performance of ISSR primers in the genetic diversity analysis of 16 lotus varieties 33 Table 4.5 Matrix of genetic similarity among 16 lotus varieties revealed by ISSR markers calculated by similarity coefficient of Sokal-Michener 34 Table 4.6 Matrix of genetic similarity among 16 lotus varieties revealed by both (RAPD and ISSR) markers calculated by similarity coefficient of SokalMichener 40 Table 4.7 Comparison of the discriminating capacity of RAPD and ISSR of 16 lotus varieties 42 Table 4.8 Matrix comparisons of Mantel test / Two-tailed test between markers 43 vii LIST OF FIGURES Figure 2.1 Distribution of Nelumbo lutea and Nelumbo nucifera (modified according to SNIGIREVSKAYA, 1964) (Đorđević-Milutinović et al., 2018) Figure 2.2 Nelumbo spp A Floating and emergent leaves, young rolled up leaf that began to unfold, flower and fruit in natural habitat B Flotant leaf detail C Emergent leaf detail.(Đorđević-Milutinović et al., 2018) Figure 2.3 Schematic illustration of random amplified length polymorphism analysis.(Das and Dash, 2018) 10 Figure 2.4 Principles of the amplification of DNA with a single oligo-nucleotide primer in ISSR marker system DNA segments delimited by the inverted simple sequence repeats (SSR) (Individual and 2) are amplified with a single ISSR primer (green) ISSR Variation that may result from insertion or deletion (Red mark) in different individuals produces PCR fragments of different sizes (blue) of the segment (Meheret et al.) 12 Figure 4.1 Results of the electrophoresis with primers OPN01 24 Figure 4.2 UPGMA cluster analysis of 16 varieties lotus with a similarity coefficient of RAPD marker Dash line indicates the mean similarity (0.792) The dendogram distributed the 16 lotus varieties in four indicated groups (I,II,III,IV) 30 Figure 4.3 PCA analysis of 16 lotus varieties based on RAPD 30 Figure 4.4 UPGMA cluster analysis of 16 varieties lotus with a similarity coefficient of ISSR marker Dash line indicates the mean similarity (0.833) The dendogram distributed the 16 lotus varieties in four indicated groups (I, II, III, IV) 36 Figure 4.5 PCA analysis of 16 lotus varieties based on ISSR 37 Figure 4.6 UPGMA cluster analysis of 16 varieties lotus with a similarity coefficient of both RAPD and ISSR marker Dash line indicates the mean similarity (0.817) The dendogram distributed the 16 lotus varieties in four indicated groups (I,II,III,IV) 38 viii REFERENCES Anderson J.A., Churchill G., Autrique J., Tanksley S and Sorrells M (1993) Optimizing parental selection for genetic linkage maps Genome Vol 36 pp 181-186 Bardakci F (2001) Random amplified polymorphic DNA (RAPD) markers Turkish Journal of Biology Vol 25 pp 185-196 Belguith H., Fattouch S., Jridi T and Ben H.J (2013) Immunopurification and characterization of a rape (Brassica napus L.) seedling lipase African Journal of Biotechnology Vol 12 pp Caballero B., Trugo L.C and Finglas P.M (2003) Encyclopedia of food sciences and nutrition: Academic Chase M.W., Byng J., Christenhusz M., Fay M., Judd W., Mabberley D., Sennikov A., Soltis D., Soltis P.S and Stevens P.F (2016) An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG IV [" Botanical Journal of the Linnean Society"] Vol 181 pp Chesnokov Y.V., Kosolapov V and Savchenko I (2020) Morphological Genetic Markers in Plants Russian Journal of Genetics Vol 56 pp 1406-1415 Dakin E and Avise J (2004) Microsatellite null alleles in parentage analysis Heredity Vol 93 pp 504-509 Das S and Dash H.R (2018) Microbial diversity in the genomic era: Academic Press Diao Y., Chen L., Yang G., Zhou M., Song Y., Hu Z and Liu J.Y (2006) Nuclear DNA C-values in 12 species in Nymphaeales Caryologia Vol 59 pp 25-30 10 Đorđević-Milutinović D., Stevanović B and Ćulafić G (2018) Nelumbo protospeciosa SAPORTA 1891 from the Berane Basin (Lower Miocene) Geoloski anali Balkanskoga poluostrva Vol 79 pp 41-55 11 Gandolfo M.A and Cuneo R.N (2005) Fossil Nelumbonaceae from the La Colonia Formation (Campanian–Maastrichtian, Upper Cretaceous), Chubut, Patagonia, Argentina Review of Palaeobotany and Palynology Vol 133 pp 169-178 12 Gilbert J., Lewis R., Wilkinson M and Caligari P (1999) Developing an appropriate strategy to assess genetic variability in plant germplasm collections Theoretical and applied Genetics Vol 98 pp 1125-1131 13 Gowthami R., Sharma N., Pandey R and Agrawal A (2021) A model for integrated approach to germplasm conservation of Asian lotus (Nelumbo nucifera Gaertn.) Genetic Resources and Crop Evolution Vol 68 pp 1269-1282 14 Gui S., Peng J., Wang X., Wu Z., Cao R., Salse J., Zhang H., Zhu Z., Xia Q and Quan Z (2018) Improving Nelumbo nucifera genome assemblies using high‐resolution genetic maps and BioNano genome mapping reveals ancient chromosome rearrangements The Plant Journal Vol 94 pp 721-734 15 Guo H., Li S., Peng J and Ke W (2007) Genetic diversity of Nelumbo accessions revealed by RAPD Genetic Resources and Crop Evolution Vol 54 pp 741-748 16 Guo X and Elston R (1999) Linkage information content of polymorphic genetic markers Human heredity Vol 49 pp 112-118 17 Han Y.-C., Teng C.-Z., Chang F.-H., Robert G.W., Zhou M.-Q., Hu Z.-L and Song Y.-C (2007a) Analyses of genetic relationships in Nelumbo nucifera using nuclear ribosomal ITS sequence data, ISSR and RAPD markers Aquatic Botany Vol 87 pp 141-146 18 Han Y.-C., Teng C.-Z., Zhong S., Zhou M.-Q., Hu Z.-L and Song Y.-C (2007b) Genetic variation and clonal diversity in populations of Nelumbo nucifera 45 (Nelumbonaceae) in central China detected by ISSR markers Aquatic Botany Vol 86 pp 69-75 19 Hess J., Kadereit J and Vargas P (2000) The colonization history of Olea europaea L in Macaronesia based on internal transcribed spacer (ITS‐1) sequences, randomly amplified polymorphic DNAs (RAPD), and intersimple sequence repeats (ISSR) Molecular Ecology Vol pp 857-868 20 Hsu J (1983) Late Cretaceous and Cenozoic vegetation in China, emphasizing their connections with North America Annals of the Missouri Botanical Garden Vol pp 490-508 21 Ismail A., Mosa K.A., Ali M.A and Helmy M (2020) Biochemical and Molecular Markers: Unraveling Their Potential Role in Screening Germplasm for Thermotolerance Heat Stress Tolerance in Plants: Physiological, Molecular and Genetic Perspectives Vol pp 47-76 22 Jarne P and Lagoda P.J (1996) Microsatellites, from molecules to populations and back Trends in ecology & evolution Vol 11 pp 424-429 23 Jia-Qing Q (2002) Cardiovascular pharmacological effects of bisbenzylisoquinoline alkaloid derivatives Acta Pharmacol Sin Vol 23 pp 1086-1092 24 Kesawat M.S and Das Kumar B (2009) Molecular markers: it’s application in crop improvement Journal of Crop Science and Biotechnology Vol 12 pp 169-181 25 Kubo N., Hirai M., Kaneko A., Tanaka D and Kasumi K (2009) Development and characterization of simple sequence repeat (SSR) markers in the water lotus (Nelumbo nucifera) Aquatic Botany Vol 90 pp 191-194 26 Les D.H., Garvin D.K and Wimpee C.F (1991) Molecular evolutionary history of ancient aquatic angiosperms Proceedings of the National Academy of Sciences Vol 88 pp 10119-10123 27 Li W., Shen J and Wang J (2005) Genetic diversity of the annual weed Monochoria vaginalis in southern China detected by random amplified polymorphic DNA and inter‐simple sequence repeat analyses Weed research Vol 45 pp 424-430 28 LI Y., Svetlana P., YAO J and LI C (2014) A review on the taxonomic, evolutionary and phytogeographic studies of the lotus plant (Nelumbonaceae: Nelumbo) Acta Geologica Sinica‐English Edition Vol 88 pp 1252-1261 29 Li Z., Liu X., Gituru R.W., Juntawong N., Zhou M and Chen L (2010) Genetic diversity and classification of Nelumbo germplasm of different origins by RAPD and ISSR analysis Scientia horticulturae Vol 125 pp 724-732 30 Lim T.K (2012) Edible medicinal and non-medicinal plants: Springer 31 Mantel N (1967) The detection of disease clustering and a generalized regression approach Cancer research Vol 27 pp 209-220 32 Masoomi-Aladizgeh F., Jabbari L., Nekouei R.K and Aalami A (2016) A simple and rapid system for DNA and RNA isolation from diverse plants using handmade kit Vol pp 33 Mattioni C., Casasoli M., Gonzalez M., Ipinza R and Villani F (2002) Comparison of ISSR and RAPD markers to characterize three Chilean Nothofagus species Theoretical and applied Genetics Vol 104 pp 1064-1070 34 Meheret F., Tesfaye K., Haileselassie T and Abate T Genetic Diversity Study of Brachiaria brizantha (A Rich) Stapf Collected from Ethiopia Using Inter-Simple Sequence Repeat (ISSR) Markers Vol pp 35 Ming R., VanBuren R., Liu Y., Yang M., Han Y., Li L.-T., Zhang Q., Kim M.-J., Schatz M.C and Campbell M (2013) Genome of the long-living sacred lotus (Nelumbo nucifera Gaertn.) Genome biology Vol 14 pp 1-11 46 36 Moreno S., Martín J.P and Ortiz J.M (1998) Inter-simple sequence repeats PCR for characterization of closely related grapevine germplasm Euphytica Vol 101 pp 117125 37 Moulin M.M., Rodrigues R., Gonỗalves L.S.A., Sudrộ C.P and Pereira M.G (2012) A comparison of RAPD and ISSR markers reveals genetic diversity among sweet potato landraces (Ipomoea batatas (L.) Lam.) Acta Scientiarum Agronomy Vol 34 pp 139-147 38 Mukherjee P.K., Saha K., Das J., Pal M and Saha B (1997) Studies on the antiinflammatory activity of rhizomes of Nelumbo nucifera Planta medica Vol 63 pp 367-369 39 Nonić M and Šijačić-Nikolić M (2021) Genetic diversity: sources, threats, and conservation Life on Land Vol pp 421-435 40 Poltronieri P and Hong Y (2015) Applied plant genomics and biotechnology: Woodhead Publishing 41 Prevost A and Wilkinson M (1999) A new system of comparing PCR primers applied to ISSR fingerprinting of potato cultivars Theoretical and applied Genetics Vol 98 pp 107-112 42 Roy S., Anwar A., Tabassum N and Islam M.N (2022) Genotypic Variability Analysis of Lotus (Nelumbo nucifera Gaertn.) from Bangladesh using RAPD and SSR Markers Plant Tissue Culture and Biotechnology Vol 32 pp 77-87 43 Sambrook J., Fritsch E.F and Maniatis T (1989) Molecular cloning: a laboratory manual: Cold spring harbor laboratory press 44 Seymour R.S and Schultze-Motel P (1996) Thermoregulating lotus flowers Nature Vol 383 pp 305-305 45 Sharma A and Sharma A (2014) Chromosome techniques: theory and practice: Butterworth-Heinemann 46 Shen-Miller J (2002) Sacred lotus, the long-living fruits of China Antique Seed Science Research Vol 12 pp 131-143 47 Sinha S., Mukherjee P.K., Mukherjee K., Pal M., Mandal S.C and Saha B (2000) Evaluation of antipyretic potential of Nelumbo nucifera stalk extract Phytotherapy Research: An International Journal Devoted to Pharmacological and Toxicological Evaluation of Natural Product Derivatives Vol 14 pp 272-274 48 Siragusa M., De Pasquale F., Abbate L and Tusa N (2006) Identification of sour orange accessions and evaluation of their genetic variability by molecular marker analyses HortScience Vol 41 pp 84-89 49 Sokal R.R (1958) A statistical method for evaluating systematic relationships Univ Kansas, Sci Bull Vol 38 pp 1409-1438 50 Souframanien J and Gopalakrishna T (2004) A comparative analysis of genetic diversity in blackgram genotypes using RAPD and ISSR markers Theoretical and applied Genetics Vol 109 pp 1687-1693 51 Trang N.T.Q., Thao T.T.H and Hong H.T.K (2019) Study on the anatomical morphology of lotus varieties (Nelumbo nucifera gaertn.) in Vietnam Plant cell biotechnology and molecular biology Vol 20 pp 95-105 52 Tytgat O., Gansemans Y., Weymaere J., Rubben K., Deforce D and Van Nieuwerburgh F (2020) Nanopore sequencing of a forensic STR multiplex reveals loci suitable for single-contributor STR profiling Genes Vol 11 pp 381 53 Verma A and Singh A (2013) Animal biotechnology: Models in discovery and translation: Academic Press 47 54 Wang K., Deng J., Damaris R.N., Yang M., Xu L and Yang P (2015) LOTUS-DB: an integrative and interactive database for Nelumbo nucifera study Database Vol 2015 pp 55 Wang Q and Zhang X (2005) Colored illustration of lotus cultivars in China China Forestry Publishing House Vol pp 56 Wang Y., Fan G., Liu Y., Sun F., Shi C., Liu X., Peng J., Chen W., Huang X and Cheng S (2013) The sacred lotus genome provides insights into the evolution of flowering plants The Plant Journal Vol 76 pp 557-567 57 Wolfe A.D., XIANG Q.Y and Kephart S.R (1998) Assessing hybridization in natural populations of Penstemon (Scrophulariaceae) using hypervariable intersimple sequence repeat (ISSR) bands Molecular Ecology Vol pp 1107-1125 58 Xiuqiang H and Junyu C (1992) Preliminary studies on biosystematic relationship between the two Nelumbo species Acta Horticulturae Sinica (China) Vol pp 59 Xue J., Zhuo L and Zhou S (2006) Genetic diversity and geographic pattern of wild lotus (Nelumbo nucifera) in Heilongjiang Province Chinese Science Bulletin Vol 51 pp 421-432 60 Yuping Z., Meilin C., Xiaodong W and Benmei X (1998) RAPD analysis of germplasm in ancient “Taizi lotus” and modern Chinese lotus Acta Botanica Sinica Vol 40 pp 163-168 61 Zhang X., Ran J., Lian T., Li Z., Yang C., Jiang X., Du H., Cui Z and Liu Y.P (2019a) The single nucleotide polymorphisms of myostatin gene and their associations with growth and carcass traits in Daheng broiler Brazilian Journal of Poultry Science Vol 21 pp 62 Zhang Y., Lu X., Zeng S., Huang X., Guo Z., Zheng Y., Tian Y and Zheng B (2015) Nutritional composition, physiological functions and processing of lotus (Nelumbo nucifera Gaertn.) seeds: a review Phytochemistry Reviews Vol 14 pp 321-334 63 Zhang Y., Nyong'A T.M., Shi T and Yang P (2019b) The complexity of alternative splicing and landscape of tissue-specific expression in lotus (Nelumbo nucifera) unveiled by Illumina-and single-molecule real-time-based RNA-sequencing DNA research Vol 26 pp 301-311 64 Zhou P., Jin Q., Qian P., Wang Y., Wang X., Jiang H., Yao D., Liu X., Liu F and Li J (2022) Genetic resources of lotus (Nelumbo) and their improvement Ornamental Plant Research Vol pp 1-16 48 APPENDIX ELECTROPHORESIS RESULTS OF THE PCR PRODUCTS Electrophoresis results of the PCR products with UBC891 Primer Electrophoresis results of the PCR products with UBC889 Primer 49 Electrophoresis results of the PCR products with UBC888 Primer Electrophoresis results of the PCR products with UBC864 Primer Electrophoresis results of the PCR products with UBC845 Primer 50 Electrophoresis results of the PCR products with UBC836 Primer Electrophoresis results of the PCR products with UBC835 Primer Electrophoresis results of the PCR products with UBC826 Primer 51 Electrophoresis results of the PCR products with UBC820 Primer Electrophoresis results of the PCR products with UBC815 Primer Electrophoresis results of the PCR products with UBC808 Primer 52 Electrophoresis results of the PCR products with UBC810 Primer Electrophoresis results of the PCR products with ISSR13 Primer Electrophoresis results of the PCR products with ISSR6 Primer 53 Electrophoresis results of the PCR products with ISSR5 Primer Electrophoresis results of the PCR products with ISSR4 Primer Electrophoresis results of the PCR products with ISSR3 Primer 54 Electrophoresis results of the PCR products with ISSR1 Primer Electrophoresis results of the PCR products with OPS10 Primer Electrophoresis results of the PCR products with APD18 Primer 55 Electrophoresis results of the PCR products with APG05 Primer Electrophoresis results of the PCR products with APH18 Primer Electrophoresis results of the PCR products with OPA05 Primer 56 Electrophoresis results of the PCR products with OPA18 Primer Electrophoresis results of the PCR products with OPC01 Primer Electrophoresis results of the PCR products with OPC03 Primer 57 Electrophoresis results of the PCR products with OPN01 Primer Electrophoresis results of the PCR products with OPS05 Primer Electrophoresis results of the PCR products with OPS08 Primer 58 Electrophoresis results of the PCR products with OPA03 Primer 59