VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FALCULTY OF BIOTECHNOLOGY GRADUATION THESIS SUBJECT: “GENETIC DIVERSITY ANALYSIS OF 15 CYCLOCODON SPP. ACCESSIONS BY RAPD MARKERS” Hanoi – 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FALCULTY OF BIOTECHNOLOGY GRADUATION THESIS SUBJECT: “GENETIC DIVERSITY ANALYSIS OF 15 CYCLOCODON SPP. ACCESSIONS BY RAPD MARKERS” Student: Dao Ngoc Hai Student Code: 620485 Class: K62CNSHE Faculty: Biotechnology Supervisor: Dinh Truong Son , PhD. Hanoi – 2022 COMMITMENT I guarantee that the graduate thesis: “Genetic diversity analysis of Cyclocodon spp.
accessions by RAPD markers” is my own research wich is done with the instruction of Mr. Dinh Truong Son, PhD. The parts where I use references in the thesis have been mentioned in the references section. The data and results presented in the thesis are completely honest.
If wrong, I will take responsibility and all the discipline that the faculty and the academy offer./2022 Student Dao Ngoc Hai i ACKNOWLEDGEMENT In order to complete this graduation thesis, in addition to my own efforts, i would like to express my sincere thanks to: Thanks to the Vietnam National University of Agriculture for supporting me about facilities with modern library system, diverse types of documents that are convenient for my research. I would like to express my deepest gratitude to my instructor, Mr. Dinh Truong Son, whose sincerity and encouragement I will never forget. This thesis would not have been possible without Mr.
Dinh Truong Son, whose guidance from the initial step in research enabled me to develop an understanding of the subject. I am thankful for the extraordinary experiences he arranged for me. It is an honor to learn from Mr. Dinh Truong Son.
I would also like to thank some students at the Department of Plant Biotechnology, Faculty of Biotechnology, Vietnam National University of Agriculture for creating favorable conditions for me to complete this thesis. Finally, i look forward to receiving comments and suggestions from the teachers to make my graduation thesis more complete./2022 Student Dao Ngoc Hai ii CONTENT COMMITMENT. iii LIST OF ABBREVIATIONS. v LIST OF TABLES.
vi LIST OF FIGURES. Rationale of the Study. Researches on Cyclocodon spp. RAPD and the applications of RAPD in genetic diversity.
Molecular markers/DNA markers. Randomly amplified polymorphic DNA (RAPD). Some Researches on genertic diversity using RAPD. MATERIALS AND METHODS.
Materials, Chemicals, tools and equipments used in the research. Chemicals, tools and equipment used in the research. Contents and Methods. RESULTS AND DISCUSSION.
DNA extraction results. Results of using RAPD molecular markers in genetic diversity analysis. CONCLUSIONS AND RECOMMENDATIONS. 37 iv LIST OF ABBREVIATIONS CTAB Cetyltrimethylammonium bromide DNA Deoxyribonucleic acid RNA Ribonucleic acid EDTA Ethylendiamin Tetraacetic Acid et al.
et alii (Latin), and others ml Milliliter PCR Polymerase chain reaction PVPP Polyvinylpyrrolidone RAPD Random Amplified Polymorphic DNA TAE Tris-acetate EDTA °C Degree Celsius % Percent spp. Species v LIST OF TABLES Table 3. 15 accessions of Cyclocodon spp. List of chemicals used in the study.
List of RAPD primers used in the research. Components of each PCR reaction. Spectrophotometric results of extracted samples. Polymorphism among 15 Cyclocodon spp.
accessions revealed by RAPD markers. PIC values and Rp values of RAPD Primers. Matrix of genetic similarity among 15 Cyclocodon spp. accessions revealed by RAPD markers calculated by similarity coefficient of Sokal-Michener.
31 vi LIST OF FIGURES Figure 2. characteristics and distributions. Results of electrophoresis with primer OPB-02. UPGMA cluster analysis of 15 Cyclocodon spp.
accessions with a similarity coefficient of RAPD marker. PCA analysis of 15 Cyclocodon spp. accessions based on RAPD markers. Rationale of the Study From ancient times to the present, medicinal plants have always played an important role in maintaining the health and well-being of human communities around the world.
Vietnam is fortunate to be located in the tropical monsoon belt with ¾ of the area being mountainous, stretching from north to south. Such natural conditions give our country a rich and diverse forest ecosystem. According to statistics, our country has nearly 12,000 species of vascular plants belonging to more than 2,256 genera, 305 families. Not only playing the role of a green lung to regulate the climate, the forest also brings a great potential for medicinal plant resources.
Medicinal plants are widely distributed throughout Vietnam with 8 key regions: Northwest, Northeast, Red River Delta, North Central, East Truong Son, South Central Coast, Central Highlands, Southeast and Mekong River Delta. Among the announced species, there are many species classified as rare such as: Panax vietnamensis, Tam That Hoang, Bach Hop, Cyclocodon spp. are relatively rare genetic resource in Vietnam. The root is used as a tonic and wine yeast.
Young leaves are edible vegetables. In areas where they are distributed, fewer and fewer individuals have been found. Although they are only recently exploited to a limited extent, deforestation for cultivation has directly damaged their habitat. In Vietnam, although studies on medicinal plants have been conducted early, the genetic diversity of Cyclocodon spp.
is not yet analyzed. Therefore, I decided to do this research. Research Objective Determination of genetic diversity of the 15 collected Cyclocodon spp. accessions using the RAPD primers.
Research Requirements • Determine the appropriate DNA extraction procedure, ensuring the suitable DNA quality for PCR reaction. • Determine the PCR procedure with RAPD marker for highly repeatable, clear DNA bands. • Evaluation of the genetic diversity of the 15 Cyclocodon accessions. Origin Cyclocodon spp.
belongs to the Campanulaceae family, Asterales order. • Subfamilia: Campanuloideae • Species: C. Distributions It is distributed mainly in: Assam, Bangladesh, Borneo, Cambodia, China South-Central, China Southeast, East Himalaya, Hainan, India, Japan, Jawa, Laos, Malaya, Maluku, Myanmar, Nansei-shoto, Nepal, New Guinea, Philippines, Sulawesi, Sumatera, Taiwan, Thailand, Tibet, Vietnam. Characteristics Cyclocodon spp.
are perennial or annual herbs that grow erect or ascending. The Stems mostly branched. Leaves are opposite, rarely whorled. Flowers solitary, terminal or axillary, or in dichasium, perfect; bracteoles present, filiform or leaflike, or absent.
Calyx partly adnate to or completely free from ovary, and thus epigynous or hypogynous for ovary; lobes 4-6, from subentire to branched. Corolla epigynous, tubular, 4-6-merous. Stamens 4-6; filaments dilated below, glabrous or ciliate at dilated part. Ovary 3-6-locular; stigma 4-6-fid; ovules exceptionally abundant; ovary inferior for corolla yet semi-inferior to superior for calyx.
Berry-like fruit. Seeds are abundant and subglobose. characteristics and distributions 4 Figure 2. Cyclocodon Lancifolius Figure 2.
Researches on Cyclocodon spp. In the world In 1789, Jussieu and his colleagues gave the scientific name the bellflower family Campanulaceae. According to Lammers and Thomas in 2011, the bellflower family is a family of plants in the order Asterales, including 2,380 species in 84 genera. This family is mainly herbaceous or shrub.
Species usually have white latex or rarely colored. Roots fibrous or rarely tuberous. This family is widely distributed around the world but is concentrated mainly in the Northern Hemisphere. In the Southern Hemisphere, South Africa is home of many species of this family.
Species in this family do not exist in the Sahara, Antarctica and northern Greenland. In 2007, Tzu-Chao Lin et al. sequenced the ITS region to evaluate the genetic relationships of 6 species of the genus Codonopsis in China, including Cyclocodon spp. The authors have also built a Phyogenetic tree which is used in taxonomy.
Nowadays, with the development of science and technology, in addition to morphological and chemical markers, DNA markers are also used in classification. In Vietnam In 2012 Dinh Doan Long evaluated the generic diversity of some Codonopsis sp accessions by using DNA Barcoding. The study identified DNA barcodes that could distinguish species of the genus Codonopsis. Genetic diversity Genetic diversity refers to the variability of various inherited traits within a species.
In a species with high genetic variability, there would be many individuals with a wide range of different traits. The ability of a population to adapt to changing conditions is linked to genetic variety. When a highly chosen and low diversity strain, such as fish populations 6 bred for aquaculture, is transferred into a natural population, the population's capacity to adapt to changes will be reduced. RAPD and the applications of RAPD in genetic diversity 2.
Classical markers Morphological markers Seed shape, flower color, growth habit, and other critical agronomic properties may all be visually distinguished using morphological markers. Morphological markers are simple to use and do not require any special equipment. They don't necessitate any advanced biochemical or molecular techniques. Breeders have successfully used such markers in breeding programs for a variety of crops.
Main disadvantages of morphological markers are: they are limited in number, influenced by the plant growth stages and various environmental factors Humans have effectively employed numerous morphological markers to examine variation for use in plant breeding from ancient times. Cytological markers Cytological markers are markers that are associated with changes in chromosomal numbers, banding patterns, size, shape, order, and location. These variations reflect differences in euchromatin and heterochromatin distributions. For example, G bands are produced by Giemsa stain, Q bands are produced by quinacrine hydrochloride and R bands are the reversed G bands.
Normal and mutant chromosomes can be distinguished using these chromosomal landmarks. Such markers can also be used in the identification of linkage groups and in physical mapping. Biochemical markers Biochemical markers, also known as isozymes, are multi-molecular versions of enzymes that are encoded by different genes but perform the same tasks. Because they are allelic variants of enzymes, biochemical markers may 7 be used to determine gene and genotypic frequencies.
Biochemical markers have been used successfully to identify genetic diversity, population structure, gene flow, and population subdivision. They are co-dominant, simple to utilize, and cost effective. They are, however, less in number, detect less polymorphism, and are impacted by different extraction procedures, plant tissues, and plant growth stages. Molecular markers/DNA markers Molecular markers are nucleotide sequences that can be evaluated using polymorphism in the nucleotide sequences of different individuals.
These polymorphisms are caused by insertion, deletion, point mutations, duplication, and translocation; however, they do not always influence gene function. A perfect DNA marker would be co-dominant, evenly distributed throughout the genome, highly repeatable, and capable of detecting increasing levels of variation. Molecular markers are classified into various groups on the basis of: • Gene action mode (co-dominant or dominant markers); • Detection method (hybridization-based molecular markers or polymerase chain reaction (PCR)-based markers); • Transmission mode (paternal organelle inheritance, maternal organelle inheritance, bi-parental nuclear inheritance or maternal nuclear inheritance). Various forms of DNA molecular markers have been designed and effectively used in genetics and breeding operations in a variety of agricultural crops.
Randomly amplified polymorphic DNA (RAPD) Random amplification of polymorphic DNA (RAPD), also known as "rapid," is a polymerase chain reaction (PCR) that amplifies random DNA segments.The scientist using RAPD generates multiple random, short primers (8–12 nucleotides) and then performs PCR on a large template of genomic 8 DNA in the hopes that fragments will amplify. A RAPD response can produce a semi-unique profile by resolving the resulting patterns. There is no need to know the DNA sequence of the targeted genome because the primers will bind somewhere in the sequence, but it is unknown where. This makes the method effective for comparing the DNA of biological systems that have received little attention from the scientific community or systems with a small number of DNA sequences to compare (it is not suitable for forming a cDNA databank).
It has several limitations in the use of degraded DNA materials since it relies on a large, undamaged DNA template sequence. Its resolving power is significantly lower than targeted, species-specific DNA comparison approaches like short tandem repeats. In recent years, RAPD has been used to characterize and trace the phylogeny of diverse plant and animal species. Limitations • Almost all RAPD markers are dominant, which means that it is impossible to tell whether a DNA segment is amplified from a locus that is heterozygous (1 copy) or homozygous (2 copies).