VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY ------- oOo ------- GRADUATION THESIS TITLE: RESEARCH ON GENETIC DIVERSITY OF NGOC LINH GINSENG (Panax vietnamensis Ha et Grushv.) BASED ON MORPHOLOGICAL TRAITS AND SSR MARKERS HANOI, 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY ------- oOo ------- GRADUATION THESIS TITLE: RESEARCH ON GENETIC DIVERSITY OF NGOC LINH GINSENG (Panax vietnamensis Ha et Grushv.) BASED ON MORPHOLOGICAL TRAITS AND SSR MARKERS Student name : Tran Huyen Trang Student code : 637435 Class : K63CNSHE Department : Biotechnology Supervisor : Associate Professor Dr. Le Hung Linh Dr. Nguyen Thi Thuy Hanh HANOI, 2022 COMMITMENT I hereby declare that this is my research project under the guidance of Associate Professor Dr. Le Hung Linh - Department of Molecular Biology, Institute of Agricultural Genetics and Dr.
Nguyen Thi Thuy Hanh - Department of Biology, Vietnam National University of Agriculture. The results and data in this thesis are truthful have not been published by anyone in any way. I confirm that all information and data from articles and sources of other authors contain full citations and references from official sources. I take full responsibility for this guarantee.
Hanoi, 05th December, 2022 Student Tran Huyen Trang i ACKNOWLEGEMENTS During the process of studying, researching and completing the thesis, I have received the help of many individuals. First, I would like to express my thanks to the lecturer in the Faculty of Biotechnology of the Vietnam National University of Agriculture for their knowledge and dedication to conveying valuable knowledge to me during my time studying at the university. Secondly, I am using this opportunity to express my deep and sincere gratitude to Associate Professor Dr. Le Hung Linh - Department of Molecular Biology, Institute of Agricultural Genetics to and Dr.
Nguyen Thi Thuy Hanh - Department of Biology, Vietnam National University of Agriculture, who took time out of their hectic schedules to listen, guide, and keep me on the right track. Their passionate guidance aided me throughout the research and writing of this thesis. I am also grateful to the Department of Molecular Biology, Institute of Agricultural Genetics, for technical support during implementation. I would not forget to express special thanks to my Dr.
Khuat Thi Mai Luong and Ms. Nguyen Thi Thuy Ngoan whose guidance, superior knowledge, and experience supported me during my study time at the thesis. Last but not least, I am grateful to my family and my friends for supporting me spiritually throughout writing this thesis and my life in general I sincerely thank you! Hanoi, 05th December, 2022 Student Tran Huyen Trang ii CONTENTS COMMITMENT .ii LIST OF TABLES. v LIST OF FIGURES.
v LIST OF ABBREVIATIONS .1 Introduction about Panax genus .2 Introduction about Ngoc Linh ginseng .1 The original of Ngoc Linh ginseng .2 Natural features of Ngoc Linh ginseng .3 Vegetable traits, growth, and development of ginseng Ngoc Linh.4 Evaluation of Genetic Diversity of Plant Species – Ginseng. MATERIAL AND METHODS .1 Evaluation of morphological traits of Ngoc Linh Ginseng .2 Building a morphological table of Ngoc Linh Ginseng .3 Build a phylogenetic tree based on the data encryption table and UPGMA subgroup type .4 Evaluation of genetic relationships of 24 Ngoc Linh ginseng samples by molecular markers. DISCUSSION AND SUGGESTION. 45 iv LIST OF TABLES Table 1.1: Panax species in the world .1: List SSR markers .2: PCR reaction components .3: 8% Polyacrylamide gel components.1: Classification of morphology describing and Encoding table of Ngoc Linh ginseng leaves over 6 years-old .2: The morphological evaluation traits of 3 subgroups in 120 individuals of Ngoc Linh ginseng .3: Total band and polymorphisms of 8 SSR markers .4: Summary of the results of evaluating the homology between 24 samples of Ngoc Linh ginseng.
38 v LIST OF FIGURES Figure 3. Leaves and petioles color of Ngoc Linh ginseng. Inflorescence morphology and inflorescence bloom .3: Ngoc Linh ginseng seed. Shape of seeds of Ngoc Linh ginseng .5: Phylogenetic tree showing the relationship of 120 individuals of Ngoc Linh ginseng based on Euclidean distance.6 : The results of a survey of 28 molecular markers in two representatives of Ngoc Linh ginseng.7 : Results PCR of 24 samples of Ngoc Linh ginseng using 8 primer, 1kb ladder.
Aphylogenetic tree describes the genetic diversity relationship between24samples of Ngoc Linh ginseng. 35 vi LIST OF ABBREVIATIONS Abbreviation Abbreviation PCR Polymerase chain reaction SSR Simple-sequence repeat µl Microliter ml Milliliter bp Base pairs DNA Deoxyribonucleic Acid vii ABSTRACT The most recently identified Panax species is Ngoc Linh Ginseng (Panax vietnamensis Ha et Grushv). Plants are renowned in Vietnam for their medical properties and are primarily employed in medicine (Le et al. Ngoc Linh ginseng has been overexploited and is practically extinct in the wild due to its beneficial effects on human health and high commercial worth.
The genetic diversity of Ngoc Linh ginseng was assessed using morphological traits and SSR markers in this study. This is to maintain, select, and produce new kinds as well as proper propagation methods to satisfy the needs of the pharmaceutical processing industry's development. The first step was to evaluate 15 morphological traits of 120 Ngoc Linh ginseng individuals, create a trait coding table in Excel, and create a taxonomy tree in NTSYSpc 2. The second step, 24 individuals were chosen based on the stem length, petiole length, and flower stalk length parameters.
The genetic diversity of the 24 Ngoc Linh ginseng samples was assessed using eight SSR molecular markers in the second stage. This suggests that some samples had morphological and genetic similarities. Keywords: Ngoc Linh ginseng, morphological traits, genetic diversity, molecular marker viii INTRODUCTION Panax vietnamensis (Ngoc Linh ginseng) is a rare medicinal plant found primarily in the Ngoc Linh mountain region. The plant can be found in the provinces of Kon Tum and Quang Nam.
Because of its great economic importance, the two municipalities have suggested numerous systems and strategies in recent years to protect and develop this valuable ginseng. The investigation of the diversity of Ngoc Linh ginseng populations in the original seed garden is a critical component in determining the homogeneity and purity of the seed source (Kim et al. 2003) or the diversity of genetic resources for work to generate new kinds (Jo et al. The first stage in assessing the genetic diversity of Ngoc Linh ginseng populations in the original seed garden is to evaluate their physical traits.
Ginseng species, on the other hand, have tremendously diverse and complex morphologies (Choi, 2008). Furthermore, based on the culture technique and natural conditions, the morphological qualities of ginseng plants can be easily adjusted (Bang et al. 2012; Ryu et al. Therefore, we undertake study on the topic: " Research on genetic diversity of Ngoc Linh Ginseng (Panax vietnamensis Ha et Grushv) based on morphological traits and SSR markers”.
The goal of this study is to determine genetic homogeneity, diversity of seed sources for breeding, selection of novel kinds, and development of high-quality plants for production. Objectives Evaluation genetic diversity based on morphological traits of Ngoc Linh ginseng. Using (SSR) molecular markers to evaluate the genetic diversity of Ngoc Linh ginseng. 1 Requirements Evaluation of the morphological traits of 120 samples of Ngoc Linh ginseng obtained from the location in Kon Tum, of the 15 morphological traits.
Evaluation of genetic relationships of 120 Ngoc Linh ginseng samples by morphological traits. Evaluation of genetic relationships of 24 Ngoc Linh ginseng samples by molecular markers.1 Introduction about Panax genus Panax is a perennial plant in the Araliaceae family that has been used for thousands of years for medicinal purposes. It has several species worldwide, with a concentration in Eastern Asia, the Central to Eastern Himalayan region, China, South-East Asia, and North America. The genus Panax consist 16 - 18 species (Wen 2001, Sharma & Pandit 2011.
Until now, the authors agreed that the genus Panax L., Araliaceae in the world, has 11 species and one variety (Table 1.1): Ginseng (Panax ginseng C. Mey); Panax pseudo-ginseng Wall; Panax notoginseng Chen; American ginseng (Panax quinquefolius L. May); narrow- leaf ginseng (Panax wangianus S. Sun); Panax bipinnatifidus Seem; Panax bipinnatifolius var.
Angustifolia (Burk) Wen); Panax stipuleanatus Tsai et Feng; Panax zingiberensis Wu et Feng; Panax trifoliatus L.; and Vietnamese Ginseng (Panax vietnamensis Ha et Grushv) (Nguyen Thuong Dong et al. Ginseng is classified into two types based on its geographical or commercial origin: Panax ginseng Asian and Panax quinquefolium American. The majority of the genus is found in Asia, from East to North Asia to the Himalayas, with only three species found in North America. Ginseng is classified into two types based on its geographical or commercial origin: Panax ginseng Asian and Panax quinquefolium American.
The majority of the genus is found in Asia, from East to North Asia to the Himalayas, with only three species found in North America.1: Panax species in the world No. Species name Common name Distribution 1 P. ginseng Korean/Asian ginseng Eastern Asia 2 P. notoginseng Sanqi ginseng SW China 3 P.
Major Pearl ginseng Japan 4 P. Angustifolius Narrow leaf pearlginseng Japan 5 P. Bipinnatifidus Yu-yeh-shen Japan 6 P. pseudo-ginseng Chiajen-shen Nepal/ E Himalayas 7 P.
zingiberensis Ginger shaped ginseng Eastern Asia 8 P. stipuleanatus Pingbiann Sanqi Eastern Asia 9 P. vietnamensis Vietnamese ginseng Vietnam 10 P. quinquefolius American ginseng North America 11 P.
trifolius Dwarf ginseng NE America (Source: http://www.org/browse/A/Araliaceae/Panax) Ginseng Panax C. Meyer, a perennial plant in the Araliaceae family, is a valuable medicinal plant that has been utilized for thousands of years. It is the most grown, studied, and costly Panax species P. ginseng, often known as Korean ginseng, is categorized into three varieties based on age and processing, namely fresh ginseng, white ginseng, and red ginseng.
Each type can be found in a range of ginseng products, including fresh sliced, powder, juice, liquid extract (tincture or boiling extract), infusion, tablet, and capsule. ginseng has been shown to have several therapeutic effects, including the treatment and or prevention of several disorders of the central nervous, immunological, endocrine, and cardiovascular systems, presenting immunomodulatory, antihypertensive, anti- inflammatory, antiallergic, antiatherosclerotic, antidiabetic and anticarcinogenic actions (Christensen, 2008). The presence of triterpenoid saponins known as ginsenosides, which are present in all Panax species, is responsible for its 2 pharmacological and biological actions. Ginsenosides differ in chemical structure and composition depending on the species, resulting in varied therapeutic benefits.
(Christensen, 2008) Vietnam has a tropical monsoon climate that is hot and humid all year, with several sub-terrains and climates. As a result, Vietnam boasts a rich species and distribution of valuable medicinal plants. Among the Panax species that grow naturally in Vietnam are the following: Vu Diep Ginseng (P., Lai Chau ginseng (P. fuscidiscus), and Ngoc Linh ginseng (Panax vietnamensis Ha et Grushv.
Vu Diep ginseng and P. stipuleanatus are widely distributed on the Hoang Lien Son mountain in two provinces Lao Cai and Lai Chau. Ngoc Linh Ginseng grows on Ngoc Linh Mountain in Kon Tum and Quang Nam provinces, while Lai Chau Ginseng is only found in Lai Chau Province. All of them are valued not only for their monetary worth, but also for their medicinal and health-promoting properties.2 Introduction about Ngoc Linh ginseng 1.1 The original of Ngoc Linh ginseng When it comes to ginseng production and processing, South Korea, the United States, Japan, and China are the most well-known.
It is in Vietnam. This ginseng species has been classified as one of the world's five most valuable ginseng species. The product is Ngoc Linh ginseng, a national treasure that is intended to vault Vietnam into the elite realm of a "ginseng powerhouse". Ngoc Linh ginseng is the world's 18th type of ginseng.
Prior to its scientific discovery, Ngoc Linh ginseng was used by central ethnic minorities in Vietnam, particularly the Xe Dang nation, as a forest root known as a pistol wormwood or a hidden medicinal plant to treat a variety of maladies using traditional treatments.