Research on genetic diversity of ngoc linh ginseng (panax vietnamensis ha et grushv) based on morphological traits and ssr markers

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY --- oOo --- GRADUATION THESIS TITLE: RESEARCH ON GENETIC DIVERSITY OF NGOC LINH GINSENG Panax vietnamensis Ha et

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

HANOI, 2022

Student name Student code Class Department

: Tran Huyen Trang : 637435

: K63CNSHE : Biotechnology Supervisor : Associate Professor Dr Le Hung Linh

Dr Nguyen Thi Thuy Hanh

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, 05thDecember, 2022

Student

Tran Huyen Trang

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

CONTENTS

COMMITMENT i

ACKNOWLEGEMENTS ii

LIST OF TABLES v

LIST OF FIGURES v

LIST OF ABBREVIATIONS vii

ABSTRACT viii

INTRODUCTION 1

PART I LITERATURE REVIEW 1

1.1 Introduction about Panax genus 1

1.2 Introduction about Ngoc Linh ginseng 3

1.2.1 The original of Ngoc Linh ginseng 3

1.2.2 Natural features of Ngoc Linh ginseng 4

1.2.3 Vegetable traits, growth, and development of ginseng Ngoc Linh 6

1.2.4 Evaluation of Genetic Diversity of Plant Species – Ginseng 9

PART 2 MATERIAL AND METHODS 14

2.1 Material 14

2.2 Methods 14

2.2.1 Morphological Evaluation 14

2.2.2 Molecular Method 14

2.2.3 Phylogenetic trees 19

PART 3 RESULTS 19

3.1 Evaluation of morphological traits of Ngoc Linh Ginseng 20

3.2 Building a morphological table of Ngoc Linh Ginseng 23

3.3 Build a phylogenetic tree based on the data encryption table and UPGMA subgroup type 29

3.4 Evaluation of genetic relationships of 24 Ngoc Linh ginseng samples by molecular markers 31

PART 4 DISCUSSION AND SUGGESTION 39

DISCUSSION 44

SUGGESTION 39

REFERENCES 40

APPENDIX 45

LIST OF TABLES

Table 1.1: Panax species in the world 2

Table 2.1: List SSR markers 16

Table 2.2: PCR reaction components 17

Table 2.3: 8% Polyacrylamide gel components 18

Table 3.1: Classification of morphology describing and Encoding table of Ngoc Linh ginseng leaves over 6 years-old 23

Table 3.2: The morphological evaluation traits of 3 subgroups in 120 individuals of Ngoc Linh ginseng 30

Table 3.3: Total band and polymorphisms of 8 SSR markers 34

Table 3.4: Summary of the results of evaluating the homology between 24 samples of Ngoc Linh ginseng 38

LIST OF FIGURES

Figure 3.1 Leaves and petioles color of Ngoc Linh ginseng 21

Figure 3.2 Inflorescence morphology and inflorescence bloom 22

Figure 3.3: Ngoc Linh ginseng seed 23

Figure 3.4 Shape of seeds of Ngoc Linh ginseng 23

Figure 3.5: Phylogenetic tree showing the relationship of 120 individuals of Ngoc Linh ginseng based on Euclidean distance A, B, C are subgroups 29

Figure 3.6 : The results of a survey of 28 molecular markers in two representatives of Ngoc Linh ginseng 32

Figure 3.7 : Results PCR of 24 samples of Ngoc Linh ginseng using 8 primer, 1kb ladder 33

Figure 3.8 Aphylogenetic tree describes the genetic diversity relationship between24samples of Ngoc Linh ginseng 35

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 2015) 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 2.1 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

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 2015) 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 2012)

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

PART I LITERATURE REVIEW

1.1 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 A Mey); Panax pseudo-ginseng Wall; Panax notoginseng Chen; American ginseng (Panax quinquefolius L.); Japanese Ginseng (Panax japonicus) A May); narrow- leaf ginseng (Panax wangianus S C 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 2007) 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

Table 1.1: Panax species in the world

3 P japonicus var Major Pearl ginseng Japan

4 P japonicus var Angustifolius Narrow leaf pearlginseng Japan

5 P.japonicus var 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

10 P quinquefolius American ginseng North America

(Source: http://www.theplantlist.org/browse/A/Araliaceae/Panax)

Ginseng Panax C.A 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 P 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

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 bipinnatifidus), P., Lai Chau ginseng (P vietnamensis var fuscidiscus), and Ngoc Linh ginseng (Panax vietnamensis Ha et Grushv.) (Le Thanh Son and Nguyen Tap, 2006) 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

1.2 Introduction about Ngoc Linh ginseng

1.2.1 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 According to research results from 1978 by the Ministry of Health of Vietnam, the rhizome of Ngoc Linh ginseng in Vietnam contains 26 saponin compounds

with known chemical structures and 24 new saponins that are not found in other types of ginseng The Korean ginseng contains approximately 25 saponins The latest published research results and chemical composition isolation extended the list of saponins in Ngoc Linh ginseng to a total of 52 types

• Scientific name: Panax vietnamensis Ha & Grushv

• Name of two parts: Panax articulatus KL Dao (1973) ex Ha et Gruskv

(1985)

• Common name: Ngoc Ling ginseng, Vietnam ginseng, Khu Nam ginseng (K5 ginseng)

• Distribution: Vietnam is one of the few countries on the world's ginseng

distribution map such as Vu Diep ginseng (P bipinnatifidus), P stipuleanatus in Lao Cai and Lai Chao, Lai Chau Ginseng (P vietnamensis var fuscidiscus) is endemic to Lai Chau Province, and Ngoc Linh ginseng (P vietnamensis) in Kon

Tum and Quang Nam Currently, Ngoc Linh ginseng has only been discovered in the Ngoc Linh Mountain area of the Quang Nam and Kon Tum provinces

1.2.2 Natural features of Ngoc Linh ginseng

Ngoc Linh ginseng is currently spread in a restricted ecological area and grows and develops well at the top of Ngoc Linh Mountain, under the canopy of primeval woods in the territory of two provinces: Kon Tum and Quang Nam This region has geographic coordinates between 14°44' and 15°13' North latitude and 107°45' to 108°10' East longitude, and it is also the southernmost limit (within 15° north latitude) of the Panax L distribution map worldwide The following are the distinctive climate traits of the Ngoc Linh Mountain area: The average total annual rainfall ranges from 2,600 to 3,200 mm, the average annual temperature is

15 - 19°C, the total annual average evaporation ranges from 670 to 770 mm, and the average humidity ranges from 85 to 90% Ngoc Linh Ginseng is typically found in protected forest areas, with a coverage level of more than 70% Ginseng, both natural and produced, grows and develops exclusively under the canopy of

primeval woods, where a dense carpet of decay exists

• Vegetation

Ngoc Linh ginseng thrives in lowland and medium ridge continuous areas (cloud forests) at a height of roughly 1500 - 2000 m above sea level, on shale and silicate rocks, under moist forests, humus, and broadleaf trees Ngoc Linh ginseng grows naturally in regions where the forest canopy has not been disturbed It is where there are often numerous dead, collapsed, rotting trees, forming a dense rotting plant layer and a porous, usually moist soil layer The canopy covers around 60 - 90% of the area Ngoc Linh ginseng regenerates at random on the edge or under the forest canopy, which has been heavily damaged and has a steep slope It is often washed away completely in the heart of mountains or rocks where there is a lot of light Despite the fact that the weather in Central Vietnam is nearing the end of the dry season and the beginning of the rainy season, Sam Ngoc Linh grows strongly in spring and summer due to its distribution at an altitude of over 1,500 m and the original vegetation in the Ngoc Linh mountain area creating

a humid environment

• Topographic traits

Ngoc Linh is Vietnam's second-highest massif, forming part of the Southern Truong Son This massif is located in the provinces of Kon Tum, Quang Nam, Quang Ngai, and Gia Lai on the northern plateau of Vietnam's Central Highlands This massif extends northwest-southeast from Ngok Lum Heo mountain to Muong Hoong mountain, Ngoc Linh, Ngoc Krinh, Ngok Tem, and Ngok Roo, with an average elevation of 800 - 2,600 m Ngoc Linh had the highest peak observed (2,598 m) The Ngoc Linh Massif is distinguished by its strong separation, steep slope, and numerous narrow and deep valleys Ngoc Linh Ginseng lives at an altitude of 1,000 - 2,400 m above sea level, according to distribution traits, where the forest cover density is over 70%, and ginseng grows and develops well

evaporation, and low temperature

• Soil traits

Ngoc Linh ginseng thrives and develops exclusively beneath the canopy

of primordial forests with a thick decaying carpet, not underground This plant may also survive in humid conditions under the primary forest canopy According

to soil nutrient statistics, the physical and chemical features, particularly the criteria of the surface layer content, are very high Ngoc Linh ginseng grows in predictable settings that do not alter easily As a result, a specific approach is required to propagate and preserve Ngoc Linh ginseng

1.2.3 Vegetable traits, growth, and development of ginseng Ngoc Linh

Ngoc Linh ginseng has two types of stems: underground and vital The underground stem consists of many segments that develop continuously during the growth of Ngoc Linh ginseng The diameter of the underground stem is 2–3

cm or more, and it is branched, horizontal, and usually above ground Stem length increased, according to the number of years of growth The underground stem is light yellow or dark yellow, with many nodes bearing scars left by the body withered over the winter, so each scar corresponds to one year of age The aerobic stem grows upright, green, or purple, with a stem diameter from 0.3 to 0.6 cm, is

smooth, about 40 to 60 cm high, sometimes up to 1m tall and often withers in winter, leaving on rhizomes almost round scars The job of the stem is to transport nutrients and simultaneously burn the top of the qi body, which carries leaves, flowers, fruits, and seeds Thus, the size of biogas plants greatly affects plant growth and development The bigger, fatter and stronger the qi body is, it creates good conditions for the transmission of nutrients and assimilation products after photosynthesis to feed the plant, and at the same time helps the leaves, flowers, fruits and seeds of the plant grow and develop good From the top node of the stem, the leaves grow, and the leaves of Ngoc Linh ginseng grow according to the law of ascending Corresponding to the phylum of the first and second years, there

is only one compound leaf; in the third year, there are two compound leaves; in the fourth year, there are three compound leaves; and from the fifth year onwards, most plants have 4 - 5 compound leaves, which are very rare plants without compound leaves Compound leaves are propeller-shaped and consisting of 3-4 compound leaves, each with 5-7 oblong leaflets, 10–14 cm long, 3–5 cm wide, two pointed ends, serrated margins, and hard hairs on both 2 sides Leaflets are often uneven, and the middle leaf is usually the largest (15 cm long, 5–6 cm wide) When the plant is three–four years old and has three compound leaves, flowers appear, growing at the highest position of the phylum Flowers are self-inflorescences with peduncles attached to the phylum, inflorescence canopy, inflorescence peduncles 15 – 30 cm long, beyond the foliage (1.5 - 2 times longer than the compound petiole)

Flowers bloom from the outside in and from the bottom up The calyx drops 1 to 2 days after blooming, and the flower canopy begins to bear the fruit The blooming season varies by climate zone but usually runs from April to June every year Therefore, when growing ginseng to obtain seeds from flowers, fruits, and seeds, it is necessary to pay attention to the flowering time of each variety, favorable weather conditions, favorable conditions for pollination, and the

number of fruits and seeds Ginseng has good, uniform ripening This is also an important characteristic in the process of selecting Ngoc Linh ginseng varieties Ngoc Linh ginseng is a slightly flattened spherical berry, 0.6 - 1.0 cm in diameter, with a stigma that exists when ripe, has a bright red color, often with irregular black dots at the top of the fruit Seeds 1 or 2, white or ivory white, 6 – 7 mm long, 5 – 6 mm wide, 2 mm thick; grain surface rough with many protrusions On the eastern slopes of Ngoc Linh in Quang Nam province, the fruitful season may

be one month slower than that on the western slopes of Kon Tum province New buds appear at the beginning of January every year At the end of October, pneumatophores die gradually, leaves fall, leaving a scar on the head of ginseng, and the plant begins to hibernate through December When harvested, only ginseng from three years of age or preferably over five years of age should be collected The winter is also the season for harvesting the best ginseng rhizomes

Growth cycle of Ginseng Ngoc Linh:

• From January to March, ginseng develops aerial stems, leaves, and flower canopy

• From April to May, the plant begins to bloom In years of early rainy weather, trees usually grow early; therefore, they bloom around the end of March

• The green fruit period lasts 3 - 4 months (from after pollination) to the end

of July, and early August is the period of ripe fruits The fruit is fully ripe in August, particularly from late ripening to October When the fruit is ripe, the rind turns from green to yellow-green and then red-orange, with large black dots accounting for 1/4 to 1/5 appearing at the top fruit

• From October to December every year, after the ripe fruit season, the leaves begin to turn yellow, and the entire aboveground part dies

1.2.4 Evaluation of Genetic Diversity of Plant Species – Ginseng

• Mophology

Panax vietnamensis (Vietnamese ginseng, Ngoc Linh ginseng) is an

endemic Panax species of Vietnam that has been evaluated by scientists and professional agencies both at home and abroad as one of the four most valuable ginseng plants in the world (American ginseng, Korean ginseng, False ginseng, and Vietnamese ginseng) Since 1973, local and international experts have undertaken extensive research and discovered that Ngoc Linh ginseng stems and roots have a significantly greater saponin content than other ginseng species Ngoc Linh ginseng possesses distinguishing traits that Korean and Chinese ginseng do not Vietnamese ginseng has been shown to have antibacterial, antidepressant, anxiety-reducing, and antioxidant qualities, as well as the ability

to enhance the effectiveness of antibiotics and diabetes medications anti-cancer,

and prevention of aging (Tran et al 2002)

Nguyen Xuan Phuc (now President) visited the Ngoc Linh ginseng farming area of Ngoc Linh Kon Tum Ginseng JSC in 2018 Ngoc Linh ginseng has been named a "Vietnam's national treasure" by Prime Minister Nguyen Xuan Phuc

Research "Genetic diversity based on morphological traits of ngoc linh

ginseng (panax vietnamensis ha et grushv.) at Nam Tra My, Quang Nam (2018) ''

(Truong et al 2018) This study assessed the uniformity and morphological traits

of Ngoc Linh Ginseng at Nam Tra My, Quang Nam, Vietnam The individuals were collected from different areas of Ngoc Linh Mountain and subjected to detailed morphological characterization Most ginseng individuals in the population were highly uniform and belonged to one genetic group The number

of leaves and leaf length and width can be used as critical morphological traits for evaluating and dividing individuals into subgroups

"Morphological characteristics of ngoc linh ginseng from Nam Tra My District, Quang Nam Province (2018)" (Truong Thi et al 2018) In this study, they revealed the morphological characteristics of 4- and 5-year-old Ngoc Linh ginseng in order to build a seedling center for the conservation and growth of high-quality plants for production in the Nam Tra My district of Quang Nam province Ginseng plants aged 4 to 5 years have one stem with a variety of morphological traits and stem, leaf, and root sizes Four-year-old plants had smaller leaves and stems than five-year-old plants, and the full flower lacked the characteristics of a Ngoc Linh ginseng semi-circle form The pigment anthocyanin was found at the top of the stem Five-year-old Ngoc Linh ginseng plants displayed the species traits: long stems, complete blossom with a semi-circular shape, and no anthocyanin coloring at the top of the stem Therefore, 5- year-old Ngoc Linh ginseng plants should be used to study their typical morphological traits

According to study subjects and projects, this is an important component

in encouraging the protection of Ngoc Linh ginseng's genetic resources, promoting local strengths, and simultaneously adjusting production, crop structure, and enhancing production Increase regional revenue, provide people with incentives to protect forests, and maintain natural resources and biodiversity

A solid and long-term legal corridor in Quang Nam province is being constructed

to encourage investment in the conservation, development, processing, and branding of Ngoc Linh ginseng

Molecular

Ngoc Linh ginseng is a species with high genetic diversity and much higher saponin content than other Panax species Due to its narrow distribution range, over-exploitation, and low natural resilience, the natural populations of Ngoc Linh ginseng have almost disappeared (Ministry of Science and Technology, 2007) The price of Ngoc Linh ginseng is high due to its multiple

uses, and there are many fake samples of this ginseng species (the fake samples

are usually other species or subspecies of the same Panax genus marketed in

Vietnam) As a result, precisely identifying Ngoc Linh ginseng species becomes increasingly challenging Because sampling does not always give standard samples, traditional taxonomy based solely on morphology is difficult to execute

in practice (with sufficient flowers and fruits) As a result, assistance from experts

in molecular biology techniques is required This is especially true for uncommon and frequently imitated species like Ngoc Linh ginseng

Gene flow, genetic drift, mutation, and natural selection are all used to assess genetic diversity (Hamrick et al 1992), and genetic structure and variation are related to a variety of elements such as distribution range, evolutionary history, life cycle, and mating systems (Frankham et al 2002) Thus, genetic diversity plays an important role in reflecting a species' resilience to changes in its environment Genetic variety in small populations may be reduced by genetic drift and increased homozygozity for common alleles (Gijbels et al., 2015), reducing future viability and evolutionary potential (Bijlsma et al., 1997) As a result, it is critical to recognize that understanding population genetic structure is critical to evaluating evolutionary traits, which can lead to the establishment of a species conservation program (Hamrick and Godt, 1996a)

Molecular techniques are now frequently utilized in the investigation of

genetic diversity, and they have aided in the conservation and management of endangered species (Bruford et al., 2017) There have been numerous scientific investigations on Ngoc Linh ginseng in Vietnam There are very few works that focus on study, classification, identification of distribution region, research on chemical composition, testing of hepatoprotective function, and chloroplast

genome decoding Population genetics of Ngoc Linh ginseng

Several studies have been conducted to investigate genetic diversity within and between plant populations The Far East Soil Institute (Russia) conducted

research on the genetic diversity of species populations in partnership with the personnel of the Department of Molecular Systematics & Conservation Genetics, Institute of Ecology & Biological Resources ISSR and AFLP were used to identify Russian ginseng (Panax ginseng) and Vietnamese ginseng (Panax vietnamensis) This is the first study on Vietnamese ginseng population genetics (Galina, 2010; Elena, 2018) studied and compared the genetic diversity of Panax ginseng CA Mayer using three distinct approaches and found low levels with allozymes and high levels with AFLP and SSR methods However, they demonstrated that employing allozymes to develop successful conservation methods proved difficult Despite the fact that AFLP markers revealed substantial genetic variety, it was hard to directly calculate allele frequencies to measure genetic variability within and between populations Because of their codominance and polymorphism, microsatellites (SSRs) have been shown to successfully describe genetic diversity Similarly, ISSR markers used in the genetic diversity research of P stipuleanatus Tsai in Vietnam and RAPD for P ginseng in Russia (Reunova et al 2014) revealed a limitation in estimations of genetic variety within populations and divergence within populations SSR studies in Panax species have been employed (Kim et al 2007; Van Dan et al 2010; Liu et al 2011; Reunova et al 2014) To study population genetics, a set of SSRs was created using expressed sequence tags (EST) We used EST-SSRs derived from P vietnamensis in this study to evaluate the genetic diversity and population

structure of P vietnamensis, as well as to provide a platform for conservation,

restoration, and sustainable exploitation of this endangered species in Vietnam

Because SSR markers are highly polymorphic and distinct to each studied species, they are regarded the most effective approach for assessing genetic diversity at the species and population levels SSRs are distinct nucleotide sequences that are derived from short, simple oligonucleotide segments and repeated several times SSR has the advantage of being a co-dominant marker

with high specificity and the ability to detect polymorphisms, which helps minimize misunderstanding in genome analysis SSRs derived from genomic transcription (EST-SSRs) can aid in species identification Furthermore, because they are located in genomic areas recognized as unique, EST-SSR markers built particularly for a species frequently exhibit a degree of well-characterized variability Because of their high specificity, EST-SSRs have been used to aid in the evaluation of breeding and classification in many economically valuable/exported species, including sweet potato (Ipomoea batatas), Cymbidium

sinense, and Russian ginseng (Panax ginseng) SSR is extensively employed in

research on genetic diversity at the species and population levels, genetic exchange, genetic structure, and population reproductive interactions (Kim 2007)

PART 2 MATERIAL AND METHODS

2.1 Material, time and place

- Material: Collecting random 120 individuals of Ngoc Linh ginseng over

6 years old in the original breed garden of Ngoc Linh Ginseng, Kon Tum province

- Time and Place:

+ Place: Department of Molecular Biology - Agricultural Genetics Institute (AGI)

+ Time: From August 2022 to December 2022

2.2 Methods

2.2.1 Morphological Evaluation

Data analysis: The data is synthesized and processed by Excel 2010 The

tree diagram is built based on Euclidean distance using NTSYSpc 2.1 software After that, evaluating the homogeneity of ginseng populations of Ngoc Linh in the original breed garden

2.2.2 Molecular Method

2.2.2.1 DNA Extraction

DNA extraction protocol by CTAB (Cetyltrimethylammonium bromide)

as described by Murray and Thompson, 1980

• Step 1: Take 200mg of fresh root sample into a 2ml Eppendorf tube and grind with liquid nitrogen to a fine powder

• Step 2: Add 1.2ml of CTAB buffer which has been heated at 65°C before, and vortex for 5-10 seconds to mix well

• Step 3: Heat in a hot bath at 65°C for 1 hour 30 minutes to 2 hours Every 5-10 min invert each Eppendorf tube to mix the solution inside

• Step 4: Centrifuge for 10 minutes at 13500 rpm to deposit the residue

• Step 5: Draw 800 µl of phenol: chloroform: Isoamyl alcohol solution into

a 1.5ml Eppendorf tube Prepare each tube corresponding to a sample to be separated

• Step 6: Aspirate 800 µl of the supernatant in step 4 into a 1.5 ml Eppendorf tube containing a solution of phenol: chloroform: Isoamyl alcohol

• Step 7: Close the cap tightly and mix gently by inverting the tube for 20 minutes at room temperature 23°C Centrifuge for 10 minutes at 13500 rpm to separate into 2 layers (phases) of the solution

• Step 8: Carefully aspirate the supernatant into a tube containing 800 µl of cold isopropanol (-20°C) and mix gently by inverting the tube for 10 min at room temperature 23°C to precipitate the DNA

• Step 9: Centrifuge for 10 minutes at 13500 rpm to deposit DNA Remove the supernatant, then dissolve the DNA precipitate with 250 µl TE at room temperature

• Step 10: Add 2.5µl DNase-free RNase, mix well and incubate at 37°C for

30 minutes

• Step 11: Add 25 l of 3M NaAc solution and mix Then add 600 µl cold ethanol ( -20°C), mix, and cool incubate at -20°C for 20 minutes to precipitate DNA

• Step 12: Centrifuge for 10 minutes at 13500 rpm to deposit DNA

• Step 13: Carefully remove the supernatant, retaining the DNA precipitate Then rinse the DNA 1-2 times with 70% (v/v) cold ethanol

• Step 14: Dry the DNA with a vacuum machine for 10-15 minutes Dissolve DNA with 25µl deionized water or TE solution and store at -20°C for long-term use

2.2.2.2 Measure DNA concentration

DNA samples were measured by NanoDrop2000 to determine the concentration and quality based on the measurement index at 260/280 nm Then proceed to dilute to a concentration of 50 ng/µl to run the PCR reaction

2.2.2.3 Polymerase chain reactions (PCRs)

8 molecular markers used for genetic analysis at the molecular level of Ngoc Linh ginseng samples (Table 2.1)

Table 2.1: List SSR markers

size (bp)

Reference

(Khuat et al 2018)

Table 2.2: PCR reaction components

The amplification reaction was performed as follows: denaturation 5 min

at 94°C, 35 cycles at 94°C 30s, primer annealing temperature 52°C to 60°C depending on each primer respectively for 45 seconds, lasting at 72°C for 2 minutes, and then ending at 72°C for 7 minutes PCR products were analyzed using 2,5% agarose gel electrophoresis Gels were stained with ethidium bromide and observed and imaged under a UV

2.2.2.4 PCR products were analyzed

PCR products were electrophoresized in 2,5% agarose gel and 8% polyacrylamide gel Gels were stained with ethidium bromide and photographed using a UVP GelDoc-It2 Imager

A, Gel agarose 2,5%

• Prepare tray, comb and 2.5% agarose gel

• In a microwave, the agarose solution was cooked until it became uniform Raise the temperature of the agarose gel to around 50°C Shake thoroughly 5µL of ethidium bromide (EtBr) at a concentration of 10 µg/ml

• • After inserting the comb into the prepared gel tray, the EtBr agarose gel mixture was poured into the gel tray Wait 45-60 minutes for the gel to solidify, paying close attention to pouring to avoid air bubbles

• The comb was withdrawn, and the gel was placed in the electrophoresis tank Pour 0.5 × TBE into the gel surface at approximately 2 mm Place the PCR product mixed with xylene cyanol into the well at a ratio of 5µl Xylene cyanol/15µl sample Prepare 0.5 × TBE buffer for the electrophoresis bath

• Wash the gel in H2O, place it in the UV scanner, and take a picture

B, 8% polyacrylamide gel

• Make the binding solution: 3μl Bind Silane + 1 ml 0.5% acetic acid in 95% ethanol The mounting solution was evenly smeared on glass plate 1 with soft paper Allow 5 minutes for drying Avoid obtaining this solution on a two glass plate Apply 3 mL of Sigmacote to the surface of the glass sheet On glass, the Sigmacote was evenly applied with dry soft paper Allow 5 minutes for drying Assemble the glass in order to pour the gel Place the comb in a gel (6–8 mm)

The gel was left to spontaneously freeze for at least 2 hours

Table 2.3: 8% polyacrylamide gel components

Complement 8% polyacrylamide gel

- Electrophoresis of PCR products on polyacrylamide gels

• The comb was pulled out of the gel The sticky gel was wiped from the top of the gel mold with distilled water

• After that, the gel was placed on a running stand Fill the buffer chamber with 2 liters of TBE 1X (10.8 g, Tris base: 0.92 g, EDTA: 5.52 g Boric acid

in 1 liter of solution) To remove air bubbles from the gel mold's upper

surface, a syringe was utilized The wells were then filled with 8 µl of each sample The gel was then operated at 100V for 2 hours After staining the gel with EtBr for 15 minutes, place it in the UV scanner and take photos

Morphological evaluation and molecular method based on using the software

NTSYS-pc (numerical taxonomy and multivariate systems) Version 2.01.